Dynamic instabilities in microtubule cytoskeleton. A phase diagram

Instabilities in the growth and depolymerization of microtubules are considered in the framework of self-organization theory. An extended reaction-diffusion model for the microtubule dynamics has been formulated. A phase diagram of microtubule cytoskeleton has been constructed, which determines the regions of stability for steady and nonstationary solutions of the model. It is shown that the instabilities in microtubule dynamics result from kinetic nonequilibrium phase transitions. On the basis of phase diagram structure, a general classification of the microtubule cytostatic regulatory factors is suggested. The problem of mutual amplification of the activity of cytostatic agents is discussed.     

Modulation of microtubule dynamics by drugs: a paradigm for the actions of cellular regulators

Microtubules are intrinsically dynamic polymers. Two kinds of dynamic behaviors, dynamic instability and treadmilling, are important for microtubule function in cells. Both dynamic behaviors appear to be tightly regulated, but the cellular molecules and the mechanisms responsible for the regulation remain largely unexplored. While microtubule dynamics can be modulated transiently by the interaction of regulatory molecules with soluble tubulin, the microtubule itself is likely to be the primary target of cellular molecules that regulate microtubule dynamics. The antimitotic drugs that modulate microtubule dynamics serve as excellent models for such cellular molecules. Our laboratory has been investigating the interactions of small drug molecules and stabilizing microtubule-associated proteins (MAPs) with microtubule surfaces and ends. We find that drugs such as colchicine, vinblastine, and taxol, and stabilizing MAPs such as tau, strongly modulate microtubule dynamics at extremely low concentrations under conditions in which the microtubule polymer mass is minimally affected. The powerful modulation of the dynamics is brought about by the binding of only a few drug or MAP molecules to distinct binding sites at the microtubule surface or end. Based upon our understanding of the well-studied drugs and stabilizing MAPs, it is clear that molecules that regulate dynamics such as Kin 1 and stathmin could bind to a large number of distinct tubulin sites on microtubules and employ an array of mechanisms to selectively and powerfully regulate microtubule dynamics and dynamics-dependent cellular functions.     

Toward reconstitution of in vivo microtubule dynamics in vitro

The transition from interphase to mitosis is marked by a dramatic change in microtubule dynamics resulting in the reorganization of the microtubule network that culminates in mitotic spindle formation. While the molecular basis for this change in microtubule organization remains obscure, it is currently thought that a balance in the activity of microtubule stabilizing and destabilizing factors regulates how dynamic cellular microtubules are. By mixing the microtubule stabilizer XMAP215 and the microtubule destabilizer XKCM1, reconstitution of in vivo-like microtubule dynamics has now been achieved in vitro.     

Beyond the GTP-cap: Elucidating the molecular mechanisms of microtubule catastrophe

Almost 40 years since the discovery of microtubule dynamic instability, the molecular mechanisms underlying microtubule dynamics remain an area of intense research interest. The “standard model” of microtubule dynamics implicates a “cap” of GTP-bound tubulin dimers at the growing microtubule end as the main determinant of microtubule stability. Loss of the GTP-cap leads to microtubule “catastrophe,” a switch-like transition from microtubule growth to shrinkage. However, recent studies, using biochemical in vitro reconstitution, cryo-EM, and computational modeling approaches, challenge the simple GTP-cap model. Instead, a new perspective on the mechanisms of microtubule dynamics is emerging. In this view, highly dynamic transitions between different structural conformations of the growing microtubule end – which may or may not be directly linked to the nucleotide content at the microtubule end – ultimately drive microtubule catastrophe.     

Balanced regulation of microtubule dynamics during the cell cycle: a contemporary view

Assembly of mitotic and meiotic spindles into an elliptical bipolar shape is an example of morphogenetic processes that involve local chromosomal regulation of microtubule dynamics for proper spatial microtubule assembly. Global microtubule dynamics during the cell cycle and local microtubule dynamics during spindle assembly are regulated by a balance between microtubule stabilizing and destabilizing factors. How a chromosome-induced phosphorylation gradient may be generated and modulate spindle microtubule assembly through balanced regulation of the activity of microtubule-associated proteins and Stathmin/Op 18 is analyzed.     

