Cell Biology Exam 2

What is an example of an endocrine signal?

hormones

What would you conclude if a 30 kDa band also appeared in Lane 1 of Fig 3A?

if I saw a 30 kDa band in lane 1, this would suggest that the wildtype alga also produce the aCD22 antibody even though they do not have the transgene, and this could suggest an error or that the wildtypes do accumulate the antibody

What is immunostaining?

immunostaining is any use of an antibody-based method to detect a specific protein in a sample. (i.e. Western blot)

When the alpha-subunit of a G protein is bound to GDP the complex is

inactive

In affinity chromatography, what binds to the column?

proteins that can attach to the molecule that is attached to the column matrix

The largest class of enzyme coupled receptors are

receptor tyrosine kinases

Polysaccharides are broken down into

simple sugars

What are flilopodia?

slender cytoplasmic projections that extend beyond the lemellipodium in migrating cells that contain actin filaments cross-linked into bundles by actin binding proteins

Describe the general composition of intracellular signaling molecules

small and hydrophobic and can cross the lipid bilayer (things like hormones and NO)

What is IP3?

small, intracellular signaling molecule that triggers the release of calcium ions from the endoplasmic reticulum into the cytosol and the calcium signals other proteins in the cytosol; produced when a G protein molecule activates membrane bound phospholipase C.

Microtubules are protofilaments built of the protein ______

tubulin

How many electrons does a hydrogen atom have?

1

How many polypeptide chains compose a typical G-protein coupled receptor (GPCR) and where might we find this protein?

1 polypeptide chain that threads across the lipid bilayer 7x

Do co-localization see if two proteins interact?

No! Co-localization does NOT test protein-protein interactions. It addresses the question of where a certain protein is located in the cell

If a cell is treated with cochicine, it is unable to divide. Explain how this drug works

1) Cochicine binds tightly to free tubulin dimers and prevents their polymerization into microtubules, so the mitotic spindle rapidly disappears and the cell stalls in the middle of mitosis because it is unable to partition the chromosomes into 2 groups, since the mitotic spindle is a microtubule based apparatus that guides the chromosomes during mitosis and the inactivation or destruction of the mitotic spindle will eventually kill dividing cells. Also, dividing cells need to divide their organelles so that the regional differences in internal membranes are maintained after division Because both the ER and Golgi are attached to microtubules, when cells are treated with cochicine, both the ER and Golgi change their location dramatically. The ER, which is connected to the nucleus, collapses around the nucleus, and the Golgi, which si not attached to any other organelle, fragments into small vesicles around the cytoplasm.

How is a cell's cytoskeleton like the bony skeleton of our bodies? How is it different?

1) It is similar, because it allows the eukaryotic cell to adopt a variety of shapes, interact mechanically with the environment and carry out coordinated movements. It helps support the cell. It is different, because it is constantly reorganized as a cell changes shape, divides, and responds to the environment. The cytoskeleton also serves as the cell's muscles, and is responsible for large-scale movements. It also helps organize compartments, unlike out bones, and is involved in the segregation of chromosomes into 2 daughter cells, and when the cell divides, pinching off the 2 cells.

Describe the function of γ-tubulin rings in the centrosome?

1) The centrosome matrix includes ring-shaped structures formed from gamma tubulin and each gamma tubulin ring complex serves as the starting point for the growth of one microtubule and the alpha-beta-tubulin dimers add to each gamma tubulin ring complex in a specific orientation with the result that the minus end of each microtubule is embedded in the centrosome and growth occurs only at the plus end that extends throughout the cytoplasm. It is very difficult to start a new microtubule from scratch, but adding alpha beta tubulin dimers to the gamma tubulin is much easier. In a living cell, the concentration of free alpha beta tubulin is too low to drive the difficult first step of assembling the initial ring, but by providing organizing sites via gamma-tubulin and keeping the concentration of free alpha beta tubulin dimers low, the cells can control where microtubules form The centrioles in the middle of the centrosome have no role in serving as the starting point (nucleation site) for new microtubules, the gamma tubulin rings alone are sufficient

Why do eukaryotic cells, especially animal cells have a large and complex cytoskeleton? List the differences between animal cells and bacterial cells that depend on the eukaryotic cytoskeleton.

1. Animal cells are much larger, more diversely shaped, and do not have a cell wall. 2. Animal cells and other eukaryotic cells have a nucleus that is shaped and held in place by intermediate filaments 3. Animal cells have a much larger genome than bacteria and this genome is fragmented into many chromosomes. For cell division, chromosomes need to be accurately distributed to the daughter cells, requiring the mitotic spindle 4. Animal cells have internal organelles. Their localization in the cell is dependent on motor proteins to move them along microtubules

Briefly outline the basic steps required to perform the assay represented in 6A-D.

1. CA-46, Ramos, and Jurkat cells were treated in triplicate with varying doses of each of the antibodies (Negative control: cycloheximide was added, which inhibits protein synthesis to kill all of the cells. Positive control: no antibodies added) 2. Each plate was incubated for the same time in the same temperature for 72 hours 3. After incubation, WST-8 (a tetrazolium salt) reagent was added and absorbance was read for each cell type and for each dose of antibody 4. Cytotoxicity was measured based on cell survival

What are the steps of colocalization using a transgene?

1. Introduce 2 transgenes or antibodies into the nucleus of the cell. One is going to be promoter + gene 1 + GFP and the other will be promoter + marker gene that is abundant in a known place + RFP. 2. Then, view the cells under the fluorescent microscope. 3. If you see red and green specks separately, this means that they do not co-localize, but if you see yellow specks, this suggests co-localization Using a transgene, we can use live cells

8 different functions of actin binding proteins.

1. Monomer sequester protein 2. Nucleating 3. Severing 4. Cross-linking 5. Capping 6. Side-binding protein 7. Myosin motor protein 8. Bundling protein

What are the steps of colocalization using an antibody?

1. Permeablize the membrane so the antibodies can get in (this kills the cells) 2. Introduce 2 antibodies, one for a marker and one for another protein. The antibodies will go find their protein and light up 3. Then, view the cells under the fluorescent microscope. 4. If you see red and green specks separately, this means that they do not co-localize, but if you see yellow specks, this suggests co-localization

What are the 3 steps scientists use to piece together intracellular signaling pathways?

1. Screen for mutants in which signaling pathways are not functioning a. Find the gene and the protein for which it encodes 2. Determine the order in which proteins act in a pathway 3. Then look at protein-protein interactions in the pathway

The Ras protein functions as a molecular switch that is set in its "on" state by other proteins that cause it to expel GDP and bind GTP. A GTPase-activating protein helps reset the switch to "off" by inducing Ras to hydrolyze its bound GTP to GDP much more rapidly than it would without encouragement. You are given a mutant cell that lacks the GTPase-activating protein. What abnormalities would you expect to find in the way Ras activity responds to extracellular signals?

1. There would be a high background level of Ras activity since it cannot be turned off efficiently 2. Because some molecules of Ras would already GTP-bound, Ras activity in response to extracellular signal would be greater than normal, but the activity will likely saturate when all Ras molecules are converted to the GTP-bound form 3. The increase in Ras activity in response to a signal would be prolonged compared to the response in normal cells

Describe 4 functions of intracellular signaling pathways.

1. They can relay the signal onward and help it spread around the cell 2. They can amplify the signal received to make it stronger so that a few extracellular signal molecules are enough to evoke a large intracellular response 3. They can detect signals from more than 1 intracellular signaling pathways and integrate them before relaying the signal onward 4. They can distribute the signal to more than 1 effector protein to create branches in the information flow diagram to evoke a complex response

The first stage of cellular respiration is what?

The breakdown of foods into simple subunits in catabolic reactions

Does glycolysis produce energy? In what form?

2 net x ATP and 2 x NADH

A mutant has an inactive form of Ras. What could cause Ras to become inactive? How could we reactivate the downstream events of this pathway?

A Ras mutant could be unable to release GDP or bind GTP, CAUSED BY A STRUCTURAL CHANGE in the GTP/GDP domain. As a result, the MAP kinase cascade will not be activated. To reactive the pathway, we could express a permanently phosphorylated form of MAP kinase kinase kinase, which would initiate the phosphorylation cascade constitutively.

The dynamic instability of microtubules allows them to grow and shrink. The rapid remodeling of microtubules is crucial to their function. How can a microtubule be both stabilized in a cell and prevented from de-polymerizing?

A microtubule can be prevented from disassembling if its plus end is stabilized by attachment to a protein, called a plus end binding protein. A microtubules minus is stabilized by its association with the centromere, but the plus end is where tubulin dimers are added and removed, so in order for the plus side to be prevented from depolymerizing, it must be bound to something

A particular type of C.elegans mutant becomes paralyzed when the temperature is raised. The mutation affects axonal transport in neurons.Propose one testable hypotheses that explain how these loss-of-function mutations, specifically affect axonal transport, and describe a technique that you would use to test your hypothesis and why you chose that technique.

A mutation in kinesin gene so that at higher temperatures, the kinesin protein cannot bind to the tubulin in microtubules to transport things down the axon, and the things that are needed for signal transmission cannot get transported down the axon, so the C. elegans is paralyzed since it cannot stimulate its muscle cells. I would to use a kymograph, because with a kymograph I can visualize movement of molecules within the cell and chart it to see if molecules do more, or do not move. I will fluorescently tag kinesin proteins by making a transgene that is promoter + Kinesin gene + GFP and I will view them under the fluorescent microscope and then take a video and I can plot their movement on a time vs. distance plot to see if the motor proteins did move at all. I will have a wildtype to compare to my mutant and compare at high and low temperatures (WT at high, WT at low, mutant at high, and mutant at low). If my hypothesis was correct, my kymograph would just show straight lines for the distance travelled over time for the fluorescently labeled kinesin proteins because they do not move.

G-protein coupled receptors are a very common type of switch used to control signal transduction. GPCRs activate G-proteins. An α-subunit of a G-protein bound to GDP is inactive, but becomes active when the GDP is replaced by GTP. This releases the ß and γ subunits, which are involved in activating signaling pathways within the cell. What process could be involved in turning a GPCR off (GTP -> GDP)?

A phosphatase could dephosphorylate the GTP bound to the G-protein, reverting the G-protein to its inactive form (bound to GDP)

Describe the function of a motor protein and explain how it works.

A protein molecule traveling by diffusion may take an extremely long time to reach its target destination, if it reached that destination at all. Motor proteins are proteins that actively transport organelles, vesicles, and other macromolecules along microtubules. Motor proteins use the energy derived from ATP hydrolysis to travel along a microtubule or actin filament in a single direction. Motor proteins attach to other cell components, and transport the cargo along the filaments. Motor proteins are dimers that have 2 globular ATP-binding heads and a single tail. The heads interact with microtubules in a stereospecific manner so that the motor protein will attach to a microtubule in only one direction. The tail of the motor protein generally binds to a cell component, and this determines the type of cargo that the motor protein can transport. The globular heads are enzymes with ATP-hydrolyzing activity, which provides the energy for driving a directed series of conformational changes in the head that enable it to move along the microtubule by a cycle of binding, release, and rebinding to the microtubule.

The same signaling molecule can elicit a different response in different cells. Explain how this works.

A signal from a cell surface receptor is conveyed to the target cell interior via intracellular signaling molecules that act in sequence. The intracellular relay system and intracellular effector proteins on which it acts vary from one type of specialized cell to another, so different cells respond to the same signal in different ways. In addition, a combination of signals can evoke a response that is different from the sum of the effects that each signal would trigger on its own. This tailoring process occurs in part because the intracellular relay signals activated by different signals interact

How is a GPCR involved in amplification of signal?

A single GPCR can activate multiple proteins within the cell before it is inactivated and as long as it's active (bound to GTP) it can interact with multiple downstream target proteins, therefore amplifying the signal. For example, one GPCR activates one G protein attached to the plasma membrane, which activates adenyl cyclase, and from there adenyl cyclase can synthesize many, many molecules of cAMP from ATP and cAMP can activate many proteins downstream

In ion-exchange chromatography, what can we use to release a molecule bound to the column?

A solvent that alters the pH of the column because depending on the pH, a protein can have a net negative, net positive, or neutral charge. If the protein was negatively charged, it would bind to a positively charged column, but if you decreased the pH of the solvent, the negatively charged protein would pick up an H+ and no longer be attracted to the positively charged column and be eluted.

Ca2+ pumps in both the plasma membrane and ER membrane maintain a low concentration of Ca2+ in the cytoplasm. You have isolated cardiac myoctyes (heart muscle cells) with abnormally high levels of Ca2+ in the cytosol and have even confirmed the presence of a base pair substitution in the gene, but this does not alter the expression and the protein is still made. You wonder what happens to the protein in the mutant myocytes. A) Propose a testable hypothesis that could explain why pump function is altered in your mutant. B) Design an experiment that would allow you to test your hypothesis.

A) I hypothesize that the pumps are made, but the base pair substitution prevents them from from incorporating into the cell membrane, and they stay in the cytosol and cannot pump the calcium back in. The base pair substitution might have changed one amino acid from a nonpolar to a polar amino acid, and if this amino acid was one that normally is in the membrane spanning part of the protein, it could not incorporate into the membrane stably. B) I will use co-localization and attach GFP to the gene for the mutant pump and the promoter for the mutant pump and also use a marker gene of a protein that I know is in the ER membrane and plasma membrane that is fused with RFP and I will insert those into the nucleus of the cell in development and after the cell is big, I will look at the cell under a fluorescent microscope to see if my hypothesis is true. If my hypothesis is true, I will see the green in a different location than the red, meaning that the calcium pump is not in the membrane. I will see different colors becasue the fluorescent proteins will fluoresce at different wavelengths and thus emit different colors

In which of the following reactions is the starred atom oxidized? A) Na*--> Na+ B) Cl* --> Cl- C) C*H3CH2OH --> CH3CHO D) CH3C*HO --> CH3COO- E) C*H2=CH2 --> CH3CH3

A) Na*--> Na+ D) CH3C*HO --> CH3COO-

Compared to the normal situation, in which actin monomers carry ATP, what do you predict would happen if actin monomers that bind a nonhydrolyzable form of ATP were incorporated into actin filaments? A. Actin filaments would grow longer. B. Actin filaments would grow shorter because depolymerization would be enhanced. C. Actin filaments would grow shorter because new monomers could not be added to the filaments. D. No change, as addition of monomers binding nonhydrolyzable ATP would not affect actin filament length.

A. Actin filaments would grow longer.

Which of the following statements about the function of the centrosome is false? A. Microtubules emanating from the centrosome have alternating polarity such that some have their plus end attached to the centrosome while others have their minus end attached to the centrosome. B. Centrosomes contain hundreds of copies of the γ-tubulin ring complex important for microtubule nucleation. C. Centrosomes typically contain a pair of centrioles, which is made up of a cylindrical array of short microtubules. D. Centrosomes are the major microtubule-organizing center in animal cells.

A. Microtubules emanating from the centrosome have alternating polarity such that some have their plus end attached to the centrosome while others have their minus end attached to the centrosome.

Cortisol, made in the adrenal gland, and regulates metabolism in the body. Insulin made in the pancreas regulates glucose uptake. What type of signaling molecules are these? A. Endocrine B. Paracrine C. Autocrine D. Contact dependent

A. Endocrine

What is the technique that separates a homogenate into different parts?

Centrifugation

Which of the following hypotheses could be addressed by running a western blot on a fraction of cell homogenate/lysate isolated using differential centrifugation? Circle all that are correct. A. Protein A is targeted for degradation in the mutant and is thus less abundant in the mitochondria B. The size of protein A is altered in a mutant of interest C. Peroxisomes are more abundant in a mutant of interest

A. Protein A is targeted for degradation in the mutant and is thus less abundant in the mitochondria B. The size of protein A is altered in a mutant of interest (Western blots do not detect organelles)

Which of the situations below will enhance microtubule shrinkage? A. addition of a drug that inhibits GTP exchange on free tubulin dimers B. addition of a drug that inhibits hydrolysis of the GTP carried by tubulin dimers C. addition of a drug that increases the affinity of tubulin molecules carrying GDP for other tubulin molecules D. addition of a drug that blocks the ability of a tubulin dimer to bind to γ-tubulin

A. addition of a drug that inhibits GTP exchange on free tubulin dimers (Tubulin dimers that are released from the shrinking microtubule exchange their bound GDP for GTP when they are back in the cytosol, becoming able to add to another microtubule that is growing)

Which is likely to occur more rapidly in response to an extracellular signal. A. changes in protein phosphorylation B. changes in proteins being synthesized

A. changes in protein phosphorylation

What are ARP complexes?

ARP complexes promote the formation of a web of branched actin filaments in lamellipodia because they form complexes that bind to the sides of existing actin filaments and nucleate the formation of new filaments, which grow out at an angle to produce side branches.

Generate a list of favorable reactions (exergonic) involved in cellular respiration.

