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Bio 353 Exam 1

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Phosphatidylserine influences a great deal of area primarily due to its large size. T/F

False

Phosphatidylserine influences a great deal of surface area primarily due to its large size. T/F

False

The empty center of a single pass transmembrane protein provides a channel for the transport of ions. T/F

False

The lipid bilayer functions to keep charged particles and fats outside of the cell. T/F

False

The primitive proto-prokaryote likely acquired motility before it lost the cell wall. T/F

False

The primitive protoprokaryote likely acquired motility before it lost the cell wall. T/F

False

Treadmilling of actin filaments only occurs in a test tube. T/F

False

When a cell is anchorage-dependent for growth, it can then go through the cell cycle when it is suspended. T/F

False

What is a filopodium: what is its purpose for the cell and what is it comprised of?

Filopodium pushes out on the plasma membrane into finger-like projections and is comprised of approximately 20 actin filaments. Contains receptors that bind to chemicals and other factors in the environment and allow the cell to explore its environment. They form focal adhesions on ventral surface and form ruffles on dorsal surface.

Explain why the plasma membrane does not rip as it extends out a lamellipodium.

Fluid Mosaic: The plasma membrane is made up of a phospholipid bilayer with hydrophilic heads pointing outward and hydrophobic tails pointing inward. The heads are polar and are not covalently bonded and this allows the membrane to stretch without rupturing and without allowing water to leak through because the tails "hate" water so much that they will do everything possible to "kick" water out.

uniporter, symporter, antiporter

carries one solute carries two solutes in same direction carries two solutes in opposite direction

In primary culture all cells survive. T/F

False

In primary culture all cells survive. True or false?

False

It is the proteins in the biological membrane that serve to partition water. T/F

False

Peroxisomes likely originated from endosymbiosis. T/F

False

Draw the membrane in a hairpin using cell shapes: cylindrical, cone, and upside down cone.

...

Starting with a living mouse explain the technology for making a cell line.

1) In a sterile environment such as a biological safety cabinet with laminar air flow, place a euthanized mouse on its back, and douse its ventral surface with 70% ethanol—importantly let the ethanol evaporate.2) Cut into the mouse using a set of sterile scissors and forceps, removing one or more of the larger muscles and place it in a sterile plate. Remove the mouse from the hood, leaving the covered plate with the muscle segments in the laminar flow hood.3) Using two sterile scalpels, dice the muscle into small (less than 1 mm diameter) chunks with opposing scalpels, moving them in an <-X -> fashion.4) Transfer these small chunks aseptically into a sterile Erlenmeyer flask that contains 0.5% trypsin and 3 mM MgCl2.5) Aseptically seal the Erlenmeyer flask and place the flask in an agitator for 5 hours at room temperature.6) After 5 hours of agitation, transfer the flask to a laminar flow hood and add medium containing serum to the contents of the flask. The serum in the medium acts as an inexpensive agent to inactivate the trypsin.7) The contents of the flask is transferred aseptically into a sterile cell culture plate and covered with the culture plate lid.8) The cell culture plate prepared at the end of the Primary Explant phase is placed in a cell culture incubator. Using aseptic technique, every 3 days half of the cell culture medium should be removed by aspirating slowly from the top of the medium. This should be replaced with fresh cell culture medium containing 10% serum.9) The cells will need to be re-fed every 3 days, and it will likely be necessary to subculture the plate (i.e., releasing the adherent cells, collecting them, and placing portions of the cells from one plate into several plates).10) A cell line is made when you aseptically take a single cell from the Secondary Culture and put it into a sterile cell culture plate with no other cells around. This culture plate contains medium with 10% serum in it. When the cell divides, all cells that form originate from that single cell. Thus, all cells are MITOTIC clones of that original cell.

List the 5 steps of directional cell walking when starting with a cell in suspension.

1. When cells are suspended from the surface they become spherical due to the disruption in the cytoskeleton. 2. When cells are added to the culture medium, they slowly settle to the bottom at different rates. 3. When they reach the bottom of the plate, the cells release many filopodia to explore their environment, which begins to stir around the medium. A filopodium is 2-10 micrometers in length and 0.2 micrometers in diameter. The filopodia move like a cockroach's antenna as they sense their environment. These cell extensions whose outer edges are the plasma membrane filled with receptors. 4. The filopodia move around, searching for signals, using their receptors. When a signal is found, the filopodia in that area of the cell stops moving because it is undergoing a signal transduction biochemical cascade that reorganizes that part of the cell. The lamellipodium extends out a larger, irregularly-sized structure that pushes out against the plasma membrane, this mechanism is caused by a network of actin filaments. And when this plasma membrane comes into contact with the substrate, 3 things happen in succession: (i) an "adhesion plaque" (super glue) is released at the site. This is where many integrin receptors bind to the substrate. (ii) Then in the center of the lamellipodium, the cell-equivalent of a cell muscle is assembled, made up of myosin and actin. (iii) Then this muscle contracts causing the cell to flatten out and pulling the back end of the cell forward in the direction of the signal. 5. The lamellipodium will extend on all sides of the initially spherical cell by repeating steps 3 and 4 until it is pancake-shaped with a bulge over the nucleus. Once directionally moving, the cell has a front and back end. The lamellipodia is at the front end, the nucleus is towards the back, and the retraction fiber is at the rear of the cell. Retraction fibers contain the cell-equivalent of superglue/adhesion plaque, thinly stretched (like a rubber band) plasma membrane with very little cytoplasm. Later, the plasma membrane breaks near the adhesion plaque, and the plasma membrane on both sides seals the break. This residual cellular material is called Microexudent.