Yeast kinetochores do not stabilize Stu2p-dependent spindle microtubule dynamics

The interaction of kinetochores with dynamic microtubules during mitosis is essential for proper centromere motility, congression to the metaphase plate, and subsequent anaphase chromosome segregation. Budding yeast has been critical in the discovery of proteins necessary for this interaction. However, the molecular mechanism for microtubule-kinetochore interactions remains poorly understood. Using live cell imaging and mutations affecting microtubule binding proteins and kinetochore function, we identify a regulatory mechanism for spindle microtubule dynamics involving Stu2p and the core kinetochore component, Ndc10p. Depleting cells of the microtubule binding protein Stu2p reduces kinetochore microtubule dynamics. Centromeres remain under tension but lack motility. Thus, normal microtubule dynamics are not required to maintain tension at the centromere. Loss of the kinetochore (ndc10-1, ndc10-2, and ctf13-30) does not drastically affect spindle microtubule turnover, indicating that Stu2p, not the kinetochore, is the foremost governor of microtubule dynamics. Disruption of kinetochore function with ndc10-1 does not affect the decrease in microtubule turnover in stu2 mutants, suggesting that the kinetochore is not required for microtubule stabilization. Remarkably, a partial kinetochore defect (ndc10-2) suppresses the decreased spindle microtubule turnover in the absence of Stu2p. These results indicate that Stu2p and Ndc10p differentially function in controlling kinetochore microtubule dynamics necessary for centromere movements.     

F11L-mediated inhibition of RhoA-mDia signaling stimulates microtubule dynamics during vaccinia virus infection

Microtubules play an important role in the transport of viral pathogens during the establishment of their infection cycles. The microtubule cytoskeleton also facilitates efficient release of newly assembled progeny at later stages of infection. However, the precise effects of viral infection on microtubule dynamics are not understood. Using live-cell imaging, we show that vaccinia virus stimulates increases in peripheral microtubule dynamics at 8 hr postinfection. Infection also increases the frequency with which microtubule tips reach the cell cortex and reduces the acetylation of peripheral microtubules consistent with their increased dynamics. These virus-induced changes in peripheral microtubule dynamics are independent of the GTPases Rac and Cdc42, which are known stimulators of microtubule dynamics in uninfected cells. They do, however, require F11L-mediated inhibition of signaling via the GTPase RhoA and its effector, mDia. We suggest that increases in peripheral microtubule dynamics and cortical targeting contribute to enhanced virus release.     

Nonequilibrium self-assembly of linear fibers: microscopic treatment of growth, decay, catastrophe and rescue

Many of the large structures of cells are constructed from fibers. These fibers self-assemble from individual proteins in a far-from-equilibrium fashion. Nonequilibrium self-assembly results in a highly dynamic process at the subcellular level that can be regulated and tuned to carry out many of the biological functions of the cell: growth, division and locomotion. We construct and analyze a nonequilibrium model of the dynamic end of a biological fiber that possesses site-resolved resolution. We solve for the steady states of this nonequilibrium system using a variational method. The results are compared to exact numerical solutions for systems with modest size. Using an effective reaction coordinate, we construct an effective potential from the steady-state distribution. The stochastic transitions of the system can be analyzed in this representation. We then apply this method to model microtubule systems. Predictions for macroscopic catastrophe, rescue and dynamic instability in the steady states are analyzed. We find that the length of the cap of the microtubule is small. The relations between the catastrophe/rescue rate and the growth rate are also discussed.     

Ipl1/Aurora-dependent phosphorylation of Sli15/INCENP regulates CPC-spindle interaction to ensure proper microtubule dynamics

