Exam 3 - Cell Biology

What causes catastrophe of the microtubule in vitro?

GTP hydrolysis

What is the role of GTP in microtubule polymerization?

GTP stabilizes the tip of the microtubule, allowing more monomers to be added.

What would happen in the treadmilling experiment if a non-hydrolyzable analogue of GTP were used?

The microtubule would treadmill until the new tubulin, with non-hydrolyzable GTP, reached the minus end, and then it would only extend at the plus end.

What is the difference between the plus and minus ends of the microtubule in in vitro experiments?

The plus end has a lower critical concentration for tubulin heterodimers.3

How would the drug taxol affect the in vitro dynamic instability and treadmilling experiments?

Taxol would stabilize the microtubules in both experiments, leading to polymerization without catastrophe.

A model helps scientists form testable hypotheses.

What hypothesis was being tested with the ΔFus3 strain?

Fus3 is required for the signal transduction pathway leading to shmoo formation.

What hypothesis was being tested with the Δformin strain?

Formin is required for the signal transduction pathway leading to shmoo formation.

In addition to testing shmoo formation in the two mutant strains of yeast, the scientists also tested shmoo formation in wild-type yeast.

What is the purpose of including wild-type yeast cells in the experiment?

to show normal shmoo formation under the experimental conditions

When designing an experiment, scientists make predictions about what results will occur if their hypothesis is correct.

One of their hypotheses was that Fus3 kinase is required for the signal transduction pathway leading to shmoo formation. If this hypothesis is correct, what result should be observed in the ΔFus3 strain?

The ΔFus3 strain should not form shmoos, and the cells should not have a red zone in their walls.

One of their hypotheses was that formin is required for the signal transduction pathway leading to shmoo formation. If this hypothesis is correct, what result should be observed in the Δformin strain?

The Δformin strain should not form shmoos, and the cells should not have a red zone in their walls.

The micrographs shown below were taken of wild-type, ΔFus3, and Δformin cells after they were stained green, exposed to mating factor, and then stained red.

For each micrograph, drag the labels to answer the questions. Labels may be used once, more than once, or not at all.

Wild Type: Red Green - Asym - Y - Y - Y

ΔFus3: Yellow - Sym - N - Y - Y

Δformin: Yellow - Sym - N - Y - Y

Fus3 kinase and formin proteins are generally distributed evenly throughout a yeast cell. Based on the model in the diagram, why does the shmoo projection emerge on the same side of the cell that bound the mating factor?

The only formin molecules that get phosphorylated and thus activated are those near the G protein-coupled receptor that binds mating factor.

What do you predict would happen if the yeast had a mutation that prevented the G protein from binding GTP?

No shmoo would form in response to mating factor.

Involved in muscle contraction

MF

Involved in the movement of cilia and flagella

MT

More important for chromosome movements than for cytokinesis

MT

More important for cytokinesis than for chromosome movements in animal cells.

MF

Most likely to remain when cells are treated with solutions of nonionic detergents or solutions of high ionic strength

IF

Structurally similar proteins are found in bacterial cells.

MT MF IF

Their subunits can bind and catalyze hydrolysis of phosphonucleotides

MT MF

Can be detected by immunofluorescence microscopy.

MT MF IF

Play well-documented roles in cell movement

MT MF

The fundamental repeating subunit is a dimer.

MT IF

Microtubules, microfilaments, and intermediate filaments all exist in a typical eukaryotic cell in dynamic equilibrium with a pool of subunit proteins.

True

Latrunculin A treatment would block intracellular movements of Listeria.

True

All of the protein subunits of intermediate filaments are encoded by genes in the same gene family.

True

The minus end of microtubules and microfilaments is so named because subunits are lost and never added there.

F

The energy required for tubulin and actin polymerization is provided by hydrolysis of a nucleoside triphosphate.

F

An algal cell contains neither tubulin nor actin.

F

All microtubules within animal cells have their minus ends anchored at the centrosome.

F

As long as actin monomers continue to be added to the plus end of a microfilament, the MF will continue to elongate.

Maybe

Provide a brief justification for the following false answer:

The minus end of microtubules and microfilaments is so named because subunits are lost and never added there.

The rate of loss is greater than the rate of addition at the minus end over time. There is no absolute absence of addition at the minus end.

