Chapter_16_Solutions - Chapter 16 The Cytoskeleton THE...

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Unformatted text preview: Chapter 16 The Cytoskeleton THE SELF-ASSEMBLY AND DYNAMIC STRUCTURE OF CYTOSKELETAL FILAMENTS DEFINITIONS 161 162 163 164 165 166 Protofilament Dynamic instability Plus end Intermediate filament Treadmilling Cytoskeleton 16 In This Chapter THE SELF-ASSEMBLY A367 AND DYNAMIC STRUCTURE OF CYTOSKELETAL FILAMENTS HOW CELLS REGULATE A378 THEIR CYTOSKELETAL FILAMENTS MOLECULAR MOTORS THE CYTOSKELETON AND CELL BEHAVIOR A384 A387 TRUE/FALSE 167 True. Each protofilament in a microtubule is assembled from subunits that all point in the same direction; thus, each protofilament has a-tubulin at one end and b-tubulin at the other. Since the protofilaments in a microtubule are aligned in parallel, a-tubulin is always at one end and b-tubulin is always at the other. True. When ATP in actin filaments (or GTP in microtubules) is hydrolyzed, much of the free energy released by cleavage of the high-energy bond is stored in the polymer lattice, making the free energy of the ADP-containing polymer higher than that of the ATP-containing polymer. This shifts the equilibrium toward depolymerization so that ADP-containing actin filaments disassemble more readily than ATP-containing actin filaments. False. In contrast to actin filaments and microtubules, which are present in all eucaryotic organisms, intermediate filaments are found only in some metazoans, including vertebrates, nematodes, and snails. Even in these organisms intermediate filaments are not required in every cell type. The nuclear lamins, which are the ancestors of the intermediate filaments, form a meshwork of protein that lines the nuclear membrane; they are much more widely distributed among eucaryotes. 168 169 THOUGHT PROBLEMS 1610 Intermediate filaments provide mechanical stability and resistance to shear stress. Microtubules determine the positions of membranous organelles and direct intracellular transport. Actin filaments determine the shape of the cell's surface and are necessary for whole-cell locomotion. A367 A368 Chapter 16: The Cytoskeleton (A) MEASUREMENTS 5 6 increase in fluorescence 41.0 mM 4 3 2 1 0 0 10.3 mM lag 10 20 30 40 50 actin (mM) Cc (B) CRITICAL CONCENTRATION 5 fluorescence intensity 4 20.5 mM 3 growth equilibrium 2 Figure 1647 Analysis of actin polymerization (Answer 1612). (A) Actin polymerization at three different actin concentrations, as indicated on the individual curves. (B) The critical concentration determined from many such experiments. The plateau values for increase in intensity of fluorescence (corrected for fluorescence of the monomers) are plotted against actin concentration. The black circles indicate the data shown in (A); white circles are additional data points from similar experiments. The critical concentration (Cc) is estimated by extrapolating to a value of zero increase in intensity. 1 0 0 30 60 90 time (seconds) 1611 Although the subunits are indeed held together by noncovalent bonds that are individually weak, there are a very large number of them, distributed among a very large number of filaments. As a result, the stress a human being exerts by lifting a heavy object is dispersed over so many subunits that their interaction strength is not exceeded. By analogy, a single thread of silk is not nearly strong enough to hold a human, but a rope woven of such fibers is. 1612 A. Phase A corresponds to a lag phase (Figure 1647A), during which actin monomers must assemble to form a nucleus for polymerization (thought to be a trimer of subunits). Formation of a nucleus (nucleation) is followed by rapid growth (phase B), as actin monomers are added to the ends of the growing filaments. At phase C, equilibrium is reached between the rate of addition of actin at the ends and its rate of release. Once equilibrium is reached, the concentration of free actin remains constant. B. If the starting concentration of actin were doubled, the lag phase would be shorter, the growth phase would be more rapid (steeper), and the mass of polymer at equilibrium would be twice as great. The experimental curves generated at twice and half the initial actin concentrations illustrate these relationships (Figure 1647A). The concentration of free actin monomers at equilibrium--the critical concentration (Cc)--would be the same regardless of the initial actin concentration. It can be estimated from the data in Figure 1647A, as shown in Figure 1647B. Reference: Carlier M-F, Pantaloni D & Korn ED (1985) Polymerization of ADP- and ATP-actin under sonication and characteristics of the ATP-actin equilibrium polymer. J. Biol. Chem. 260, 65656571. 1613 The critical concentration is the concentration of actin at which filaments initially form (Figure 1648). Above the critical concentration the mass of free actin remains constant. Two tubulin dimers have a lower affinity for each other (because of a more limited number of interaction sites) than a tubulin dimer has for the end of a microtubule. At the end of an existing microtubule there are multiple possible interaction sites, both end-to-end as the tubulin dimers add to a protofilament, and side-to-side as they bind to adjacent protofilaments in the microtubule lattice. Thus, to initiate a microtubule from scratch, enough tubulin dimers have to come together and remain bound to one another for long enough for other tubulin molecules to add to them. Only when several tubulin dimers have already assembled will the binding of the next subunit be favored. mass filament monomer 1614 Cc actin concentration Figure 1648 Critical concentration (Cc) of actin (Answer 1613). THE SELF-ASSEMBLY AND DYNAMIC STRUCTURE OF CYTOSKELETAL FILAMENTS (A) SHEET (B) MICROTUBULE A369 Figure 1649 Interactions between protofilaments composed of ab-tubulin dimers (Answer 1615). (A) A sheet of protofilaments. An example of homotypic lateral interactions between ab-tubulin dimers in the protofilaments is shown on the right. (B) A microtubule. An example of heterotypic lateral interactions between ab-dimers at the seam is shown on the right. b a a b b a b a lateral homotypic interactions seam lateral heterotypic interactions 1615 The heterotypic interactions between the protofilaments are likely to be weaker than the homotypic interactions between them. If the interactions between a-tubulin and b-tubulin were stronger than the homotypic interactions, the protofilaments would preferentially align so that heterotypic interactions, rather than homotypic ones, were maximized. If the two sets of interactions were the same strength, the arrangement of protofilaments might be mixed within the same microtubule, or two different types of microtubule--with protofilaments aligned either by homotypic or heterotypic lateral interactions--might be observed. You can imagine the formation of the microtubule as building a sheet of protofilaments that curl into a microtubule by forming the seam (Figure 1649). Reference: Desai A & Mitchison TJ (1997) Microtubule polymerization dynamics. Annu. Rev. Cell Dev. Biol. 13, 83117. 1616 Subunit 1 will add faster to the right end of the polymer than to the left end, subunit 2 will add to both ends at equal rates, and subunit 3 will add faster to the left end of the polymer. A difference in growth rates at the two ends reflects a change in conformation of the free subunit as it adds to the polymer. For example, subunit 1 can add to the right end of the polymer through an existing binding site (its pointed end) and change conformation later. To add to the left end, however, it must make the conformational change before or during addition. For the simple polymerization described here, both ends must grow or shrink; there is no concentration of subunit that can allow one end to grow while the other shrinks (or stays the same length). This is because the conformations of the subunits at the two ends of the polymer are identical and they involve identical contacts. You could not tell from which end a free subunit derived. Thus, the DG for subunit loss, which determines the equilibrium constant for subunit association at an end, must be the same for both ends. The ends of the shrinking microtubule are visibly frayed, and the individual protofilaments appear to come apart and curl as the end depolymerizes. This micrograph therefore suggests that the GTP cap (which is lost from shrinking microtubules) holds the protofilaments properly aligned with each other, perhaps by strengthening the side-to-side interactions between ab-tubulin dimers when they are in their GTP-bound form. The rapidly growing microtubules, by contrast, have nonfrayed ends. The one on the left in Figure 165A has an end that appears cylindrical. The one at right, however, has a different kind of end, one that may reflect the pattern of addition of ab-tubluin to a growing end. Reference: Chretien D, Fuller SD & Karsenti E (1995) Structure of growing microtubule ends: Two-dimensional sheets close into tubes at variable rates. J. Cell Biol. 117, 13111328. 1617 A370 Chapter 16: The Cytoskeleton 1618 A. The microtubule is shrinking because it has lost its GTP cap; that is, the tubulin subunits at its end are all in their GDP-bound form. GTP-loaded tubulin subunits from solution will still add to this end, but they will be short-lived--either because they hydrolyze their GTP or because they fall off as the microtubule rim around them disassembles. If enough GTP-loaded subunits are added quickly enough to cover up the GDP-containing tubulin subunits at the microtubule end, then a new GTP cap can form and regrowth will be favored. B. The rate of addition of GTP-tubulin will be greater at higher tubulin concentrations. The frequency with which shrinking microtubules switch to the growing mode will therefore increase with increasing tubulin concentration. The consequence of this regulation is that the system is self-balancing. The more microtubules shrink (resulting in a higher concentration of the free tubulin), the more frequently microtubules will start to grow. As microtubules grow, the concentration of free tubulin will fall and the rate of GTPtubulin addition will slow down. At some point GTP hydrolysis will catch up with new GTP-tubulin addition, the GTP cap will be destroyed, and the microtubule will switch to the shrinking mode. C. If only GDP were present, microtubules would continue to shrink and eventually disappear, because tubulin dimers with bound GDP have very low affinity for each other and will not add stably to microtubules. D. If a GTP analog that cannot be hydrolyzed were present, microtubules would continue to grow until all free tubulin subunits had been used up. 1619 Severing a microtubule in the middle would generate new plus and minus ends, both of which would lack a GTP cap. The b-tubulin subunits in the middle of a microtubule will have already hydrolyzed their GTP cap and thus will have bound GDP. Thus, the simple notion that ends with GTP caps grow and ends without caps shrink leads to the expectation that the newly exposed plus and minus ends will both shrink. In reality, in this sort of experiment the plus end shrinks, as expected, but the minus ends are stable and immediately resume a slow rate of polymerization. These results may indicate that a GTP cap is important for growth at a plus end but is not important for stability and growth at a minus end. Reference: Walker RA, Inou S & Salmon ED (1989) Asymmetric behavior of severed microtubule ends after ultraviolet-microbeam irradiation of individual microtubules in vitro. J. Cell Biol. 108, 931937. 1620 By analogy with Ras, tubulin can be thought of as its own GAP, accelerating GTP hydrolysis by the formation of intersubunit contacts during polymerization. A few examples of the differences between bacteria and animal cells are listed below. This is by no means a complete list. 1. Animal cells are much larger, diversely shaped, and do not have a cell wall. Cytoskeletal elements are required to provide mechanical strength and shape in the absence of a cell wall. 2. Animal cells, and all other eucaryotic cells, have a nucleus that is shaped and held in place by intermediate filaments; the nuclear lamins attached to the inner nuclear membrane support and shape the nuclear membrane, and a meshwork of intermediate filaments surrounds the nucleus and spans the cytosol. 3. Animal cells can move by a process that requires a change in cell shape. Actin filaments (and myosin motor proteins) are required for these activities. 