ATP -> ADP Glucose -> Pyruvate -> acetyl CoA NADH -> NAD+ FADH -> FAD (Oxidation reactions)

Explain why all of the actin monomers in the cell don't polymerize and the cell is able to keep a reserve of monomers available

About half of the actin in a cell is assembled into filaments, and the other half remains as actin monomers in the cytosol, and the concentration of actin is much higher than the concentration required for purified actin monomers to polymerize spontaneously, but cells contain small proteins that bind to actin monomers in the cytosol to prevent them from adding to the ends of actin filaments and play a crucial role in regulating actin polymerization.

In the mitochondria, pyruvate gets converted to what?

Acetyl-CoA

With each turn of the citric acid cycle, what gets oxidized?

Acetyl-CoA is oxidized because it is donating hydrogen atoms to NAD+ and the rest of the molecule is just CO2 and that is released by our bodies when we breathe

Differentiate between dynamic instability and treadmilling

Actin filaments treadmill, which is where actin monomers are being added to one side and being removed from the other side, and when they treadmill, they stay approximately the same size. Microtubules undergo dynamic instability, which is when they add tubulin dimers to the plus side, and then all of a sudden the tubulin dimers get removed from that same side, and when they are growing and shrinking, the growth or shrinkage is very drastic

Explain how a signaling cascade with Ras protein and MAP kinases relay a message from the extracellular space to the nucleus

Activated RTKs recruit and activate many intracellular signaling proteins, one of them being Ras, which is a GTP-binding protein that is bound by a lipid tail to the plasma membrane on the cytosolic face. Ras resembles the alpha subunit of a G protein and functions as a molecular switch in the same way, it is active when it is bound to GTP and inactive when it is bound to GDP. First, an adaptor protein docks on a particular phosphotyrosine on the activator protein on the RTK, and recruits Ras guanine nucleotide exchange factor. Ras-GEF encourages Ras to exchange GDP for GTP to activate it. When it is active, Ras initiates a phosphorylation cascade. One of the molecules Ras activates is MAP kinase kinase kinase, which phosphorylates MAP kinase kinase, which activates MAP kinase and at the end of the signaling cascade, MAP kinase phosphorylates certain transcription regulators, altering their ability to control gene transcription.

What are similarities and differences between the reactions that lead to the activation of G proteins and the reactions that lead to the activation of Ras?

Activation in both cases depends on proteins that catalyze GDP-GTP exchange on the G protein or the Ras protein. Activated GPCRs perform this function directly on G proteins, enzyme-linked receptors assemble multiple signaling proteins into a signaling complex when the receptors are activated by phosphorylation, one of them being an adaptor protein that recruits a guanine nucleotide exchanger that fulfills the function for Ras

The most frequent target enzymes for G proteins are what?

Adenyl cyclase, which produces cyclic AMP and phospholipase C, which produces IP3 and DAG

Explain how adrenaline stimulates glycogen breakdown in skeletal muscle cells.

Adrenaline binds to GCPR, changing the conformation of the GPCR, which alters the conformation of a G protein, which decreases the G protein's affinity for GDP, allowing it to release GDP and bind to GTP. The binding of GTP activates the G protein. The alpha subunit of the activated G protein switches on adenylyl cyclase, causing an increase in the synthesis of cyclic AMP from ATP. Cyclic AMP activates cyclic AMP-dependent protein kinase A, which is normally held inactive in a complex with a regulatory protein, but the binding of cyclic AMP to the regulatory protein forces a conformational change that releases the active kinase. Activated PKA catalyzes the phosphorylation of phosphorylase kinase by hydrolyzing ATP to ADP and transfers the phosphate to phosphorylase kinase, activating it, which hydrolyzes another ATP to ADP and transfers this phosphate to another target protein, and this target protein breaks down glycogen to glucose.

How does the binding of adrenaline to the GPCR promote an interaction between Gα and adenylyl cyclase?

Adrenaline, the signaling molecule binds to the GPCR at the membrane. GPCR changes conformation and can interact with the Galpha to induce it to release GDP and bind to GTP. When the G protein's alpha subunit binds to GTP, it is in a conformation that is activated now and it can interact with adenyl cyclase

Yeast cells can grow both in the presence of O2 and in its absence. Under which of the 2 conditions could you expect the cells to grow better?

Aerobically. Under anaerobic conditions, they cannot perform oxidative phosphorylation and therefore they have to produce all of their ATP by glycolysis, which is less efficient.

Understand the mechanisms that recycle postglycolytic NADH molecules under aerobic conditions

After glycolysis, under aerobic conditions, the NADH molecule travels to the inner mitochondrial membrane and donate their electrons, which release energy as they are passed from one protein to the other and go from a state of higher energy to a lower one. In releasing electrons, NADH is converted back to NAD+ which is available to be used again in glycolysis.

From figure 3, if the scientists in this experiment had not built the Flag peptide into their recombinant proteins, how else could they have recognized their proteins of interest? Could they have used alternate antibodies to recognize other parts of their proteins? Why or why not?

Alternate antibodies that target the specific chimeric immunotoxins, but they likely do not exist and antibodies would need to be developed in an animal and purified and validated, which would cost a lot of money. If there is a specific antibody for aCD22 or PE40, it is possible that they could use those, but they would need to make sure that the antibody binds to the specific domains that they incorporated into the immunotoxin since they only used certain pieces of the full proteins to make the immunotoxins, not the full proteins themselves

What is acetyl-CoA?

An activated carrier that donates the carbon atoms in its acetyl group to the citric acid cycle; the acetyl group is linked to coenzyme A by a thirster bond that releases a lot of energy when hydrolyzed

What is a high energy electron?

An easily transferred electron; they are more loosely bound to the donor molecule

Activation of a GPCR can lead to transcription. Describe how an extracellular signal acting on a GPCR could alter the activity of a regulatory protein via phosphorylation of a transcription factor

An extracellular signal molecule binds to GCPR, changing the conformation of the GPCR, which alters the conformation of a G protein, which decreases the G protein's affinity for GDP, allowing it to release GDP and bind to GTP. The binding of GTP activates the G protein. The alpha subunit of the activated G protein switches on adenylyl cyclase, causing an increase in the synthesis of cyclic AMP from ATP. Cyclic AMP activates cyclic AMP-dependent protein kinase A, which is normally held inactive in a complex with a regulatory protein, but the binding of cyclic AMP to the regulatory protein forces a conformational change that releases the active kinase. Activated PKA catalyzes the phosphorylation of serines and threonines on specific intracellular proteins, altering their activity. PKA moves into the nucleus and phosphorylates an inactive transcription factor, activating it, which then activate transcription of certain genes.

What are phospho-antibodies?

Antibodies that detect the phosphorylated versions of proteins and this can help determine the status of signaling molecules in the cell

In the electron transport chain, H+ must be pumped against their concentration gradient into the inner membrane space which requires energy, where does this energy come from?

As the electrons get dumped off at the membrane and shuttled off between proteins, they go from a high energy state and go to a lower energy state as they move through the electron transport chain in the membrane and they lose energy, and the protons are pumped across the membrane

Which of these reactions will occur only if a second energetically favorable reaction is coupled? A) Glucose + O2 --> CO2 + H2O B) CO2 + H2O --> glucose + O2 C) Nucleoside triphosphate --> DNA D) Nucleotide bases --> nucleoside triphosphates E) ADP + P --> ATP

B) CO2 + H2O --> glucose + O2 D) Nucleotide bases --> nucleoside triphosphates E) ADP + P --> ATP These reactions are all going to a higher-order structure that is more complicated and has higher energy bonds than the starting materials

What is the term used to describe the thick soup of cell extract produced when cells are broken open? A) Supernatant B) Homogenate C) Pellet D) Sample

B) Homogenate

Your classmate ran a western blot, but had nothing appear in either lane. Which of the following is a plausible explanation for how they messed up? A) They forgot to add a blocking agent B) They forgot to add a primary antibody, and only used a secondary C) They forgot to add a secondary and only used a primary D) They used the pellet instead of the supernatant after their centrifugation E) They ran the wild type and mutant samples on the same lane F) They forgot to treat the samples with SDS and mercaptoethanol

B) They forgot to add a primary antibody, and only used a secondary C) They forgot to add a secondary and only used a primary D) They used the pellet instead of the supernatant after their centrifugation (Forgetting SDS would give them an unequal charge distribution, so the charge would affect how much they transfer, but we would still see bands)

Consider the mechanism by which actin and tubulin polymerize. Which of the items below does not describe something similar about the polymerization mechanisms of actin and microtubules? A. Although both filaments can grow from both ends, the growth rate is faster at the plus ends. B. Depolymerization initiates at the plus ends of filaments. C. Nucleotide hydrolysis promotes depolymerization of filaments. D. Free subunits (actin and tubulin) carry nucleoside triphosphates.

B. Depolymerization initiates at the plus ends of filaments. (in a concentrated solution of pure tubulin, tubulin dimers will add to either end of a growing microtubule, but they add more rapidly to the plus end)

Which of the following separates proteins according to size? A. Ion-exchange chromatography B. Gel-filtration chromatography C. Affinity chromatography

B. Gel-filtration chromatography

The endothelial cells that line blood vessels release nitric oxide locally to neighboring cells of the smooth muscle A. Endocrine B. Paracrine C. Autocrine D. Contact dependent

B. Paracrine (NO has a really short half life because it gets broken down very fast and can only reach neighboring cells)

Cheryl is performing SDS-PAGE gel electrophoresis but she forgets to heat her samples with SDS. Which of the following occurs as a result of Cheryl's mistake? A. Proteins separate by charge instead of by molecular mass B. Polypeptides do not unfold and thus migrate at slower rates through the gel C. Proteins do not migrate through the gel D. All of the above may occur as a result of Cheryl's mistake

B. Polypeptides do not unfold and thus migrate at slower rates through the gel

Why would you expect to activate RTKs by exposing the exterior of cells to antibodies that bind to the respective proteins? How about for GPCRs?

Because each antibody has 2 antigen binding sites, it can cross link the 2 receptors and cause them to cluster on the cell surface, which will likely activate RTKs, which are activated by dimerization because the individual kinase domains of the receptors phosphorylate adjacent receptors in the cluster. The activation of GPCRs is more complicated, because the ligand has to induce a conformational change

in the citric acid cycle, the acetyl group of acetyl-CoA is linked to a larger carbon skeleton before the carbons are oxidized to CO2. Why do you think this is?

Because the function of the citric acid cycle is to harvest the energy released during the oxidation, it is advantageous to break the overall reaction into as many steps aspkossible, and using a compound with more carbons, it is possible to generate more intermediates

What is Bimolecular Fluorescence Complementation?

BiFC is a technique to see if protein A interacts with protein B. In order to do this, we will take protein A and fuse it to the N-terminus of GFP, and take protein B and fuse it to the C-terminus of GFP, then look under the fluorescent microscope and see if you see a signal. If you do see signal, this means that the proteins interact and that they have brought together the 2 halves of GFP, because the halves alone cannot fold properly, and thus cannot function, but when the halves are brought together, they will be able to fold properly and the fluorophore can be activated In addition, the intensity of the fluorescence emitted is proportional to the strength of the interaction, with stronger levels of fluorescence indicating close or direct interactions and lower fluorescence levels suggesting interaction within a complex. This also allows us to see where the proteins interact as well.

Taxanes are a class of cancer fighting drugs which block the dynamic instability of the microtubules in the mitotic spindle. Why would it be beneficial to stabilize these filaments? What is the role of dynamic instability during cell division?

By blocking the instability of the microtubules, taxanes are able to restrict uncontrolled cell division. By restricting the mobility of the filaments, they are not able to coordinate the movements needed to drive division including movement of the cytosol and localization of organelles.

DNP is a small molecule that renders membranes permeable to protons. In the 1940s, small amounts of this toxic compound were given to patients to induce weight loss. DNP was effective at melting away the pounds and promoting the loss of fat reserves. How might it cause such loss? As an unpleasant side reaction, patients had an elevated temperature and sweated profusely during the treatment. Why?

By making membranes permeable to protons, DNP removes the proton gradient across the inner mitochondrial membrane. Cells continue to oxidize food molecules to feed high-energy electrons in the electron transport chain, but H+ ions pumped across the membrane flow back into the mitochondria in a futile cycle. As a result, the energy of these electrons cannot be tapped to drive ATP synthesis and is instead released as heat. Patients who have been given small doses lose weight because their fat reserve are used more rapidly to feed the electron transport chain and the whole process simply wastes energy as heat. If protons were allowed to simply flow across the innermitochondrial membrane, the energy would not be harvested by ATP synthesist and it would be liberated as heat since the energy must go somewhere, and normally it is harvested to transfer the protons across the gradient, which requries energy, and then reharvested when the protons are allowed back into the matrix, but since there is no gradient, energy is not needed and it will be lost as heat

Receptor tyrosine kinases (RTKs) respond to a dimer signal to form a phosphorylation complex. When in conjunction, two RTKs phosphorylate each other and other signaling proteins. RTKs generally activate Ras, which has several downstream targets. Phosphorylation requires energy. Why might the RTKs phosphorylate each other before they begin to phosphorylate other signaling proteins?

By using ATP to phosphorylate each other, this activates them and the RTKs are capable of binding their substrates and have the energy needed to phosphorylate them (stored in their phosphates).

T/F: The extracellular signal molecule, acetylcholine has different effects on different cell types in an animal and often binds to different cell-surface receptor molecules on different cell types

True.

NADH pills can be purchased over the counter, and are often taken by sufferers of chronic fatigue syndrome. How might these pills benefit a CFS patient? A) They will increase energy by creating more FADH2 B) They will decrease the rate of glycolysis C) They would increase the number of electrons provided to the electron transport chain D) They would decrease the rate of alcoholic fermentation

C) They would increase the number of electrons provided to the electron transport chain (NADH molecules are activated carriers that move electrons to the ETS)

Which of the following is used to purify proteins? A. Differential centrifugation B. SDS-page gel electrophoresis C. Chromatography D. Homogenization

C. Chromatography

Which of the following statements about the cytoskeleton is false? A. The cytoskeleton is made up of three types of protein filaments. B. The cytoskeleton controls the location of organelles in eukaryotic cells. C. Covalent bonds between protein monomers hold together cytoskeletal filaments. D. The cytoskeleton of a cell can change in response to the environment.

C. Covalent bonds between protein monomers hold together cytoskeletal filaments.

Which of the following is NOT a characteristic of an antibody used in the process of western blotting? A. Specific to a protein of interest B. Attached to an enzyme that converts a color-less substrate to a colored substrate C. Migrates through the SDS-PAGE gel D. All of the above are characteristics of antibodies used in western blotting.

C. Migrates through the SDS-PAGE gel

The lab you work in has discovered a previously unidentified extracellular signal molecule called QGF, a 75,000-dalton protein. You add purified QGF to different types of cells to determine its effect on these cells. When you add QGF to heart muscle cells, you observe an increase in cell contraction. When you add it to fibroblasts, they undergo cell division. When you add it to nerve cells, they die. When you add it to glial cells, you do not see any effect on cell division or survival. Given these observations, which of the following statements is most likely to be true? A. Because it acts on so many diverse cell types, QGF probably diffuses across the plasma membrane into the cytoplasm of these cells. B. Glial cells do not have a receptor for QGF. C. QGF activates different intracellular signaling pathways in heart muscle cells, fibroblasts, and nerve cells to produce the different responses observed. D. Heart muscle cells, fibroblasts, and nerve cells must all have the same receptor for QGF.

C. QGF activates different intracellular signaling pathways in heart muscle cells, fibroblasts, and nerve cells to produce the different responses observed. (A protein would not diffuse across the plasma membrane becasue it is polar and made of amino acids and it is a large molecule)

The following happens when a G-protein-coupled receptor activates a G protein. A. The β subunit exchanges its bound GDP for GTP. B. The GDP bound to the α subunit is phosphorylated to form bound GTP. C. The α subunit exchanges its bound GDP for GTP. D. It activates the α subunit and inactivates the βγ complex.

C. The α subunit exchanges its bound GDP for GTP.

The growth factor Superchick stimulates the proliferation of cultured chicken cells. The receptor that binds Superchick is a receptor tyrosine kinase (RTK), and many chicken tumor cell lines have mutations in the gene that encodes this receptor. Which of the following types of mutation would be expected to promote uncontrolled cell proliferation? A. a mutation that prevents dimerization of the receptor B. a mutation that destroys the kinase activity of the receptor C. a mutation that inactivates the protein tyrosine phosphatase that normally removes the phosphates from tyrosines on the activated receptor D. a mutation that prevents the binding of the normal extracellular signal to the receptor

C. a mutation that inactivates the protein tyrosine phosphatase that normally removes the phosphates from tyrosines on the activated receptor

Which of the following statements is correct? Kinesins and dyneins ____________________. A. have tails that bind to the filaments. B. move along both microtubules and actin filaments. C. often move in opposite directions to each other. D. derive their energy from GTP hydrolysis.