Dynamic microtubules facilitate chromosome arrangement before anaphase, whereas during anaphase microtubule stability assists chromosome separation. Changes in microtubule dynamics at the metaphase-anaphase transition are regulated by Cdk1. Cdk1-mediated phosphorylation of Sli15/INCENP promotes preanaphase microtubule dynamics by preventing chromosomal passenger complex (CPC; Sli15/INCENP, Bir1/Survivin, Nbl1/Borealin, Ipl1/Aurora) association with spindles. However, whether Cdk1 has sole control over microtubule dynamics, and how CPC-microtubule association influences microtubule behavior, are unclear. Here, we show that Ipl1/Aurora-dependent phosphorylation of Sli15/INCENP modulates microtubule dynamics by preventing CPC binding to the preanaphase spindle and to the central spindle until late anaphase, facilitating spatiotemporal control of microtubule dynamics required for proper metaphase centromere positioning and anaphase spindle elongation. Decreased Ipl1-dependent Sli15 phosphorylation drives direct CPC binding to microtubules, revealing how the CPC influences microtubule dynamics. We propose that Cdk1 and Ipl1/Aurora cooperatively modulate microtubule dynamics and that Ipl1/Aurora-dependent phosphorylation of Sli15 controls spindle function by excluding the CPC from spindle regions engaged in microtubule polymerization.     

Filopodial actin bundles are not necessary for microtubule advance into the peripheral domain of Aplysia neuronal growth cones

Filopodial actin bundles guide microtubule assembly in the growth cone peripheral (P) domain and retrograde actin-network flow simultaneously transports microtubules rearward. Therefore, microtubule-end position is determined by the sum of microtubule assembly and retrograde transport rates. However, how filopodia actually affect microtubule assembly dynamics is unknown. To address this issue we quantitatively assessed microtubule and actin dynamics before and after selective removal of filopodia. Filopodium removal had surprisingly little effect on retrograde actin-flow rates or underlying network structures, but resulted in an approximate doubling of peripheral microtubule density and deeper penetration of microtubules into the P domain. The latter stemmed from less efficient coupling of microtubules to remaining actin networks and not from a change in microtubule polymer dynamics. Loss of filopodia also resulted in increased lateral microtubule movements and a more randomized microtubule distribution in the P domain. In summary, filopodia do not seem to be formally required for microtubule advance; however, their presence ensures radial distribution of microtubules in the P domain and facilitates microtubule transport by retrograde flow. The resulting dynamic steady state has interesting implications for rapid microtubule-positioning responses in the P domain.     

Redox-dependent regulation of end-binding protein 1 activity by glutathionylation

Cytoskeletal proteins are susceptible to glutathionylation under oxidizing conditions, and oxidative damage has been implicated in several neurodegenerative diseases. End-binding protein 1(EB1) is a master regulator of microtubule plus-end tracking proteins(+TIPs) and is critically involved in the control of microtubule dynamics and cellular processes. However, the impact of glutathionylation on EB1 functions remains unknown. Here we reveal that glutathionylation is important for controlling EB1 activity and protecting EB1 from irreversible oxidation. In vitro biochemical and cellular assays reveal that EB1 is glutathionylated. Diamide, a mild oxidizing reagent, reduces EB1 comet number and length in cells, indicating the impairment of microtubule dynamics. Three cysteine residues of EB1 are glutathionylated, with mutations of these three cysteines to serines attenuating microtubule dynamics but buffering diamide-induced decrease in microtubule dynamics. In addition, glutaredoxin 1(Grx1) deglutathionylates EB1, and Grx1 depletion suppresses microtubule dynamics and leads to defects in cell division orientation and cell migration, suggesting a critical role of Grx1-mediated deglutathionylation in maintaining EB1 activity.Collectively, these data reveal that EB1 glutathionylation is an important protective mechanism for the regulation of microtubule dynamics and microtubule-based cellular activities.     

The interplay of the N- and C-terminal domains of MCAK control microtubule depolymerization activity and spindle assembly

Spindle assembly and accurate chromosome segregation require the proper regulation of microtubule dynamics. MCAK, a Kinesin-13, catalytically depolymerizes microtubules, regulates physiological microtubule dynamics, and is the major catastrophe factor in egg extracts. Purified GFP-tagged MCAK domain mutants were assayed to address how the different MCAK domains contribute to in vitro microtubule depolymerization activity and physiological spindle assembly activity in egg extracts. Our biochemical results demonstrate that both the neck and the C-terminal domain are necessary for robust in vitro microtubule depolymerization activity. In particular, the neck is essential for microtubule end binding, and the C-terminal domain is essential for tight microtubule binding in the presence of excess tubulin heterodimer. Our physiological results illustrate that the N-terminal domain is essential for regulating microtubule dynamics, stimulating spindle bipolarity, and kinetochore targeting; whereas the C-terminal domain is necessary for robust microtubule depolymerization activity, limiting spindle bipolarity, and enhancing kinetochore targeting. Unexpectedly, robust MCAK microtubule (MT) depolymerization activity is not needed for sperm-induced spindle assembly. However, high activity is necessary for proper physiological MT dynamics as assayed by Ran-induced aster assembly. We propose that MCAK activity is spatially controlled by an interplay between the N- and C-terminal domains during spindle assembly.     