Transmembrane proteins of alpha helices most frequently span the membrane ...... or ....... times.

1;7

How many amino acids are required to cross plasma membrane in an alpha helix?

22-24

How many amino acids are required to span a typical membrane?

22-24

The sodium/potassium ATPase removes ___________ ions from the cell and pumps ______________ ions into the cell against their respective concentration gradients.

3 Na; 2 K

The number of amino acids in an actin monomer is:

375

Human cells are kept at 37°C. Do you think a change to the temperature by 3°C would be more hazardous to the cell line if it were added, or subtracted? In other words, is a temperature of 40°C or 34°C more likely to result in detrimental effects?

40°C is a more dangerous temperature for cells. Cells can be cooled without suffering appreciable damage - in fact, cells are often frozen for long-term storage and re-animated when needed. Enough thermal energy, however, will permanently denature the enzymes and other cellular proteins resulting in cell death.

Human cells are kept at 37°C. Do you think a change to the temperature by 3°C would be more hazardous to the cell line it were added, or subtracted? In other words, is a temperature of 40°C or 34°C more likely to result in detrimental effects?

The thickness of the membrane is .................. nm.

The thickness of the plasma membrane is _____.

5 nm

A normal mouse fibroblast cell, when not contact inhibited, can divide approximately _______ times before becoming senescent.

The diameter of the actin filament is ...... nm.

Explain two methods of making a cell line in detail? Assume in this answer you are starting with the end of secondary culture moving into making up the cell line. (alternate response)

A cell line is made when you take a single cell from the Secondary Culture and put it in a plate all by itself in a culture plate with medium in it, thus all cells are MITOTIC clones of that original cell. There are two methods of taking a single cell from the Secondary Culture. One method is visual using micromanipulation and the other is mathematical using serial dilution. There are two methods that can be used to make a cell line. The first, serial dilution, is really inexpensive, but is more of a statistical approach. It first involves taking the secondary culture dish and trypsinizing the cells. Once the cells in the dish are not latched on to the bottom anymore, a pipette will be used to suck up 10 mL of the cell medium, theoretically containing about 100 cells. If there is 10 mL of 100 cells, and you took 1 mL of the solution containing the 100 cells, it would have about 10 cells in that 1 mL. You would then take a second test tube with 9 mL of buffer, and add that 10 cells in 1 mL to that. You would then have 10 cells in 10 mL of the solution. Then, you would take 1 mL at a time and drop it into 10 different culture dishes. Statistically, you should have 1 cell in each 1 mL on each plate. The next method is micromanipulation. This method requires a really expensive machine. Starting with the secondary culture, you would take it and put it under a microscope. You would then take a fine pipette and push the cell and bump it a little bit, so that it is no longer latched onto the plate. It is important to do this gently so that the membrane is not breached. Once the cell is free, the cell can be sucked up in a micropipette, while still being watched under the microscope, and placed in a separate dish. The advantage to using this method is that the cell can always be watched while performing the micromanipulation procedure, and you can probably even identify which cell you are placing in the other dish. For both procedures, once the cells are alone in the dish, you are going to want to add in a conditioned medium, which is a medium containing nutrients from a medium and ligands from other cells, so that the cell doesn't think it is lonely and die.

Explain two methods of making a cell line in detail? Assume in this answer you are starting with the end of secondary culture moving into making up the cell line.

A cell line is made when you take a single cell from the Secondary Culture and put it in a plate all by itself in a culture plate with medium in it, thus all cells are MITOTIC clones of that original cell. There are two methods of taking a single cell from the Secondary Culture. One method is visual using micromanipulation and the other is mathematical using serial dilution.-------------------------------------------------------------------------------There are two methods that can be used to make a cell line. The first, serial dilution, is really inexpensive, but is more of a statistical approach. It first involves taking the secondary culture dish and trypsinizing the cells. Once the cells in the dish are not latched on to the bottom anymore, a pipette will be used to suck up 10 mL of the cell medium, theoretically containing about 100 cells. If there is 10 mL of 100 cells, and you took 1 mL of the solution containing the 100 cells, it would have about 10 cells in that 1 mL. You would then take a second test tube with 9 mL of buffer, and add that 10 cells in 1 mL to that. You would then have 10 cells in 10 mL of the solution. Then, you would take 1 mL at a time and drop it into 10 different culture dishes. Statistically, you should have 1 cell in each 1 mL on each plate. The next method is micromanipulation. This method requires a really expensive machine. Starting with the secondary culture, you would take it and put it under a microscope. You would then take a fine pipette and push the cell and bump it a little bit, so that it is no longer latched onto the plate. It is important to do this gently so that the membrane is not breached. Once the cell is free, the cell can be sucked up in a micropipette, while still being watched under the microscope, and placed in a separate dish. The advantage to using this method is that the cell can always be watched while performing the micromanipulation procedure, and you can probably even identify which cell you are placing in the other dish. For both procedures, once the cells are alone in the dish, you are going to want to add in a conditioned medium, which is a medium containing nutrients from a medium and ligands from other cells, so that the cell doesn't think it is lonely and die.