Provide a brief justification for the following false answer:

The energy required for tubulin and actin polymerization is provided by hydrolysis of a nucleoside triphosphate.

Hydrolysis of the ATP bound to actin and the GTP bound to tubulin usually occurs during monomer polymerization, but the polymerization process still occurs even if the ATP or GTP is replaced by a nonhydrolyzable analogue.

Provide a brief justification for the following false answer:

An algal cell contains neither tubulin nor actin.

Algae are eukaryotes and therefore possess the same kinds of cytoskeletal components as other eukaryotic cells.

Provide a brief justification for the following false answer:

All microtubules within animal cells have their minus ends anchored at the centrosome.

Most do, but not all. In addition, in ciliated or flagellated cells, the minus ends of microtubules can be anchored at the basal body, which also serves as a microtubule-organizing center.

Provide a brief justification why the following answer can be either true or false:

As long as actin monomers continue to be added to the plus end of a microfilament, the MF will continue to elongate.

The statement is true if the monomer concentration is above the overall critical concentration but false otherwise.

Small vesicles containing pigment inside of pigmented fish epidermal cells aggregate or disperse in response to treatment with certain chemicals. When nocodazole is added to cells in which the pigment granules have been induced to aggregate, the granules cannot disperse again.

Pigment granule dispersal is a microtubule-dependent process.

When an animal cell is treated with colchicine, its microtubules depolymerize and virtually disappear. If the colchicine is then washed away, the MTs appear again, beginning at the centrosome and elongating outward at about the rate (1μm/min) at which tubulin polymerizes in vitro.

The centrosome serves as a microtubule-organizing center in vivo, and all of the microtubules radiating from the centrosome apparently have the same polarity.

Extracts from nondividing frog eggs in the G2 phase of the cell cycle were found to contain structures that could induce the polymerization of tubulin into microtubules in vitro. When examined by immunostaining, these structures were shown to contain γ-tubulin.

The extracts appear to contain structures that are functionally equivalent to centrosomes (as evidenced by the presence of γ-tubulin), which nucleate micro-tubule growth.

family proteins polymerization is very fast.

With Arp2/3 and WASP

family proteins polymerization is very slow.

Without Arp2/3 or WASP

With Arp2/3 polymerization increases, but not as much as with Arp2/3 and WASP family proteins.

With Arp2/3 with Arp2/3 and WASP

Which phase of microtubule polymerization in vitro would be affected most by adding short microtubules to unpolymerized GTP-tubulin?

lag phase

Which of the following is true about the structure of the microtubule wall?

α-β-tubulin heterodimers are arranged with the same orientation in each protofilament, and protofilaments are arranged with same polarity in the microtubule wall

CapZ

prevents assembly and disassembly at microfilament plus ends.

desmin keeps muscle myofibrils in register

nucleation formation of tubulin oligomers

myosin subfragment 1 (S1) decorates actin microfilaments

gelsolin breaks and caps actin filaments

microvilli increase cell surface area

treadmilling assembly and disassembly on opposite ends of the same filament

tropomodulin prevents loss of subunits from filament minus end

lamin forms scaffold underlying nuclear envelope

MAPs stabilizes and organizes microtubules

EB1 binds to microtubule plus ends

plectin crosslinks microtubules to intermediate filaments

axonemal microtubules is in cilia and flagella

phosphoinositides binds to profilin and CapZ

filamin connects crisscrossing actin filaments into 3D networks

Arp2/3 complex nucleates actin filament branches

desmin keeps muscle myofibrils in register

nucleation formation of tubulin oligomers

myosin subfragment 1 (S1) decorates actin microfilaments

gelsolin breaks and caps actin filaments

microvilli increase cell surface area

treadmilling assembly and disassembly on opposite ends of the same filament

tropomodulin prevents loss of subunits from filament minus end

lamin forms scaffold underlying nuclear envelope

MAPs stabilizes and organizes microtubules

EB1 binds to microtubule plus ends

plectin crosslinks microtubules to intermediate filaments

axonemal microtubules is in cilia and flagella

phosphoinositides binds to profilin and CapZ

filamin connects crisscrossing actin filaments into 3D networks

Arp2/3 complex nucleates actin filament branches

Intermediate filaments

can be composed of the protein vimentin.

