MCB 252 Exam 3

What are the hallmarks of Cancer Cell growth?

Inappropriate growth, metasis and divison

You have a solution of G-actin and AMPPNP. Initially your solution contains no filaments. You add Mg++ to your solution, wait five minutes and examine your reaction by electron microscopy. You find that there are now a substantial number of F-actin filaments. What can you conclude about the initial concentration of G-actin in your solution prior to the addition of Mg++? Which one of the following answers best fits your observations?

Initial concentration of G-actin was above the Cc.

What is nucleation?

Initial step of the formation of actin, three monomers must collide in perfect orientation

Are membranes rigid?

No

Do terminally differentiated cells divide?

No

Is actin a homogeneous polymer?

No

Does the shape of the plasma membrane dictates the shape of a cell?

No the cytoskeleton does

What do cancer cells start off as?

Normal cells

Which drug stabilizes actin filaments?

Phalloidin

Profilin binds to which end of an actin monomer?

Plus

If you are given a monoclonal antibody and a polyclonal antibody against a given protein, which do you think is more likely to have blocking activity (to inhibit the function of the given protein)?

Polyclonal antibodies are more likely to have blocking function than monoclonal antibodies. The idea is that antibody binding to some surfaces of the protein (some epitopes) will inhibit its function (have blocking activity) whereas binding to other surfaces (epitopes) of the protein will not have blocking activity. Since polyclonal antibodies bind to multiple surfaces of a protein (multiple epitopes) they have a higher probablitiy of binding a surface (epitope) that blocks function than is a monoclonal antibody that binds to only one surface (epitope).

In lecture we discussed an experiment that utilized a plastic bead coated with the ActA protein. What conclusion did we come to based on the results of that experiment?

Polymerization of actin is sufficent to move a membrane

What is the neural tube?

Precursor to brain and spinal cord

What is apoptosis?

Programmed cell death

A red blood cell has what in it?

Spectrin

What type of cells, once formed, typically do not undergo further cell divisions?

Terminally differentiated cells

Which of the following is/are often properties of protein domains?

That they are N- C- opposites

The cleft of actin is hidden on which side?

The + end

The cleft of actin is exposed on which side?

The - end

In lecture we talked about the rationale of why some proteins are conserved over the course of evolution. What were three reasons we gave why actin is conserved?

"Because" it is essential (to the cell) "Because" it has many functions "Because" it interacts with many actin binding proteins

The barbed end of an actin filament is the

+ end

The pointed end of an actin filament is the

- end

What are the properties of cells within a tissue?

1. Adopt defined shape 2. Express specific set of genes 3. Carry out specific functions 4. Replace dead cells with new ones 5. Remain distinct from cells in neighboring tissues 6. Work together

In lecture we talked about an in vitro reaction that involved the ActA protein bound to one side of a plastic bead. What two conclusions did draw from that experiment?

1. That ActA can trigger actin polymerization (comet tail formation) 2. That actin polymerization is sufficient for movement of the bead. More formally, but not necessary for the point: that the energy of polymerization of actin filaments can move the bacterium, and by inference may be sufficient to move the leading edge of the cell (the lamellipodium) forward.

How many cells are in an adult?

100 trillon

The concentration of actin monomers in a cell is approx what the cc?

100X

Approximately how many RAS superfamily members are there in humans?

160

Actin filaments contain how many strands?

2

How many cell types are in humans?

210 cell types

1/2 turn of an actin filament is how many nm?

36

What is the distance that is equal to one full turn of an actin filament?

72 nm (36*2)

What is a downstream target of the Rac pathway?

A WASp family member and the Arp2/3 complex

In order to determine the structure of a protein by X-ray crystallography you need which of the following? A. The protein of interest needs to be purified. B. You need to know the amino acid sequence of the protein of interest. C. You need to know the approximate shape of the protein from electron microscopy. D. Both A and B E. All of the above (A, B, and C)

A and B

What is an assay?