C. often move in opposite directions to each other.

Which of the following methods would be the most suitable to assess levels of expression of your target protein in different cell types? A. gel-filtration chromatography B. gel electrophoresis C. western blot analysis D. ion-exchange chromatography

C. western blot analysis (Gel electrophoresis would not necessarily work, since it would show all the proteins, not the target protein)

With each turn of the citric acid cycle, what gets released into the environment?

CO2

The Warburg hypothesis has recently gained attention in cancer research as evidence continues to build on the idea that cancer cells inside solid tumors generate their energy mainly through glycolysis. Why would cancer cells in tumors might lower mitochondrial respiration and take advantage of glycolysis?

Cancer cells in tumors are not exposed to oxygen, so they cannot undergo oxidative phosphorylation to make ATP (in the mitochondria), so they use solely glycolysis

What are capping proteins and what is their function in cellular migration?

Capping proteins are found at the plus end of the actin filaments, and they are removed so that actin can grow out at the leading edge of the lamellipodia

The breakdown of foods into simple subunits are what type of reactions?

Catabolic; releases energy

How does cell motility occur?

Cells activate ARP complexes, which promote the formation of a web of branched actin filaments in lamellipodia because they form complexes that bind to the sides of existing actin filaments and nucleate the formation of new filaments, which grow out at an angle to produce side branches. Then, they remove capping proteins on the plus ends of the actin filaments, so that actin can grow out at the leading edge, and then actin is polymerized to push out the leading edge and proteins facilitate this whole process.

At the leading edge of a crawling cell, the plus ends of actin filaments are located close to the plasma membrane and actin monomers are added at these ends pushing the membrane outward to form lamellipodia or filopodia. What holds the filaments at the other ends to prevent them from just being pushed into the cell interior?

Cells contain actin binding proteins that bundle and cross link actin filaments. The filaments extending from lamellipodia and fliopodia become firmly connected to the filamentous meshwork of the cell cortex, providing the mechanical anchorage required for the growing rodlike filaments to deform the cell membrane

Most receptor tyrosine kinases activate ________

a GTPase called Ras

What process is used to purify proteins?

Chromatography

What technique would allow us to simply separate our favorite protein from the homogenate once cells are homogenized?

Chromatography

What are cilia? What kinds of cells have cilia? Why are they important?

Cilia is a hairlike structure made of microtubules that is found on the surface of many eukaryotic cells. When it is present, its rhythmic beating can drive the movement of fluid over the cell surface. Each cilia contains a core of microtubules arranged in a bundle that grow from a cytoplasmic basal body, which is the organizing center. Cilia and flagella's microtubules are arranged in a 9 + 2 array, where there are 9 outer microtubules, each carrying 2 rows of dynein molecules. The dynein is attached by its tail to one microtubule and its two heads interact with an adjacent microtubule to generate a sliding force between the 2 microtubules. Because of the links that hold adjacent microtubule doublets together, the sliding force between adjacent microtubules is converted into the bending motion in the cilium.

A particular type of C.elegans mutant becomes paralyzed when the temperature is raised. The mutation affects axonal transport in neurons. You suspect that things cannot be transported down the axon because there is a mutation in the gene that codes for kinesin, making it so that the kinesin protein cannot bind to the tubulin that makes up the microtubules. How could you test this hypothesis?

Co-IP

The cholera toxin prevents GTP hydrolysis by Galpha in a canonical GPCR signaling pathways that regulates CFTR via phosphorylation by PKA. In the presence of the cholera toxin, cAMP levels increase by 100-fold, over activating PKA. Phosphorylated CFTR is active, over-active in the case of cholera. You have isolated mice with mutations downstream of Galpha that do not show symptoms of cholera when exposed to the toxin. You decide to examine the ability of Galpha to interact with adenyl cyclase. What technique could you use to do this? Describe this technique with respect to your experiment.

Co-IP 1) Lyse cells 2) Add antibodies that recognized Galpha 3) Add proteins beads that are conjugated to a protein that bind to the antibody 4) Centrifuge the lysate, to pellet the protein complex of interest 5) Do western blot to visualize what actually precipitated

What is co-localization?

Co-localization answers the question of "where in the cell is my protein located?" It identifies the presence of two or more fluorescent labels in the same place in a cell, tissue, or organelle using a fluorescent microscope to see if two things are in the same place in the cell, but just because they are in the same place in the cell does not mean that they interact

Adipocytes store fatty acids as triacylglycerol, a molecule of glycerol esterified to three long chain fatty acids. Lipase, the enzyme that hydrolyzes triacylglycerols to one molecule of glycerol and three long chain fatty acids, is found mainly in adipocytes and is active in the presence of the signaling molecule, adrenaline. It is phosphorylated by Protein Kinase A (PKA) and is catalytically active only when phosphorylated. You are studying a line of cultured adipocytes that express the G-protein-coupled receptor is coupled to G. What would a gain of function mutation in the gene that encodes PKA result in?

Constitutively low levels of TAGs, because if there is a gain of function in PKA, it will always be on and always phosphorylating lipase, so we do not even have to consider what is happening upstream. We do not even need adrenaline to activate this signaling pathway most likely

What is contact-dependent signaling?

Contact-dependent signaling does not require the release of signaling molecules but rather occurs directly through physical contact between two cells. The emitting cell's signaling molecule is membrane bound, and it interacts with the membrane bound receptor on the target cell

How do proteins move from the polyacrylamide gel onto nitrocellulose?

Current is applied to flow the protein molecules from the gel to attach to the membrane; the positive electrodes are on the side of the membrane and the negative electrodes are on the side of the gel and the current will flow from negative to positive and as the current flows, the proteins flow to the nitrocellulose membrane

The locomotion of fibroblasts in culture is immediately halted by the drug cytochalasin (affects actin), whereas colchicine causes fibroblasts to cease to move directionally and extending lamellipodia in seemingly random directions. The injection of fibroblasts with antibodies to vimentin (intermediate filaments) has no effect on their migration. what do these observations suggest to you about the involvement of the 3 types of cytoskeletal filaments in fibroblast locomotion?

Cytochalasin interferes with actin filament formation and its effect on cell demonstrates the importance of actin in cell locomotion. Colchicine shows that microtubules are required to give the cell a polarity that then determines which head becomes the leading edge. In order for motile cells to move in one direction, they must have a defined front and rear. The injection of antibodies to vimentin that bind the antigen and prevent it from interacting with other cell components shows that the intermediate filaments are not required for the maintenance of cell polarity or motile machinery

The hydrolysis of GTP to GDP carried out by tubulin molecules ________________. A. provides the energy needed for tubulin to polymerize. B. occurs because the pool of free GDP has run out. C. tips the balance in favor of microtubule assembly. D. allows the behavior of microtubules called dynamic instability.

D. allows the behavior of microtubules called dynamic instability.

During nervous-system development in Drosophila, the membrane-bound protein Delta acts as an inhibitory signal to prevent neighboring cells from developing into neuronal cells. Delta is involved in ________ signaling. A. endocrine B. paracrine C. neuronal D. contact-dependent

D. contact-dependent

For both actin and microtubule polymerization, nucleotide hydrolysis is important for ______. A. stabilizing the filaments once they are formed. B. increasing the rate at which subunits are added to the filaments. C. promoting nucleation of filaments. D. decreasing the binding strength between subunits on filaments.

D. decreasing the binding strength between subunits on filaments.

In an energetically unfavorable reaction, is the entropy increased or decreased?

Decreased

What is the question(s) the authors attempt to answer through the experimentation represented in figure 6?

Do the algal-produced immunotoxin kill B-cell lymphoma cell, and only B-cell lymphoma cells.

Microtubules play a large role in cellular division by forming the mitotic spindle, a structure which relies heavily on dynamic instability. What is dynamic instability?

Dynamic instability is the concurrent assembly and disassembly of the microtubules at the plus end. Unlike treadmilling, there is only one end where this is possible, so the length can increase or decrease.

Which of the following is a shared characteristic of both GPCRs and enzyme coupled receptors? A) Cytoplasmic domains act as enzymes B) Cytoplasmic domains phosphorylate downstream target proteins directly C) Display ligand binding domain on the inner surface of the plasma membrane D) Multiple transmembrane segments E) None of the above

E) None of the above

In an experiment to test the physiological effects of caffeine on aerobic respiration, you plan to measure the amount of radioactively labeled CO2 expelled from mice exercising on a wheel. Which atom and in which molecule will you label to allow you to carry out this experiment? A. Oxygen atoms in glucose B. Atmospheric oxygen atoms C. Carbon atoms in glucose D. A or B E. A or C

E. A or C

Describe the main difference between enzyme-coupled receptors and GPCRs.

Enzyme coupled receptors are transmembrane proteins that display their ligand binding domains on the outer surface of the plasma membrane, but instead of associating with a G protein, the cytoplasmic domain either acts as an enzyme itself or forms a complex with another protein that acts as an enzyme, whereas for GPCRs, the G protein is the enzyme.

Even if favorable, some reactions have a substantial activation energy, how can activation energy be diminished?

Enzymes

What if a cell that had a mutant G-protein that could only bind GDP and not GTP had a mutant form of PKA which was always active?

Even though adenylyl cyclase would not be able to create cAMP, PKA would still be able to phosphorylate phosphorylase kinase so the downstream events would always happen. Therefore, as long as glycogen is present, it would be constitutively broken down.

T/F: Antibodies can identify organelles

False!

T/F: Some enzyme-catalyzed reactions cease completely if their enzyme is absent

False, because enzymes enhance the rate, but do not change the equilibrium point of the reaction

T/F: Cells having an intermediate filament network that cannot be depolymerized would die

False, cells could not divide without rearranging their intermediate filament network, but many long-lived cells have stable intermediate filaments that do not depolymerize

T/F: The oxidation of sugar molecules by the cell takes place according to the following reaction C6H12O6 + 6O2 --> 6CO2 + 6H2O + energy. All of the energy produced is in the form of heat

False, if this were the case, the reaction would be useless for the cell and no chemical energy would be harvested in a useful form to be used for metabolic processes

T/F: The oxidation of sugar molecules by the cell takes place according to the following reaction C6H12O6 + 6O2 --> 6CO2 + 6H2O + energy. Many steps of the oxidation of sugar molecules involve reaction with O2.

False, just the last step

T/F: Because endocrine signals are broadcast throughout the body, all cells will respond to the hormonal signal.

False, the cell must have an appropriate receptor

T/F: GTP is hydrolyzed by tubulin to cause the bending of the flagella

False, to cause bending, ATP is hydrolyzed by the dynein motor proteins that are attached to the outer microtubules in the flagellum

T/F: The oxidation of sugar molecules by the cell takes place according to the following reaction C6H12O6 + 6O2 --> 6CO2 + 6H2O + energy. None of the energy produced is in the form of heat

False. No energy conversion process can be 100% efficient since the entropy in the universe has to increase and for most reactions that is accomplished by releasing heat

T/F: Because hormones like cortisol are broadcast throughout the body, all cells will respond to them

False. Not all cells have the same receptors

T/F: The electron transport chain generates an electrical potential across the membrane because it moves electrons from the inter membrane space into the matrix

False. The potential is due to protons that are pumped across the membrane from the matrix to the inter membrane space. Electrons remain bound electron carriers in the inner mitochondrial membrane

If the cell response of the receiver cells is altered protein function, what is the speed of that response?

Fast

The pathway that occurs after adrenaline binds to the GPCR and initiates glycogen breakdown, does it happen fast or slow?

Fast since no new protein synthesis is involved

In Fig 3B, the band in lane 4 forms at approximately 190 kDa. As in Fig 3A, lane 4 represents the recombinant protein aCD22CH23PE40. Hypothesize why that protein forms a band at 100 kDa in one gel, and a band at 190 kDa in the other.

Figure 3B represents a non-reducing gel, which means that the disulfide bond between the homodimer would not be reduced (split) in this immunotoxin as it was in figure 3A, the reducing gel, so in 3B, it represents the entire aCD22CH23PE40, whereas in 3A, it only represented half of aCD22CH23PE40.

What is the purpose of a flow cytometry analysis?

Flow cytometry is a laser based technology employed in cell counting, cell sorting, biomarker detection by suspending cells in a liquid and passing them through a laser

Does glycolysis consume energy? In what form?

For each molecule of glucose that enters glycolysis, 2x ATP are needed to provide the energy to prepare the sugar to be split. Glucose will not just spontaneously combust into pyruvate, so we need a little bit of energy to break the glucose molecule because even energetically favorable reactions have activation energy

What is a GTP cap?

Found at the end of rapidly growing microtubules are tubulin dimers associated with GTP that bind more strongly to their neighbors

Actin is a nucleotide binding protein. How does ATP hydrolysis affect the stability of actin polymers in cells?

Free actin monomers carry a tightly bound ATP. The actin molecule can hydrolyze ATP to ADP as soon as it is incorporated into the filament, reducing the strength of binding between monomers, decreasing stability of the polymer. Nucleotide hydrolysis promotes nucleotide depolymerization, helping the cell to disassemble its microtubules and actin filaments after they have formed.

What proteins are the target of 1/3 of all drugs?

G-protein coupled receptors

Are the components produced, used, both, or neither in glycolysis? Glucose CO2 H2O ATP ADP + P NADH NAD+

Glucose: Used CO2: Neither H2O: Neither ATP: Used and produced (a little made) ADP + P: Used and produced NADH: Produced NAD+: Used

Are the components produced, used, both, or neither in the electron transport chain? Glucose CO2 H2O ATP ADP + P NADH NAD+

Glucose: neither CO2: neither H2O: produced ATP: A LOT produced ADP + P: used NADH: Used NAD+: Produced

Are the components produced, used, both, or neither in the citric acid cycle? Glucose CO2 H2O ATP ADP + P NADH NAD+

Glucose: neither CO2: produced H2O: Neither ATP: GTP is produced ADP + P: neither NADH: Produced NAD+: Used

The 2nd stage of cellular respiration is what?

Glycolysis

How is a GPCR involved in the integration of multiple signals?

GPCRs can work in conjunction with other signaling molecules to induce a joint change in the cell. Multiple GPCRs receiving individual signals, but then the downstream target proteins in both of those pathways interacting later to produce one cellular effect.

What molecule is necessary to activate Ras?

GTP

Proteins do not always remain intact when they are run through Western blots. Consequentially, some bands on Western blots may contain only parts of the intended proteins, and may not accurately reflect the size of those molecules. In this paper, the researchers claim that the band indicated by the blue arrow in Fig 3B contains a product assembled of aCD22CH23PE40 and a degradation product of the same protein that lacks the antibody binding domain. Compare the homodimer to what the degradation product might look like, and consider that one antibody binding domain should weigh about 30 kDa. Does the researchers' explanation sufficiently account for the extra band in lane 4, at about 150 kDa? Why or why not?

Given that the researchers said that the band indicated by the blue arrow in figure 3B contains a product assembled of aCD22CH23PE40 and a degradation product of the same protein that lacks the antibody binding domain, the data displayed in the figure makes sense, since the intermediate product is abut 150 kDa, and the antibody binding domain that it is missing is about 30 kDa, and the fully assembled homodimer is around 180-190 kDa.

Identify the input and output of each stage (glycolysis, citric acid cycle, and ETC) in cellular respiration

Glycolysis: glucose + ATP + NAD+ + ADP + P NADH + pyruvate + ATP Citric Acid Cycle: acetyl CoA + FAD + NAD+ + GDP + P NADH + FADH2 + GTP + CO2 Electron Transport Chain: NADH + FADH2 H2O + NAD+ + FAD + ATP

Differentiate between hormones which originate from endocrine cells and local mediators which participate in paracrine signaling

Hormones are "public" signals that broadcast the signal throughout the whole body through the blood. Paracrine signals, instead of entering the bloodstream, diffuse locally through the ECF and remain in the neighborhood of the cell that secreted them to locally mediate neighboring cells

The cholera toxin prevents GTP hydrolysis by Galpha in a canonical GPCR signaling pathways that regulates CFTR via phosphorylation by PKA. In the presence of the cholera toxin, cAMP levels increase by 100-fold, over activating PKA. Phosphorylated CFTR is active, over-active in the case of cholera. You find that G alpha and adenyl cyclase do interact with your mutant mouse. Propose an alternative hypothesis that addresses both the identity of the gene mutated and the consequence that the mutation has on protein function with respect to signaling to CFTR and name an experimental technique that you could use to test this alternative hypothesis

Hypothesis: CFTR in mutant mice cannot be phosphorylated because a mutation that encodes the protein PKA that cannot phosphorylate CFTR, and thus the signaling pathway will be shut down Western blot using a phosphoantibody that can detect the phosphorylated version of CFTR, and compare that to the wildtype. The wildtype mice that have an active PKA and are exposed to cholera would have phosphorylated CFTR, but in the mutant mice, even when they are exposed to cholera, they cannot phosphorylate CFTR.