Which of the following proteins would be a good drug target for interfering with microfilament network breakdown?

gelsolin

Which of the following is not associated with the bundle of microfilaments supporting each microvillus in small intestine mucosal cells?

fascin

Propagation of an action potential in a skeletal muscle cell links the signal from a motor neuron to contraction of the muscle cell. An action potential in a muscle cell is propagated by the same mechanism as in neurons, the sequential opening and closing of voltage-gated Na+ and K+ channels in the plasma membrane. However, in muscle cells, the topography of the plasma membrane is quite different than in neurons, and this difference is critical to the function of muscle cells.

Which of the following statements correctly describe(s) T tubules and their role in conducting action potentials in muscle cells?

Without T tubules, the muscle cell would not be able to contract.

T tubules carry action potentials into the interior of the muscle cell via voltage-gated Na+ and K+ channels.

T tubules are infoldings of the plasma membrane that encircle the myofibrils and are in contact with the sarcoplasmic reticulum.

In a relaxed muscle, the myosin heads of the sarcomeres' thick filaments are extended and ready to bind to the actin strands of the thin filaments. But this binding does not occur until an action potential is triggered in the muscle cell. An action potential results in the release of Ca2+ ions from the sarcoplasmic reticulum into the cytosol of the muscle cell.

As the concentration of Ca2+ rises in the cytosol, so does the concentration of Ca2+ in the sarcomeres. In response to changes in the Ca2+ concentration in the sarcomeres, two protein components of the thin filaments, troponin and tropomyosin, control access to actin's myosin-binding sites. In this way, Ca2+ concentration in the cytosol and sarcomeres regulates muscle contraction.

Which of the following statements correctly describe(s) the relationship between Ca2+ concentration in the cytosol and the response in the sarcomere?

Select all that apply.

Decreasing Ca2+ concentration causes dissociation of Ca2+ from troponin.

Increasing Ca2+ concentration causes movement of tropomyosin, exposing myosin-binding sites on actin.

Which of the following statements correctly describes why a series of closely spaced action potentials causes a sustained contraction rather than a series of closely spaced twitches?

When a series of action potentials is closely spaced, there is not sufficient time for Ca2+ uptake into the sarcoplasmic reticulum between action potentials, and Ca2+ remains bound to troponin throughout the series.

Which muscle type is involved in the function of the digestive tract and blood vessels?

Smooth

How does cardiac muscle differ from the other types of muscle?

It contains branched cells.

True or false? Myofibrils are the alternating light-dark units that produce the banded appearance of muscle fibers.

False

Which molecules form the thick filaments of sarcomeres?

Myosin

Which of the following interactions is the molecular basis of muscle contraction?

Myosin and thin filaments.

Which step constitutes the power stroke of muscle contraction?

The phosphate ion is released, and the myosin head moves back to its original position.

Which of the following statements about the stimulation of muscle cells is true?

An action potential in a muscle cell ultimately results in the release of calcium ions into the cell.

Frog skeletal muscle consists of thick filaments that are about 1.6μm long and thin filaments about 1μm long.

Part A

What is the length of the A band and the I band in a muscle with a sarcomere length of 3.2μm?

The length of the A band is 1.6μm, and the length of the I band is also 1.6μm.

Describe what happens to the length of both bands as the sarcomere length decreases during contraction from 3.2 to 2.0μm.

The length of the A band remains fixed at 1.6μm, and the length of the I band decreases from 1.6μm to 0.4μm.

The H zone is a specific portion of the A band. If the H zone of each A band decreases in length from 1.2 to 0μm as the sarcomere length contracts from 3.2 to 2.0μm, what can you deduce about the physical meaning of the H zone?

The H zone corresponds to that portion of the thick filament length that is not overlapped by thin filaments.

What can you say about the distance from the Z line to the edge of the H zone during contraction?

The distance remains constant during contraction.

Observation of flagella in the biflagellate alga, Chlamydomonas reinhardtii, indicates that particles move toward the tips of flagella at a rate of 2.5μm/min, but the particles moving back toward the base of flagella move at 4μm/min. How do you explain this difference in rate of movement?

Particles use different motors with different velocities to move outward and inward.