4. Animal cells have a much larger genome than bacteria; this genome is fragmented into many chromosomes. For cell division, chromosomes need to be accurately distributed to the daughter cells, which requires the microtubules that form the mitotic spindle. 5. Animal cells have internal organelles. Their localization in the cell depends on motor proteins that move them along microtubules. The long-distance 1621 THE SELF-ASSEMBLY AND DYNAMIC STRUCTURE OF CYTOSKELETAL FILAMENTS travel of membrane-enclosed vesicles along microtubules in an axon, which can be up to a meter long in the case of the nerve cells that extend from your spinal cord to your feet, provides a remarkable example. 1622 The evolution of actins and tubulins is constrained not only by the requirement that they bind to one another, but also by the necessity that they interact with a large number of other proteins that bind to the same or overlapping sites on their surfaces. A mutation in actin that results in a desirable change in its interaction with one protein might cause undesirable changes in its interactions with a half-dozen other proteins that bind at or near the same site. These multiple interactions constrain the evolution of most of the surfaces of actins and tubulins. By contrast, the proteins that bind to actin filaments and microtubules need only preserve their filament-binding sites--which are, in fact, the portions of their structures that are most conserved--and the binding sites for the limited number of other proteins they interact with. This reduced constraint allows them considerably more evolutionary freedom. The building blocks--soluble subunits--of the three types of filaments are the basis for their polarity differences. The building blocks for actin filaments (an actin monomer) and microtubules (ab-tubulin) have polarity-- distinct ends--and thus form a polymer with distinct ends when they are linked together. By contrast, the building block of intermediate filaments is a symmetric tetramer with identical ends. Thus, when these subunits are linked together, the ends of the resulting filament are also identical. Cells that migrate rapidly from one place to another, like amoebae (A) and sperm cells (E), do not, in general, need intermediate filaments in their cytoplasm, since they do not develop or sustain large tensile forces. Plant cells (F) are pushed and pulled by the forces of wind and water, but they resist these forces by means of their rigid cell walls, rather than by their cytoskeleton. Epithelial cells (B), smooth muscle cells (C), and the long axons of nerve cells (D) are all rich in cytoplasmic intermediate filaments, which prevent them from rupturing as they are stretched and compressed by the movements of surrounding tissues. The disulfide bonds that cross-link keratin filaments in skin cells form after the cells have died. In the absence of cellular metabolism to maintain the reducing environment characteristic of a living cell, a dead cell's contents quickly become oxidized. It is in this postmortem environment that the keratin filaments become cross-linked by disulfide bonds. It is surprising that so many knockouts of genes for intermediate filaments have little effect in mice. The amino acid sequence of vimentin, for example, is 98% identical in hamster, chicken, mouse, and human, implying an important function, yet mouse knockouts appear entirely normal. The absence of an effect of knockout of such a conserved gene is usually interpreted in terms of a `backup' system that compensates for the loss. In the case of intermediate filament genes, the backup system may be other intermediate filaments. That the combined knockout of vimentin and GFAP does have a phenotype--defective astrocytes--suggests that these two intermediate filaments back each other up in astrocytes. Astrocytes express genes for three intermediate filament proteins-- vimentin, GFAP, and nestin, whose properties have been studied using the pure proteins. Nestin cannot form intermediate filaments on its own, which presumably explains its inability to compensate for the loss of vimentin and GFAP Vimentin can form intermediate filaments, but only with nestin or . GFAP as a partner, and GFAP can form somewhat abnormal intermediate filaments on its own. Thus, these three proteins appear to cooperate in the formation of correct intermediate filaments in astrocytes: disruption of one can be tolerated, but not the ablation of two. References: Herrmann H & Aebi U (2000) Intermediate filaments and their associates: multi-talented structural elements specifying cytoarchitecture and cytodynamics. Curr. Opin. Cell Biol. 12, 7990. A371 1623 1624 1625 1626 A372 Chapter 16: The Cytoskeleton Eliasson C, Sahlgren C, Berthold CH, Stakeberg J, Celis JE, Betsholtz C, Eriksson JE & Pekny M (1999) Intermediate filament protein partnership in astrocytes. J. Biol. Chem. 274, 2399624006. 1627 Cell division depends on the ability of microtubules to polymerize and to depolymerize. During mitosis, cells first depolymerize most of their microtubules and then repolymerize them to form the mitotic spindle. Taxoltreated cells are prevented from depolymerizing their existing microtubules, and thus cannot form a mitotic spindle. Colchicine-treated cells cannot polymerize new microtubules, and thus are also prevented from forming a mitotic spindle. On a more subtle level, both drugs would block the dynamic instability of microtubules and thus would interfere with the workings of the mitotic spindle, even if one could be formed. These results, and those from other studies, make it highly unlikely that the effects of acrylamide on neurofilaments are responsible for acrylamide neurotoxicity. If the original hypothesis were correct, then acrylamide toxicity would have been expected to be absent in mice that are missing neurofilaments. References: Stone JD, Peterson AP Eyer J & Sickles DW (2000) Neurofilaments , are nonessential elements of toxicant-induced reductions in fast axonal transport: pulse labeling in CNS neurons. Neurotoxicology 21, 447457. Sickles DW, Pearson JK, Beall A & Testino A (1994) Toxic axonal degeneration occurs independent of neurofilament accumulation. J. Neurosci. Res. 39, 347354. 1628 CALCULATIONS 1629 A growth rate of 2 mm/min (2000 nm/60 sec = 33 nm/sec) corresponds to the addition of 4.2 ab-tubulin dimers [(33 nm/sec) (ab-tubulin/8 nm) = 4.17 dimers/sec] to each of 13 protofilaments, or about 54 ab-tubulin dimers/sec to the ends of a microtubule. Reference: Detrich WH, Parker SK, Williams RC, Nogales E & Downing KH (2000) Cold adaptation of microtubule assembly and dynamics. J. Biol. Chem. 275, 3703837047. 1630 A. The average time for a small molecule such as ATP to diffuse across a cell 10 mm (103 cm) in diameter is t = x 2/2D = (103 cm)2/2 (5 106 cm2/sec) = 0.1 sec Similarly, a protein molecule takes 1 second and a vesicle 10 seconds, on average, to travel 10 mm. B. The diffusion of long, cytoskeletal filaments is even slower than that of membrane vesicles; hence, it would take much longer to rearrange the cytoskeleton by diffusion. In addition to time, there is also the problem of length: polymerization allows filaments to be constructed to fit. Finally, if the long cytoskeletal elements were to rearrange by diffusion, they would become hopelessly entangled with one another. DATA HANDLING 1631 A. The plus end of the myosin-decorated filament is defined as the one that grows more rapidly and, thus, has the longer newly synthesized actin segment, which is the end on the left in Figure 166. The minus end grows more THE SELF-ASSEMBLY AND DYNAMIC STRUCTURE OF CYTOSKELETAL FILAMENTS slowly; hence, it is the end on the right in Figure 166. The myosin heads form arrowheadlike structures on an actin filament, with the point corresponding to the minus end and the barb corresponding to the plus end. If you examine the myosin-decorated actin filament in Figure 166, you should see a chain of about 15 of these arrowheadlike structures. This easy way of visualizing the two ends is why the ends of an actin filament are commonly referred to as `pointed' or `barbed.' B. If the mixture were diluted below the critical concentration of actin monomers, the actin filaments would depolymerize. The plus end, the one with the longer tail, would depolymerize more rapidly. The plus end is the more dynamic end, always polymerizing and depolymerizing more rapidly than the minus end. C. During depolymerization, actin monomers dissociate exclusively from the ends because fewer noncovalent bonds hold them in place. A terminal actin monomer is held in place by two sets of interactions: those between it and the next monomer in the chain and those between it and the actin monomers in the adjacent chain. In addition to these two sets of interactions, an internal actin monomer is held in place by noncovalent bonds to a second actin monomer in the same chain (Figure 1650). References: Pollard TD, Blanchoin L & Mullins RD (2000) Molecular mechanisms controlling actin filament dynamics in nonmuscle cells. Annu. Rev. Biophys. Biomol. Struct. 29, 545576. Oosawa F (2001) A historical perspective of actin assembly and its interactions. Res. Prob. Cell Diff. 32, 921. 1632 These observations show that ab-tubulin dimers are oriented in microtubules with b-tubulin exposed at the plus end and a-tubulin facing the minus end. Because the GTP that is bound to the a-tubulin monomer is physically trapped at the dimer interface, it is never hydrolyzed or exchanged. By contrast, the GTP in b-tubulin is hydrolyzed and can be exchanged. Thus, when a microtubule is exposed to GTP-coated fluorescent beads, the GTP can bind to the b-tubulin subunits exposed at the end of the microtubule. Finding the fluorescent beads at the plus ends indicates that the ab-tubulin dimer must be oriented with the b-tubulin monomer at the plus end. The presence of the beads only at one end, and not all along the microtubule, indicates that GTP can be exchanged only at the exposed ends. Similarly, the presence of gold beads coated with antibodies specific for atubulin at the minus ends indicates that the ab-tubulin dimer must be oriented with the a-tubulin monomer exposed at the minus end. The presence of beads only at one end indicates that the portion of a-tubulin with which the antibody reacts is buried at the interface between adjacent ab-tubulin dimers, and thus is available only at the end. References: Mitchison TJ (1993) Localization of an exchangeable GTP binding site at the plus end of microtubules. Science 261, 10441047. Fan J, Griffiths AD, Lockhart A, Cross RA & Amos LA (1996) Microtubule minus ends can be labeled with a phage display antibody specific to a-tubulin. J. Mol. Biol. 259, 325330. Nogales E, Whittaker M, Milligan RA & Downing KH (1999) High-resolution model of the microtubule. Cell 96, 7988. 1633 A. The two ends of an individual microtubule appear to behave independently of one another. One end can grow while the other shrinks, and both ends B A A A373 B A Figure 1650 Interactions between actin subunits in an actin filament (Answer 1631). Shading indicates defined interactions between the subunits, which are designated A and B to distinguish between the two actin protofilaments. B A374 Chapter 16: The Cytoskeleton can grow or shrink at the same time. Furthermore, the transitions between growth states at the two ends do not correlate with one another in any obvious way. B. The GTP-cap hypothesis predicts that the faster-growing end, which has the longer GTP cap, should be more stable than the slower-growing end, which has a shorter GTP cap. Thus, a fast-growing end should persist in a growth state longer than a slow-growing end; that is, a fast-growing end should switch from a growth state to a shrinking state less frequently than a slowgrowing end. (The hypothesis says nothing about how frequently a shrinking end, which does not have a cap, will be converted into a growing end.) The experimental results appear, if anything, to run counter to the predictions of the GTP-cap hypothesis. The growth periods at the plus ends do not seem to be significantly longer (they actually appear somewhat shorter) than the growth periods at the minus ends. Thus, these results do not support this version of the GTP-cap hypothesis. In cells, proteins other than tubulin may bind to GTP caps and help to stabilize fast-growing ends. Reference: Horio T & Hotani H (1986) Visualization of the dynamic instability of individual microtubules by dark-field microscopy. Nature 321, 605607. 