Explain the geography of actin filaments. Explain the geography of stress cable, filapodia and lamelliapodia in a cell, and locomoting in a culture plate.

Actin filaments are seen under the plasma membrane in the cell periphery in both motile and non-motile cells. For their geography, the cell will want to use as little energy as possible, so the stress cables, the filopodia, and the lamellipodia are located very close to each other. After the cell uses its filopodia (which are a bundle of actin filaments) to sense the direction it wants to go, it will then extend its lamellipodia (using actin filaments) to place new adhesion plaque on the substrate to pull itself forward. The stress cables are a bundle of actin filaments that are the cell equivalent of a muscle, with Myosin II, that interact with anti-parallel contractions that cause the cell to walk. Because of the geography of these items within the cell, the cell is able to limit the amount of ATP it uses.

What causes a myosin II molecule to be able to form a bipolar fiber?

Calcium binds to calmodulin, then the calcium-bound calmodulin binds to myosin light chain kinase, activating it, which leads to phosphorylation of one light chain on each head group, causing a conformational change in myosin II into a bipolar fiber.

Lipids can move freely in the plane of the membrane because they are covalently linked. T/F

False

Describe the physical and chemical properties of the monomer and polymer of actin filaments. Explain how a) actin filaments assemble and disassemble. Classify a) actin filaments, based on their ability to treadmill.

Actin filaments diameter = 7nm and can be various lengths, depending on the number of monomers present making up the actin filament into a helix. Monomer = 42kD molecular weight and composed of 375 amino acids When actin monomer has ATP present, it is very polar and will be attracted to other monomers. Assembly can only happen if the monomer concentration is high enough. ATP portion will be polar. In vitro, there is a rate-limiting step, termed nucleation, which must occur first with 3 monomers with the same polarity. This is followed by elongation of the actin filament, which is not rate-limiting if the monomer concentration is above the threshold level. In vivo, the cell has associated proteins form "pre-assembled nucleation sites" tethered to the inner leaflet of the plasma membrane because of the geography of the cell. If the concentration of the actin monomer is above the "critical concentration" of 0.2 micromolar, growth of the filament will occur on both sides of the nucleated actin filament in vitro. In vitro, as actin filament growth occurs, the monomer concentration reduces; when it reaches the critical concentration, a chemical equilibrium is established. At this time, one end of an actin filament can be detected as the assembly end while the other end of the filament is a disassembly end. In vitro and In vivo, at chemical equilibrium, a phenomenon called treadmilling occurs for actin filaments; this happens when the filament does not grow in length, but monomers assemble on one side while they disassemble on the other side.

Describe the size and shape of an actin filament.

Actin filaments have diameter of 7 nm and can be various lengths in the cell, depending on the number of monomers present in the linear array of actin filaments and shaped like a helix. The monomers are proteins that have a globular/dumbbell shape. The monomer has a molecular weight of 42 kD and is composed of 375 amino acids.

Phospholipids are amphipathic. What does this mean?

Amphipathic phospholipids are molecules with both hydrophilic and hydrophobic regions.

Explain how myosin II can be made into a tool by scientists using biotechnology. And explain how this can be used to determine the orientation of an actin filament in: 1. stress cables 2. filopodia

As part of actin filaments, myosin II can be used to find out where actin filaments are located or to study actin-myosin interactions. stress cables: in stress cables we have anti-parallel bundles of actin filaments; hence the myosin barbs will point in opposite directions along the two filaments. Filopodia: In filopodia, the barbs will all be directed at the tip as that is the + end of the actin (polymerizing or growing end).

Typical quantity of carbons in a phospholipid tail?

Between 14-24 carbons

Indicate the difference between phosphatidyl choline and sphingomyelin.

Both PC and SM have choline heads; however, SM has a sphingosine backbone instead of a glycerol backbone. The tail in PC is provided by the two fatty acids ester bonded to glycerol; in SM it is provided by one amide-bonded fatty acid and by the sphingosine hydrocarbon tail itself. PC has phosphodiester and ester bonds; SM has phosphodiester and amide bonds.

Flip flopping between the inner leaflet and outer leaflet of the membrane is a frequent event. T/F

False

Transmembrane Proteins:

Can move laterally and cannot flip 180 in the membrane

An unsaturated site in a phospholipid can do what...........

Cause a KINK, which reduces flexibility and the ability to kick out water. The more kinks there are, the higher the probability of leakage

Explain what cell permeabilization is

Cell permeabilization is a process that uses detergent to make holes in the plasma membrane of a cytologically fixed cell.

Explain why specific amino acids are necessary in transmembrane proteins to permit their function, and are the amino acids different in a protein floating freely in the cytoplasm.