Adipocytes store fatty acids as triacylglycerol, a molecule of glycerol esterified to three long chain fatty acids. Lipase, the enzyme that hydrolyzes triacylglycerols to one molecule of glycerol and three long chain fatty acids, is found mainly in adipocytes and is active in the presence of the signaling molecule, adrenaline. It is phosphorylated by Protein Kinase A (PKA) and is catalytically active only when phosphorylated. You are studying a line of cultured adipocytes that express the G-protein-coupled receptor is coupled to G. What would mutation in the cytosolic domain of the G protein coupled receptor that disrupts its ability to interact with other proteins in the cell result in?

If the GPCR cannot interact with other proteins in the cell, it cannot activate the G protein, and if the G protein is not activated, it cannot activate adenyl cyclase, which cannot make cyclic AMP, which will not be able to activate PKA. If PKA is not activated, it will not phosphorylate lipase, and lipase will not be catalytically active, therefore there will be constitutively high levels of TAG. There would be no downstream signaling

How do you make a kymograph?

Label something of interest with a fluorophore in cells, then take a movie using a fluorescent microscope of these things moving in a cell over time, then their movement can be plotted on a graph of time v. distance travelled

Huntington's disease is a neurodegenerative disorder caused by an expansion of polyglutamine peptide in the amino terminal of the cytosolic protein, huntingin (HTT). This disease leads to motor abnormalities, cognitive dysfunction, and psychiatric effects. You recently joined a research team t figure out how the disease form of HTT (dHTT-polyQ) causes HD. The current hypothesis is that the amino fragment of dHTT-polyQ after cleavage translocates to the nucleus from the cytoplasm and through binding the transcription factor p53, they activate expression of apoptotic genes in neurons. In healthy individuals p53 is expressed prevalently but does not activate the same genes. Your team has a mouse model of HD that expresses dHTT-polyQ in neurons and a healthy mouse designated as WT. How could you test this hypothesis and what would you expect to see?

I would test this hypothesis by doing a co-immunoprecipitation and then running a wester blot. I will use wild type and HD cells, and lyse them and isolate the total protein. Then, I will add an antibody to both cell lines that recognizes dHTT-polyQ and another experiment I will IP with an anti-p53 antibody, and then have beads that are conjugated to a protein that recognizes that antibody. Then, I will add beads that are conjugated to a protein that binds to the antibody. Then I will centrifuge the sample and since the beads are dense, they will go to the bottom and I can remove everything that is not attached to the bead. I will run a western blot and analyze the proteins. I will expect that for my mutant cells, since dHTT-polyQ binds p53, in my western blot for the IP with the anti-dHTT polyQ samples, I will also be able to detect p53, and in my IP with the anti-p53 samples, I will also detect anti-dHTTpolyQ. In my wild type cells, I will not detect any dHTT-polyQ, since that is the mutant version and they do to have that, and I also will not detect and p53 in that sample, because even though they do have p53, when I IP with anti-dHTT-polyQ, there is nothing to bind to p53, but when I IP with anti-p53, I will expect to only get a p53 band, but no dHTT-polyQ band. For tubulin, my control, I will not get any bands because tubulin does not bind t o either of those proteins, and since all of the other proteins that didn't attach to the beads were washed away, it will not have been run in my SDS PAGE

Ca2+ pumps in both the plasma membrane and ER membrane maintain a low concentration of Ca2+ in the cytoplasm. You have isolated cardiac myoctyes (heart muscle cells) with abnormally high levels of Ca2+ in the cytosol and have even confirmed the presence of a base pair substitution in the gene, but this does not alter the expression and the protein is still made. You hypothesize that the base pair substitution made it so the protein that needs to interact with the pump for the pump to work properly cannot interact. How would you test this?

I would use BiFC.

Ca2+ pumps in both the plasma membrane and ER membrane maintain a low concentration of Ca2+ in the cytoplasm. You have isolated cardiac myoctyes (heart muscle cells) with abnormally high levels of Ca2+ in the cytosol and have even confirmed the presence of a base pair substitution in the gene, but this does not alter the expression and the protein is still made. You hypothesize that the base pair substitution made it so the protein cannot fold properly, and it got degraded. How would you test this?

I would use Western blot comparing the wild type to the mutant, and use antibodies against the Ca2+ pump. If this hypothesis is true, I would expect little to no band in the mutant

Dynamic instability causes microtubules to grow or shrink rapidly. Consider an individual microtubule that is currently shrinking. What would happen if a solution contained an analog of GTP that could not be hydrolyzed?

If GTP is present but cannot be hydrolyzed, microtubules will continue to grow until all free tubulin subunits have been used up

What would be the outcome of a disease causing bacterium that produces a toxin that prevents Gα from hydrolyzing bound GTP

If Galpha cannot hydrolyze bound GTP, it will not be able to turn GTP into GDP and it will be permanently activated

Describe what you would expect to find in a well with low absorbance value after doing an MTT assay. Indicate relative population size of cells, color of medium, and other molecules present.

If a well had low absorbance, we would expect the opposite, since a low absorbance means that there was not a lot of colored salt, and if there is not a lot of colored salt this means that there was not much enzyme produced and if there was not much enzyme produced there was either not a lot of cells, or very inactive cells.

Dynein arms in a cilium are arranged so that, when activated, the heads push their neighboring outer doublet outward toward the tip of the cilium. Why would no bending motion of the cilium result if all dyne molecules were active at the same time? What pattern of dynein activity can account for the bending of a cilium in one direction?

If all the dynein arms were equally active, there would be no significant relative motion of one microtubule to thee other as required for bending, thus a few ciliary dynein molecules must be activated selectively on one side of the cilium. As they move their neighboring microtubules toward the tip of the cilium, the cilium bends away from the side containing the activated dyneins

From figure 4, what would happen if Lane 1 showed a band? What conclusions would scientists draw from this?

If lane 1 showed a band, this would mean that it was the CD22 antibody, not necessarily the exotoxin that transfers biotin to eEF2, or it might mean that something else in solution ribosylates eEF2, it is not just PE40.

If no O2 is available, all components of the mitochondria electron transport chain will accumulate in their (reduced/oxidized) form. Why?

If no O2 is available, all components of the mitochondrial ETC will accumulate in their reduced form because the electrons derived from NADH and FADH2 enter the chain and cannot be transferred to O2

Dynamic instability causes microtubules to grow or shrink rapidly. Consider an individual microtubule that is currently shrinking. What would happen if only GDP were present in the solution, no GTP?

If only GDP were present, microtubules would continue to shrink and eventually disappear because GDP-tubulins have a very low affinity for each other and will not stably add to microtubules

Explain how the concentration of actin monomers in cells affects the rate at which polymers grow and shrink.

If the concentration of free actin monomers is very high, an actin filament will grow rapidly and add monomers to both ends. At intermediate concentrations of free actin, actin monomers add to the plus end at a rate faster than the bound ATP can be hydrolyzed, so the plus end will grow. At the minus end, ATP is hydrolyzed faster than new monomers can be added, because ADP-actin destabilizes the structure and the filament loses subunits from its minus end at the same time it adds them to the plus end. If the concentration of actin is low in the cells, the actin filaments will depolymerize since actin-ADP cannot associate as tightly to their neigbors as actin-ATP and if the actin monomers are not being added as fast, ATP will be hydrolyzed before another monomer can be added and they will rapidly depolymerize from both sides

If you want to determine the order of proteins in a signaling pathways that includes Ras, how could you do this?

If you have protein Z, you can see if it acts upstream or downstream of Ras by expressing an inactive, mutated form of the protein in cells, and seeing if the wild type function can be rescued with a continuously active form of Ras. If it can be rescued, it means that Ras is downstream of the protein Z, but if it cannot be rescued, it means that Ras is upstream of protein Z

What is the purpose of using a marker gene in co-localization?

If you use a marker where you know where the protein found, it can help you identify certain places in the cell when it fluoresces because if not, it is hard to tell what is what within the cell

Describe what you would expect to find in a well with high absorbance value after doing an MTT assay. Indicate relative population size of cells, color of medium, and other molecules present.

In a well with high absorbance values, we would expect a lot of cells that are very active, since they are making a lot of reducing enzymes that color the salt, and the more colored salt there is, the less the light passes through because it was absorbed. The medium would be highly colored with a lot of reducing enzyme in it

In Figure 2C, why are there bands in lanes 2, 3 and 4, but not in lane 1? What conclusions do the researchers draw from this gel

In figure 2C, we see bands in lanes 2, 3, and 4 since these are the three cell types that contain the aCD22 scFv gene downstream of the psbA 5' UTR, and since they did PCR analysis with a primer designed to recognize the aCD22 scFv gene and the psbA 5' UTR, they would only expect to get bands in the algae that had been transformed. Lane 1 is the wildtype algae cells that have not been transformed, and so they do not have the aCD22 scFv gene for the primers to recognize. The researchers can draw the conclusion that the antibody genes have been successfully incorporated into the chloroplast genome in the place that they wanted them to be incorporated.

In Figure 2D, why do all of the lanes have the same lower band? How does this band serve as a control?

In figure 2D, the lower band is the gene for the ribosomal 16s rRNA sequence, and this is seen in all of the lanes because they all have this gene and it was not replaced in any of the strains. This serves as a positive control to show that their PCR worked, and the fact that we do not see bands for the psbA gene in lanes 2, 3, and 4 is not because of some error with the experimental technique, but because they really are not there.

When the drug dinitrophenol is added to mitochondria, the inner membrane becomes more permeable to protons. In contrast, when the drug nigericin is added to the mitochondria, the inner membrane becomes permeable to K+. How does the electrochemical proton gradient change in response to dinitrophenol and how does it change in response to nigericin?

In response to dinitrophenol, the electrochemical proton gradient is removed because H+ ions that are pumped to one side of the membrane flow back freely and no energy to drive ATP synthesis can be stored across the membrane. In response to nigericin, K+ will be driven into the matrix by the electrical potential of the inner membrane sine it is negative inside and the influx of positive charged K+ will abolish the membrane's electrical potential, but because the pH gradient will not be changed by nigericin, only part of the driving force that makes it energetically favorable for H+ ions to flow back in the matrix is lost

How does kinesin move along the microtubule?

In solution, both of the kinesin heads contain tightly bound ADP. When one head encounters a microtubule, it binds tightly and the binding causes ADP to be released from the attached head and ATP enters the site and the nucleotide exchange triggers the neck linker to whip the other head forward to the next binding site. The attached trailing head then hydrolyzes ATP and releases phosphate and the neck linker unzippers. Then, the leading head releases its ADP, binds to ATP and zippers its neck linker to the catalytic core, which whips the trailing head forward and the cycle repeats

Where is chemical energy stored in macromolecules?

In the bonds

Where does glycolysis take place?

In the cytosol

Where does oxidative phosphorylation happen?

In the inner mitochondrial membrane

In cells that can grow both aerobically and anaerobically, fermentation is inhibited by the presence of O2. Why?

In the presence of O2, oxidative phosphorylation converts most of the cellular NADH to NAD+ and fermentation requires NADH

Predict what might happen if we created a G-protein which could only bind to GDP and not GTP.

In this situation, the G-protein would not be able to function because it would be permanently inactive and none of the downstream events would be initiated.

In an energetically favorable reaction, is the entropy increased or decreased?

Increases and so does disorder

Epidermis bullosa simplex is a genetic condition that renders cells unable to resist the mechanical stress that occurs when cells are stretched, causing the skin to be fragile and blister easily. Which of the three protein filaments of the cytoskeleton are affected by this condition and why?

Intermediate filaments because they are involved in helping the cells withstand mechanical stress

There are no known motor proteins that move on intermediate filaments. Why?

Intermediate filaments have no polarity and their ends are chemically indistinguishable. It would be difficult for a motor protein to bind a filament and go a defined direction

Explain in your own words what the fluorescent shift in Figure 5A demonstrates

It demonstrates that the immunotoxins bind to the CA-46 T cells and because there is a fluorphore conjugated to one of the antibodies, it produces a different fluorescent color

Under aerobic conditions, what happens to pyruvate?

It gets converted to acetyl CoA

What is a kymograph?

It is a graph of spatial position vs. time, with time on the y axis and distance traveled on the X axis that can allow us to visualize transport in cells

You have successfully identified a gene required for cell division in yeast called Cdc2. Yeast with loss-of-function mutations in Cdc2 cannot divide at warm temperatures. You are particularly interested in the protein encoded by Cdc2 because your data suggests that the causative mutation does not affect gene expression. In other words, you observed no difference in the relative abundance of RNA transcript in the mutant. What is a protocol that could test the hypothesis that the sub-cellular localization of the protein is altered in the mutant and thus it will be located in a different part of a mutant cell relative to that of a wild-type cell.

It is a protein involved in cell division, so the wildtype is in the nucleus To determine the sub-cellular localization of the altered protein, we will use a co-localization technique with antibodies. 1) We have 2 different plates of yeast cells with growth media. The first plate is the cells with the wildtype protein and the second plate has cells with the mutant protein GROWN IN AN INCUBATOR SINCE THE MUTANT IS TEMPERATURE SENSITIVE 2) First we will need to get antibodies that recognize and bind to the proteins (they will bind to both the wildtype and the mutant because the antibody recognizes such a small part of the protein that it probably will not differentiate between the wildtype and mutant that were made in a rabbit, and an anti-rabbit antibody that is tagged with a red fluorophore 3) Because the protein is involved in cell division and we know that the wildtype protein is in the nucleus, we will get an anti-transcription factor antibody conjugated to a green fluorophore. 4) We will permeablize the cells and allow the antibodies to enter and bind to their specific proteins 5) We will view the cells with a fluorescent microscope and the wildtype cell will serve as our control and we should see green and red colors together and in the mutant cells we should see the red in the nucleus, but green outside of the nucleus and this will tell us that the mutant is located in the incorrect part of the cell and that is why it is not working. To determine the sub-cellular localization of the altered protein, we will use a co-localization technique with a transgene 1) We have 2 different plates of yeast cells with growth media grown in an incubator.First plate is the cells with a transgene inserted into the nucleus that was promoter + WT Cdc2 + GFP. Second plate has cells with a transgene inserted into the nucleus that was promoter + mutated Cdc2 + GFP 2) Because the protein is involved in cell division and we know that the wildtype protein is in the nucleus, we will get an anti-transcription factor antibody conjugated to a red fluorophore. 3) We will view the cells with a fluorescent microscope and the wildtype cell will serve as our control and we should see green and red colors together and in the mutant cells we should see the red in the nucleus, but green outside of the nucleus and this will tell us that the mutant is located in the incorrect part of the cell and that is why it is not working.

What is the role of cyclic AMP in the glycogen breakdown pathway?

It is a secondary messenger molecule that activates PKA and thus amplifies the signal because it can activate a bunch of PKAs

Describe what a tetrazolium salt assay is

It is colorimetric assay for assessing cell metabolic activity and can be used to measure cytotoxicity when exposed to a treatment molecule. In an MTT assay, growth medium and cells are plated, and then treatment molecules are added to the cells. Then, to see if the treatment molecule had any effect on the cells, tetrazolium salts are added to the plates. Tetrazolium salts are colorless when alone, but when the salt is exposed to electron acceptors, like those associated with reducing enzymes in cells, the salt reduces to form a colored dye, which limits the light that can pass through. Then, the plates are processed with a spectrophotometer that broadcasts light through the plate and an absorbance value is collected for the wells. The more dye produced, the higher the absorbance value and the number of active cells in each can be quantified according to the amount of dye produced.

All of the energy in cellular respiration goes where?

It is either lost as heat to increase disorder of the universe or it is stored high energy electrons that are transferred top activated carriers

From figure 2, why is it essential to the overall experiment that the researchers select the algae that have been successfully transformed?

It is important to select the algae that have been successfully transformed because they need to know which strains to use for their experiment and which strains that they can make conclusions off of.

Nitric oxide (NO) freely crosses the plasma membrane of endothelial cells but acts only locally on cells nearby. Why doesn't NO act as a signal to cells at a greater distance?

It is quickly converted to nitrates and nitrites by reacting with water and oxygen outside of the cell

What makes a good activated carrier?

It is small, it has a high energy bond that when broken, releases a lot of energy, and energy is easily exchangeable

What are examples of paracrine signals?

Nitric oxide and neurotransmitters

Correct the following statement, and say how you could support your answer experimentally. "The oxygen consumed during the oxidation of glucose in animal cells is returned as part of CO2 to the atmosphere"

It is true that oxygen atoms are released as part of CO2 into the atmosphere, but the CO2 released from cells does not contain the specific oxygen atoms that were consumed as part of the oxidative phosphorylation process and converted to water. You can prove this directly by incubating living cells in an atmosphere that contains O18 labeled water and in an experiment, you will find that the CO2 released contain the naturally occurring O16 isotope, therefore the oxygen atoms that are released in the CO2 molecules do not come directly from the atmosphere ,but from the organic molecules that the cell has oxidized as fuel

What does cholera toxin do?