1634 A. An enzyme-catalyzed reaction reaches a plateau when the enzyme becomes saturated with substrate. Beyond that point an increase in substrate concentration cannot increase the rate of the reaction, because the enzyme is already working at maximum capacity. In contrast, growth of an actin filament does not saturate. Each time a monomer is added to the filament, a new site for addition of the next monomer is created. Addition of new monomers occurs through productive collisions with the end of the filament. The number of productive collisions increases linearly with the concentration of actin monomers. B. At concentration A, both ends would shrink. At concentration B, the minus end would shrink and the plus end would be unchanged. At concentration C, the plus end would grow and the minus end would shrink. At concentration D, the plus end would grow and the minus end would remain unchanged. At concentration E, both ends would grow, with the plus end growing faster than the minus end. The critical concentration is the concentration of actin at which an end neither shrinks nor grows. For plus ends the critical concentration is concentration B (0.12 mM); for the minus ends the critical concentration is concentration D (0.62 mM). At any concentration between these two critical concentrations the filament would exhibit treadmilling. At concentration C the plus end would grow at exactly the same rate as the minus end would shrink, giving treadmilling with no change in length of the filament. References: Pollard TD, Blanchoin L & Mullins RD (2000) Molecular mechanisms controlling actin filament dynamics in nonmuscle cells. Annu. Rev. Biophys. Biomol. Struct. 29, 545576. Pollard TD (1986) Rate constants for the reactions of ATP- and ADP-actin with the ends of actin filaments. J. Cell Biol. 103, 27472754. 1635 A. You should expect the amino acid changes to strengthen primarily the lateral interactions in the microtubule lattice; that is, interactions between tubulin subunits in adjacent protofilaments. The surest way to strengthen the lattice is to strengthen the weakest interactions. The interactions between the a- and b-tubulin subunits in the ab-tubulin dimer are the strongest interactions in the lattice; the dimer is so stable that it is the predominant form of tubulin in a cell. Increasing the affinity between adjacent dimers in a protofilament would tend to increase the stability of the microtubule; however, the appearance of microtubule ends undergoing catastrophic shrinkage--flayed ends with curled protofilaments (see Figure 165B)--indicates that interactions within protofilaments are stronger than THE SELF-ASSEMBLY AND DYNAMIC STRUCTURE OF CYTOSKELETAL FILAMENTS interactions between protofilaments. Thus, interactions between the protofilaments are the weakest interactions in the lattice. Therefore, it is not surprising that the amino acid changes in the tubulins in notothenioid fish strengthen interactions between adjacent protofilaments, thereby increasing the overall stability of the microtubules. B. It seems highly unlikely that notothenioid fish cells could exist if they had a stable microtubule cytoskeleton. In normal cells, microtubules are constantly shifting, and they undergo a dramatic rearrangement during mitosis when the cytoskeletal microtubules disassemble to form the mitotic spindle. It seems inconceivable that fish cells could divide with a fixed microtubule architecture. It is much more likely that notothenioid microtubules are just as dynamic in cells as normal microtubules, but that accessory proteins control the instability. Reference: Detrich WH, Parker SK, Williams RC, Nogales E & Downing KH (2000) Cold adaptation of microtubule assembly and dynamics. J. Biol. Chem. 275, 3703837047. 1636 This simple purification procedure takes advantage of the properties of tubulin and microtubules. At 0C, microtubules dissociate into ab-tubulin dimers, which remain in the supernatant when subjected to high centrifugal force. In the presence of GTP at 37C, the tubulin dimers polymerize into microtubules, which are large enough to form a pellet when centrifuged. This repetitive, two-step procedure purifies tubulin away from all other cell components. Large cell components are discarded each time the supernatant is saved; small cell components, including other proteins, are discarded each time the pellet is saved. Reference: Sloboda RD, Dentler WL & Rosenbaum JL (1976) Microtubuleassociated proteins and the stimulation of tubulin assembly in vitro. Biochemistry 15, 44974505. 1637 A. Although the end points for polymerization and ATP hydrolysis were the same, the initial rate of ATP hydrolysis was less than the initial rate of polymerization. (Compare the slopes of the two curves in Figure 169 at short times.) At the time when all the actin was polymerized (about 30 seconds), less than half the ATP was hydrolyzed. It is the difference in initial rates that your advisor noticed, and, as he said, it proves that actin polymerization can occur in the absence of ATP hydrolysis. B. Since the rate of polymerization is faster than the rate of ATP hydrolysis, newly added actin subunits must still retain bound ATP Since the bound ATP . is not hydrolyzed until some time after assembly, growing actin filaments have ATP caps. Once an ATP-actin monomer has bound to a filament, the ATP can be hydrolyzed, giving rise to the bound ADP found interior to the ATP caps. Reference: Carlier M-F, Pantaloni D & Korn ED (1984) Evidence for an ATP cap at the ends of actin filaments and its regulation of the F-actin steady state. J. Biol. Chem. 259, 99839986. 1638 In BHK-21 cells the entire vimentin network depolymerizes in preparation for mitosis and then reassembles afterwards (see Figure 1610A). Note the absence of obvious filaments during the two phases of mitosis and their clear presence in the daughter cells. By contrast, in PtK2 cells the vimentin network remains largely intact until late cytokinesis, when the portion of the network in the connecting cytoplasmic bridge is finally `dissolved.' Thus, in these cells only a small portion of the vimentin network is disassembled during mitosis. It is unclear how these two quite different strategies are accomplished. Reference: Yoon M, Moir RD, Prahlad V & Goldman RD (1998) Motile properties of vimentin intermediate filament networks in living cells. J. Cell Biol. 143, 147157. A375 A376 Chapter 16: The Cytoskeleton 1639 A. Treatment with alkaline phosphatase reduces the number of spots to three--one for each lamin--because it removes all phosphates from the individual lamins. Lamin molecules of the same type migrate at different positions because they carry different numbers of attached phosphate groups. The charges on the phosphates alter the total charge on the lamins, thereby changing their migration under conditions that are sensitive to charge. B. The untreated mixture of lamins from interphase and mitotic cells shows most clearly the total number of different types of lamin molecules present overall. For example, lamin C shows up as four equally spaced spots in the untreated mixture. The spot farthest to the right in Figure 1611 (most positively charged) corresponds to the one lamin C spot that is present when all phosphates have been removed by alkaline phosphatase. Thus, that spot is derived from lamin C molecules that carry zero phosphate groups. The equal spacing between adjacent spots of lamin C suggests that they correspond to molecules that carry one, two, or three phosphates (although in principle they could carry two, four, or six phosphates, or any such multiple). A comparison of the interphase pattern with the mixture indicates that lamin C molecules in interphase cells carry zero or one phosphate. Similarly, lamin C molecules from mitotic cells carry two or three phosphates. The same analysis indicates that lamin A molecules carry zero or one phosphate in interphase cells and three phosphates in mitotic cells. (The gap in the pattern in the mixture indicates that lamin A molecules rarely, if ever, carry two phosphates.) Analysis of lamin B molecules indicates that they carry zero phosphates in interphase cells and one (or perhaps two) phosphates in mitotic cells. C. 35S-Methionine was used instead of 32P-phosphate in order to see the positions of all the lamins, even those that do not carry phosphates. If 32P-phosphate had been used to label the cells, the three spots visible after alkaline phosphatase treatment would not have been present. D. Although these results are highly suggestive that phosphorylation plays an important role in the disassembly of the lamin network, they stop short of proving the point. There are many examples of cellular proteins whose state of phosphorylation can change without any apparent alteration in function. Site-directed mutagenesis of phosphorylation sites in lamins has now demonstrated conclusively that phosphorylation is essential for disassembly of the nuclear lamina during mitosis. References: Ottaviano Y & Gerace L (1985) Phosphorylation of the nuclear lamins during interphase and mitosis. J. Biol. Chem. 260, 624632. Ward GE & Kirschner MW (1990) Identification of cell cycle-related phosphorylation sites on nuclear lamin C. Cell 61, 561577. Heald R & McKeon F (1990) Mutations of phosphorylation sites in lamin A that prevent nuclear lamina disassembly in mitosis. Cell 61, 579589. Peter M, Nakagawa J, Dore M, Labb JC & Nigg EA (1990) In vitro disassembly of the nuclear lamina and M phase-specific phosphorylation of lamins by cdc2 kinase. Cell 61, 591602. 1640 A. Figure 1613 shows that cytochalasin B interferes with filament assembly by stopping actin polymerization at the plus end, the preferred end for the addition of monomers. One plausible mechanism to explain this inhibition is that cytochalasin B binds to the plus end of the actin filament and physically blocks the addition of new actin monomers. This mechanism can also account for the viscosity measurements. Since growth at the minus end is unaffected, the filaments continue to grow, but much more slowly. The slower growth rate explains the slower increase in viscosity in the presence of cytochalasin B. The lower viscosity at the plateau THE SELF-ASSEMBLY AND DYNAMIC STRUCTURE OF CYTOSKELETAL FILAMENTS indicates that the actin filaments are shorter in the presence of cytochalasin B. The filaments are shorter when they are growing only from the minus ends because the critical concentration for assembly at the minus end is higher than the critical concentration for assembly at the plus end. B. An actin filament normally grows at different rates at the plus and minus ends. This observation indicates that the monomer probably undergoes a conformational change upon addition to an actin filament. If all subunits, assembled and free, were identical in conformation, the rates of growth at the two ends should be the same (see Problem 1616). Reference: MacLean-Fletcher S & Pollard TD (1980) Mechanism of action of cytochalasin B on actin. Cell 20, 329341. 1641 The gold particles in Figure 1651B follow parallel helical paths that are staggered relative to one another, just like the two protofilaments in an actin filament. The spacing of gold particles (5.5 nm per particle) also matches the spacing of actin subunits in the actin filament. In Figure 1651C the positions of the gold particles have been mapped onto the surface of the actin filament. Reference: Steinmetz MO, Stoffler D, Hoenger A, Bremer A & Aebi U (1997) Actin: From cell biology to atomic detail. J. Struct. Biol. 119, 295320. 