A biochemical test or reaction

Some antibodies inhibit the function of the protein that they bind; whereas other antibodies do not inhibit the function of the protein that they bind. What is the general term described in lecture for an antibody that inhibits the function of the protein that it binds?

A blocking antibody

In lecture we discussed methods for purifying proteins. What is the starting material we discussed?

A cell Extract

What type of experiment initially revealed that actin filaments are polar?

A decoration experiment. More specifically binding of myosin S1 head fragments, which are arrow-shaped, to actin filaments revealed that all the myosin arrowhead-shaped proteins pointed in one direction.

What is the difference between a monoclonal antibody and a polyclonal antibody?

A monoclonal antibody is a single type of antibody that recognizes a single epitope on the antigen. A polyclonal antibody is a mixture of different types of antibodies with each type recognizing a different epitope on the antigen. Monoclonals are generated from immortalized cell lines and consequently there is a relatively unlimited supply of a given monoclonal antibody. In contrast, polyclonal antibodies are derived from the blood of live animals and therefore the supply of polyclonals lasts only as long as the animal is alive.

In lecture we discussed a version of the figure below. Which of the following comments are TRUE about the relationship between A and B in this figure? A---] B

A negatively regulates A inhibits B A inhibits B, and A negatively regulates B A negativley refulates B and in the absence of A B will function.

What is at the minus end of actin?

ADP

What is at the plus end of actin?

ATP

What is the role of ATPase in Actin?

ATP hyrdolysis is not required for actin filaments

What does pluripotent mean?

Ability of a stem cell to develop into many different cell types

Where is T-tubule located?

Across the sarcomere

Until the last few years, actin was thought to reside in the cytoplasm exclusively. However, it was recently found that at least some actin is also found in the nucleus. What is at least one role of actin in the nucleus that was discussed in lecture?

Actin is a subunit of some chromatin remodeling complexes.

What experiment described in lecture lead to the idea that actin polymerization is necessary for formation of movement of listeria in vivo?

Addition of latrunculin prevented the actin polymerization and movement of listeria.

In Xenopus laevis, proper tissue development requires which of the following? A. Regulated cell division B. Signaling between cells C. Apoptosis D. Both A and B E. All of the above (A, B, and C)

All of the above

Which of the following contain actin family members? A. The "hubs" of the cytoskeleton that allow for deformation and reformation of the shape of red blood cells B. At least some chromatin remodeling complexes C. The protein complex that is the target of Listeria ActA protein D. Both A and B E. All of the above (A, B, and C)

All of the above

In addition to an X-ray diffraction pattern, what other information is needed to generate a high-resolution three-dimensional structure of a protein?

Amino Acid sequence of the protein.

During column chromatography you need to identify which fractions contain your protein of interest. What do you need to identify those fractions?

An Assay, Biochemical test

Why is it important for cells to be in contact with eachother?

Because there is certain relationships that they hold

Troponin

Binds Ca, acts on tropomyosin

Latrunculin

Binds actin monomers and prevents their polymerization

Purify the proteins involved and study their behavior in vitro. Recapitulate the process in a test tube.

Biochemistry

Which statements are true: Statement 1: Apoptosis can occur in response to cellular damage. Statement 2: Apoptosis can occur in response to normal developmental events.

Both

What is metastasis?

Cancer cells leaving a tumor and invading other parts of the body

Cells control when and where (within a cell) and what type of actin structures are assembled. Part of that process is to actively regulate the nucleation of actin filaments. Another part of that process is to inhibit random nucleation of filaments at times or in places where they are not wanted. How do cells inhibit filament formation at sites of random nucleation?

CapZ is active and distributed throughout the cytoplasm. When actin filaments are randomly nucleated without the aid of regulated mechanisms (such as by formin or Arp2/3) CapZ binds the plus end of those filaments and blocks further assembly. Those filaments are disassembled from the minus end. The plus ends of filaments that are nucleated by formin or Act2/3 are protected from capping by CapZ.

Which protein can cap the plus end of actin filaments?

Capz

Watch the process in a microscope. Follow the dynamics of particular proteins in a living cell. Does inhibiting the function of a specific protein in a living cell block the process you are interested in?