Cells are made of proteins and amino acids are the building block of protein. Stretches of transmembrane proteins have amino acids with hydrophobic side groups exposed externally. The hydrophobic portions would be localized to interact with the hydrophobic portion of the membrane (the hydrophobic tails of the lipids), while the hydrophilic portions would interact with the portions of the membrane exposed to the aqueous environment and the head groups of the lipid through hydrogen bonding. For amino acids found in protein that is floating in the cytoplasm, the hydrophobic portions would be clustered into the interior of the protein; surrounded on all sides by other parts of the protein. The hydrophobic areas hold parts of the protein together by their repulsion of water.

Describe how cholesterol protects the lipid bilayer from freezing.

Assume to a sample of a purified phospholipid you have added cholesterol. The entire sample is in the liquid crystal state. What happens as you progressively drop the temperature of the sample 1°C an hour until you reach -5°C? Describe the events that will happen.

Cholesterol depresses the freezing point of the lipids by keeping the fatty acid tails shifting around the cholesterol as they try to shift from the liquid crystal to gel state when the external temperature is lowered. As the temperature is lowered, the cholesterol will continue to try to keep up the fluidity of the membrane. While the phospholipid is in the liquid crystal state it will be able to continue to serve as a barrier to water. The temperature will become too cold, the fluidity of the membrane will decrease too much, bringing the phospholipid into the gel state. As the phospholipid enters the gel state, those portions will allow water to seep through.

What are the limitations of the technology for making a cell line.

Depending on the strategy, it is either expensive (micromanipulation) or it may not have the right number of cells and of an unknown type (serial dilution). However, it is necessary to isolate the cell in one of these manners otherwise the cell will likely die; if it doesn't think it has neighbors, it usually dies.

Address the multiple roles of cholesterol in the plasma membrane.

Depresses freezing point by keeping fatty acid tails shifting around the cholesterol as they try to shift from the liquid crystal to gel state. Prevents from getting overheated and having lipids from flying out of the plasma membrane b/c they have too much kinetic energy Suppresses melting point

Flip flopping between the inner leaflet and the outer leaflet of the membrane is a frequent event. T/F

False

Why would the evolutionary time line of the eukaryotic cell require a loss of the cell wall before the process of endosymbiosis?

Engulfing another organism by phagocytosis is not possible with a cell wall. The cell wall would have inhibited the smaller cell from being engulfed into the larger one. Without a cell wall, the smaller cell could be absorbed.

Starting with a living mouse explain the technology for making a cell line.

Euthanize the mouse. Starting with the mouse placed on its back, douse it with 70% ethanol and allow it to evaporate. Open and pin the skin with a cut from the chest to groin and a cut across shoulders and hips using sterile forceps and scissors. Cut the same pattern in the body wall with new sterile forceps and scissors. Remove one or more of the larger muscles and place into a sterile plate with new sterile forceps and scissors. Remove the mouse from the hood. With two new sterile scalpels in an x-shaped fashion, dice the muscle into less than 1 mm diameter chunks and transfer the chunks into an Erlenmeyer flask containing 0.5% trypsin and 3 mM mgCl2. After aseptically sealing the flask, place in an agitator for 5 hours at room temperature. Then, transfer the flask to a laminar hood and add medium containing serum. Transfer the contents into a sterile cell plate and cover. Every three days for 1-2 weeks, aseptically remove half of the cell culture medium and replace with fresh. At the end of the time period, plate is tipped into a sterile waste container to drain the medium and discard in biohazard waste. The culture lid is placed back on the plate. For the next several weeks, refeed the cells every three days and subculture plates. Once a single cell is aseptically taken from secondary culture using serial dilution or micromanipulation and placed onto a sterile cell culture plate with 10% serum, allow it to grow. The cells on the cell line are mitotic clones of the original single cell.

"Flip flopping" between the inner leaflet and outer leaflet of the membrane is a frequent event. T/F

False

Actin filaments will only assemble in parallel bundles. T/F

False

Aseptic technique is no longer as important after a cell line is established. T/F

False

Explain gated transport for a transmembrane protein.

Gated transport involves protein channels that selectively open when stimulated by a ligand in the case of ligand-gated channels, or a voltage threshold in the case of voltage-gated channels

In culture, the cell often switches from breaking down glucose to breaking down ______.

Glutamine

In culture, the cell often switches from breaking down glucose to breaking down _________.

Glutamine

In a phospholipid, the fatty acid chains are linked to the .................. by .................. bonds.

Glycerol ester

In a phospholipid, the fatty acid chains are linked to the .................. by .................. bonds.

Glycerol; ester

What are the limitations a healthy tissue cell faces when undergoing successive cell division in vitro and in vivo

In vitro means outside of the body. Limitations nutrients, lack of space (because they can feel contact inhibition). In vivo mean within the organism. Limitations include getting nutrients (by capillaries), getting rid of waste, having space to grow.

Explain how the binding of ATP affects cytoskeletal monomers. Explain how the function of cytoskeletal networks can change depending on reversible binding of cytoskeleton associated proteins

Hydrolysis of ATP to ADP helps identify the assembly vs the disassembly end with an ATP "cap" on the assembly and an ADP "cap" on the disassembly. With ATP present, the monomer is very polar and promotes self-assembly into the actin filament. When ATP is hydrolyzed to ADP, it changes the conformation of the monomer making it less suited to fit into an actin filament. Position of ATP gives the monomer a polarity that sets up the polarity of the actin filament and the concentration of monomers has to be high enough to initiate the assembly of monomers. When concentration is reached, disassembly begins.