1642 A. Phalloidin increases the growth rate of actin filaments by eliminating the off rate. Because the slopes of the lines in Figure 1615A are identical, kon is the same in the presence and absence of phalloidin. The y intercept (koff) is dramatically altered, from about 12 molecules/sec in the absence of phalloidin to 0 molecules/sec in its presence. These results suggest that the off rate is zero in the presence of phalloidin. B. The results in Figure 1615B confirm the interpretation in part A. Actin filaments made in the absence of phalloidin disassemble as expected when diluted in the absence of actin monomers. Filaments made in the presence of phalloidin, however, are rock-solid stable, as expected if the off rate were zero. C. The critical concentration for actin assembly is the concentration of actin at which no growth occurs. In the absence of phalloidin, this point occurs at about 1 mM. In the presence of phalloidin, it occurs at an actin concentration of 0 mM. This result is also consistent with phalloidin reducing the off rate to zero: when phalloidin is present, the filament will grow at any concentration of actin. D. Phalloidin interferes with actin assembly by binding to the filament to prevent dissociation of actin subunits. The requirement for a 1:1 molar mixture suggests that phalloidin is required stoichiometrically with actin but does not tell you whether it binds to free monomers or to each actin subunit in the filament. The stability of phalloidin-treated filaments upon dilution indicates that phalloidin binds to the filaments. It is thought that phalloidin binds to actin subunits and locks them in place. (A) (B) (C) A377 11 nm 11 nm Figure 1651 Relationship of phalloidin to actin monomers in an actin filament (Answer 1641). (A) An actin filament with gold-tagged phalloidin at low contrast. (B) The same filament as in (A) but at high contrast. (C) The mapped positions of the gold particles on the surface of an actin filament. The positions of the gold particles are indicated by arrows. A378 Chapter 16: The Cytoskeleton Reference: Coluccio LM & Tilney LG (1984) Phalloidin enhances actin assembly by preventing monomer dissociation. J. Cell Biol. 99, 529535. 1643 A. The fluorescence in Figure 1616B reaches a plateau when all the actin has been converted to monomers. The plateau is not at zero because the monomers themselves exhibit a low level of fluorescence. B. The humps in the depolymerization curves (see Figure 1616B) show that the rates of depolymerization increase with time in the presence of swinholide A. Since the rate of depolymerization depends on the number of ends, an increase in rate is consistent with an increase in the number of ends. This feature of the depolymerization curves supports the idea that swinholide A severs actin filaments, thereby increasing the number of ends. It is not consistent with the idea that swinholide A brings about depolymerization through mass-action effects by binding to actin subunits, which predicts a linear loss of fluorescence with time. C. Multiple molecules of swinholide A are required to sever an actin filament. This conclusion is based on the data in Figure 1616C, which shows that the increments in swinholide A concentration have progressively larger effects on depolymerization. Were a single molecule of swinholide A required, the relationship would have been linear, with each increment in swinholide A concentration producing the same increment in depolymerization rate. Reference: Bubb MR, Spector I, Bershadsky AD & Korn ED (1995) Swinholide A is a microfilament disrupting marine toxin that stabilizes actin dimers and severs actin filaments. J. Biol. Chem. 270, 34633466. HOW CELLS REGULATE THEIR CYTOSKELETAL FILAMENTS DEFINITIONS 1644 1645 1646 1647 Centrosome Cell cortex g-Tubulin ring complex (g-TuRC) Centriole TRUE/FALSE 1648 False. Although centrosomes, the major microtubule-organizing centers in almost all animal cells, do contain centrioles, a number of microtubuleorganizing centers in plants, animals, and fungi do not. The common feature of all microtubule-organizing centers is an electron-dense matrix that usually contains g-tubulin, which is used to nucleate microtubules. False. Most of the identified proteins that bind to the ends of actin filaments and microtubules cap the ends and prevent further polymerization. There are exceptions, however. For example, XMAP215 binds to the ends of microtubules in a way that stabilizes them without inhibiting continued polymerization, and catastrophe factors such as kinesin 13 bind to ends to destabilize them and promote rapid shrinkage. 1649 THOUGHT PROBLEMS 1650 Once the first lateral association has occurred, the next ab-dimer can bind much more readily because it is stabilized by both lateral and longitudinal HOW CELLS REGULATE THEIR CYTOSKELETAL FILAMENTS LINEAR GROWTH LATERAL ASSOCIATION A379 Figure 1652 Rapid addition of ab-tubulin dimers to nucleation structure (Answer 1650). contacts (Figure 1652). The formation of a second protofilament stabilizes both protofilaments, allowing the rapid addition of new ab-tubulin dimers to form adjacent protofilaments and to extend existing ones. At some point the initial sheet of tubulin curls into a tube to form the microtubule. Reference: Leguy R, Melki R, Pantaloni D & Carlier M-F (2000) Monomeric g-tubulin nucleates microtubules. J. Biol. Chem. 275, 2197521980. 1651 The centrosome nucleates a three-dimensional, star-burst array of microtubules that grow until they encounter an obstacle, ultimately the plasma membrane. Dynamic instability of the microtubules, coupled to the requirement for equal pushing of oppositely directed microtubules, eventually positions the centrosome in the middle of the cell. One way to think about the notion of equal and opposite forces is to realize that the microtubules are not absolutely rigid structures. Imagine pushing an object with a short steel rod versus a very long one; the short rod transmits force effectively, but the long rod will bend, delivering less force. The same principle may operate inside the cell, with microtubules of equal length delivering the same force. When all the oppositely directed microtubules emanating from a centrosome are the same length, the centrosome will be in the center of the cell. Any actin-binding protein that stabilizes complexes of two or more actin monomers will facilitate the initiation (nucleation) of a new filament. The actin-binding proteins must not block the ends required for filament growth. Both g-TuRC and the ARP complex nucleate growth by binding at the minus end of microtubules and actin filaments, respectively, allowing rapid growth at the plus end. Both contain subunits that are evolutionarily related to the subunits of the filaments they nucleate: g-tubulin for g-TuRC, and Arp2 and Arp3, which are related to actin, for the ARP complex. g-TuRC most often nucleates microtubule growth deep within the cell cytoplasm, whereas the ARP complex most frequently nucleates actin filaments near the plasma membrane. The ARP complex can bind to the side of an actin filament and thereby build a web-like network, whereas g-TuRC does not promote branching. In cells, most of the actin subunits are bound to thymosin, which locks actin into a form that cannot hydrolyze its bound ATP and cannot be added to either end of a filament. Thymosin reduces the concentration of free actin subunits to around the critical concentration. Actin subunits are recruited from this inactive pool by profilin, whose activity is regulated so that actin polymerization occurs when and where it is needed. The advantage of such an arrangement is that the cell can maintain a large pool of subunits for explosive growth at the sites and times of its choosing. Since centrosomes nucleate growth of microtubules by binding to the minus end, all the free ends would be plus ends. As a consequence, the ends would 1652 1653 1654 1655 A380 Chapter 16: The Cytoskeleton behave uniformly--like plus ends (see Figure 167A). The minus ends would be stably associated with the centrosome. Since MAPs tend to stabilize microtubules against disassembly, they would be expected to reduce the frequency of switching between the two growth states and extend the length of time they remain in the growing state. (This is the result that is observed in the actual experiment. The switches in the growth state are abolished and growth is smooth and continuous until the steady-state length is reached, after which the length remains constant.) Reference: Horio T & Hotani H (1986) Visualization of the dynamic instability of individual microtubules by dark-field microscopy. Nature 321, 605607. 1656 Cofilin binds preferentially to actin with bound ADP. When cofilin binds to ADP-containing actin filaments, it introduces strain into the filament, which makes it easier for the filament to be severed and for ADPactin subunits to dissociate. Because polymerization is faster than ATP hydrolysis, the newly added subunits are resistant to depolymerization by cofilin. Thus, cofilin efficiently dismantles older filaments in the cell, which contain more ADPactin. Lamellipodia help cells to crawl across solid surfaces. In the absence of filamin, cells do not crawl properly; instead, they protrude disorganized membrane blebs. The loss of filamin in melanoma cells restricts their ability to invade new sites, making them less likely to metastasize. Cancer cells that metastasize are extremely difficult to eradicate. Thus, the loss of filamin means that the melanoma cells can be eliminated with treatment: bad news for them, good news for the patient. Katanin cleaves microtubules all along their length. The fragments that form therefore contain GDP-tubulin at their exposed ends and rapidly depolymerize. Katanin thus provides a very quick means for destroying existing microtubules. 1657 1658 CALCULATIONS 1659 A. Centrosomes lower the critical concentration by providing nucleation sites for microtubule growth. Nucleation sites make it easier to start new microtubules; moreover, they protect the bound end from disassembly. Thus, once started, a microtubule is more likely to persist. In the absence of such a nucleation site, it is much more difficult to start a microtubule, and both ends serve as sites for disassembly. B. The shapes of the curves in the presence and absence of centrosomes differ because of the nature of the assays used to detect polymerization. In the absence of centrosomes (see Figure 1618A), the assay was for total polymer formed, which depends only on the concentration of added ab-tubulin. Thus, it increases indefinitely in a linear fashion with increasing concentration of tubulin. In the presence of centrosomes (see Figure 1618B), the assay was the number of microtubules per centrosome. Since each centrosome has a limited number of nucleation sites (about 60 for the centrosomes used in this experiment), the measurement must reach a plateau at high tubulin concentrations. C. A concentration of ab-tubulin dimers of 1 mg/mL corresponds to 9.1 mM. mmol tubulin 1000 mL [tubulin] = 1 mg tubulin 1.1 105 mg tubulin L mL = 9.1 103 mmol/L = 9.1 103 mM, or 9.1 mM This value is below the critical concentration for microtubule assembly in the absence of centrosomes. Thus, without a nucleation site for growth, HOW CELLS REGULATE THEIR CYTOSKELETAL FILAMENTS commonly provided by the centrosome, a cell would have no microtubules. This simple consideration probably explains why the majority of microtubules originate from centrosomes in animal cells. Reference: Mitchison T & Kirschner M (1984) Microtubule assembly nucleated by isolated centrosomes. Nature 312, 232237. 1660 A. The decrease in the lag time for assembly of microtubules in the presence of g-tubulin indicates that g-tubulin monomers accelerate the nucleation event in microtubule polymerization. Assembly in the presence of g-tubulin occurs more rapidly because there are more sites of polymerization, as a result of the more efficient nucleation by g-tubulin. B. The critical concentration in the absence of g-tubulin is a combination of the critical concentrations for the plus and minus ends. g-Tubulin lowers the critical concentration by capping the minus end, preventing polymerization and depolymerization from that end. Thus, the critical concentrations in the presence of g-tubulin represents the critical concentration for the plus end, which is lower than that for the minus end (discussed for actin assembly in Problem 1634) and lower than the combination of critical concentrations measured in the absence of g-tubulin (see Figure 1619B). The greater extent of polymerization in the presence of g-tubulin (see Figure 1619A) occurs because growth occurs at the plus ends, which have a lower critical concentration. As a result, they reach a greater extent of polymerization before they come into equilibrium with the concentration of free ab-tubulin. C. The Kd for g-tubulin binding to microtubules is about 0.1 nM, which represents very tight binding. The Kd can be estimated from the slope of the line in Figure 1619C, which is 6.2/0.64 nM (the bound/free value decreases by 6.2 units for an increase in bound g-tubulin of 0.64 nM). Thus, 1 = 6.2 Kd 0.64 nM 0.64 nM Kd = or 0.1 nM 6.2 Since microtubules were present at 0.62 nM and the x-intercept is 0.64 nM g-tubulin, one g-tubulin monomer is bound per microtubule, presumably to the minus end. This result, along with the others in this problem, suggests that one g-tubulin monomer can nucleate a microtubule. Reference: Leguy R, Melki