Cell Biology

What is symmetric division?

Cells that give off the same cell type

What is assymmetric division?

Cells that give off two different cell types

What is column chromatography?

Column chromatography is when a solution that contains the mixture under analysis is dripped on the column containing the solid phase (the intact resolving matrix). Usually the solid phase is glass. Glass, which is silicon dioxide and has a net dipole moment, is polar. Therefore, polar parts of a compound adhere and move slower than nonpolar parts.

A steady state reaction requires

Constant input of energy

How do cells define and maintain their shape?

Cytoskeleton

Which protein nucleates the formation of branched actin filaments?

The Arp2/3

Which of the following does not require one or more motor proteins for movement? A. Muscle contraction B. Contraction of a contractile bundle that is part of a stress fiber C. Cytoplasmic streaming D. Movement of Listeria in the cytoplasm of eukaryotic cells E. All of the above require one or more motor proteins.

D

Which end does actin grow slower at?

The minus end

As long as ATP is present in an in vitro actin assembly reaction that is at steady state, which of the following will be occurring?

Disassembly at the minus end, Treadmilling, and assembly at the plus end

Which term below most specifically refers to the type of allele that revealed the role of Rac in actin filament formation?

Dominant

What connects to the transmembrance protein to the actin?

ERM family members

What do ERM family members do?

ERM family members link the actin cytoskeleton to a membrane by connecting to transmembrane(or membrane associated) proteins.

From which germ layer does the neural tube form during Xenopus development?

Ectoderm

The neural tube is formed from cells of which of the three primary tissue types?

Ectoderm

What are the three primary tissue types (also known as germ layers) in early development?

Ectoderm, Mesoderm and Endoderm

What are the three primary tissues?

Ectoderm, mesoderm, endoderm

What types of cells is/are pluripotent?

Embyonic Stem Cells

What is the difference between Embryonic and Adult Stem Cells?

Empryonic are pluripotenet

Which property of eukaryotic gene regulatory regions, as discussed in lecture, allowed for the evolution complex regulatory regions?

Enhancers and Splicing occurs over a distance

What is steady state?

Equal ratio of on and off of monomers

Is this experiment measuring reactions that are at equilibrium or reactions that are undergoing net assembly (or disassembly)?

Equilibrium

Actin is the most abundant protein in

Eukaryotes

In lecture we discussed the evolution of eukaryotic genes. The evolution of some (most) human genes was described as being dependent on some parts of the genes to be able to "act over a distance." What are two parts of human genes that were described as being able to act over a distance?

Exons and enhancers

In wound healing, what triggers the activation of platelets? More specifically, where are the signaling molecules located that lead to platelet activation?

Exposure to the extracellular matrix results in activation of platelets. The signaling molecules that activate platelets are in the extracellular matrix.

What is ECM?

Extra Cellular Molecules

As discussed in lecture, the contractile bundles (sarcomeres) found in muscle cells most likely appeared in evolution before the contractile bundles found in stress fibers.

False

In lecture, we discussed the modular construction of some genes and the evolution of genes (and proteins) by the addition of domains. As described in lecture, this process occurs in prokaryotes and eukaryotes.

False

T/F As described in lecture, movement of the bacterium Lysteria monocytogenes in eukaryotic cells is dependent on actin filaments and on a motor protein that moves along those filaments.

False

T/F As discussed in lecture, the shape of the plasma membrane dictates the shape of a cell.

False

Where are stem cells divided?

In the stem cell niche/crypt

As described in lecture, because actin filaments are helical, any motor protein that moves along those filaments must follow a helical path.

False, they would be all wound up

CDC42

Filopodia

Which proteins allow for the arrangement of actin filaments into parallel bundles of filaments?

Fimbrin and alpha-actinin

Antibody staining involves which type of cells?

Fixed

Why do cell have different shapes?

For different functions

You are studying movement of listeria monocytogenes in vivo. You take pictures of moving lysteria and measure the length of the comet tails in tissue culture cells. You now inject the cells with phalloidin. What do you expect to happen as a result? Explain your reasoning.