Why would a pharmaceutical company want to use a cell line rather than a primary culture or a secondary culture?

If a researcher or pharmaceutical company were studying the effects of a drug that would lower cholesterol production in the liver (i.e., LDL, often referred to as bad cholesterol), then ideally the researcher/pharmaceutical company would want to be studying liver cells (cell type name, hepatocytes). When a cell line is made from a single hepatocyte cell (with conditioned medium), then for a period of time the phenotype (based on differential gene expression) will be that of hepatocytes. A primary or secondary culture would have a mixed population of cells that could include skeletal muscle cells, fibroblasts, macrophages and other white blood cells. Results from studies of drugs on this mixed population of cells types would not produce meaningful results.

Why is it important to know the limitation of a technology?

In order to get accurate results when using technology and be able to test your hypothesis. Also, knowing the limitations of technology can help improve that technology for future research.

From the following, where would a proline residue be least likely to be?

In the middle of an alpha helix

How would the evolution of internal membrane compartments, such as the endoplasmic reticulum affect the fitness of the cell?

Internal membrane compartments: Nuclear envelope for DNA Endoplasmic Reticulum formed peroxisomes, which played a role in removing toxic oxygen in ancient earth These enclosed compartments, like the nuclear envelope, ER, Golgi apparatus, vesicles, would allow different chemicals, including enzymes, to be concentrated inside of them and allowed chemical reactions to occur more efficiently. Important: They partition water and partition the things that are soluble in water

In the context of biomolecular machines, what limits the size and number of structures that can be constructed?

It is limited by the number of biomolecular machines that can be held within a single-celled organism. In other words, within a single factory, the cell, you can only put so many biomolecular machines. If a cell has to do everything, find a mate, food, shelter, etc. for life to continue the cell is overcrowded with biomolecular machines and is size limited.

Explain chemically gated channels.

Ligand-gated ion channels are a group of transmembrane ion-channel proteins which open to allow ions such as Na+, K+, Ca2+, and/or Cl− to pass through the membrane in response to the binding of a chemical messenger (i.e. a ligand), such as a neurotransmitter.

Explain how determination of lipid melting point contributes to knowledge concerning a lipid membrane and assess the limitations of this technology.

Measures the amount of calories or energy it takes to make a lipid go from the gel state to a liquid crystal state. Also that there is a point where lipid membranes go through an endothermic reaction, they go through a plateau, where the temperature goes unchanged, and more energy needs to be added to push the lipids out of the endothermic reaction and go through the phase change from the "gel state" to the "liquid crystal state". In a living system, the lipids in the biological membrane are in the liquid crystal state, and it is in this state that they are a barrier to water. Limitations of technology: you can only analyze five or 10 lipids at a time. If you have 10 in there, you get so many peaks you can't distinguish the individual peaks from each other. Ask an engineer to do this: you identify a limitation in technology, you design something to give you better data. So that's what you'd do as your next step. If you do this, science will progress faster.

Explain how subcellular localization/geography of related components conserves ATP and could have increased fitness of cells.

Membrane anchors can tether specific proteins close to the membrane, typically close to a site where that protein is going to be needed, which is subcellular localization. By pre-positioninig proteins/enzymes in a region where they are going to be used, the cell avoids waiting for diffusion or some sort of active transport to carry that enzyme to the needed location.

Of the following, select the 3 most crucial ions required for maintaining proper osmolarity and membrane potential?

Na, K, Cl

What are the three most crucial ions required for maintaining proper osmolarity and membrane potential?

Na, K, Cl

Is the order of transformation or aneuploidy important to the formation of a cancer cell? Y/N

Describe how a normal cell locomotes in a cell culture plate; contrast with mechanisms used by cancer cells to locomote in a culture plate.

Normal cells are anchorage-dependent and experience contact inhibition, they stop moving in the direction of stimulus to their filopodia/lamellipodia (if possible, they even retract). Normal cells become pancake-shaped in interphase and spherical in M phase. Cells change shape with contact to each other and signals stoppage of cell cycle in which slight rounding reduces or inhibits protein synthesis. Cancer cells are anchorange-independent and do not experience contact inhibition. They will continue to grow over the surrounding cells and keep a spherical shape throughout all phases of cell growth. Cancer cells become anchorage-independent due to a transformation and become immortal due to aneuploidy (addition or subtraction of one chromosome). These events can happen in either order.

Synthesize an understanding of what is required to maintain a healthy cell line in the laboratory.

Normally, when cells are cohabitating in a dish, they exocytosis ligands into the medium. The ligands are captured by different cells' receptors, letting those cells know they have neighbors. If you were to isolate one of those cells, it would eventually die due to lack of ligands contacting its receptor. To prevent that from happening, you must keep adding conditioned media which contains ligands and growth factors emitted from a separate dish full of cells. Also, when handling the cell line, you must make sure to utilize aseptic procedures.

Synthesize an understanding of what is required to maintain a healthy cell line in the laboratory.

Normally, when cells are cohabitating in a dish, they exocytosis ligands into the medium. The ligands are captured by different cells' receptors, letting those cells know they have neighbors. If you were to isolate one of those cells, it would eventually die due to lack of ligands contacting its receptor. To prevent that from happening, you must keep adding conditioned media which contains ligands and growth factors emitted from a separate dish full of cells.Also, when handling the cell line, you must make sure to utilize aseptic procedures.