Stepwise Reconstitution of Interphase Microtubule Dynamics in Permeabilized Cells and Comparison to Dynamic Mechanisms in Intact Cells

Microtubules in permeabilized cells are devoid of dynamic activity and are insensitive to depolymerizing drugs such as nocodazole. Using this model system we have established conditions for stepwise reconstitution of microtubule dynamics in permeabilized interphase cells when supplemented with various cell extracts. When permeabilized cells are supplemented with mammalian cell extracts in the presence of protein phosphatase inhibitors, microtubules become sensitive to nocodazole. Depolymerization induced by nocodazole proceeds from microtubule plus ends, whereas microtubule minus ends remain inactive. Such nocodazole-sensitive microtubules do not exhibit subunit turnover. By contrast, when permeabilized cells are supplemented with Xenopus egg extracts, microtubules actively turn over. This involves continuous creation of free microtubule minus ends through microtubule fragmentation. Newly created minus ends apparently serve as sites of microtubule depolymerization, while net microtubule polymerization occurs at microtubule plus ends. We provide evidence that similar microtubule fragmentation and minus end–directed disassembly occur at the whole-cell level in intact cells. These data suggest that microtubule dynamics resembling dynamics observed in vivo can be reconstituted in permeabilized cells. This model system should provide means for in vitro assays to identify molecules important in regulating microtubule dynamics. Furthermore, our data support recent work suggesting that microtubule treadmilling is an important mechanism of microtubule turnover.     

Microtubule treadmilling: what goes around comes around

“I'll see it when I believe it” Daniel Mazia Microtubules are centrally involved in many essential cell functions, including mitosis, vesicle motility, and the control of morphogenesis. Further, they appear to be involved in the control of cell cycle progression. To carry out these tasks properly, microtubules assume a protean array of different stability states and degrees of organization and they respond rapidly to requirements of the cell by modification of their organization and stability. In the typical fibroblast cell in culture, microtubules rapidly exchange their subunits with tubulin in the cytoplasmic pool, and control of this rapid turnover appears to be essential to their intrinsic capacity to perform such tasks as the separation of chromosomes in mitosis. Microtubules are not simple equilibrium polymers, but rather, they are capable of unusual nonequilibrium dynamic behaviors. One such behavior, termed treadmilling, involving the intrinsic flow of subunits from one polymer end to the other, is created by differences in the critical subunit concentrations at the opposite microtubule ends. Treadmilling was considered by many to be an in vitro dynamic behavior that did not play an important role in microtubule function in cells. However, recent evidence has established that treadmilling is a major in vivo mechanism underlying the dynamics of microtubule arrays. BioEssays 20 :830–836, 1998. © 1998 John Wiley & Sons, Inc.     

Regulation of microtubule dynamics by kinesins

The simple mechanistic and functional division of the kinesin family into either active translocators or non-motile microtubule depolymerases was initially appropriate but is now proving increasingly unhelpful, given evidence that several translocase kinesins can affect microtubule dynamics, whilst non-translocase kinesins can promote microtubule assembly and depolymerisation. Such multi-role kinesins act either directly on microtubule dynamics, by interaction with microtubules and tubulin, or indirectly, through the transport of other factors along the lattice to the microtubule tip. Here I review recent progress on the mechanisms and roles of these translocase kinesins.     