Temperature-sensitive mutations in a kinesin II required for intraflagellar transport (IFT) have been identified in Chlamydomonas. Such mutations only lead to defects when the temperature is raised above a certain threshold, called the restrictive temperature. When algae with fully formed flagella are grown at the restrictive temperature, their flagella degenerate. What can you conclude about the necessity of IFT from this experiment?

IFT seems to be necessary for ongoing maintenance of flagellar structure.

Based on your knowledge of the directionality of microtubule motors and the information in part B, where would you predict that the plus ends of flagellar microtubules are? State your reasoning.

Kinesins transport cargoes toward the plus ends of microtubules, so the plus ends should be at the tip of the flagellum.

What effects would you predict on a sperm flagellum to which AMPPNP was added? In your explanation, please be specific about what molecule's function would be inhibited and what the effect on overall flagellar function would be.

AMPPNP would inhibit flagellar dynein, causing cessation of flagellar bending.

When researchers incubated purified vesicles, nerve cytosol from squid giant axons, and MTs in the presence of AMPPNP, the vesicles bound tightly to the microtubules but did not move. Scientists then used AMPPNP to promote its tight binding to MTs, and the MTs with bound proteins were collected by centrifugation. The main protein purified in this way promotes movement of vesicles away from the cell body, where the nucleus resides. What was this protein?

kinesin

How can the wave-like motion seen in the beating of sperm flagella be created?

Some dynein molecules must be exerting force while those on the opposite side of the tail must be inactive.

Stress fibers of nonmuscle cells contain contractile bundles of actin and myosin II. For stress fibers to contract or develop tension, how would actin and myosin have to be oriented within the stress fibers?

There would have to be regions of antiparallel actin filaments (with some actin filaments attached to myosin in the opposite orientation).

Which of the following accurately describes the nucleotide dependence of cilia and flagella structure and function?

The structures of cilia and flagella are dependent on GTP, whereas the functions of cilia and flagella are dependent on ATP.

What would be the likely outcome if the radial spokes inside cilia were destroyed and the cilia attempted to move?

The bending of the cilia would cease, and the microtubules inside the cilia would slide past one another.

Which of the following would result if an inhibitor of myosin light-chain kinase (MLCK) were added to a culture of smooth muscle cells?

There would be no muscle contraction.

Flow of vesicles from the ER to the Golgi involves all of these structures EXCEPT __________.

kinesin

Axonemal dyneins are

associated with cilia and flagella.

A tubule: complete 13 protofilament microtubule

B tubule: incomplete 10−11 protofilament microtubule

sidearms: generates force to slide microtubules past one another

radial spokes: project inward at each doublet

nexin: links adjacent doublets

A tubule: complete 13 protofilament microtubule

B tubule: incomplete 10−11 protofilament microtubule

sidearms: generates force to slide microtubules past one another

radial spokes: project inward at each doublet

nexin: links adjacent doublets

Addition of ATP to isolated axonemes causes localized bending. What can be done to the isolated axonemes to cause the outer doublets to slide past each other unrestrained, lengthening the overall structure instead of causing bending?

remove the interdoublet nexin links

Which of these cell junctions form a barrier to the passage of materials?

tight junctions

The primary role of _____ is to bind animal cells together.

desmosomes

_____ aid in the coordination of the activities of adjacent animal cells.

Gap (communicating) junctions

Connexin

gap junctions

E-cadherin

adherens junctions

Desmocollins

desmosomes

desmosomes

plasmodesmata

Annulus

plasmodesmata

α-Catenin

adherens junctions

Claudins

tight junctions

Associated with filaments that confer either contractile or tensile properties.

adhesive junctions (A)

Sites of membrane fusion are limited to abutting ridges of adjacent membranes.

Seal membranes of two adjacent cells tightly together.

tight junctions (T)

Require the alignment of connexons in the plasma membranes of two adjacent cells.

gap junctions (G)

Found exclusively in plant cells.

plasmodesmata (P)

Allow the exchange of metabolites between the cytoplasms of two adjacent cells.

G and P

Apical surface is the surfaces of intestinal epithelial cells that face the lumen of the intestine. basolateral surface is the surfaces of intestinal epithelial cells that face the circulatory system.

Fibronectins occur widely throughout supporting tissues and body fluids, whereas laminins are found mainly in the basal laminae.