For the tails to get longer. Phalloidin stabalizes microtubulues

Which protein "travels" with the growing plus end of an actin filament?

Formin

How many domains does formin have? What are their names and what does each do?

Formin has three types of domains, the FH1 domain, FH2 domain and RBD domain. The FH2 domain is a split ring that can nucleate linear actin filament formation, catalyzes elongation elongation and moves with the plus end. The FH1 domains (formin contains multiple FH1 domains) contain profilin binding sites and act to "load" actin monomers onto the growing plus end. The RBD domain binds activated Rho (Rho in the GTP form). When not bound to activated Rho, formin is in an inactive conformation, binding of Rho-GTP to the RBD domain activates Rho.

Depolymerization occurs when

G-actin<Cc

Polymerization occurs when

G-actin>Cc

In 2008 the Nobel Prize in Chemistry went to the researchers involved in the development of GFP as an experimental tool for visualization of proteins and expression patterns. What is the advantage discussed in lecture of using GFP rather than immunofluorescence?

GFP allows visualization of living cells and organisms.

In lecture we discussed fluorescence microscopy and the use of immunofluorescence and GFP. What does GFP stand for? Describe the types of questions immuno-flourescence and GFP can be used to answer, and whether each can be used in whole live organisms, in individual live cells growing in cell culture, or only on fixed (dead) samples.

GFP stands for green fluorescent protein. Both techniques can be used to determine the sub-cellular localization of a given protein of interest and both can be used to determine which cells in an organism produce that protein. Immunofluorescence is typically performed on cells or tissue samples which are permeabilized and fixed on a microscope slide and are therefore not alive. In other cases antibodies can be injected into individual living cells in culture but not into all the cells of a living organism. Since GFP fusion constructs (fusion genes) can be transformed into organisms to generate transgenic organisms, it is possible to visualize GFP expression patterns in all cells in living organisms.

How are gene and protein families formed?

Gene Duplication and Divergence

What process was described in lecture as generating families of genes (and therefore families of proteins)?

Gene Duplication and Divergence

transgenics

Genes of one organism inserted into another organism

Isolate a mutant organism that is defective in the process.

Genetics

What is the Basal Lamina?

Gives the tissues and cells extra integrity

What are the signals to survive called?

Growth Factors

How was RNA pol 2 purified?

In vitro assay

A high charge will come off at which salt concentration?

High

Which step in the ATPase cycle of myosin II results in rotation of the head into the "cocked" position?

Hydrolysis of ATP to ADP+Pi

In lecture we discussed the use of speckle microscopy. What did the speckled microscopy experiment that we discussed actually show?

In the experiment we discussed, speckle microscopy was used to show that actin filaments treadmill in vivo.

What is ion exchange chromatography (what it is used for)? Explain how it works. What is the purpose of the salt gradient in ion exchange chromatography?

Ion exchange chromatography is a version of column chromatography that is used for protein purification. Typically a cell extract is prepared by breaking open cells in a test tube and removing all the non-soluble (membranous) material. As a result, the extract contains all the soluble proteins. One can purify individual proteins from the mixture of proteins in the extract by separating the proteins based on their physical and chemical properties. Ion exchange columns contain charged residues (either positively or negatively charged). As a result proteins that have regions of opposite charge will bind to the column; whereas proteins that do not have opposite charges on their surface will not stick to the column and will pass through the column. The proteins that bind to the column are eluted by a salt gradient. The charged salt ions compete for binding to the charged residues on the column beads and displace the bound proteins. Proteins that are bound to the column by fewer charges are eluted at lower salt concentrations than proteins that are bound to the column by many charges. As a result, different proteins are separated into different fractions that elute from the column.

What types of column chromatography are there?

Ion-Exchange, Gel-Filtration

In lecture we discussed how our figures are misleading. How is it misleading, and what should it show?

It is misleading in that, as drawn, it looks like profilin activates the conversion of ATP to ADP. In reality, and what it should show, is that profilin is an exchange factor that promotes the dissociation of ADP from actin monomers and promotes the subsequent binding of ATP to the actin monomers.