When the 1 mm2 tissue fragment is placed in the Erlenmeyer flask to loosen the cells in the tissue (primary explant), what were the components of the medium?

PBS 0.5% with trypsin and 3mM MgCl2

When the 1 mm^2 tissue fragment is placed in the Erlenmeyer flask to loosen the cells in the tissue (primary explant), what were the components of the medium?

PBS 0.5% with trypsin and 3mM with MgCl^2

Explain the geography of membrane anchors.

Refers to the placement of specialized proteins to where they are most effective. Proteins are present in this membrane to provide channels across this lipid barrier; proteins are also there to bind to ligands and allow the cell to make responses to environmental stimuli. Membrane lipids make up the portion of the biological membrane that a) partitions water and b) compartmentalizes enzymes to increase the efficiency of chemical reactions.

Describe how myosin II can be made into a tool to analyze the differing polarities of actin filaments in the cell.

Researchers were able to cleave off a large part of the carboxyl-terminal end of the heavy chains of myosin II, which is hydrophobic; thereby leaving the amino terminal head, which is polar. Knowing the polarities of the heavy chains of myosin II allowed researchers to cleave off the end they did not want, so they could concentrate on obtaining the polarity they did want for the experiment.

Explain how transmembrane proteins function to make channels in the plasma membrane and how they work with a) diffusion, b) passive transport, c) active transport.

Some transmembrane proteins let specific ions across channels. The uniporter permits one component to move through the channel. The symporter permits two components to move through in the same direction, and the antiporter permits two components to move across the membrane in the opposite direction. Diffusion doesn't require a transmembrane protein. Substances pass through plasma membrane from an area of high concentration to lower concentration gradient. Open channels move components down their concentration gradient is passive transport, and doesn't use ATP. Active transport: molecule is moved against its concentration gradient and uses ATP/energy.

Explain how noncovalent interactions between lipids in the biological membrane could have improved the fitness of cells of cells (in both multicellular organism and single cell organisms) to respond to differing environmental stimuli.

The absence of covalent bonds between lipids in the plasma membrane improved the fitness of cells because the plasma membrane is able to stretch without breaking, like when retracting a fiber or lamellipodium. The lack of covalent interaction allows for the sliding motion of the lipids known as the "fluid mosaic" in the plasma membrane.

Contrast the benefits of having versus lacking a cell wall- why would it be an evolutionary advantage to eukaryotes to lose the cell wall?

The cell wall provided protection to early life. It would prevent the cell from rupturing if, for example, it was saturated with too much water.Loss of the cell wall changed the surface of the cell, exposing the outer boundary of life: the plasma membrane. For example, a cell surrounded by a cell wall is not going to allow for locomotion, to be able to extend a filopodia or lamellipodia in any functional way as it would be blocked by the cell wall. Also, phagocytosis of large chunks of food would also be blocked because large chunks of food would not be able to move across the cell wall.Overall, the loss of the cell wall is thought to permit a flexibility at the cell surface/plasma membrane that enabled the evolution of both the endomembrane system and the endosymbiotic presence of other, smaller cells evolving into mitochondria and chloroplasts.

Describe the evolution of the primitive, proto-eukaryotic cell from the primitive, proto-prokaryotic cell.

The cell wall was lost which allowed two separate processes-- endosymbiosis and endomembrane. Under the endosymbiosis theory, other cells were incompletely digested leading to the formation of a couple of organelles. Under the endomembrane theory, invaginations of plasma organelle led to other internal membrane structures.

Why would the evolutionary time line of the eukaryotic cell require a loss of the cell wall before the process of endosymbiosis?

The cell wall would have inhibited the smaller cell from being engulfed into the larger one. Without a cell wall, the smaller cell could be absorbed.Engulfing another organism by phagocytosis is not possible with a cell wall.

Why would the evolutionary timeline of the eukaryotic cell require a loss of the cell wall before the process of endosymbiosis?

What happens in primary culture?

Explain how lipid packing contributes to the partitioning of the aqueous components of the cell.

The phosphate head group is hydrophilic ("waterloving") because it is polar, enabling it to form hydrogen bonds with water. By contrast, the two fatty acid tails are hydrophobic ("water-fearing") because they are nonpolar and do not form hydrogen bonds with water. The shape of the structure is determined by the bulkiness of the head group relative to the hydrophobic tails.

What happens in primary culture?

The cells that can walk out of the tissue chunk walk out. Other cells (like red blood cells in capillaries) can also drift out of the tissue chunk, but they don't adhere to the plate. A key thing that happens during Primary Culture is that a lot of cells die a) Cells in the center of the tissue chunk will die in the absence of the functioning circulatory system because they can't get nutrients oxygen or get rid of waste andCO2. b) Some cells were damaged by the trypsin digestion and can't recover. c) Some cell types cannot survive without special growth factors that are not provided by the 10% serum. These cell types either die or become very lethargic and cannot undergo cell division.

Actin filaments ______________.

The correct answer is not listed

Explain how social systems of cells increased the fitness of the organism.