It prevents the G protein from hydrolyzing GTP, locking the G protein in its active state and continuously stimulates adenyl cyclase.

What is the function of PKA and how is this function related to that of phosphorylase kinase?

It takes an ATP and hydrolyses it to ADP and transfers the phosphate to the inactive phosphorylase kinase, which activates it, which then phosphorylates its target protein downstream, and that downstream protein breaks down glycogen to glucose

Predict what might happen if Galpha could not hydrolyze bound GTP

It will be permanently activated and constantly send signals throughout the cell. The amount of time that the alpha subunit remains switched on determines how long a response lasts. The timing is controlled by the behavior of the alpha subunit, which has GTPase activity and it eventually hydrolyzes GTP to GDP and returns the whole G protein to its original, inactive conformation

Would the outcome of this experiment from figure 5 change if they used different types of B-cells (other than CA-46 and Ramos

It would not change, since CD22 is a surface antigen that is found on the surface of B cells

How might a non-hydrolysable analog of ATP affect the function of kinesin?

It would not move

2 protein kinases function sequentially in an intracellular signaling pathway. If either kinase contains a mutation that permanently inactivates its function, no response is seen in cells when an extracellular signal is received. A different mutation in K1 makes it permanently active, so that in cells containing that mutation a response is observed in the absence of an extracellular signal. You observe a double mutant cell that contains K2 with the inactivating mutation and K1 with the activating mutation. You observe that the response is seen in the absence of an extracellular signal. In the normal pathway, does K1 activate K2 or does K2 activate K1?

K2 activates K1. If K1 is permanently activated, a response would be observed regardless of the status of K2, but if K1 activated K2, this could not happen

When cells enter mitosis, their existing array of cytoplasmic microtubules has to be rapidly broken down and replaced with the mitotic spindle that forms to pull he chromosomes into the daughter cells. The enzyme katanin is activated during the onset of mitosis and chops microtubules into short pieces. What do you suppose is the fate of the microtubule fragments created by katanin?

Katanin breaks microtubules along their length and at positions remote from their GTP caps. The fragments that form therefore contain GDP-tubulin at their exposed ends and rapidly depolymerizes. Katanin provides a quick means of destroying existing microtubules

You are a hydrogen atom in glucose molecule. Today you finally get to take the adventure you have been waiting for and participate in cellular respiration. Your first step is glycolysis! Write a summary of your travels, where you go, and who you meet.

Kids, let me tell you how I met your mother. As a Hydrogen ion, life was peaceful and stable in the Glucose family. However, everything changed the day glycolysis began. NAD invaded our community, ripping me away from the Carbon and Oxygen molecules I called family. NAD forced me to integrate with their customs, now calling themselves NADH because my new arrival, and we traveled until reaching the electron transport chain. Apparently not needing me anymore, they drop me off at the Mitochondrial membrane, leaving me to rot away. Only then I saw a beautiful thing, it couldn't be.... but it was! An electron! I chased it blindly, and accidentally was transported across the membrane to the intermembrane. Looked like I wasn't the only one that made this mistake, and many of family members, Hydrogen ions that I recognized from living on glucose, were there as well. However, there were too many of us and I had to get out; you know how family reunions are. Going back and forth searching for an escape back across the membrane, I discover a transport protein called 'ATP Synthase', and immediately I barrel through it. In my frantic crossing, I believe I even went so fast I turned some sort of turbine, which apparently powered the whole place and attached phosphate groups to ADP and made ATP; looks like they owe me now. This long adventure gave me a new perspective on things however, and I now sought to settle down. It was here I that met your mother, a sultry Oxygen molecule, where I made a bond in both matrimony and covalence."

Which lane in Fig. 2C is the control, and how does it serve as a control?

Lane 1 serves as a control because it is the wildtype and since they are running the gel with samples they obtained through the PCR analysis using primers specific to the aCD22 scFv gene and the psbA 5' UTR, they should not get a band for the wildtype because it does not contain the antibody gene and this serves as a negative control to prove that they did not get any false positive results with their transgenic algal strains.

What are flagella? What kinds of cells have flagella? Why are they important?

Longer versions of cilia that are capable of propelling a cell through a fluid medium with its rhythmic beating. Cilia and flagella's microtubules are arranged in a 9 + 2 array, where there are 9 outer microtubules, each carrying 2 rows of dynein molecules. The dynein is attached by its tail to one microtubule and its two heads interact with an adjacent microtubule to generate a sliding force between the 2 microtubules. Because of the links that hold adjacent microtubule doublets together, the sliding force between adjacent microtubules is converted into the bending motion in the cilium.

What method should you use to break open plant cells?

Mechanical force to shear the cells since the cell walls are very strong

The signaling molecule for contact dependent signaling is where?

Membrane bound

Discuss why it is that GPCRs can be activated by when a molecule binds to them, but why RTKs must dimerize to be activated.

Membrane proteins that span the membrane many times, like GPCRs can undergo a conformational change upon ligand binding that can be sensed on the other side of the membrane, because upon ligand binding, the transmembrane helices will rearrange with respect to one another, that will be sensed on the cytosolic side of the membrane because a change in the arrangement of the cytoplasmic loops. Proteins that only span the membrane one time cannot transmit a conformational change across the membrane and require oligomerization because no rearrangements in the membrane are possible

What type of cytoskeleton element is made of hollow tubes made of globular protein subunits?

Microtubules

How do vesicles move around the cell?

Microtubules and motor proteins or actin and myosin

The structural polarity of microtubles is crucial for their assembly and function in cells. What is structural polarity?

Microtubules are structurally polar, meaning that in each protofilament that makes up the microtubule, they are all the same and have alpha tubulin on end one, and beta tubulin on the other end, making it so that each side of a microtubule is entirely alpha tubulin or entirely beta tubulin and because of this, the tubulin molecule as a whole has structural polarity. In this sense, polarity doe not indicate electrical charge, but it is to indicate that in a solution, tubulin dimers add more rapidly to the plus end because the 2 sides are chemically and functionally distinct. This polarity is crucial for the assembly of microtubules and their function once they have been formed, because without it, they could not do things like guide intracellular transport

Compare the structure of the filament between microtubules and actin filaments.

Microtubules are tubular and hollow and actin filaments are helical

Compare the nucleotide bound to microtubules and actin filaments

Microtubules bind GTP/GDP and actin filaments bind ATP/ADP

Explain the role that microtubules play in the transport and trafficking of vesicles throughout the cell?

Microtubules create a system of tracks within the cell that vesicles can be transported on via kinesins and dyneins.

Compare filament behavior between actin and microtubules

Microtubules display dynamic instability, while actin filaments display treadmilling

Compare growth on the ends between microtubules and actin filaments

Microtubules only grow from the plus end, while actin filaments grow from both ends

Compare where they lose subunits on the ends between microtubules and actin filaments

Microtubules only lose subunits from the plus ends because the minus ends are anchored at the centrosome, but actin filaments can lose subunits from both ends

What is the role of a blocking agent in western blotting?

Milk is applied to cover most of the nitrocellulose membrane so the antibody doesn't bind to unspecific region of the membrane and because the antibody is so specific to the target, it will not let any blocking prevent it from binding. The nitrocellulose paper has a very high affinity for proteins (which makes sense since we could transfer proteins to it), and we want to block any unoccupied sites on the membrane

A mutant form of constitutively active Ras was originally identified in cancer cells. How do you think mutant Ras function differs from wild-type Ras functionally in cancer cells and why would this lead to uncontrolled cell division?

Mutant Ras is always bound to GTP, and can continually signal downstream targets even in the absence of a signaling molecule. Once RTKs are activated via binding of a signal molecule and phosphorylation, they recruit an adaptor protein, which recruits Ras GEF, which activates Ras and they recruit MAP kinase kinase kinase, and activates MAP kinase kinase, which activates MAP kinase, which can phosphorylate a transcription regulator and cause cell division.

What are the motor proteins that travel along actin?

Myosin

What is the ADP-ribotransferase reaction equation from figure 4?

NAD+—ADP+—Biotin + eEF2 ---(PE40)----> eEF2- ADP+-Biotin + NAD+

Which molecules shuttle high energy electrons to the electron transport chain?

NADH and FADH2

Outline the three processes that occur when a white blood cell is crawling out of the blood to an infected tissue.

Neutrophils are white blood cells that migrate out of the blood into infected tissues when they smell small molecules released by bacteria. The chemotactic molecules bind to receptors on the neutrophil cell surface and trigger changes in actin filament assembly that help direct the cells toward the bacteria. a. The cell pushes out protrusions at its front driven by actin polymerization. The leading edge of the crawling cell extends a thin lamellipodia, which contains a dense meshwork of actin filaments, oriented so that most of the filaments have most of their + ends close to the plasma membrane. Some cells also extend filopodia, which also contain actin filaments with + ends pointing out. The lamellipodia and filopodia are motile structures that form and retract fast, b. Protrusions adhere to the surface over which the cell is crawling because when the lamellipodia and filopodia touch down on favorable surface, they stick c. Rest of the cell drags itself forward by traction on these anchorage points

Receptors are proteins that recognize and respond to specific signals. Do all cells have the same receptors?

No, by producing only a limited set of receptors out of thousands that are possible, a cell restricts the types of signals that can affect it

Does the kinesin always take the same route through the cell?

No, the cytoskeleton is changing constantly and microtubules are constantly being built and broken down, so therefore kinesin has to take different routes

Do intermediate filaments have unique polarity?

No, the ends are indistinguishable from each other because the filaments are built by the assembly of tetramers made from 2 coiled coil dimers and each dimer points in the opposite direction, so they are identical.

A particular type of C.elegans mutant becomes paralyzed when the temperature is raised. The mutation affects axonal transport in neurons. In this situation, does temperature cause the mutation?

No. These worms have a gene that is mutated and they show the mutated phenotype at the temperature. temperature does not cause the mutation, but makes it show the mutation that it would not normally show

In the electron transport chain, what molecule ultimately accepts electrons?

O2 to form H2O

Why is horseradish peroxidase conjugated to secondary antibodies?

Once a substrate is added, the enzyme converts the substrate into a product and the product that appears is colored

Knowing that TAS2R2 is a receptor for cyclohexamine, how could Dr. Dorian test to see if the ligand binding site of the mutated protein is dysfunctional?

One possible solution is to use affinity chromatography with beads conjugated to cyclohexamine. Because TAS2R2 would bind to the cyclohexamine, it would slowly precipitate out in relationship to other proteins and could be isolated* from the wild type. Then, by running another chromatograph with the mutant sample, he could see if the protein precipitates in the same time frame (if at all). If the protein does not separate from the rest of the sample, then the mutation likely affects the binding site. If it precipitates like the WT, then the mutation occurs somewhere else in the protein. Then, a western blot would be performed to verify that TAS2R2 was the protein that separated

Suppose that the actin molecules in a cultured skin cell have been randomly labeled in such a way that 1 in 10,000 molecules carries a fluorescent marker. What would you expect to see if you examined the lamellipodium of this cell through a fluorescent microscope? Assume the microscope can detect single fluorescent molecules.

Only fluorescent actin molecules assembled in filaments are visible since unpolymerized actin molecules diffuse so rapidly that they will produce a dim uniform background. If you watch the movement, you will see individual fluorescent spots move steadily back from the leading edge toward the interior of the cell because actin monomers are added to filaments at the plus end and lost from the minus end, so actin monomers move through the actin filaments by tread milling

To remain a local stimulus, paracrine signal molecules must be prevented from straying too far from their origin. How could this be accomplished?

Paracrine signals could either be degraded by extracellular enzymes or are taken up by neighboring target cells.

Intracellular signaling molecules are often switched off by what?

Phosphatases

What does it mean to purify proteins?

Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture

In ion-exchange chromatography, what binds to the column?

Proteins of the opposite charge of the beads

In gel-filtration chromatography, what binds to the column?

Proteins that can fit into the pores; smaller molecules

During fermentation in yeast and in muscle cells , what gets oxidized and what gets reduced?

Pyruvate gets reduced to ethanol/lactate, and this oxidizes NADH to NAD+ so the yeast/muscle cell can continue to undergo glycolysis and survive under the anaerobic conditions

Assume that pyruvate containing radioactively labeled C14 in its carboxyl group is added to a cell extract that can support oxidative phosphorylation. Which molecules should contain the vast majority of the C14 that was added?

Pyruvate is converted to acetyl-CoA, and the labeled C14 atom is released as C14O2 gas

Explain why cyclic AMP must be broken down rapidly in a cel to allow for rapid signaling?

Rapid breakdown keeps the intracellular cAMP concentrations low. The lower the cAMP levels are, the larger and faster the increase achieved upon activation of adenyl cyclase, which makes new cAMP.

List the downstream effects of Ras activation.

Ras activates MAP kinase kinase kinase, which phosphorylates MAP kinase kinase, which phosphorylates MAP kinase, which activates a variety of proteins by phosphorylation.

Receptor tyrosine kinases (RTKs) phosphorylate tyrosines on target intracellular proteins when activated but they are not fully activated by binding a signaling molecule. Explain how full activation is achieved by receptor kinases

Receptor tyrosine kinases are enzyme-coupled receptors with a cytoplasmic domain that functions as a tyrosine protein kinase, which phosphorylates particular tyrosines on specific intracellular signaling proteins. Receptor tyrosine kinases are only single pass transmembrane proteins, and single pass transmembrane proteins are poorly suited to transmit a conformational change across the bilayer, so with RTKs, the binding of an extracellular signal molecule causes 2 RTKs to come together in the plasma membrane to form a dimer. The signal molecule can either physically link them (like the case if it was a dimer) or cause conformational changes that would link them. This pairing brings the 2 intracellular tails of the receptors together, activating their kinase domains so that each receptor tail phosphorylates the other, and the phosphorylation triggers a transient intracellular signaling pathway

Receptor tyrosine kinases (RTKs) respond to a dimer signal to form a phosphorylation complex. When in conjunction, two RTKs phosphorylate each other and other signaling proteins. RTKs generally activate Ras, which has several downstream targets. What forms of signaling mechanisms could RTKs be considered?

Relay, amplification

After purifying proteins through chromatography, how can you assess purity?

SDS PAGE

How is a GPCR involved in signal relay?

Signal relay is the spreading the signal onward throughout the cell. GPCRs process extracellular signals and relay them to signaling pathways within the cell via activation of a G protein.

What is signal transduction?

Signal transduction is the processes whereby one type of signal is converted to another. In a typical communication between cells, the signaling cell produces a type of extracellular signal molecule that is detected by the target cell. Target cells have proteins called receptors that recognize and respond specifically to the signal molecule. Signal transduction begins when the receptor on the target cell gets an incoming extracellular signal and converts it to the intracellular signaling molecules that alter cell behavior.

What are endocrine signals?

Signals that target cells at a distance. They travel through the blood to reach all parts of the body (hormones)

What happens when dynein gets to the very end of the - end?

Since dynein can only walk in one direction, kinesin, which is attached to its cargo at the back will take it back to the plus end of the microtubule, and then dynein can start to walk back up

What happens when kinesin gets to the very end of the + end?

Since kinesin can only walk in one direction, dynenin, which is attached to its cargo at the back will take it back to the minus end of the microtubule, and then kinesin can start to walk back up

In Fig 3A, bands form at approximately 30 kDa, 75 kDa and 100 kDa. The researchers already know the approximate molecular weights of the proteins they hoped to create, because they know the sizes of the constituent antibody and exotoxin protein domains. How does Fig 3A provide evidence to support the researchers' claims that the algal chloroplasts are producing the proteins?

Since the researchers know the sizes of the antibody and the exotoxin protein domain, they know where they are supposed to appear on the gel and when they saw that the proteins had migrated as they are supposed to, they could confirm that they accumulated in the transgenic alga.

What is DAG?

Small messenger molecule produced by the cleavage of membrane inositol phospholipids in response to extracellular signals. It is a lipid molecule that stays in the plasma membrane to help recruit and activate protein kinase C.

Four methods to break open cells:

Sonication Detergent Mechanical force to shear cells Cells are forced through a small hole using high pressure to break open cells

What are the functions of stable microtubules in cells?

Stable microtubules are found in differentiated cells and they maintain the organization of the differentiated cell and they serve as tracks.

Trypan blue is a dye that stains the cytoplasm of cells, and it is used to indicate whether cells in a culture or assay are living or dead. The dye can only enter dead cells through their ruptured cell membranes. It has no effect on living cells, because their membranes are intact and their cytoplasm is not exposed. How is staining using Trypan blue different from the method used in the paper?