A dominant negative mutation displays which of the following phenotypes?

LOF

Rac

Lamellipodia

Which of the following statements is TRUE? 1. Large proteins elute from a gel filtration column before small proteins. 2. Small proteins elute from a gel filtration column before large proteins.

Large proteins elute from a gel filtration column before small proteins. They don't get distracted by all the little holes they can fit through. The fatties make it out first.

GFP allows for the imaging of what type of cells?

Live

A low charge will come off at which salt concentration?

Low

How many derivatives of GFP are there?

Many

What did they add to G-actin to cause assembly?

Mg++

You have a solution of G-actin and ATP and wish to study actin assembly. What do you do to your solution to get the G-actin monomers to assemble into filaments?

Mg++

Actin is a subunit of what?

Microfilaments

What is elongation?

Monomers are randomly diffusing around

All filaments are ____ stranded.

Multi

Can you determine the structure of a protein without knowing its amino acid?

NO

Which end of an actin filament has a higher concentration?

The minus end

What is the result of different Cc?

Treadmilling

In lecture we discussed a process that facilitates exon shuffling and is required for exon shuffling to be frequent enough to be a mechanism for the evolution of modular genes and proteins. What process was that?

Splicing

Titin acts as a

Spring

What does multipotent mean?

Stem cells with limited potential to develop into many types of differentiated cells

Rho

Stress Fibers

What are the 4 global responses to signals?

Survive, grow and divide, differentiate or die

What best describes the fate of the cloned DNA used in a transfection experiment?

The DNA remains on a plasmid and is maintained as an episome (non-chromosomal DNA) for a day or two and is then lost.

The Zebra fish is a model organism used to study vertebrate development. One feature of Zebra fish that is that particularly useful is that they are transparent during the early stages of development and therefore you can visualize whole live fish (and all their cells) in a microscope. However, it is often difficult to clearly distinguish cell types apart and to follow the length of long cells such as neurons. You are interested in how the nervous system develops and wish to view all the neurons in a zebra fish as it develops. Describe a way that you could readily identify and visualize all the neurons in a living zebra fish. Assume a gene has already been identified that is transcribed in all neurons and is not transcribed in any other cell types.

The GFP needs to be bound to the protein prior to the cell type being expressed. So if you add the GFP to the end of the gene and then since the cell in the neurons are only transcribed in that specific cell type it will only "glow" were the GFP protein is expressed which was after the GFP was made at the end of the gene.

What do almost all cancer cells not have?

The ability to preform apoptosis

Secondary antibodies bind to which region of a primary antibody?

The constant region

What is the neural tube created by?

The ectoderm

The length of which region of myosin determines the step size?

The lever arm

As discussed in lecture, profilin binds to actin monomers to promote the association of that monomer with the plus end of an actin filament and prevents that monomer from adding to the minus end of a filament. To which end of the monomer does profilin bind?

The plus end

Which end does actin grow faster at?

The plus end

Which end of actin filaments within sarcomeres is associated with (attached to) Z discs?

The plus end

What is fractionation?

The separation by physical and chemical properties

What determines the shape of a cell? If a cell needs to change its shape what must it do?

The shape of the cytoskeleton determines the shape of a cell. If a cell needs to change its shape it need to change the shape of its cytoskeleton.

You are watching Listeria moving around inside two cells. You inject one cell with phalloidin. What are you most likely to expect to see happen in the injected cell?

The tail to become longer

In lecture we discussed how the data plotted here was collected. Is the data displayed in this figure collected from a single experiment in a single test tube, or was it collected from multiple test tubes?

This experiment was carried out in multiple test tubes, each of which started with a different concentration of actin monomers.

In lecture we talked about a series of experiments involving transfection of mutant alleles of Rho, Rac and Cdc42 into tissue culture cells. Were those mutant alleles dominant or recessive to wild-type? Why was it important that the alleles were the type they were (dominant or recessive) as opposed to the other type?