The division of labor allows the whole organism to do more than the sum of its parts. When cells work together in a social system there is a division of labor, allowing for each cell to focus on its own task whereas if a cell was working alone, it would have multiple responsibilities (looking for a mate, food, shelter, etc) thus lowering the effectiveness.

How would the evolution of internal membrane compartments, such as the endoplasmic reticulum affect the fitness of the cell?

The enclosed membrane compartments would partition water and things soluble in water. Later, they would compartmentalize enzymes to increase the efficiency of chemical reactions. Additionally, some of the membranes would later specialize to form the endoplasmic reticulum that gave rise to a new type of protein synthesis from membrane-bound polyribosomes to create a special class of proteins. This also became the site of new lipid synthesis as well as the site where preassembled carbohydrate trees were added onto a subset of the special proteins.

What are the limitations of cell culture technology?

The limitations of cell culture technology are accuracy and expenses. With serial dilution, is it fairly inexpensive to perform, however, a person is relying on statistical methods to ensure that 1 cell is in 1 mL on each plate. Sometimes there may be 2 cells on one plate and 0 on the other. With micromanipulation, the cell is in view at all times and it is very easy to keep track of the cell, where it is going, and maybe even be able to identify which cell it is. The downside of micromanipulation is that it requires very expensive equipment. Your choices are either expensive equipment or having to deal with tasks like serial dilutions that are not expensive, but mathematically there could be some errors becomes you don't know how many cells you have in each test tube and you don't have a purified cell line.

When a lipid goes from the gel state to the liquid-crystal state what occurs?

The lipid is going through a phase transition caused by an endothermic reaction due to temperate increase. Kinetic energy causes rapid movement of the fatty acid tail of the lipid.

How would the evolution of a mitochondrion affect the fitness of the cell?

The mitochondria is the site where most of the cellular energy is produced; it allows cells to deal with energy production and usage to fight against entropy and maintain order.

Explain the geography of actin filaments as it relates to a living cell.

The pre-assembled nucleation sites are tethered to the inner leaflet of the plasma membrane, actin filaments are greatly enriched in at the cell periphery both in motile cells and in non-motile cells (non-motile cells would exist in epithelium). In non-motile cells from an epithelium (such as might exist in the enterocytes of the gut), the geography of actin filaments is also seen at the cell periphery, including in the microvilli at the apical surface of these cells.

What difference in results will you observe if you compared the melting point of two samples? Sample 1 has is made of a single type of phospholipid. Sample 2 has 35 different types of phospholipids in them.

The results would be different. Sample 1 would probably have a steady increase, plateau, and increase again. Sample 2 there are too many different phospholipids and there will be too many peaks you can't distinguish the individual peaks from each other. We can only analyze up to 5-10 phospholipids at a time.

Explain the lipid membrane: what are the two basic functions of a membrane as a cellular structure , and why are they so important?

The two basic functions of a membrane as a cellular structure are to partition water and those components soluble in water and to compartmentalize enzymes to increase the efficiency of chemical reactions.It is important because it allows different compartments to have different functions and have differing levels of solubility

An unsaturated site in a phospholipid can do what...........

The unsaturated site, which is seen as a kink in the fatty acid tail, can reduce flexibility and decrease the ability to kick water out. It can also create pockets where water can leak through and increase the probability of this happening.

Draw a figure to illustrate what a normal diploid cell must undergo to become a cancer cell and include the mechanisms discussed in class for these alterations.

These 2 events can happen in either order and both must happen to be cancerous. Immortality because of aneuploidy, which is loss or addition of a single chromosome. Anchorage-independent for growth because of transformation, from a mutation in a single gene.

Explain how phospholipids establish a cohesive membrane without covalent bonding between the phospholipids, and the role of unsaturated sites.

They are not covalently linked. The fluid mosaic structure allows the membrane to stretch. The hydrophilic end is hydrophilic because it can hydrogen bond with water while the hydrophobic end is hydrophobic because it cannot hydrogen bond with water. The love of water on 1 side and hate for water on the other is what establishes a cohesive membrane. The lack of covalent bonding is what makes the cell flexible, allowing for the cell to stretch without breaking. The unsaturated sites are caused by a double bond, making it stronger, but can cause kinks in the Tail increasing the likelihood of water leaking through.

Distinguish between transmembrane proteins and peripheral membrane proteins in a biological membrane.

Transmembrane proteins span both leaflets of the plasma membrane. Transmembrane proteins either contain different amino acids as they pass through the hydrophobic portion of the membrane, or they wrap in a way to hide the hydrophilic side groups away from the hydrophobic portion of the membrane.Peripheral membrane proteins bind at the surface of the membrane through hydrogen bonding or charge interactions.