R, Pantaloni D & Carlier M-F (2000) Monomeric g-tubulin nucleates microtubules. J. Biol. Chem. 275, 2197521980. A381 (A) AXONEME (+) axoneme () (B) CENTROSOME (+) (+) (+) centrosome (+) DATA HANDLING (+) (+) 1661 A. Microtubules assembled on flagellar axonemes are extensions of the microtubules already present in the axonemes. Therefore, the polarity of growth is fixed: the plus end of the axoneme will nucleate a microtubule that has its plus end free for the addition of new subunits. The newly assembled microtubule therefore has its plus end pointing away from the axoneme and its minus end attached to the axoneme (Figure 1653A). B. The plus end of the microtubule must grow faster since microtubules with free plus ends (attached to the plus end of the axoneme) are longer than those with free minus ends (attached to the minus end of the axoneme). C. For axonemes, where the plus and minus ends can be distinguished, it is clear that the growth rate at the plus end is faster than at the minus end, since the microtubules attached to the plus end are longer than those attached to the minus end. It is this difference in growth rates that allows one to decide the polarity of growth nucleated by centrosomes and kinetochores. Microtubules nucleated on centrosomes have lengths that indicate their plus ends are free. Thus, centrosomes nucleate microtubule growth by (C) KINETOCHORE (+) kinetochore chromosome () Figure 1653 Polarities of microtubules (Answer 1661). (A) Nucleation on a flagellar axoneme. (B) Nucleation on a centrosome. (C) Nucleation on a kinetochore. A382 Chapter 16: The Cytoskeleton Figure 1654 Model for nucleation of a microtubule by monomeric g-tubulin and for capping growth at the minus end (Answer 1662). g-tubulin subunit seed addition blocked binding to the minus end of the microtubule (Figure 1653B). Kinetochores, on the other hand, have a bimodal distribution of lengths, which suggests that some microtubules are attached by their minus ends (the longer ones) and some are attached by their plus ends (the shorter ones) (Figure 1653C). Reference: Mitchison T & Kirschner MW (1985) Properties of the kinetochore in vitro. I. Microtubule nucleation and tubulin binding. J. Cell Biol. 101, 755765. 1662 The reduced number of ab-tubulin dimers required for nucleation in the presence of monomeric g-tubulin, coupled with the tight binding of g-tubulin to b-tubulin, suggests the model for nucleation shown in Figure 1654. Tight binding of g-tubulin to b-tubulin would permit the addition of an abdimer above it (toward the plus end) by lateral interactions with the adjacent ab-dimer and by longitudinal interactions with g-tubulin. The longitudinal interactions would be between g-tubulin and the a-tubulin of the ab-dimer. Since minimal interaction with free a-tubulin was detected in the blot (see Figure 1621B), these interactions in the microtubule might be very weak; however, they could be stronger if the conformation of a-tubulin were altered when it was incorporated into the microtubule lattice. In order for g-tubulin to terminate growth at the minus end, you would need to postulate that ab-dimers cannot be added below g-tubulin or to the left, as shown in Figure 1654. Reference: Leguy R, Melki R, Pantaloni D & Carlier M-F (2000) Monomeric g-tubulin nucleates microtubules. J. Biol. Chem. 275, 2197521980. 1663 Whether g-TuRC is present or not makes no difference to the lengths of the bright segments at the plus ends of the microtubules (and, as expected, they tend to be much longer that those at the minus ends). However, g-TuRC shifts the distribution of bright segments at the minus ends, suggesting that it blocks (or at least retards) growth at that end. g-TuRC appears to cap the minus end and, from there, to nucleate growth. You might reasonably ask why there are any bright segments at all at the minus ends. Although 50% have very short, or nonexistent bright segments, the remainder have a distribution of lengths that is not much different from that observed in the absence of g-TuRC. Some of these microtubules may have nucleated spontaneously, g-TuRC may have dissociated from some, or some microtubules may have been broken. Reference: Zheng Y, Wong ML, Alberts B & Mitchison T (1995) Nucleation of microtubule assembly by a g-tubulin-containing ring complex. Nature 378, 578583. HOW CELLS REGULATE THEIR CYTOSKELETAL FILAMENTS (A) PLUS END BRANCHING (B) SIDE BRANCHING A383 capping protein Figure 1655 Expectations of two models for the branching of actin filaments induced by the binding of the ARP complex (Answer 1664). (A) ARPcomplex binding at the plus end. (B) ARP-complex binding to the side. ARP complex 1664 The two alternatives make different predications about the kinds of structures that should be generated. As shown in Figure 1655A, if the ARP complex were to bind to plus ends, the capped actin filament might not be a substrate for ARP binding, in which case no branches would be seen. Alternatively, the ARP complex might bind to the capped structure, in which case a kinked filament would be generated (Figure 1655A). If the ARP complex were to bind instead to the sides of the filament, then the plus end cap would be irrelevant and a typical branched structure would be generated (Figure 1655B). The results of such experiments revealed highly branched structures, supporting the idea that the ARP complex binds to the sides of actin filaments. References: Amann KJ & Pollard TD (2001) The ARP2/3 complex nucleates actin filament branches from the sides of preexisting filaments. Nat. Cell Biol. 3, 306310. Higgs HN & Pollard TD (2001) Regulation of actin filament network formation through ARP2/3 complex: Activation by a diverse array of proteins. Annu. Rev. Biochem. 70, 649676. 1665 A. ActA alone has no effect on actin polymerization. The ARP complex stimulates the rate of actin polymerization but does not substantially decrease the delay (lag) before polymerization begins (see Figure 1624B), which is a measure of rate of nucleation. Thus, the absence of an effect on the lag indicates that the ARP complex does not efficiently nucleate actin polymerization under these conditions. The combination of ActA and the ARP complex dramatically stimulates nucleation (decreases the lag time) and enhances the rate of polymerization. The increase in rate may be a consequence of accelerated nucleation, which would generate many more ends, and hence faster polymerization rates. B. The ActA protein stimulates nucleation of new actin filaments by the ARP complex, so that actin polymerization occurs in the immediate vicinity of the bacterium (since ActA is attached to the bacterial surface). The actin polymerization is oriented so that the growing ends--the plus ends--are pointed toward the bacterium. In this orientation the growing ends can `push' on the bacterium and move it forward (much as the meshwork of actin filaments pushes on the plasma membrane at the leading edge of a lamellipodium). How the actin filaments actually push the bacterium is not certain. A thermal rachet provides one plausible way to think about it. Thermal motion allows enough separation between the bacterium and the ends of the nucleated filaments to permit actin to polymerize at the end, and the end then acts like a ratchet to prevent backward motion of the bacterium. The actin meshwork is likely anchored in some way to the cell's cytoskeleton, which prevents its own backward movement. Thus, the bacterium is continually A384 Chapter 16: The Cytoskeleton moved forward by random thermal motion and by the unidirectional polymerization of actin. As it moves forward, the bacterium triggers more nucleation sites in its wake, thereby perpetuating its movement. References: Dramsi S & Cossart P (1998) Intracellular pathogens and the actin cytoskeleton. Annu. Rev. Cell Dev. Biol. 14, 137166. Welch MD, Rosenblatt J, Skoble J, Portnoy DA & Mitchison TJ (1998) Interaction of human Arp2/3 complex and the Listeria monocytogenes ActA protein in actin filament nucleation. Science 281, 105108. 1666 Protein 1 lowers the critical concentration, allowing actin filaments to polymerize at a low concentration of actin, as is typical of plus ends. Thus, protein 1 must cap the minus end, like the ARP complex does. Protein 2, which raises the critical concentration, must cap the plus end, like CapZ. Note that in the absence of either protein the critical concentration is a balance of those for the plus and minus ends. 1667 A. The observation that Dictyostelium strains with a defect in either a-actinin or gelation factor have normal motility and development, whereas the double mutant strains have profound problems, suggests that each protein alone can substitute for the functions normally provided by the other. Although a-actinin and gelation factor presumably serve somewhat different roles in Dictyostelium, they also provide a mutual backup system. This appears to be common in higher organisms. As reverse genetics is used to knock out more and more genes, it is becoming clear that many genes provide overlapping functions that can partially substitute for one another (see, for example, Problem 1626). B. The most peculiar observation in these experiments is that the amoebae of the doubly mutant strain move perfectly well, even though the two major actin-binding proteins are defective. This may mean that there are still other actin-binding proteins in Dictyostelium that function sufficiently well to allow amoebae to move, but not well enough to permit the multicellular slug to move properly. Reference: Witke W, Schleicher M & Noegel AA (1992) Redundancy in the microfilament system: abnormal development of Dictyostelium cells lacking two F-actin cross-linking proteins. Cell 68, 5362. MOLECULAR MOTORS DEFINITIONS 1668 1669 1670 Dynein Myosin Kinesin TRUE/FALSE 1671 True. An individual myosin II molecule, with its two motor domains and tail, would be insufficient to slide actin filaments past each other efficiently. By polymerizing into bipolar filaments, however, the motor domains at each end of the filament are properly arranged to slide oppositely oriented actin filaments past each other. False. The centrosome, which establishes the principal array of microtubules in most animal cells, nucleates microtubule growth at the minus end. Thus, the plus ends of the microtubules are near the plasma membrane, and the 1672 MOLECULAR MOTORS minus ends are buried in the centrosome at the center of the cell. This orientation of the array requires that plus end-directed motors be used to transport cargo to the cell periphery and that minus end-directed motors be used for cargo delivery to the center of the cell. A385 THOUGHT PROBLEMS 1673 Intermediate filaments have no polarity; their ends are indistinguishable. It would therefore be difficult for a hypothetical motor protein bound to the middle of the filament to sense a defined direction. Such a motor protein would be equally likely to attach to the filament facing in one direction as the other. The known molecular motors all move in one direction along a filament of defined polarity, allowing them to move toward their intended destinations. 1674 A. Motor proteins are unidirectional in their action; nearly all kinesins move toward the plus end of a microtubule and dyneins always move toward the minus end. Thus, if dynein molecules, for example, were attached to the coverslip, only those individual molecules that were correctly oriented relative to the microtubule that settles on them could attach to it, exert force, and propel it forward. B. On a bed of dynein motors, microtubules will always move plus end first over the coverslip. The dynein motors `walk' toward the minus end; thus, since the motors are fixed, the microtubule moves plus end first. C. The protein on the coverslip is a plus end-directed motor. Since the bead, which marks the minus end of the microtubule, is moving forward, the motor must be walking toward the opposite end--the plus end. Reference: Fan J, Griffiths AD, Lockhart A, Cross RA & Amos LA (1996) Microtubule minus ends can be labeled with a phage display antibody specific to a-tubulin. J. Mol. Biol. 259, 325330. 