TACC3 is a microtubule plus end–tracking protein that promotes axon elongation and also regulates microtubule plus end dynamics in multiple embryonic cell types

Microtubule plus end dynamics are regulated by a conserved family of proteins called plus end–tracking proteins (+TIPs). It is unclear how various +TIPs interact with each other and with plus ends to control microtubule behavior. The centrosome-associated protein TACC3, a member of the transforming acidic coiled-coil (TACC) domain family, has been implicated in regulating several aspects of microtubule dynamics. However, TACC3 has not been shown to function as a +TIP in vertebrates. Here we show that TACC3 promotes axon outgrowth and regulates microtubule dynamics by increasing microtubule plus end velocities in vivo. We also demonstrate that TACC3 acts as a +TIP in multiple embryonic cell types and that this requires the conserved C-terminal TACC domain. Using high-resolution live-imaging data on tagged +TIPs, we show that TACC3 localizes to the extreme microtubule plus end, where it lies distal to the microtubule polymerization marker EB1 and directly overlaps with the microtubule polymerase XMAP215. TACC3 also plays a role in regulating XMAP215 stability and localizing XMAP215 to microtubule plus ends. Taken together, our results implicate TACC3 as a +TIP that functions with XMAP215 to regulate microtubule plus end dynamics.     

Distinct roles of PP1 and PP2A-like phosphatases in control of microtubule dynamics during mitosis.

Assembly of a mitotic spindle requires the accurate regulation of microtubule dynamics which is accomplished, at least in part, by phosphorylation-dephosphorylation reactions. Here we have investigated the role of serine-threonine phosphatases in the control of microtubule dynamics using specific inhibitors in Xenopus egg extracts. Type 2A phosphatases are required to maintain the short steady-state length of microtubules in mitosis by regulating the level of microtubule catastrophes, in part by controlling the the microtubule-destabilizing activity and phosphorylation of Op18/stathmin. Type 1 phosphatases are only required for control of microtubule dynamics during the transitions into and out of mitosis. Thus, although both type 2A and type 1 phosphatases are involved in the regulation of microtubule dynamics, they have distinct, non-overlapping roles.     

Positive feedback interactions between microtubule and actin dynamics during cell motility

The migration of tissue cells requires interplay between the microtubule and actin cytoskeletal systems. Recent reports suggest that interactions of microtubules with actin dynamics creates a polarization of microtubule assembly behavior in cells, such that microtubule growth occurs at the leading edge and microtubule shortening occurs at the cell body and rear. Microtubule growth and shortening may activate Rac1 and RhoA signaling, respectively, to control actin dynamics. Thus, an actin-dependent gradient in microtubule dynamic-instability parameters in cells may feed back through the activation of specific signalling pathways to perpetuate the polarized actin-assembly dynamics required for cell motility.     

Tea2p kinesin is involved in spatial microtubule organization by transporting tip1p on microtubules

The positioning of growth sites in fission yeast cells is mediated by spatially controlled microtubule dynamics brought about by tip1p, a CLIP-170-like protein, which is localized at the microtubule tips and guides them to the cell ends. The kinesin tea2p is also located at microtubule tips and affects microtubule dynamics. Here we show that tea2p interacts with tip1p and that the two proteins move with high velocity along the microtubules toward their growing tips. There, tea2p and tip1p accumulate in larger particles. Particle formation requires the EB1 homolog, mal3p. Our results suggest a model in which kinesins regulate microtubule growth by transporting regulatory factors such as tip1p to the growing microtubule tips.     

CLIP-170/tubulin-curved oligomers coassemble at microtubule ends and promote rescues

BACKGROUND: CLIP-170 is a microtubule binding protein specifically located at microtubule plus ends, where it modulates their dynamic properties and their interactions with intracellular organelles. The mechanism by which CLIP-170 is targeted to microtubule ends remains unclear today, as well as its precise effect on microtubule dynamics. RESULTS: We used the N-terminal part of CLIP-170 (named H2), which contains the microtubule binding domains, to investigate how it modulates in vitro microtubule dynamics and structure. We found that H2 primarily promoted rescues (transitions from shrinkage to growth) of microtubules nucleated from pure tubulin and isolated centrosomes, and stimulated microtubule nucleation. Electron cryomicroscopy revealed that H2 induced the formation of tubulin rings in solution and curved oligomers at the extremities of microtubules in assembly conditions. CONCLUSIONS: These results suggest that CLIP-170 targets specifically at microtubule plus ends by copolymerizing with tubulin and modulates microtubule nucleation, polymerization, and rescues by the same basic mechanism with tubulin oligomers as intermediates.