Staining using Trypan blue is different from the method used in this paper, because the staining in this paper will show a color when the cells are alive, because the tetrazolium salt will appear colored only when cells are alive, because they are the only cells that are producing metabolites, whereas Trypan blue will have a greater color when cells are dead, because the dye can only enter dead cells through their ruptured cell membrane

Describe the process of co-immunoprecipitation

Step 1: lyse cells with a detergent to create a homogenate Step 2: Centrifuge the cells, and take the supernatant Step 3: Add an antibody that recognizes a certain protein X. Step 4: Add beads that are conjugated to a certain protein that has an affinity for the antibody Step 5: Centrifuge the sample, and since the beads are dense, they will go to the bottom and take with them the protein complex, and we can get rid of everything that did not attach to the bead. Step 6: Start to prepare a western blot Step 7: Denature the proteins with SDS, mercaptoethanol, and heat, and this will allow all of the proteins to dissociate from the antibodies and the beads Step 8: We will load the total protein into the polyacrylamide gel and run the gel (Lane 1: ladder; Lane 2: WT; Lane 3: Mutant) Step 9: After the run, we will transfer the proteins from the gel to the nitrocellulose paper Step 10: We will use milk to prevent any nonspecific blocking Step 11: After the transfer, we will use a primary antibody and a secondary antibody conjugated to a fluorophore 9) Develop the film We will have to use different antibodies to recognize different proteins, but it will allow us to see which proteins immunoprecipitated together and which were interacting

Explain how taxol works to treat cancer cells?

Taxol binds tightly to microtubules and prevents them from losing subunits, but new subunits can still be added, so the microtubules can only grow, not shrink, but Taxol still arrests the cell in the middle of mitosis, because it is unable to partition the chromosomes into 2 groups, since the mitotic spindle is a microtubule based apparatus that guides the chromosomes during mitosis, and in order for the mitotic spindle to function, microtubules must be able to assemble and disassemble and the inactivation or destruction of the mitotic spindle will eventually kill dividing cells. In addition, the formation of the mitotic spindle requires the prior depolymerization of other cellular microtubules to free up the tubulin to build the spindle.

Control primers for the 16S rRNA region of the chloroplast genome were used for validation that the PCR was successful. Why was the 16s rRNA region a good control and for what was it used to control?

The 16S rRNA sequence is a good control because it is a very conserved gene among all chloroplast DNA. It is used as a control to ensure the PCR was done correctly, because if this band did not appear, the PCR would have probably been a fail

What is glycolysis?

The 2nd stage of catabolism that is a chain of reactions that splits molecules of glucose into 2 pyruvate molecules, and since the electrons in a pyruvate molecule are at a lower energy state than those in a glucose molecule, it releases energy in the form of ATP and NADH.

What is the difference between the B-cells represented by the red peak and the B-cells represented by the blue peak from figure 5?

The B cells represented by the red peak are the B cells without the antibody and the B cells represented by the blue peak are the B cells with the antibody. The cells represented by the red peak were not incubated with the antibodies to produce a baseline.

Explain how ATP production in the mitochondria follows the First Law of Thermodynamics.

The First Law of Thermodynamics states that energy is neither created nor destroyed. ATP production the mitochondria occurs primarily in the ETC, a system which utilizes a gradient (potential energy) of protons to oxidize hydrogen. This oxidation generates the energy ATP Synthase relies on to add a phosphate to ADP to make ATP. This process is oxidative phosphorylation.

From figure 3, when the researchers designed the genes that would code for their scFv protein and immunotoxin proteins, they included the sequence for a small polypeptide called a Flag-tag. In this experiment, the Flag peptide does not contribute to the immunotoxins' ability to recognize B-cells or kill eukaryotic cells. Why did the researchers incorporate a Flag peptide into aCD22, aCD22PE40, and aCD22CH23PE40?

The Flag peptide can be used if there is not an antibody to recognize their immunotoxins, they can include the sequence for Flag in their immunotoxin construct so that they can use an anti-Flag antibody as their antibody in their western blot analysis to determine if the recombinant proteins accumulated in the transgenic algal strains

Entire nucleotides can also bind to intracellular signaling proteins and alter their function. How is the function of a GTP binding protein regulated by GTP hydrolysis?

The GTP bound form of a protein turns it on, and the GDP bound usually turns it off (not universal, but usually the case)

How does the absence of the upper band in Fig 2D, in lanes 2-4 support the researchers' conclusions?

The absence of the upper band in lanes 2-4 in figure 2D proves homoplasticity, meaning that the three experimental strains had all of their psbA genes replaced with the immunotoxin genes and this is important because chloroplasts have many (~80) copies of their genome, and it is important that each of their psbA genes had been replaced with the immunotoxin gene to stably express the desired gene

What is reduction?

The addition of hydrogen/gain of electrons

During movement, muscle cells require large amounts of ATP to fuel their contractile apparatus. These cells contain high levels of creatine phosphate, which has a standard free-energy change for hydrolysis of its phosphate bond of -10.3 kcal/mole. Why is this a useful compound to store energy?

The amount of free energy stored in the phosphate bond in creatine phosphate is larger than that of the bonds in ATP, so hydrolysis of creatine phosphate can be directly coupled with the phosphorylation of ATP because the ∆G for the reaction is still negative, so it will proceed to producing ATP. The enzyme, creatine kinase couples these reactions

How is the amount of time that a response lasts to a signal pathways controlled in the GPCR pathway?

The amount of time that the alpha and beta-gamma subunits remains switched on and are relaying signals determines how long the response lasts. The timing is controlled by the alpha subunit, which has intrinsic GTPase activity and will eventually hydrolyze bound GTP to GDP and return the whole entire G protein to its original, inactive conformation

Write a brief summary highlighting the most important findings associated with Figure 5.

The antibodies bind specifically to B cells because they have the CD22 surface antigen that the antibodies recognize and the antibodies do not bind nonspecifically

What is the question(s) the authors attempt to answer through the experimentation represented in figure 5?

The authors are attempting to answer the question of if the algal produced immunotoxins can bind specifically to their targets, and only their targets

From figure 5, what conclusion do the authors form from the results of the assay?

The authors conclude that the immunotoxins bind specifically to the CA-46 and Jurkat B cells, their targets, and they do not bind to Ramos T cells, which are not their targets, showing that they have specificity

In figure 4, how do the authors know that the bands observed represent ribosylated-biotinylated eEF2 and not αCD22PE40 or αCD22CH23PE40?

The authors know that the bands observed represent ribosylated-biotinylated eEF2 and not αCD22CH23PE40 because the size of the band is not the size of αCD22CH23PE40 and in addition they know that it is not αCD22PE40 or αCD22CH23PE40 because they used an anti-Biotin antibody, and this antibody would only recognize things that have biotin attached, and neither αCD22PE40 nor αCD22CH23PE40 are biotinylated, so the bands on this western blot would not appear for them.

What is the question(s) the authors attempt to answer through the experimentation represented in figure 2?

The authors wanted to see if they could successfully insert the immunotoxin gene into the chloroplast genome and they wanted to see if their chloroplasts were homoplastic which is important to stably express the desired gene

In Figure 3B, what would the formation of only one band at 100 kDa in lane 4 indicate?

The band at 100 kDa in figure 3B represents a monomeric aCD22CH23PE40, suggesting that at least some of the immunotoxins do not form the dimers.

In figure 4, what was the control for the assay? What lane represents the control in the figure?

The control in this assay is just the aCD22 antibody, which is not expected to ribosylate eEF2 since it does not contain the PE40 exotoxin, and that can be seen in the western blot that there is no biotinylated eEF2 in lane 1.

Explain how GTP can control microtubules.

The dynamic instability of microtubules stems from the intrinsic capability of tubulin dimers to hydrolyze GTP. Each free tubulin dimer contains one GTP molecule tightly bound to the beta-tubulin, which hydrolyzes GTP to GDP shortly after the dimer is added to the growing microtubule. The GDP remains tightly bound to the beta-tubulin. When polymerization is proceeding rapidly, the tubulin dimers add to the end of the microtubule faster than the GTP they carry is hydrolyzed, resulting in the end of the rapidly growing microtubules to be composed entirely of GTP-tubulin dimers, which form the GTP-cap. GTP-associated dimers bind more strongly to their neighbors in the microtubule than do the dimers that bear GDP, and they pack together more efficiently, so the microtubule will grow.

NAD+—ADP+—Biotin + eEF2 --PE40--> eEF2- ADP+-Biotin + NAD+ This is the reaction equation for the reaction that PE40 contributes to. What role does each molecule serve in the reaction and/or assay?

The eEF2-ADP is the product of the enzymatic reaction and biotin serves as the antigen to be able to probe/detect for the ADP-ribosylated elongation factor in the western blot. The immunotoxins aCD22PE40 and aCD22CH23PE40 have PE40 domains that act as the enzyme, whereas aCD22 alone has no enzyme activity.

The inner mitochondrial membrane serves as a device to convert what to what?

The energy contained in the high energy electrons of NADH and FADH2 into the high energy phosphate bond of ATP molecules

True or False? All of the proteins on an SDS-PAGE gel are transferred to nitrocellulose paper when one is performing a western blot.

True

Without a mechanism of harnessing the energy release by the energetically favorable transfer of electrons from NADH to O2, what would happen to this energy in the electron transport chain? How do cells deal with this?

The energy would be liberated as heat. Cells are able to recover as much of this energy as possible because the enzyme complexes in the electron transport chain use the energy to pump protons across the inner mitochondrial membrane into the intermembrane space.

Antibodies can recognize and bind to antigens which can be other antibodies in some cases. In Figure 5, what antigen does the immunotoxin recognize and what recognizes the immunotoxin

The immunotoxin recognizes the CD22 antibody, which is a B cell surface molecule. Anti-endothelian receptor A antibodies produced in a rabbit recognize the immunotoxin and anti-Rabbit DyLight antibodies recognize the Anti-endothelian receptor A antibody.

From figure 5, what would the conclusion be if the Jurkat cells had a fluorescent shift similar to that of the B cells?

The immunotoxins bind nonspecifically

Why were the experiments from figure 6 necessary? More specifically, why was the in vivo assay, represented by 6E, necessary?

The in vivo assay represented by 6E was necessary to make sure that the immunotoxins could kills the cells and inhibit their proliferation, meaning that the PE40 exotoxin was active. The immunotoxins could have an effect on tumor progression in animal models

Electron micrographs show that the mitochondria in heart muscle have a much higher density of cristae than mitochondria in skin cells. Why?

The inner mitochondrial membrane is the site of oxidative phosphorylation and it produces most of the cell's ATP. Cristae are porins of the mitochondrial membrane that are folded inward, and mitochondria that have a higher density of cristae have a larger area of inner membrane and a greater capacity to carry out oxidative phosphorylation. Heart muscles expend a lot of energy during contractions, whereas skin cells have a smaller energy demand. An increased density of cristae increases ATP production capacity of the heart muscle cell.

From figure 2, why do you think that the kanamycin resistance gene would need to be present in all plasmids?

The kanamycin resistance gene was present in all plasmids because they could plate the algae on plates with the antibiotic, kanamycin to select for the algae that were successfully transformed with the recombinant immunotoxin genes

Why might a cell's response to a signal be slow?

The length of time a cell takes to respond to an extracellular signal depends on what needs to happen once the message has been received. For cell growth and division, when triggered, it takes a long time, in part because the response to the signals requires change in gene expression and production of new proteins

Dynamic instability causes microtubules to grow or shrink rapidly. What must happen at the end of the microtubule that is currently shrinking to stop shrinking and to start growing again?

The microtubule is shrinking because it has lost its GTP cap. GTP loaded tubulin subunits from solution will still add to this end, but they will be short lied because either they hydrolyze GTP or because they fall off as the microtubule rim around them disassembles. If GTP loaded subunits are added quickly enough to cover up the GDP containing subunits at the microtubule end, a new GTP cap can form and growth is favored

Where does the citric acid cycle happen?

The mitochondriaal matrix

If some cell-surface receptors can rapidly signal to the nucleus by activating latent transcription factors at the plasma membrane, why do most cell surface receptors use long, indirect signaling cascades to influence gene transcription in the nucleus?

The more steps there are in an intracellular signaling pathway, the more places the cell has the regulate the pathway, amplify the signal, integrate signals from different pathways and spread the signal along divergent pathways

Genes that code for mutant forms of RTK are introduced into cells. The cells also express their own normal form of the receptor from their normal genes, although the mutant genes are constructed so that the mutant RTKs are expressed at higher concentration than the normal RTKs. What would be the consequence for introducing the mutant RTK gene that codes for an RTK that lacks its extracellular domain?

The mutant RTK that lacks its extracellular ligand-binding domain is inactive and cannot bind extracellular signals and its presence has no consequence for the function of the normal RTK

Genes that code for mutant forms of RTK are introduced into cells. The cells also express their own normal form of the receptor from their normal genes, although the mutant genes are constructed so that the mutant RTKs are expressed at higher concentration than the normal RTKs. What would be the consequence for introducing the mutant RTK gene that codes for an RTK that lacks its intracellular domain?

The mutant RTK will be inactive, but its presence will block signaling by normal receptors. When a signal molecule binds to either receptor, it will induce their dimerization and 2 normal receptors will have to come together to activate each other by phosphorylation. In the presence of an excess of mutants, the normal receptor cannot be activated because it is a mutant and lacks a kinase domain

GPCRs activate G proteins by reducing the strength of GDP binding to the G protein. This results in rapid dissociation of the bound GDP, which is then replaced by GTP which is present in the cytosol in much higher concentrations than GDP. What might be a consequence of a mutation in the alpha subunit of a G protein that reduced its affinity for GDP without significantly changing its affinity for GTP?

The mutated G protein would be almost constantly activated, because GDP would dissociate spontaneously, allowing GTP to bind even in the absence of an activated GPCR.

What is the control of the experiment in figure 5? Why was this sample selected as the control?

The negative control is the use of Jurkat T cells, because they do not have the CD22 proteins on their surface, so the immunotoxins are not expected to bind

What are GTP-tubulin dimers added?

The plus end

A cell's polarity is a reflection of what?

The polarized system of microtubules inside of the cell which help position organelles in their required location

What 3 things does a Western blot detect?

The presence, abundance, and size of proteins

How is a primary antibody different than a secondary antibody?

The primary antibody directly binds to the target protein, but it is not a reporter, it is just for the proper binding. The secondary antibody is the reporter antibody that binds to the primary antibody and the secondary antibody is also attached to an enzyme, and once a substrate is added, a product appears that is colored

After the researchers select algae that are capable of growing on kanamycin plates, they use PCR to identify several lines of transgenic algae that been transformed correctly—with the new gene inserted into the correct places in the chloroplast genome. In Figure 2C, what sequence of the plasmid are the primers designed to recognize?

The primers are designed to recognize the aCD22 scFv (single chain antibody that recognizes the CD22 antigen) gene and the psbA 5' UTR to make sure that they were integrated into the psbA locus because that was the wildtype gene that the recombinant immunotoxin genes were replacing

Dynamic instability causes microtubules to grow or shrink rapidly. Consider an individual microtubule that is currently shrinking. How would a change in the tubulin concentration affect the switch from a shrinking to a growing microtubule?

The rate of addition of GTP-tubulin will be greater at higher tubulin concentrations. The frequency with which shrinking microtubules switch to the growing mode will therefore increase with increasing tubulin concentration. The consequence of this regulation is that the system is self-balancing, the more microtubules shrink, the higher concentration of free tubulin, and the more frequently microtubules will start to grow again, and the more that they grow, the lower the concentration of free tubulin, and so tubulin addition will slow down, and GTP hydrolysis will catch up with new GTP-tubulin addition, the GTP cap will be destroyed and the microtubule will switch to shrinking mode

The bands in lanes 2 and 3 are the same in Figs 3A and 3B. Explain why there is there no difference between the aCD22 and aCD22PE40 bands under reducing and non-reducing conditions.

The reason that there is no difference between the aCD22 and aCD22PE40 bands under reducing and non-reducing conditions is because aCD22 and aCD22PE40 do not have any disulfide bonds that are reduced, and so therefore they will not change under reducing conditions

What is oxidation?

The removal of hydrogen/loss of electrons and this usually releases energy

From figure 4, describe the contents of Lanes 2 and 3. What results did the experimenters expect to see, given this assay? You should consider the MW of eEF2 when thinking of your response

The researchers expected to see a band around 92 kDa, which is what they saw because 92 kDa is the weight of eEF2, and if biotin is conjugated to eEF2, their anti-Biotin antibody would show a band around 92 kDa if they were attached.

What is the question(s) the authors attempt to answer through the experimentation represented in figure 4?

The researchers try to answer the question of if exotoxin A is enzymatically active in the immunotoxin.

Gel-filtration chromatography separates molecules according to their size. Smaller molecules diffuse faster in solution than larger ones, yet smaller molecules migrate more slowly through a gel filtration column than larger ones. Explain the paradox. What would happen at rapid flow rates?