1675 The investigators knew which end of the microtubule was attached to the gold beads because they determined the direction of motion on a bed of plus-end directed kinesin motors. The microtubules were observed to move bead-end first (see Figure 1626, which are video frames from these experiments). Since kinesin motors propel the microtubules with their minus ends forward (by walking toward the plus end), the gold beads must be at the minus ends of the microtubules. If you designed your experiment using a minus end-directed motor such as dynein, you would have observed the gold bead at the trailing end of the microtubule. Reference: Fan J, Griffiths AD, Lockhart A, Cross RA & Amos LA (1996) Microtubule minus ends can be labeled with a phage display antibody specific to a-tubulin. J. Mol. Biol. 259, 325330. 1676 A. In each cycle, the chemical free energy that drives the cycle is provided by hydrolysis of ATP. Although ATP hydrolysis is a common source of chemical free energy, it is not the only one. The free energy in a Na+ ion gradient, for example, drives active transport of sugars in animal cells, and GTP hydrolysis powers the movements of ribosomes during protein synthesis. The mechanical work accomplished during muscle contraction is the motion of actin thin filaments relative to myosin thick filaments. The mechanical work done during active transport of Ca2+ is the pumping of ions outside the cell, against their concentration gradient. B. Actin is bound tightly and then released in each cross-bridge cycle during muscle contraction; Ca2+ is bound tightly and then released during its active transport. In the diagram in Figure 1628A, actin is tightly bound to myosin at each point where the two are in contact. The binding of ATP to the myosin head A386 Chapter 16: The Cytoskeleton converts it to a weakly binding form, allowing it to detach from actin. (Although each of these steps is shown separately in the diagram, the binding of ATP is thought to initiate a conformational change, which in turn reduces the affinity of myosin for actin, thereby promoting the detachment of actin and the completion of the conformational change.) In the diagram in Figure 1628B, Ca2+ is tightly bound to the transport protein when it is on the inside of the cell (upper drawing) but only weakly bound when it faces the outside of the cell (lower drawing). Although the tightness of binding is not immediately apparent in the diagrammatic representation, it follows from the concept of active transport. Since the pump transports Ca2+ against its concentration gradient, the pump must have a high affinity for Ca2+ on the inside of the cell (so that Ca2+ can be bound effectively at its low intracellular concentration) and a low affinity for Ca2+ on the outside of the cell (so that Ca2+ can be released effectively at its high external concentration). C. In both cycles the `power stroke' is the conformational change indicated on the right side of the cycles as drawn in Figure 1628. The `return stroke' in each case is the conformational change indicated on the left side of the drawings. Reference: Eisenberg E & Hill TL (1985) Muscle contraction and free energy transduction in biological systems. Science 227, 9991006. DATA HANDLING 1677 A. The differences in landing rates at low densities of two-headed and oneheaded kinesins indicate that multiple one-headed kinesin motors are necessary to move a microtubule, in contrast to the situation with two-headed kinesin motors. At a high motor protein density the landing rates for both motors are about the same. The landing rate for two-headed kinesin declines linearly with density, whereas the landing rate for one-headed kinesin drops abruptly at lower densities. This behavior indicates that a single two-headed kinesin is sufficient to move a microtubule, but that several one-headed kinesins (foursix according to the authors) are required. A one-headed kinesin can bind a microtubule, but when it lets go to take the next step the microtubule floats away. Thus, several one-headed kinesins are required so that some can hold onto the microtubule while others release and rebind. B. Two heads are better than one. In principle, a single kinesin motor with two heads could move a vesicle for long distances along a microtubule track because it holds on with one `hand,' while it releases and rebinds with the other. A one-headed motor would lose its way each time it released the microtubule to take a step. Reference: Hancock WO & Howard J (1998) Processivity of the motor protein kinesin requires two heads. J. Cell Biol. 140, 13951405. 1678 A. The unidirectional movement of kinesin along a microtubule is driven by the free energy of ATP hydrolysis. ATP binding and hydrolysis are coupled to a series of conformational changes in the kinesin head that bring about the unidirectional stepping of the kinesin motor domains along the microtubule. B. In the first trace, the kinesin moves 80 nm in 9 sec at an average rate of about 9 nm/sec. In the second trace, the kinesin moves 80 nm in 5 sec at an average rate of about 16 nm/sec. These rates are about 100-fold slower than the in vivo rates because the experimental conditions (ATP concentration and force exerted by the interference pattern) were adjusted to slow the movements of kinesin so individual steps could be observed. C. As can be seen in Figure 1630B, the two kinesin molecules each took 10 steps to move 80 nm, indicating that the length of an individual step is about 8 nm. D. Since the step length and the interval between b-tubulin subunits along a THE CYTOSKELETON AND CELL BEHAVIOR microtubule protofilament are both 8 nm, a kinesin appears to move by stepping from one b-tubulin to the next along a protofilament. Because kinesin has two domains that can bind to b-tubulin, it presumably keeps one domain anchored as it swings the other domain to the next b-tubulinbinding site--much like a person walking along a path of stepping-stones. E. The data in Figure 1630B contain no information about the number of ATP hydrolyzed per step. Other experiments by these same investigators suggest that hydrolysis of one ATP does not cause multiple steps. By lowering ATP concentrations to slow movement along the microtubule, the investigators showed the same sort of stepping patterns as in Figure 1630B, although on a longer time scale. If hydrolysis of a single ATP could cause multiple steps, a clustering of steps might have been expected under these experimental conditions. None of these experiments rule out the possibility that more than one ATP might need to be hydrolyzed for each step. Reference: Svoboda K, Schmidt CF, Schnapp BJ & Block SM (1993) Direct observation of kinesin stepping by optical trapping interferometry. Nature 365, 721727. A387 THE CYTOSKELETON AND CELL BEHAVIOR DEFINITIONS 1679 1680 1681 1682 1683 Rho protein family Axoneme Myofibril Lamellipodium Flagellum TRUE/FALSE 1684 False. The entry of Ca2+ through the voltage-sensitive Ca2+ channels in Ttubules is not sufficient, by itself, to trigger rapid muscle contraction. Instead, this initial burst of Ca2+ opens Ca2+-release channels in the sarcoplasmic reticulum, which flood the cytoplasm with Ca2+, initiating rapid muscle contraction by binding to troponin C. True. In resting muscle, the troponin IT complex pulls tropomyosin out of its normal binding groove in actin, so that it interferes with the binding of myosin heads. Binding of Ca2+ to troponin C alters its conformation, which forces troponin I to release its hold on actin; this allows tropomyosin to slip back into its preferred position, thereby exposing binding sites for the myosin heads. True. A site of bacterial infection is a source of bacterial proteins, some of which have retained the N-formylmethionine used for the initiation of bacterial protein synthesis. As these proteins are degraded, N-formylated peptides are released and diffuse outward from the source, generating a gradient that can be sensed by neutrophils via membrane receptors. The binding of N-formylated peptides triggers changes in the cytoskeleton that allow the neutrophil to travel up the gradient to the site of infection. 1685 1686 THOUGHT PROBLEMS 1687 Kinesin-1 molecules need to be highly processive in order to accomplish their biological function of transporting organelles over long distances. For A388 Chapter 16: The Cytoskeleton example, a kinesin molecule can transport a mitochondrion all the way down a nerve axon. Their high processivity translates into high efficiency of transport. By contrast, it is essential for muscle function that myosin II molecules not be processive. Because myosin II motors in skeletal muscle always function as part of a large array, it doesn't matter that individual motors let go; others will always be bound. In fact, if myosin II bound to actin tightly enough to be highly processive, it would inhibit muscle contraction, whose speed depends on the low processivity of its motors. 1688 Both filaments are composed of subunits of protein dimers that are held together by coiled-coil interactions. Moreover, in both cases, the dimers polymerize through their coiled-coil domains into filaments. Intermediate filament dimers assemble head to head to generate symmetric building blocks that are joined end to end to create a filament that has no polarity. By contrast, asymmetric myosin molecules are assembled into a polar chain, two of which join tail to tail to form the bipolar myosin filament. As a result, all myosin molecules in the same half of the myosin filament are oriented with their heads pointing in the same direction. This polarity is necessary for them to be able to develop a contractile force in muscle. 1689 D. The sarcomeres become shorter. Upon contraction, the Z discs move closer together. Neither actin nor myosin filaments contract: they slide past one another. 1690 A. The locations of the striated muscle components in the electron micrograph are illustrated schematically in Figure 1656A. a-Actinin is a component of the Z disc, titin links the myosin II filaments to the Z disc, and nebulin binds along the length of each actin filament. B. The micrograph in Figure 1631B shows a hypercontracted muscle. The light band has entirely disappeared, and a new band, caused by the overlap of actin filaments, has appeared in the middle of the sarcomere. The relationship between the two electron micrographs in Figure 1631 is shown schematically in Figure 1656. 1691 Successive actin molecules in an actin filament are identical. After the first troponin molecule had bound to the actin filament, there would be no way a second troponin could recognize every seventh actin subunit in a naked actin filament. Tropomyosin, however, binds along the length of an actin filament, spanning precisely seven subunits and providing a molecular ruler that measures the length of seven actin monomers. Troponin becomes (A) RELAXED light band Z disc -actinin dark band myosin (thick filament) light band actin (thin filament) nebulin (+) (-) (-) (+) titin actin filaments overlap (B) HYPERCONTRACTED sarcomere Figure 1656 Schematic diagrams of electron micrographs in Figure 1631 (Answer 1690). (A) Relaxed muscle. (B) Hypercontracted muscle. THE CYTOSKELETON AND CELL BEHAVIOR localized by binding to actin at the end of each tropomyosin molecule, and thus is present every seventh actin subunit. 1692 ATP hydrolysis by the myosin motor domain is required for filament sliding in muscle contraction, and hydrolysis by the ATP-dependent Ca2+ pump is required to pump Ca2+ out of the cytosol, to allow the myofibrils to relax. (A) UPWARD BEND A389 1693 A. The components of the flagellum and their locations in Figure 1632 are listed below. 1. Inner sheath 2. Radial spoke 3. A microtubule 4. Inner dynein arm 5. B microtubule 6. Singlet microtubule 7. Nexin 8. Outer dynein arm B. The A and B microtubules of the outer doublets and the central pair of singlet microtubules are composed of a- and b-tubulin. 1694 One pattern of dynein activity that could account for the planar bending of an axoneme is depicted in Figure 1657. The axonemes shown in this figure are oriented with their tips below the plane of the page. If the dynein arms on just the left half of the axoneme were active (arrows in Figure 1657A), the cilium would bend upward toward the top of the page. This is difficult to imagine in three dimensions, but consider it step by step. First, the dynein arms push their neighbor doublets toward the tip of the axoneme, so the doublets are being pushed below the plane of the page. Second, the doublet at the top of the diagram in Figure 1657A will be pushed the farthest below the page because its total displacement is the sum of incremental displacements produced by all four active dynein arms. Third, the doublet that moves the farthest defines the `inside' of the bend (see Figure 1640). Therefore, since the top doublet moves the farthest, the axoneme will bend upward (toward the top of the page) when the dynein arms on the left half of the axoneme are active. The same reasoning argues that the axoneme will bend downward (toward the bottom of the page) if the dynein arms on the right half of the axoneme are active and the ones on the left half are passive (Figure 1657B). The actual pattern of dynein activity that gives rise to planar bending is not yet known. The two central singlet microtubules are natural candidates for regulatory elements: they are surrounded by nonidentical proteins; they contact different subsets of outer doublets; and they are linked (indirectly) to the two sets of dynein arms used in the model proposed above. Reference: Satir P & Matsuoka T (1989) Splitting the ciliary axoneme: implications for a "switch-point" model of dynein arm activity in ciliary motion. Cell Motil. Cytoskel. 