The glycocalyx is a carbohydrate-rich zone juxtaposed between the plasma membrane and the ECM of many types of animal cells.

Collagen is responsible for the strength of the ECM, whereas elastin imparts elasticity and flexibility to the ECM.

The groups mediate cell-cell recognition and adhesion; the two groups of proteins can be distinguished from each other functionally because of the calcium requirement of cadherins but not IgSFs.

These are transmembrane proteins that serve as receptors for ECM proteins such as collagen, fibronectin, and laminin; selectin is the best-characterized integrin.

Apical surface is the surfaces of intestinal epithelial cells that face the lumen of the intestine. basolateral surface is the surfaces of intestinal epithelial cells that face the circulatory system.

Fibronectins occur widely throughout supporting tissues and body fluids, whereas laminins are found mainly in the basal laminae.

The glycocalyx is a carbohydrate-rich zone juxtaposed between the plasma membrane and the ECM of many types of animal cells.

Collagen is responsible for the strength of the ECM, whereas elastin imparts elasticity and flexibility to the ECM.

The groups mediate cell-cell recognition and adhesion; the two groups of proteins can be distinguished from each other functionally because of the calcium requirement of cadherins but not IgSFs.

These are transmembrane proteins that serve as receptors for ECM proteins such as collagen, fibronectin, and laminin; selectin is the best-characterized integrin.

Fibronectin

It can bind to fibrin (important during blood clot formation), and can bind to collagen or heparin sulfate proteoglycans for attaching it to the ECM.

The RGD-containing cell-binding domain is crucial for allowing cells to attach to this molecule, because this is the site that is bound by integrins on the cell surface.

Laminin

A collagen IV binding site allows it to be attached to the basal laminae (where the molecule is predominantly found).

It has an integrin-binding site specifc for _6_1 integrins.

Both Fibronectin and Laminin

It has multiple domains that allow to attach to other ECM proteins.

Historically, an important strategy for disrupting the adhesion of integrins to their ligands is by using a synthetic peptide that mimics the binding site on the ECM molecule to which the integrin attaches. In the case of fibronectin, the amino acid sequence is arginine-glycine-aspartate (when written using the single letter designation for each amino acid, this sequence becomes RGD). Explain why addition of such synthetic peptides would disrupt binding of cells to their normal substratum.

When the integrins are bound to RGD peptides, their receptors will be unavailable for binding to FN (or LN), and thus cells will be inhibited from binding to the ECM.

Loewenstein and colleagues injected cells with fluorescent molecules of different molecular weights, and a fluorescence microscope was then used to observe the movement of the molecules into adjacent cells. When molecules with molecular weight of 1926 were injected, they did not pass from cell to cell, but molecules with molecular weight of 1158 did.

The gap junctions that connect adjacent cells in insect salivary glands allow the passage of small molecules (molecular weight up to at least 1158 daltons), whereas larger molecules (1926 daltons) do not flow between cells.

Microglia (support cells in the brain) are connected by gap junctions. When the fluorescent dye Lucifer yellow is injected into individual microglia that are part of a large group of cells, dye passes to other cells. When microglia are treated with the calcium ionophore, 4Br-A23187, there is a dramatic decrease in dye passage between cells.

"The passage of molecules between cells can be regulated by the intracellular Ca2+ content. When the Ca2+ concentration is increased in an individual cell, the movement of fluorescent molecules into that cell is increased, suggesting that gap junctions are open under these conditions.

Scurvy is a disease that until the nineteenth century was common among sailors and others whose diets were deficient in vitamin C (ascorbic acid). Individuals with scurvy suffer from various disorders, including extensive bruising, hemorrhages, and breakdown of supporting tissues. Ascorbic acid serves as a reducing agent responsible for maintaining the activity of prolyl hydroxylase, the enzyme that catalyzes hydroxylation of proline residues within the collagen triple helix, which is required for helix stability.

Part A

Based on this information, postulate a role for hydroxyproline in collagen triple helices.

Hydroxylated proline residues, but apparently not non-hydroxylated proline residues, but apparently not unhydroxylated proline residues, stabilize the collagen triple helix. The hydroxyl groups of this amino acid form interchain hydrogen bonds that help stabilize the assembled triple-stranded helix.