The slower migration of smaller molecules through a gel-filteration column occurs because smaller molecules have acces to more spaces in the porous beads that are packed into the column. It is important to have a slow flow rate to give the smaller molcules enough time to diffuse into the spaces inside of the beads. At rapid flow rates, all molecules will move rapidly around the beads and small and large molecules will exit together from the column and the proteins would not be separated

How does GTP hydrolysis made the microtubules shrink?

The structure of the tubulin dimer is changed when they hydrolyze GTP and then are bound to GDP, so they cannot pack as tightly and become destabilized and the dimers fall off

In Figure 2D, there are two bands in lane 1. What fragment of DNA does the upper band represent?

The upper band represents the psbA gene that the researchers replaced with the immunotoxin gene in the transgenic algae strains.

The whole point of the citric acid cycle is to do what?

The whole point of the citric acid cycle is to produce a lot of NADH & FADH2 to go to the electron transport chain; moving energy in the form of electrons

What is placed in Lane 1, and why is it an effective control for the experiment represented by Fig 3A and 3B?

The wildtype algae strains with no transgenic DNA is placed into lane 1, and this is an effective negative control for figure 3 because they do not expect to get any bands in this lane since these chloroplasts do not have the gene for the immunotoxin, and showing that there is no band here proves that the bands in 2, 3 and 4 are not false positives

In Figures 3A and 3B, why are there bands in lanes 2, 3 and 4, but not in lane 1? What conclusions do the researchers draw from this gel?

There are bands in lanes 2, 3, and 4 because these are the transgenic algal strains and have the gene for the immunotoxin protein with the sequence for Flag peptide attached that the antibodies recognize, but in the wildtype algae cells, they do not have the gene for the immunotoxin protein, so they will not have anything for the anti-Flag antibodies to recognize. The researchers can conclude that the transgenic algae's chloroplasts accumulate the immunotoxin and that it is not a false positive since there are none in the wildtype lane

Why is there no shift observed for the Jurkat cell sample from figure 5? Why was this cell line included in the assay?

There is no shift for the Jurkat cell sample because the antibodies do not bind to them, so there will be no difference in the fluorescent detected by the flow cytometer. This cell line was included as a negative control, and to show that the immunotoxins do not bind nonspecifically

Adipocytes store fatty acids as triacylglycerol, a molecule of glycerol esterified to three long chain fatty acids. Lipase, the enzyme that hydrolyzes triacylglycerols to one molecule of glycerol and three long chain fatty acids, is found mainly in adipocytes and is active in the presence of the signaling molecule, adrenaline. It is phosphorylated by Protein Kinase A (PKA) and is catalytically active only when phosphorylated. You are studying a line of cultured adipocytes that express the G-protein-coupled receptor is coupled to G. What would happen if you get your cells to take up a bunch of non-hydrolyzable GTP?

There would be high levels of TAGs in the absence of adrenaline, but as soon as you receive adrenaline and activate the signaling pathway, there will be constitutively low levels of TAGs because the Gα would be constantly active

T/F: Some activated carrier molecules can transfer both energy and a chemical group to a 2nd molecule

True, ATP can donate both chemical bond energy and a phosphate group

The molecules that shuttle the high energy electrons to the electron transport chain, FADH2 and NADH carry the high energy electrons that originally came from where?

These high energy electrons originally came from the glucose that we ate

Why were Rag−/− × gc−/− mice selected for the assay represented by 6E?

These mice were selected, because they lack an adaptive immunity and natural killer cells, which are important in the immune system response and natural killer cells can destroy cancer cells. If these mice were used, there would not be the additional variable of the mouse's own immune system reacting to the tumors, and they would know that the sole reason for tumor death would be the immunotoxin Experimental Analysis

Give an example of an organelle that is localized to a particular region in the cell and explain how it gets to its final destination

These organelles get to their final destination via motor proteins walking on the microtubule filaments and positioning them in their required location and a cell's polarity is a reflection of the polarized system of microtubules in cells. In cells of the immune system, cells that secrete a lot of antibodies have their Golgi close to the site of secretion. Nerve cells must move functional mitochondria to the axon terminus and worn out mitochondria from the axon terminus to the nerve cell body. Melanosomes are moved out of melanocytes so that pigments can be distributed in the epidermis

Nuclear receptors bind hormones and can be found in the cytosol and in the nucleus. What is the function of an activated nuclear receptor?

They act as transcription regulators in the nucleus. When bound by a hormone, the receptor undergoes a conformational change that activates the protein, allowing it to promote or inhibit transcription of specific target genes. Each hormone acts on a different receptor and each receptor acts at a different set of regulatory sites in DNA

Where in the cell are intermediate filaments found?

They are found in the cytoplasm, forming a network surrounding the nucleus and extending out to the cell periphery and are anchored to the plasma membrane by desmosomes. They are also found in the nucleus where they form the nuclear lamina network that underlies and strengthens the nuclear envelope

What happens to NADH and FADH2 in oxidative phosphorylation?

They are oxidized to NAD+ and FAD

How do actin filaments differ from microtubules?

They are thinner, more flexible, and shorter than microtubules, and there are many more of them so that the total length of actin filaments in a cell is many times greater than the total length of all of the microtubules. Actin filaments are also rarely found in isolation in the cell and they are generally found in cross linked. Actin filaments also differ from microtubules, in that microtubules tend to undergo more drastic changes in length than do actin filaments because microtubules undergo dynamic instability, while actin filaments can undergo treadmilling.

Explain the similarities and differences in polymerization and de-polymerization in actin filaments and microtubules?

They both add to the plus end, but actin can add to both the plus and minus end Microtubules depolymerize from the plus end. The loss of the GTP cap at the plus end of microtubules causes de-polymerization and we do not see that as much in actin Actin depolymerizes from the minus end When the triphosphate nucleotide, the tubulin or actin can pack more tightly and associate more closely with their neighbors

What is the purpose of the anti-rabbit DyLight antibody in figure 5?

They contain a fluorophore, so they can produce a fluorescent shift when subject to flow cytometry analysis.

There are 3 different tools that a researcher could use to visualize neurons in a mouse's brain that fluoresce! To obtain the image, what could they use?

They could express a transgene that was: Promoter of neuron gene + coding region of neuron gene + GFP They could use a fluorescent microscope if the neurons were stained with fluorescent dyes. Use antibodies conjugated to a fluorophore that recognize a protein that is abundant in and localized to neurons

What are protein phosphatases?

They dephorphorylate other proteins, which typically inactivates them

Cell crawling requires protrusion of the leading edge and retraction of the trailing edge. How do cells use actin to retract their rear ends?

They have severing proteins that breaks down the network at the rear end, to depolymerize actin

What do actin filament do?

They help the cell crawl

Why do protons move freely across the inner mitochondrial membrane back into the matrix during oxidative phosphorylation?

They move down their electrochemical gradient. Once the protons accumulate in the inner membrane space, they readily move across the membrane into the matrix and move down their electrochemical gradient, and as they do that, they move through a protein, ATP synthase, and ATP is synthesized

What are protein kinases?

They phosphorylate other proteins which typically activates them

What is the main function of an intermediate filament

They provide the cell with mechanical strength and allow the cell to withstand the mechanical stress that occurs when cells are stretched. They are the toughest and most durable of the cytoskeletal filaments.

Why did experimenters use two types of B-cells?

They used B-cell lines, which are immortalized and can be really messed up/irregular compared to B-cells found naturally in the body. It is necessary to show more than one type because if they only used one type of B-cell line, we could not automatically assume that the immunotoxin bound to, and killed the cells because it was a B-cell or because it was irregular and may be a cell-line specific response. Showing more than one B-cell line AND a T-cell line let's us know that what they are claiming is true

In figure 3, why did the researchers run all of the proteins from their different algae cells through SDS-PAGE?

They wanted to separate the proteins by size

What specific questions do the authors attempt to answer through the experimentation represented by figure 3A? What additional question do they attempt to answer with figure 3B?

They were trying to determine that the immunotoxin proteins could be accumulated in the chloroplasts. In 3B, they wanted to make sure that the homodimer antibody could be assembled in the chloroplasts because in 3B they ran a non-reducing western blot, which will keep the disulfide bond linkage between the two chains.

From figure 4, why was this experiment necessary? What conclusion do the authors form from the results of the assay

This experiment was necessary because in order for the immunotoxins to be effective against the B cells, they exotoxin must be enzymatically active and be able to inhibit the transcription factor and kill the cancer cells. The conclusion that the authors formed from the results is that exotoxin is enzymatically active in the immunotoxin because as can be seen on the western blot, when an anti-Biotin antibody was used to probe for eEF2-Biotinylated ADP, they could see that on the western blot, the location at which eEF2 is supposed to be located, the anti-Bitotin antibody gave a signal, showing that most likely Biotin is attached to eEF2.

From figure 5, why was this experiment necessary? Why is flow cytometry a good technique for this experiment?

This experiment was necessary to make sure that the immunotoxins produced in the algal chloroplasts are capable of recognizing their target cells and binding to them, and not binding to any nonspecific cells that they are not supposed to bind to. Flow cytometry is a good technique for this experiment because it can differentiate between different fluorescent shifts given off by cells that have antibodies attached, and cells that do not have antibodies attached.

There is a mutation in Ras where it cannot hydrolyze GTP. Is this a loss of function or gain of function mutation? Would this be dominant or recessive?

This is a gain of function since Ras has a new function that it can promote cell division without being activated by a signaling molecule. Gain of function mutations tend to be dominant and only 1 copy of the mutant Ras will be needed to have the signaling pathway on all of the time

Consider the equation light energy + CO2 + H2O --> sugars + O2 + heat energy Would you expect this reaction to occur in a single step? Why must heat be generated in the reaction?

This is the equation for photosynthesis, which occurs as a set of individual reactions that are catalyzed by individual enzymes. This reaction does not occur in a single step, because if it was accomplished in 1 step, it would generate more enegy than can be stored in a carrier molecule. Because sugars are more complicated than CO2 and H2O, the reaction generates a more ordered state inside the cell, and because of the 2nd law of thermodynamics, the increase in order must be accompanied by a greater increase in disorder, which occurs because heat is generated at many steps on the long pathway

T/F: The oxidation of sugar molecules by the cell takes place according to the following reaction C6H12O6 + 6O2 --> 6CO2 + 6H2O + energy. In cells, the reaction takes place in more than 1 step

True, if it had occurred in only 1 step, then all of the energy would be released at once and it would be impossible to harvest it efficiently to drive other reactions, such as ATP synthesis

T/F: Both GTP-bound alpha subunits and nucleotide free beta-gamma subunits, but not GDP-bound, fully assembled G proteins, an activate other molecules downstream of GPCRs

True.

T/F: Lamellipodia and filopodia are feelers that a cell extends to find anchor points on the substratum that it will then crawl over .

True.

Parts A-D represent an in vitro experiment involving the CA-46, Ramos, and Jurkat cell lines. What makes this an in vitro assay?

This was in vitro because it was not in animals, it was on plates.

What is the role of a detergent and centrifugation in the first step of a western blot?

To break down the cell membrane and the cytoplasm contents can come out, and the centrifuge gives the supernatant and a pellet, with the pellet containing the cell fragments and cellular debris and the supernatant containing all of the proteins

You have successfully identified a gene required for cell division in yeast called Cdc2. Yeast with loss-of-function mutations in Cdc2 cannot divide at warm temperatures. You are particularly interested in the protein encoded by Cdc2 because your data suggests that the causative mutation does not affect gene expression. In other words, you observed no difference in the relative abundance of RNA transcript in the mutant. What is a protocol that could test this hypothesis: The protein is targeted for degradation in the mutant and thus is less abundant or even absent in mutant yeast.

To determine if the protein is targeted for degradation in the mutant and less abundant, we will run a western blot. 1) I will grow 2 plates of cells in an incubator, one mutant yeast grown on plates with growth media and one wildtype yeast grown IN AN INCUBATOR SINCE THE MUTANT IS TEMPERATURE SENSITIVE 2) We will split open the cells using sonication to create a homogenate 3) We will centrifuge the homogenate to separate the pellet and the supernatant 4) We will use pipettes to remove the supernatant and heat the supernatant with SDS and mercapoethanol to denature the proteins and make them negatively charged. It is important to denature the proteins so that they travel through the gel based on size, and their folding does not impact this 5) We will load the total protein into the polyacrylamide gel and run the gel (Lane 1: ladder; Lane 2: WT; Lane 3: Mutant) 6) After the run, we will transfer the proteins from the gel to the nitrocellulose paper 7) We will use milk to prevent any nonspecific blocking 8) After the transfer, we will use a primary antibody that recognizes and binds to the proteins (they will bind to both the wildtype and the mutant because the antibody recognizes such a small part of the protein that it probably will not differentiate between the wildtype and mutant that were made in a rabbit, and a secondary anti-rabbit antibody that is tagged with a red fluorophore 9) Develop the film 10) After developing the film, I will look to see if there are thicker bands for the wildtype, suggesting there is more protein in the wildtype than the mutant

What is the point of moving protons back across the inner mitochondrial membrane in the ETS?

To generate ATP, which requires energy. ATP synthase takes ADP, attaches a phosphate, and makes ATP

At first glance, the final steps of fermentation appear to be unnecessary, the generation of lactate or ethanol does not produce any extra energy for the cell. Why would cells that grow in the absence of oxygen need to produce these two molecules?

To keep glycolysis going, cells need to regenerate NAD+ from NADH.

T/F: Different signals originating from the plasma membrane can be integrated by cross-talk between different signaling pathways inside the cell

True

T/F: Tyrosine phosphorylation serves to build binding sites for other proteins to bind to RTKs

True

T/F: Without actin, cells can form a functional mitotic spindle and pull their chromosomes apart, but they cannot divide

True. Actin is needed to make the contractile ring that causes the physical cleavage between 2 daughter cells, whereas the mitotic spindle that partitions the chromosomes is composed of microtubules

T/F: The electrochemical proton gradient consists of 2 components: a pH difference and an electrical potential

True. Both components add to the driving force that makes it energetically favorable for H+ to flow back into the matrix

T/F: Kinesin moves ER membranes along microtubules so that the network of ER tubules becomes stretched throughout the cell

True. In the absence of microtubules, the ER collapses towards the cell center

T/F: The oxidation of sugar molecules by the cell takes place according to the following reaction C6H12O6 + 6O2 --> 6CO2 + 6H2O + energy. The energy is produced by a process that involves the oxidation of carbon atoms

True. The carbon atoms in CO2 are fully oxidized

T/F: IP3 is produced directly by cleavage of an inositol phospholipid without incorporation of an additional phosphate group

True. The inositol phospholipid that is cleaved to produce IP3 contains 3 phosphate groups, one of which links the sugar to the diacylglycerol lipid. IP3 is generated by a simple hydrolysis

T/F: The oxidation of sugar molecules by the cell takes place according to the following reaction C6H12O6 + 6O2 --> 6CO2 + 6H2O + energy. Some organisms carry out the reverse reaction

True...photosynthesis

True or False? A single RTK can interact with multiple intracellular signaling proteins at the same time

True; often as many as 10-20 and some of them become phosphorylated and activated upon binding to the receptor and propagate the signal, but some function as scaffolds to couple the receptor to other signaling proteins to help build the active signaling complex

In this paper, the researchers used a tetrazolium salt as an indicator of cell viability. This compound is clear when it is injected into the wells of the cell assay, and turns a color when it reacts with living cells' metabolites. Cells produce more metabolites when they are healthy. If they are dying, they produce fewer metabolites, and if they are dead, they produce no metabolites. What is the benefit of using tetrazolium salt to determine cell viability over a dye like Trypan Blue?

Trypan Blue will only show if the cell is dead, not dying, whereas using tetrazolium salts, we can see how active the cells are if they are alive. Trypan Blue can be used to see if the treatment kills cells, whereas tetrazolium salts can see if the treatment inhibits cells and to what degree and if it kills them.

Cells analyzed by flow cytometry can exhibit nonspecific fluorescence. How was this experiment from figure 5set up to minimize the impact of non-specific fluorescence?

Two curves were shown for each graph to compare a baseline fluorescence to the fluorescence exhibited when the antibodies were attached, so the difference between the two can be seen

Why do you suppose it is much easier to add tubulin to existing microtubules than to start a new microtubule from scratch? How do gamma tubulins in the centrosome overcome this hurdle?

Two tubulin dimers have a lower affinity for each other because of a more limited number of interaction sites than a tubulin dimer has for the end of a microtubule where there are multiple possible interaction sites, both end-to-end tubulin dimers adding to the protofilament and side-to-side of the tubulin dimers interacting with tubulin subunits in adjacent protofilaments forming the ringlike cross section. To initiate a microtubule from scratch, enough tubulin dimers have to come together and remain bound to each other for long enough for other tubulin molecules to add to them, so this will rarely happen. Centrosomes contain preassembled rings of gamma tubulin to which the tubulin dimers can bind

G protein pathways are also able to regulate transcription. To do this, PKA must enter the nucleus. Consider a mutant in which PKA is always active after it has been exposed to cAMP. What would be the outcome if the gene being transcribed increased hair growth?