14, 345358. 1695 During protrusion, cells extend actin-rich structures--filopodia, lamellipodia, or pseudopodia--in front of them. During attachment, the actin cytoskeleton in the extended structures makes connections with the substratum. During traction, contraction of the anchored actin cytoskeleton pulls the bulk of the cytoplasm forward. The ability of cytochalasin B, which interferes with actin filament formation, to stop locomotion demonstrates the critical importance of actin to cell movement. The experiment with colchicine shows that microtubules are required to give the cell a polarity, which then determines the end of the cell that becomes the leading edge. In the absence of microtubules, cells still go through the motions normally associated with cell movement, such as the extension of lamellipodia, but in the absence of a defined cell polarity, these are futile efforts that occur indiscriminately in all directions. (B) DOWNWARD BEND Figure 1657 One possible pattern of dynein activity that could produce planar bending of an axoneme (Answer 1694). (A) Upward bend. (B) Downward bend. Arrows indicate active dynein arms. 1696 A390 Chapter 16: The Cytoskeleton Injection of an antibody against vimentin had no effect, suggesting that intermediate filaments are not required for the maintenance of cell polarity or for the motile machinery. When bound, an antibody often interferes with the function of its target protein by preventing it from interacting properly with other cell components. You would need to know more about the specific antibody used to know how much weight to give the conclusions; however, it is a common observation that such antibodies collapse the network of intermediate filaments without disrupting cell locomotion. 1697 The minus ends of the growing actin filaments are anchored to the rest of the actin cytoskeleton, which supports the growing actin filaments and allows them to push on the membrane without simply sliding back into the cell's interior. The solution to the problem at the plus end is not so straightforward. Once the filament contacts the membrane, there would be no room for a new subunit to fit onto the end of the growing chain. It is thought that random thermal motions briefly expose the plus end of the filament, allowing a new subunit to be added. By taking advantage of these small windows of opportunity, actin polymerization acts as a ratchet to capture random thermal motions. It is unclear what motions the actin ratchet is capturing. It could be that membranes `breathe' thermally, allowing polymerization. Alternatively, the actin filament may bend elastically, moving the plus end sufficiently to allow subunit addition. Injection of activated Rac triggers actin polymerization over the entire membrane periphery, forming essentially one giant lamellipodium (see Figure 1633C). Injection of activated Rho promotes the bundling of actin filaments with myosin II filaments to form stress fibers (see Figure 1633B), which associate with other proteins at focal contacts. Injection of activated Cdc42 triggers actin polymerization and bundling to form filopodia (see Figure 1633D). Reference: Hall A (1998) Rho GTPases and the actin cytoskeleton. Science 279, 509514. 1698 1699 The unidirectional motion of a lamellipodium results from the nucleation and growth of actin filaments at the leading edge of the cell and depolymerization of the older actin meshwork more distally. Cofilin plays a key role in differentiating the new actin filaments from the older ones. Because cofilin binds cooperatively and preferentially to actin filaments containing ADPactin, the newer filaments at the leading edge, which contain ATP-actin, are resistant to depolymerization by cofilin. As the filaments age and ATP hydrolysis proceeds, cofilin can efficiently disassemble the older filaments. Thus, the delayed ATP hydrolysis by filamentous actin is thought to provide the basis for a mechanism that maintains an efficient, unidirectional treadmilling process in the lamellipodium. 16100 Since all the hooks curve in the same (clockwise) direction, all the microtubules have the same orientation. If there were a mixture of microtubule orientations--as there would be in a cross section of a dendrite, for example--some of the microtubules would have hooks that curved in the opposite (counterclockwise) direction. (Note that you would never expect to see a single microtubule with some hooks curved in one direction and other hooks curved in the opposite direction.) 16101 Kinesin motors use microtubules as tracks to deliver organelles and materials to nerve endings. The similar neuropathies that develop in mice and humans with only one functional copy of the gene for the kinesin motor KIF1B suggest that half the normal number of these motors is not sufficient to keep up with the needs of the nerves. Reference: Zhao C, Takita J, Tanaka Y, Setou M, Nakagawa T, Takeda S, Yang HW, Terada S, Nakata T, Takei Y, Saito M, Tsuji S, Hayashi Y & Hirokawa N (2001) Charcot-Marie-Tooth disease type 2A caused by mutation in a microtubule motor KIF1Bb. Cell 105, 587597. THE CYTOSKELETON AND CELL BEHAVIOR A391 CALCULATIONS 16102 It would take a vesicle an average of 109 seconds--nearly 32 years--to diffuse the length of a 10 cm axon: t = x2/2D = (10 cm)2 2 (5 108 cm2/sec) = 109 second, or 31.7 years 16103 The mitochondrion is about 12 times faster. It moves at 106 body lengths/day. The swimmer moves at 100 body lengths/1.75 min, which is 8.2 104 body lengths per day. DATA HANDLING 16104 A. The presence of ATP in the suspension buffer did not cause contraction because there Ca2+ was absent. In the absence of Ca2+, troponin and tropomyosin block the myosin-binding sites on actin, thereby preventing contraction. B. Contraction upon removal of ATP is perhaps the most difficult result to understand. After all, when Ca2+ is absent, how can myosin bind to actin? One clue is the slow rate of contraction. The open and closed configurations of the myosin-binding sites on actin are in equilibrium with one another. In the absence of Ca2+, the equilibrium is far in the direction of the closed configuration. Nevertheless, the equilibrium nature of the switch guarantees that new sites will continually be exposed. As they become available, the sites are bound by myosin-ADP, and when the myosin head loses its bound ADP it undergoes a conformational change, which generates tension. In the , absence of ATP to promote dissociation and relaxation, the resulting myosinactin complex is trapped. (This complex is known as the `rigor' complex because it is the principal cause of rigor mortis in a corpse.) The tension accumulates as more myosin heads become involved. Presumably, myosin also binds at some background level in normal resting muscle (in the absence of Ca2+), but ATP continually dissociates the myosin heads, thereby keeping the muscle relaxed. C. The muscle fiber relaxes suddenly upon illumination by laser light because the ATP released from its `cage' binds to the myosin heads, causing their dissociation from actin. The release of all the myosin cross-bridges allows the actin filaments to return to their original resting position. Reference: Goldman YE, Hibberd MG, McCray JA & Trentham DR (1980) Relaxation of muscle fibers by photolysis of caged ATP. Nature 300, 701705. 16105 Sketches representing sarcomeres at each of the arrows in Figure 1637 are shown in Figure 1658. As illustrated in these pictures, the increase in tension with decreasing sarcomere length in segment I is due to increasing numbers of interactions between myosin heads and actin. In segment II, actin begins to overlap with the bare zone of myosin, yielding a plateau at which the number of interacting myosin heads remains constant. In segment III, the actin filaments begin to overlap with each other, thereby interfering with the optimal interaction of actin and myosin and producing a decrease in tension. In segment IV, the spacing between the Z discs is less than the length of the myosin thick filaments, causing their deformation and a precipitous drop in muscle tension. Reference: Gordon AM, Huxley AF & Julian FJ (1966) The variation in isometric tension with sarcomere length in vertebrate muscle fibres. J. Physiol. 184, 170192. A392 Chapter 16: The Cytoskeleton 3.6 1.0 Z disc 1.6 1.0 I Figure 1658 Schematic diagrams of sarcomeres at the points indicated by arrows in Figure 1637 (Answer 16105). Numbers refer to lengths in micrometers. 2.2 2.0 II III 1.6 IV 1.3 16106 In the electron micrograph in Figure 1638, the central pairs of microtubules are all oriented identically. The identical orientations suggest that some structural feature of the axoneme constrains the direction of bending; that is, that cilia are designed to bend in a particular way. In fact, the orientation of the central pair of microtubules correlates with the direction of bending, which is always in the plane drawn between the two central microtubules. To make this relationship clearer, imagine that the axonemes shown in cross section in Figure 1638 extend straight up out of the page. The plane between the central pair of microtubules would also extend upward and run roughly from the top of the page to the bottom. The axonemes could bend toward the top or bottom of the page--staying within the plane--but not to the left or right, for example, which would break the plane. If the microtubules of the central pair were aligned parallel to one another throughout the length of the cilium, then all bending in the power stroke and in the return stroke would be in one plane. It is not uncommon for the central pair of microtubules to be twisted around one another, in which case the direction of bending rotates around the axis of the cilium as the bend propagates up the cilium. The consequence of this arrangement is a unidirectional power stroke (which depends only on the orientation of the central pair at the base of the cilium, where bending is initiated) and a helical return stroke as the bend moves to the tip of the cilium. 