Those mutant alleles were dominant to wild-type. It was important the mutant alleles be dominant because they were transfected into cells that contained two wild-type alleles of the gene of interest. If the mutant allele were recessive, the cells would still have displayed the wild-type phenotype because they contained two wild-type copies of the gene of interest in addition to the mutation in the transfected copy of the gene.) Hence, only dominant alleles could be used in this type of experiment).

Why do cells need signals to just survive?

To keep cells different from one another ( ex:shot induces skin cells into muscle cells)

Which direction does myosin move?

Towards the plus end

As described in lecture, a polyclonal antibody contains many different antibodies, each of which recognizes a distinct epitope on the surface of an antigen.

True

As described in lecture, all cytoskeletal polymers are multi-stranded.

True

Exon shuffling occurs at the level of DNA in the genome; whereas, splicing occurs at the level of RNA.

True

T/F As described in lecture, protein family members typically have similar structure and function.

True

T/F As discussed in lecture, at least some types of cell movement are dependent on changes in cell shape.

True

T/F As discussed in lecture, at one time or another virtually every amino acid on the surface of actin is thought to contact another protein.

True

T/F As discussed in lecture, in an in vitro reaction, actin filaments assemble faster at the plus end than at the minus end.

True

T/F As discussed in lecture, many aspects of development and tissue formation can be viewed as converting sheets of cells into tubes, balls and tubular solid structures.

True

T/F Steady-state reactions require the continuous input of energy to remain at steady state.

True

True or Fale: Human cells need to receive signals in order to survive.

True

True or false? At least some types of cell movement are dependent on changes in cell shape.

True

We've talked extensively about the fact that actin polymerization can move the plasma membrane outward, away from the center of the cell. As discussed in lecture actin polymerization can also be used in endocytosis to move vesicles from the plasma membrane toward the center of the cell.

True

When performing column chromatography to purify a protein of interest, one needs an assay to identify which fractions that contain the protein of interest. True or False: As discussed in lecture, we use a different assay for each different protein that we purify (For instance, the assay for protein A will be different than the assay for protein B which will be different than the assay for protein C, etc).

True

What type of cells can stem cells beocme?

Virtually any type of cell

What is the importance of having so many cell types?

We have 210 programs of transcription, that include different ways to turn on and off genes

Give an example of apoptosis during development:

Webbing of toes or removal of a tail

transformation

When DNA is introduced into cells in tissue culture and is stably maintained in that population of cells as a result of the DNA integrating into a chromosome in those cells.

transfection

When DNA is introduced into cells, and that DNA is only transiently maintained in those cells

In lecture, we talked about the idea that actin is highly conserved over the course of evolution. What does highly conserved mean?

When a protein is highly conserved it means that there is very little sequence variation in that protein from species to species.

Can any aspects of development and tissue formation can be viewed as converting sheets of cells into tubes, balls and tubular solid structures?

Yes

Do alpha and beta subunits in hemoglobin have similar structures?

Yes

Does metastasis of cancer cells involve an active process of cell migration?

Yes

In lecture we discussed the fact that actin plays a role in phagocytosis. Based on that discussion, do you expect that addition of latrunculin to cells would stop phagocytosis?

Yes

Is polymerization reversable?

Yes

Can ADP-actin monomers form F-actin polymers in the absence of ATP in vitro? Based on your answer to this question, what important property can be inferred about actin polymerization?

Yes, ADP-actin monomers can make F-actin polymers in vitro in the absence of ATP. From this observation we can infer that the energy of ATP hydrolysis is not necessary for actin polymerization.

When a stem cell divides, the result is

a stem cell and a cell that can become specialized

A muscle fiber is an

individual muscle cell

Profilin

is a nucleotide exchange factor for actin

At critcal concentration there is

monomers

Below critical concentration there is more

monomers

Above critical concentration there is

monomers and filamenrs

Do cancer cells divide out of range with normal division?

no

Cofilin

promotes disassembly of actin filaments

Nebulin

ruler

Where is calcium stored?

sarcoplasmic reticulum

Thymosin β4

sequesters actin monomers