Until the signal molecule binds to the GPCR, stimulating the later production of cAMP, there would be no change as the pathway would not be active. Once the molecule signals the pathway to start, producing cAMP, PKA will move into the nucleus and begin initiating transcription. The PKA cannot be turned off, so the transcription will be constitutive and hair growth will occur.

CFTR is a transmembrane protein that moves Cl- ions down their concentration gradient. In people with cystic fibrosis, this protein is mutated, and is often not in the plasma membrane as it should be, and instead remains in the cytoplasm. How could you test this hypothesis?

Use co-localization. Use recombinant DNA technology to conjugate the gene for GFP to the promoter and gene for protein CFTR. Introduce the transgene into wild type and mutant cells. Fix cells and then incubate with a anti-protein X primary antibody (made in mouse) against a protein known to be ubiquitous in the plasma membrane (this protein will serve as my marker) Incubate cells with a anti-mouse secondary antibody conjugated to a red fluorophore Look at cells under a fluorescent microscope. If my hypothesis is correct then I will see a red membrane around each cell and green within the cell, not at the membrane If my hypothesis is incorrect then I will see a red/green membrane (red & green overlap)

You want to test a hypothesis that a mutation prevents protein Y and protein Z from interacting. How could you do that?

Use recombinant DNA technology to conjugate one half of gene for GFP to the promoter and gene for protein Y, use recombinant DNA technology to conjugate the other half of the gene for GFP to the promoter and gene for protein Z. Introduce the transgenes into cells. Look at cells under a fluorescent microscope. If my hypothesis is correct then I will see: no fluorescence If my hypothesis is incorrect then I will see: green fluorescence

CFTR is a transmembrane protein that moves Cl- ions down their concentration gradient. In people with cystic fibrosis, this protein is mutated, and is being degraded. How could you test this hypothesis?

Use sonication to homogenize your samples of wild type and mutant cells (although keep the two samples separate). Use centrifugation to remove large, dense cell debris from the homogenate. Keep the supernatant and discard the pellet. Treat the samples with SDS in order to solubilize the proteins and coat them with a negative charge. Treat samples with mercaptoethanol in order to reduce disulfide bonds or other covalent linkages. Run samples in separate lanes of a PAGE. Transfer proteins from polyacrylamide-gel to nitrocellulose membrane. Use a blocking agent to prevent nonspecific interactions between the antibodies and the nitrocellulose paper. Incubate with a anti-protein X primary antibody (made in a donkey). Wash and then incubate with a anti-donkey secondary conjugated to a red fluorophore. Examine the membrane If my hypothesis is correct then I will see: a red band in the wild type lane and no band in the corresponding spot on the mutant lane. If my hypothesis is in correct then I will see: a red band in the wild type lane and a red band at the same position in the mutant lane.

What is co-immunoprecipitation?

Used to show binding between 2 or more proteins. Small scale affinity purification of target protein and its binding protein partners. It can also be used when we know that 2 proteins interact, it can be used to isolate both of them together

Dr. Dorian thinks that TAS2R2 relies on an allosteric activator, but does not know what the activator is. He believes that the activator is a protein in the SNF family, and has antibodies for several SNF targets. Describe at least one way to determine which protein is the allosteric activator and explain how you would read the results.

Using a double antibody labeling system, he could try using fluorescence microscopy to see if the TAS2R2 and SNF proteins co-localize within the cell.

Lost in his daydreams, Dr. Dorian accidently mixes up his TAS2R2 and several other taste receptor proteins! Describe one way that Dr. Dorian could isolate TAS2R2. How could he verify that TAS2R2 is not still in the remaining protein sample?

Using affinity chromatography. He would need to make sure that the other proteins did not also bind to cyclohexamine. Then he would run the total protein through the column, and after everything else flowed through, he could wash the column with a solvent of a different pH, and changing the pH will release TAS2R2 from the beads by altering interactions. In order to show that TAS2R2 is not in the remaining protein, a western blot could be run on the remaining protein. If no bands for the TAS2R2 show up, then it has been fully extracted. We would have a ladder in the first lane, a control in the 2nd lane with TAS2R2 and the sample

How many signals can a certain receptor respond to?

Usually only uno

Do we find larger proteins on the reduced or non-reduced Western blots?

We find larger proteins under non-reducing conditions, meaning that in the non-reduced conditions there were disulfide bonds that kept them folded so they could not travel as far, or because the disulfide bond was holding together 2 polypeptide chains to create a larger protein. Under reducing conditions, we will only have proteins migrate at a rate that affects their molecular weight, not at a rate that affects their molecular weight and folding

You the scientist at a time when the Ras signaling pathway was still unknown and are working to uncover novel proteins in the Ras signaling pathway so you have obtained a strain of wild-type cells that proliferate in the presence of a signaling molecule known to activate the Ras pathway. In order to identify proteins that act in the Ras signaling pathway you conduct a genetic screen. To do this you mutagenize wild-type cells and culture them in the presence of the signaling molecule. You screen for mutants that do not proliferate normally (assume the cells divides some but proliferation is minimal). You identify a mutant, sequence the genome of your mutant, and find a mutation in the Ras gene. You are not surprised because you already knew Ras was involved in this signaling pathway. The next mutant that you identify contains a mutation in a previously unknown protein, and this mutant does not proliferate in the presence of a signaling molecule. How will you figure out whether this protein X acts upstream or downstream of Ras in the signaling pathway?

We will take cells that express constitutively active Ras in the mutant cells, and if we find that the cells do proliferate, this suggests that the protein acts upstream of Ras since if it was downstream, Ras would activate it, but it would not be able to pass along the signal

For figure 3, why was Western blotting an appropriate lab method to answer the experimental question?

Western blotting is an appropriate lab method to answer the experimental question of if the recombinant proteins can accumulate in transgenic algal strains, because with western blotting they can use antibodies to probe for their specific protein (the immunotoxin protein conjugated with FLAG peptide) to make sure that it is specifically their immunotoxin that is expressed. In addition, because western blotting shows the sizes of the proteins, they could make sure that all of the correct domains were formed and with the non-reducing western blot, they could see if the disulfide bond in their homodimer was formed

Ca2+ is an important signaling molecule in the cell. Describe how an activated GPCR leads to the release of Ca2+ from the ER.

When a GPCR gets activated, it activates a G protein that activates the membrane bound enzyme, phospholipase C. Once activated, phospholipase C propagates the signal by cleaving a lipid molecule, inositol phospholipid, from the plasma membrane and generates 2 messenger molecules, IP3 and DAG. IP3 is a water soluble molecule that is released into the cytosol, where it binds to and opens calcium channels in the ER. Calcium rushes out causing a rise in the cytosolic calcium level, which signals other proteins. DAG is a lipid that remains embedded in the plasma membrane and helps recruit and activate protein kinase C, which needs to bind calcium to become active. Once activated, protein kinase C phosphorylates other proteins.

Explain how G-protein coupled receptor activation transmits a signal from the extracellular space to through the cytosol.

When an extracellular signal molecule binds to a GPCR, the receptor protein undergoes conformational changes that enable it to activate a G protein on the other side of the plasma membrane, leading to transmission of the signal. The alpha subunit of a G protein is bound to GDP, and it is inactive. When an extracellular signal molecule binds to its receptor, the altered receptor activates a G protein by causing the alpha subunit to decreases its affinity for GDP, which is exchanged for a GTP molecule. Now, the 2 activated parts of the G protein, the alpha subunit and beta subunit can interact directly with target proteins in the plasma membrane, which may relay the signal to other destinations in the cell. The amount of time that the alpha and beta subunits remain on is controlled by the behavior of the alpha subunit, because it has intrinsic GTPase activity and will eventually hydrolyze its GTP to GDP to return the G protein to the inactive conformation

Why does heat denature proteins?

When the heat rises, so does the kinetic energy, so the atoms move around and break the weak hydrogen bonds

Why do yeast make alcohol?

When they do not have any oxygen, they need to turn NADH into NAD+ so that glycolysis can continue going on and they can make ATP, because without oxygen, they cannot undergo oxidative phosphorylation to make ATP, so they can only make ATP with glycolysis. Glycolysis uses NAD+, and so if they just keep having glycolysis going on, they will run out of NAD+, and so they can replenish it during fermentation

Why is a nitrocellulose membrane used in western blotting?

When we use a probe, the probe can bind to different regions of the gel, but gel is really fragile and the probing and the reactions necessary cannot take place on the gel and it has a high affinity for proteins

Can a cell have more than one copy of the same receptor?

Yes

Can glycolysis happen without oxygen?

Yes

Can cargo be transferred between motor proteins?

Yes, for example, myosin can take kinesin's cargo and travel along actin tracks

Can a cell have many different types of receptors?

Yes, to allow the cell to be simultaneously sensitive to many different extracellular signals, and a combination of signals can evoke a response that is different from the sum of the effects that each signal would trigger on its own. This tailoring process occurs in part because the intracellular relay signals activated by different signals interact

What are lamellipodia?

a dynamic sheetlike extension on the surface of a cell that has a cytoskeletal protein actin projection on the leading edge and can be used to propel a cell forward

Chromatography

a matrix of small beads that separates proteins according to charge, size, hydrophobicity, or ability to bind to a specific molecule.

What is a centrosome?

a site that organizes an array of microtubules that radiate outward through the cytoplasm. The centrosome consists of a pair of centrioles surrounded by a protein matrix, and the matrix includes a ton of ring shaped gamma-tubulins, which serve as the nucleation site for the growth of one microtubule, because alpha-beta tubulin dimers add to each gamma tubulin ring and grow from there

What is cyclic AMP?

a small intracellular signaling molecule generated from ATP in response to hormonal stimulation of cell surface receptors and it AMPLIFIES THE SIGNAL. It is water soluble and can carry the signal throughout the cell

In affinity chromatography, what can we use to release a molecule bound to the column?

a solvent that alters the pH of the column to change the shape of the protein

In gel filtration chromatography, what can we use to release a molecule bound to the column?

a solvent that washes the molecule out of the pores

What is a centromere?

a specialized DNA sequence that allows duplicated chromosomes to be separated during M phase that can be seen as the constricted region of the mitotic chromosome

Which of the following types of cells would you expect to contain a high density of intermediate filaments in their cytoplasm and why? a) Amoeba proteus b) skin epithelial cell c) smooth muscle cell in the digestive tract d) E. coli e) nerve cell in the spinal cord f) sperm cell

a) not necessarily since they migrate rapidly from place to place and do not develop or sustain large tensile forces b) lots of cytosolic intermediate filaments to prevent them from rupturing as they are stretched and compressed by the movements of their surrounding tissues c) lots of cytosolic intermediate filaments to prevent them from rupturing as they are stretched and compressed by the movements of their surrounding tissues d) none e) lots of cytosolic intermediate filaments to prevent them from rupturing as they are stretched and compressed by the movements of their surrounding tissues f) not necessarily since they migrate rapidly from place to place and do not develop or sustain large tensile forces

NO signals from the blood vessels cause smooth muscles to relax; however, these signals can only cross a short distance as NO will be converted to more stable forms if exposed in the extracellular space. a. What type of signaling is this? b. If a smooth muscle cell culture were exposed to an environment rich in NO, what would be the result? Would you expect different results if this experiment was conducted in a culture plate vs a large flask?

a. Paracrine b. In an NO rich environment, we expect smooth muscle cells to relax. This effect would be greater in a smaller vessel, such as a plate, whereas larger distances would be difficult for the NO to cross without converting to more stable forms so you would see much lower levels of relaxation.

What type of cytoskeleton element is made of helical polymers of a globular protein?

actin

Actin filaments are branched and bound by _____________ to allow the filopodia and lamellipodia to reach out

actin binding proteins

After what step of cellular respiration is the glucose molecule fully oxidized?

after the citric acid cycle

Proteins are broken down into __________

amino acids

What is adenyl cyclase?

an enzyme that catalyzes the formation of cyclic AMP from ATP; important component in some intracellular signaling pathways

What is protein kinase C?

an enzyme that phosphorylates target proteins in response to a rise in DAG and calcium ions

What kind of protein is used as a detector to probe the target protein in a western blot?

antibody

What are autocrine signals?

are produced by the emitting cell and affect that same cell via its own receptors (immune cells)

Enzymes enable unfavorable reactions by doing what?

by coupling them with highly favorable reactions. For example, a kinase molecule will transfer a phosphate group to an ADP molecule at the end of glycolysis when a phosphate group is released from pyruvate

All of the proteins (what scientists call the "total protein") in cells that were homogenized are loaded into an SDS-PAGE gel. Although the proteins are all the same_______ (charge/size) they differ in _______ (charge/size) and thus move through the gel at different rates.

charge; size

Microtubules grow and shrink, polymerizing and depolymerizing rapidly within cells. Their behavior is described as _____________

dynamic instability

In living cells, most oxidation reactions are what?

energy releasing

What is phospholipase C?

enzyme associated with the plasma membrane that gets activated by a G protein and that generates 2 small messenger molecules, DAG and IP3, in response to activation. Once it is activated, it cleaves a lipid molecule in the plasma membrane, an inositol phospholipid, and generates IP3 and DAG

What is cyclic AMP dependent protein kinase?

enzyme that phosphorylates target proteins in response to a rise in intracellular cyclic AMP. This enzyme is normally inactive and held in its inactive state by a regulatory protein, but when cyclic AMP binds to the regulatory protein, it forces a conformational change that releases the inhibition and releases the active kinase

Fats are broken down into _________

fatty acids and glycerol

Activated RTKs are phosphorylated which allows them to do what?

interact with intracellular cytosolic proteins.

What type of cytoskeleton element are strong ropelike protein filaments?

intermediate filaments

What are the different types of beads that can be used in column chromatography?

ion-exchange, gel-filtration, and affinity

What would be the outcome of a mutation in Gα that prevents the dissociation of GDP

it would be permanently inactivated and could not initiate signaling cascades because GTP would never get the ability to bind to Galpha

Intracellular signaling molecules are often switched on by what?

kinases

Cells that have been homogenized and centrifuged will be found in the________ (supernatant/pellet) of the test tube.

pellet

In an energetically favorable reaction, is the ΔG positive or negative?

negative

What is an example of contact dependent signaling?

notch signaling

Food molecules are ________ to produce ATP.

oxidized

The _________ end of a microtubule extends outward toward the plasma membrane of a cell.

plus

The motor protein kinesin travels toward the ________ end while the motor protein dynein travels towards the ______ end.

plus; minus

In an energetically unfavorable reaction, is the ΔG positive or negative?

positive

What is homogenization?

preparation of a suspension of cell constituents from tissue by physical treatment in a liquid.

Differential centrifugation

repeated centrifugation at progressively high speeds to fractionate cell homogenates into their components.

Tubulin is a GTP binding protein. Microtubules with GDP bound tubulin tend to __________ because the dimmers do not associate as tightly with each other while microtubules with GTP bound tubulin tend to_________.

shrink; grow

High energy electrons are derived from _____________ and are transported via activated carriers _______________ to the ETS, which converts the energy in these electrons to the phosphate bonds in the _________ molecules.

sugars and fats; NADH and FADH2; ATP

What are paracrine signals?

target cells locally, or within the vicinity of the emitting cell (neurotransmitters, nitric oxide)

What does it mean if energy is easily exchangeable in activated carriers?

the bonds are easily broken

Sonication

the use of high frequency sound waves to split open a cell

How do receptor tyrosine kinases become activated?

they get phosphorylated

Are extracellular signaling molecules hydrophobic or hydrophilic?

usually large and too hydrophilic to cross the plasma membrane (like proteins)

You have a mutant with a missense mutation that you hypothesize is at a site that is phosphorylated in wild-type cells. Design an experiment that would allow you to determine if this amino acid is indeed phosphorylated.

we will run a western blot. 1) I will grow 2 plates of cells in an incubator, one wildtype and one mutant 2) We will split open the cells using sonication to create a homogenate 3) We will centrifuge the homogenate to separate the pellet and the supernatant 4) We will use pipettes to remove the supernatant and heat the supernatant with SDS and mercapoethanol to denature the proteins and make them negatively charged. It is important to denature the proteins so that they travel through the gel based on size, and their folding does not impact this 5) We will load the total protein into the polyacrylamide gel and run the gel (Lane 1: ladder; Lane 2: WT; Lane 3: Mutant) 6) After the run, we will transfer the proteins from the gel to the nitrocellulose paper 7) We will use milk to prevent any nonspecific blocking 8) After the transfer, we will use a primary antibody that that is a phosphoantibody and a secondary antibody that recognizes the phosphoantibody that is conjugated to a fluorophore 9) Develop the film 10) After developing the film, I will look to see if there are thicker bands for the wildtype, suggesting there is more phosphorylated

A widely used analytical technique used to detect proteins in a sample of homogenate or cell extract

western blotting