A limitation of cell culture technology is the loss of histotype. T/F

True

Actin filaments associate with a nucleotide triphosphate. T/F

True

An evolutionary advancement occurred when organisms became multicellular and acquired a division of labor. T/F

True

Beta pleated sheets can provide a unique environment on one side of the sheet relative to the other side of the sheet because of the types of side groups that can accumulate there. T/F

True

Membrane anchors set up geography in a cell. T/F

True

Membrane anchors set up the geography of a cell. T/F

True

One method for making a cell line could involve micromanipulation. T/F

True

Peroxisomes increased the fitness of the primitive protoprokaryote by removing toxic oxygen in the ancient earth. T/F

True

Peroxisomes likely originated from the endomembrane system. T/F

True

Preassembled nucleation sites in a living cell are equivalent to intracellular preference for actin. T/F

True

Preassembled nucleaton sites in a living cell are equivalent to the intracellular preference for actin filaments. T/F

True

Single-celled organisms have a limited number of biomolecular machines within them. T/F

True

Submembrane cytoskeletons can provide shape to a cell. T/F

True

The endomembrane system supposedly resulted in the formation of the nuclear envelope, endoplasmic reticulum, Golgi apparatus and primary lysosomes. T/F

True

The endomembrane system supposedly resulted in the formation of the nuclear envelope, endoplasmic reticulum, Golgi apparatus, and primary lysosomes. T/F

True

The medium in the primary explant contains PBS made 0.5% with trypsin and 3 mM with MgCl2. T/F

True

The presence of 2 disulfide bonds near each other is a good thing when a stretch of protein is to be immobilized. T/F

True

The presence of two disulfide bonds near each other is a good thing when a stretch of protein is to be immobilized. T/F

True

Unsaturated sites in the fatty acid tails of phospholipids are places that water can use to leak though the membrane. T/F

True

Unsaturated sites in the fatty acid tails of phospholipids are places that water can use to leak through the membrane. T/F

True

Actin filaments will ____________.

Two of the above: undergo treadmilling in a test tube AND in a living cell.

Describe how the presence of ATP contributes to actin filament assembly and polarity.

When ATP is present in the monomer, the monomer is very polar, so it easily attaches itself to other monomers. Self-assembly into an actin filament occurs as long as the monomer concentration is high enough. Elongation phase continues until the concentration of monomers is at a steady state.

Describe the mechanisms of how a normal cell locomotes in a cell culture plate.

When a cell reaches the bottom of the medium it begins to release filopodia. Once the filopodia comes into contact with a signal, that region stops moving, and a signal transduction occurs. Lamellipodia are then released and an adhesion plaque (super glue) is formed between the plasma membrane and the substrate. A muscle is formed of actin and myosin which then contracts, flattening the cell, allowing it to locomote. The cell repeatedly releases lamellipodia that form an adhesion plate and muscle that allow the cell to be flattened out and pull itself around/locomote directionally.

Focusing on actin monomers and actin filaments: explain what occurs when a cell reverses its direction by 180 degrees.

When a cell reverses its direction by 180 degrees, there is a reverse displacement for the actin and myosin filament. There is increase in the movement and contraction of the filaments in direction towards each other. Due to the complete 180 degree reversal, due to the presence of the extended N-terminus in the myosin, it will tend to block the complete 180 degree swing of other myosin and hence it might lead to interruption in the function of muscles. Due to the reversal, the actin monomer tends to fall in the same direction and hence it tends to make the muscle more polar.

What is the main cause of a peptide chain being hydrophobic or hydrophilic?

Whether a peptide chain is hydrophobic or hydrophilic is based on the R-groups on each amino acid. Some amino acids are very non-polar (hydrophobic) and some are very polar/hydrogen bonding (hydrophilic)

Transmembrane proteins _________.

can move laterally and cannot flip 180˚ in the membrane

Draw actin monomer and actin filament and label all parts, molecular weight, etc

actin filament diameter = 7nm; shaped like helix; include assembly and disassembly (addition/minus); 37nm = complete rotation in helix monomer mw = 42 kD; 375 amino acids; made of beta and gamma isoforms (cell-equivalent of muscle); ATP physically linked to monomer which causes polarity

Integrin receptor binding results in the recruitment of _________.

actin filaments

Contact inhibition is a subset of ______________________________.

anchorage dependence for growth.

Filopodia contain receptors that sense the environment and a. all filopodia stop moving when the cell is about to extend a lamellipodium b. as a first step in walking the lamellipodium forms before the filopodium c. a lamellipodium extends only from filopodia that continue to move d. two of the above e. none of the above

e. none of the above

If you had a phospholipid whose melting point was 12°C, what might happen if you mixed it 1:1 with a lipid that had a melting point of 24°C. a. melting pt would shift to the left (melt at lower temp) b. melting pt would shift to the right (melt at higher temp) c. half would melt at 12 and half would melt at 24 d. it would never melt e. none of the above

e. none of the above

When a lipid goes from the gel state to the liquid crystal state, the reaction is ________.

endothermic

The number of carbons in the esterified fatty acid tails is ............ and ranges between............ and ................

even 14-24

The number of carbons in the esterified fatty acid tails is ............ and ranges between............ and ................

even; 14-24

In a phospholipid the fatty acid chains are linked to ____ by ____ bonds.

glycerol; ester

Hydrolysis of ATP to ADP:

helps identify the assembly vs the disassembly end

The hydrophilic head group is hydrophilic because it can ..................... bond.

hydrogen

What limits the growth of a tumor?

invasion by capillaries

Intracellular preference for actin filaments :

is a way to look at the geography of the cell and show the efficiency of the cell by using the least amount of ATP

Intracellular preferences for actin filaments

is a way to look at the geography of the cell and use the least amount of energy.

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