16107 A. If the flagellum bends into a semicircle, then the `inside' doublet will protrude beyond the `outside' doublet by a length equal to the difference in the perimeters of the semicircles they form (see Figure 1640). Since the perimeter of a semicircle is pr, the difference in length between the `outside' and `inside' perimeters will be p(r + 180 nm) pr, which equals p 180 nm, or about 565 nm. Thus the `inside' doublet protrudes beyond the `outside' doublet by 565 nm. This distance corresponds to about 70 tubulin dimers. It may come as a mild surprise that this calculation is independent of the radius of the semicircle. Since radius is irrelevant, the flagellum could be wrapped half way around the world and the result would be the same: the `inside' doublet would still protrude 565 nm beyond the `outside' doublet. B. The length of the stretched nexin molecule can also be calculated from simple geometric principles. The stretched nexin molecule is the hypotenuse of a right triangle with a base equal to 30 nm (the distance between the THE CYTOSKELETON AND CELL BEHAVIOR adjacent doublets) and a side whose length is the difference between the perimeters of the semicircles formed by the adjacent doublets (which can be calculated as described in part A). The length of the side is p(r + 30 nm) pr, which equals p 30 nm, or about 94 nm. The hypotenuse of the right triangle is the square root of the sum of the squares of the two sides. Thus, the stretched nexin molecule is [(30 nm)2 + (94 nm)2]0.5, which equals about 99 nm. This calculation suggests that nexin molecules can stretch to more than three times their normal length. Independent experimental measurements verify this striking elasticity. C. The different pattern of bending in the double mutant occurs because bends with opposite `signs' alternate with one another in the aberrant stroke. In the wild-type stroke, the bending is always `positive' (to the right in Figure 1639A). By contrast, in the double-mutant stroke the positive bend that initiates the power stroke (see Figure 1639B) is followed by a `negative' bend at the beginning of the return stroke (at the base of flagellum 6 in Figure 1639B). These alternating cycles of positive and negative bending give the double-mutant stroke its characteristic appearance. Wild-type flagella must suppress negative bending in some manner. This suppression is essential for generating the highly asymmetric stroke that is required for effective motility in Chlamydomonas. The lack of suppression of negative bending in the double mutants suggests that a normal function of the radial spokes and central microtubules is to inhibit negative bending. References: Brokaw CJ, Luck DJL & Huang B (1982) Analysis of the movement of Chlamydomonas flagella: the function of the radial-spoke system is revealed by comparison of wild-type and mutant flagella. J. Cell Biol. 92, 722732. Gibbons IR (1981) Cilia and flagella of eucaryotes. J. Cell Biol. 91, 107s124s. 16108 A. If the affected gene controlled the synthesis of the missing proteins in the mutant flagella, then none of those proteins would be present in the gametes from the mutant. After fusion, the mutant flagella would be repaired by the addition of unlabeled components from the nonradioactive wild-type gametes, since all protein synthesis was inhibited. As a result, the autoradiograph of the electrophoretic pattern would look exactly like the mutant alone. By contrast, if the affected gene encoded a protein whose assembly into the axoneme must precede the addition of other proteins, all the proteins except the defective one would be present in functional form in gametes from the mutant. Since the mutant was grown in radioactive medium, these proteins would be labeled. Thus, after fusion the mutant flagella would be repaired by the addition of a mixture of labeled components from the mutant gametes and unlabeled components from the wild-type gametes. The only exception would be the protein encoded by the defective gene: it would come entirely from the wild-type gametes and would therefore be unlabeled. Under these circumstances the autoradiograph of the electrophoretic pattern would look like that from the wild-type gametes with a single missing spot. The missing spot would correspond to the product of the defective gene. B. Analysis of second-site, intragenic revertants also can distinguish between the possibilities. If the affected gene encoded a product that is part of the assembled axoneme, then some revertants (those with an altered number of charged amino acids) should produce patterns with one spot at a new location (usually slightly left or right of the normal position, due to an effect on the isoelectric point of the protein). If the affected gene controlled the synthesis of the 17 missing proteins, then none of the revertants would have an altered pattern (since none of the component proteins are directly affected by mutation). This approach is less satisfactory than the first, since an unaltered pattern does not allow one to conclude that the defective gene controls synthesis--it may be that not enough revertants were examined. A393 A394 Chapter 16: The Cytoskeleton In conjunction, these two methods for analyzing flagellar mutants have proven enormously powerful. Most mutants, Pf14 included, affect assembly directly as judged by both assays; that is, they encode a protein that forms part of the axoneme structure. Moreover, the two methods agree on which protein is encoded by the defective gene: the unlabeled spot in dikaryon analysis corresponds to the shifted spot in revertant analysis. Reference: Luck DJL (1984) Genetic and biochemical dissection of the eucaryotic flagellum. J. Cell Biol. 98, 789794. 16109 A. The ascending portions of the plots in Figure 1642 are more consistent with growth at the tip of an acrosomal process than with growth at the base. All six points on the ascending portion of the plot of length versus square root of time fall on a straight line; however, no more than three or four points lie on a straight line on the ascending portion of the plot of length versus time. These results indicate that the rate of growth of the process slows down in the manner expected for a diffusion-controlled reaction, and they suggest that the addition of new subunits occurs at the tip. Independent experiments, using myosin decoration, indicate that the tip of the process is the plus end, where growth is expected to occur. B. The slower rates of growth at the beginning and end of the acrosomal reaction are not surprising. Growth is slow at the beginning presumably because it takes a short time for the subunits to diffuse from their site of storage to the site of assembly. Growth slows down (and essentially stops) at the end because the supply of subunits is exhausted. Reference: Tilney LG & Inoue S (1982) Acrosomal reaction of Thyone sperm. II. The kinetics and possible mechanism of acrosomal process elongation. J. Cell Biol. 93, 820827. 16110 A. If the oligomers were subjected to cycles of phosphorylation and dephosphorylation as they moved down the axon, then they would move back and forth between the motor and the neurofilaments, depending on their phosphorylation state. An oligomer that spends a high fraction of its time in the dephosphorylated form would mostly be associated with kinesin and would therefore keep moving. It would be at the leading edge of the transport wave. By contrast, an oligomer that is phosphorylated most of the time would spend more of its time unattached to kinesin, and thus would move down the axon more slowly. The farther the oligomers travel down the axon, the greater would be the difference between the fastest and the slowest ones; hence, the broader the transport wave would be. B. Oligomers at both the leading edge and the trailing edge of the transport wave would be expected to move at the same rate when they were attached to kinesin. When they were not attached to the motor, they would be stationary. Thus, both oligomers would be expected to move and stop as they traveled along the axon. The difference is that an oligomer at the trailing edge would have spent more of its time stationary because it was phosphorylated and attached to the neurofilament for more of the time. Note that the position of an oligomer--at the leading or trailing edge of the transport wave--indicates its past history of stopping and starting, but does not predict its future behavior. References: Yabe JT, Pimenta A & Shea TB (1999) Kinesin-mediated transport of neurofilament protein oligomers in growing axons. J. Cell Sci. 112, 37993814. Shea TB & Yabe J (2000) Occam's razor slices through the mysteries of neurofilament axonal transport: Can it really be so simple? Traffic 1, 522523. 16111 Extension and contraction of this filopodium were regulated by the rate of actin polymerization. The rate of retrograde flow was constant at about THE CYTOSKELETON AND CELL BEHAVIOR 1 mm/min, whereas the rate of actin polymerization varied from 0 to 2 mm/min. The movements of the tip correlated with the rate of actin polymerization, extending when the polymerization rate was high and retracting when the polymerization rate was low. Observations on many individual filopodia support this general conclusion. Reference: Mallavarapu A & Mitchison T (1999) Regulated actin cytoskeleton assembly at filopodium tips controls their extension and retraction. J. Cell Biol. 146, 10971106. 16112 A. In vesicular transport a monomeric GTPase shuttles between the donor membrane and the target membrane. In the donor membrane the monomeric GTPase is converted from its inactive, GDP-bound form to its active, GTP-bound form by a GEF. At the target membrane the monomeric GTPase is converted back to its inactive, GDP-bound form by a GAP that stimulates the GTPase to hydrolyze the bound GTP to GDP. As shown in Figure 1659, an analogous scheme can be proposed for the delivery of critical proteins from the donor compartment (the cytosol) to the target (the bud site). In the cytosol the Bud5 GEF converts the Bud1 monomeric GTPase to its active, GTP-bound form. The active form binds critical cytosolic proteins and delivers them to the bud site, where the Bud2 GAP converts the GTPase to its inactive form, which then releases the bound protein at the bud site. In this way it might be possible to construct a protein complex at the bud site that could capture microtubules and orient cytoskeletal elements to establish the cell polarity required for bud formation. B. Although the scheme illustrated in Figure 1659 could account for the development of cell polarity, it leaves open the question of how a bud site is initially selected. The scheme begins with the Bud2 protein already at the bud site, but how did it get there? The initial events that account for bud-site selection are unclear. References: Chant J & Herskowitz I (1991) Genetic control of bud site selection in yeast by a set of gene products that constitute a morphogenetic pathway. Cell 65, 12031212. Chant J, Corrado K, Pringle JR & Herskowitz I (1991) Yeast BUD5, encoding a putative GDP-GTP exchange factor, is necessary for bud site selection and interacts with bud formation gene BEM1. Cell 65, 12131224. Park H-O, Chant J & Herskowitz I (1993) BUD2 encodes a GTPase-activating protein for Bud1/Rsr1 necessary for proper bud-site selection in yeast. Nature 365, 269274. 16113 These experimental results clearly support a role for N-WASp in the Cdc42initiated polymerization of actin and, by implication, in the natural rearrangements of the actin cytoskeleton in cells as a result of Cdc42 activation. The inability of the mutant H208D to restore actin polymerization indicates that a direct interaction between Cdc42 and N-WASp is a critical feature of critical protein Bud1-GTP GTP Bud5 critical Bud1-GTP protein Bud1-GDP Pi critical Bud1-GTP protein Bud2 Bud SITE critical protein Bud2 Bud SITE GDP A395 Figure 1659 Plausible scheme for delivery of critical protein from cytosol to bud site (Answer 16112). A396 Chapter 16: The Cytoskeleton MICROSPIKE Figure 1660 A model for Cdc42activated polymerization of actin (Answer 16114). pleckstrin homology PH A ic id GTPase binding G CYTOSOL ARP G V C A C fil in V verprolin homology PIP2 PH binding Cdc42 active N-WASp VCA domain ac inactive N-WASp 16114 A. From Figure 1646B, it is clear that both Cdc42-GTPgS and vesicles with PIP2, in addition to the APR complex, are required to activate N-WASp fully for actin polymerization. Cdc42-GTPgS alone activates N-WASp partially, but vesicles with PIP2 do not stimulate at all. Only when both components are present is the activity of N-WASp equal to that of its C-terminal VCA segment. B. Because the C-terminal VCA segment of N-WASp has full activity, it must be the only portion of N-WASp actually involved in actin polymerization. The rest of N-WASp appears to function as an inhibitor of the C-terminal segment. The function of Cdc42 and PIP2 is to relieve that inhibition, freeing the C-terminal segment to stimulate actin polymerization. A speculative model that includes these ideas and incorporates the known functions of the domains is shown in Figure 1660. Reference: Rohatgi R, Ma L, Miki H, Lopez M, Kirchhausen T, Takenawa T & Kirschner MW (1999) The interaction between N-WASp and the Arp2/3 complex links Cdc42-dependent signals to actin assembly. Cell 97, 221231. co the activation pathway. The inability of the mutant Dcof to restore actin polymerization indicates that the cofilin domain of N-WASp is required for actin polymerization. The cofilin domain would allow N-WASp to bind to actin filaments, or perhaps to the actin-like subunits of the ARP complex, suggesting that such binding is essential for N-WASp-mediated actin polymerization. Reference: Rohatgi R, Ma L, Miki H, Lopez M, Kirchhausen T, Takenawa T & Kirschner MW (1999) The interaction between N-WASp and the Arp2/3 complex links Cdc42-dependent signals to actin assembly. Cell 97, 221231. ...
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This note was uploaded on 01/07/2011 for the course BIOLOGY 7.012 taught by Professor Ericlander during the Spring '04 term at MIT.

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