Compartment volume influences microtubule dynamic instability: a model study

Microtubules (MTs) are cytoskeletal polymers that exhibit dynamic instability, the random alternation between growth and shrinkage. MT dynamic instability plays an essential role in cell development, division, and motility. To investigate dynamic instability, simulation models have been widely used. However, conditions under which the concentration of free tubulin fluctuates as a result of growing or shrinking MTs have not been studied before. Such conditions can arise, for example, in small compartments, such as neuronal growth cones. Here we investigate by means of computational modeling how concentration fluctuations caused by growing and shrinking MTs affect dynamic instability. We show that these fluctuations shorten MT growth and shrinkage times and change their distributions from exponential to non-exponential, gamma-like. Gamma-like distributions of MT growth and shrinkage times, which allow optimal stochastic searching by MTs, have been observed in various cell types and are believed to require structural changes in the MT during growth or shrinkage. Our results, however, show that these distributions can already arise as a result of fluctuations in the concentration of free tubulin due to growing and shrinking MTs. Such fluctuations are possible not only in small compartments but also when tubulin diffusion is slow or when many MTs (de)polymerize synchronously. Volume and all other factors that influence these fluctuations can affect MT dynamic instability and, consequently, the processes that depend on it, such as neuronal growth cone behavior and cell motility in general.     

The cytoskeleton of neurites after microtubule depolymerization

We previously reported a positive correlation between the number of cold-stable microtubules (MTs) remaining after cold treatment of cat sympathetic nerve and the extent to which the original uniform polarity orientation of axonal MTs was recapitulated after rewarming (J cell biol 99 (1984) 1289). We interpreted these data to indicate that cold-stable fragments, part of larger, generally labile MTs, could act as seeds to organize MT assembly in axons. We report here a direct investigation of the form of cold-stable MTs in neurites of PC-12 cells using two-dimensional reconstruction of serial thin sections. Our data provides strong support for our previous interpretation. The number of MTs in cold-treated neurites was 2-3 times as great while the total length of polymer was approximately half that in control neurites. The average length of MTs in cold-treated neurites was 7-10 times lower than in control neurites. We observed that treatments that depolymerize axonal microtubules cause a marked increase in the number of membranous elements within the axoplasm. This may, however, be a non-specific result of an insult to the axon, since such changes have also been observed in severed, regenerating nerve fibres. We observed that neuroblastoma neurites respond to MT-depolymerization stimuli by developing lateral filopodia similar to those observed in chick dorsal root ganglion cells. Ultrastructural observation of detergent-lysed, whole mounted neuroblastoma (Neuro 2A) cells indicated that the cytoskeleton remaining after MT depolymerization splayed out perpendicular to the long axis of the neurite. That is, we were able to observe many more cytoskeletal 'ends' after MT depolymerization. The concomitant production of filopodia and the splaying of the cytoskeleton after MT depolymerization supports the hypothesis put forward by Wessels et al. (Exp cell res 117 (1978) 335) that the presence or absence of cytoskeletal ends regulates which region of the cell surface is involved in motile behaviour.     

The highly elongated Drosophila mechanosensory bristle - a new model for studying polarized microtubule function

Microtubules (MTs) are polar polymers that can facilitate asymmetric distribution of cell components, a process important for polarized cell growth. The highly elongated and polarized Drosophila mechnosensory bristle cytoplasm is filled with short MTs that constitute a significant component of the shaft cytoplasm. Inhibition of MT assembly affects biased axial growth in the bristle and highlights the importance of MTs for this process. We demonstrate that the vast majority of bristle MTs are organized in a polarized manner, minus-ends out. We also show that genetic disruption of the MT polarity affects the polar distribution of cell components and leads to an alteration in the biased axial shape of the bristle shaft. Thus, we suggest that the asymmetric organization of the MT population within the bristle cell shaft is necessary for the proper axial elongation of this cellular extension. We would also like to emphasize the benefits of using the bristle cell as a model for studying MTs and MT-binding proteins because changes to this cytoskeletal component result in easily recognized at the phenotypes.     

Augmin-dependent microtubule nucleation at microtubule walls in the spindle

The formation of a functional spindle requires microtubule (MT) nucleation from within the spindle, which depends on augmin. How augmin contributes to MT formation and organization is not known because augmin-dependent MTs have never been specifically visualized. In this paper, we identify augmin-dependent MTs and their connections to other MTs by electron tomography and 3D modeling. In metaphase spindles of human cells, the minus ends of MTs were located both around the centriole and in the body of the spindle. When augmin was knocked down, the latter population of MTs was significantly reduced. In control cells, we identified connections between the wall of one MT and the minus end of a neighboring MT. Interestingly, the connected MTs were nearly parallel, unlike other examples of end–wall connections between cytoskeletal polymers. Our observations support the concept of augmin-dependent MT nucleation at the walls of existing spindle MTs. Furthermore, they suggest a mechanism for maintaining polarized MT organization, even when noncentrosomal MT initiation is widespread.     

Motor-dependent microtubule disassembly driven by tubulin tyrosination

In cells, stable microtubules (MTs) are covalently modified by a carboxypeptidase, which removes the C-terminal Tyr residue of α-tubulin. The significance of this selective detyrosination of MTs is not understood. In this study, we report that tubulin detyrosination in fibroblasts inhibits MT disassembly. This inhibition is relieved by overexpression of the depolymerizing motor mitotic centromere-associated kinesin (MCAK). Conversely, suppression of MCAK expression prevents disassembly of normal tyrosinated MTs in fibroblasts. Detyrosination of MTs suppresses the activity of MCAK in vitro, apparently as the result of a decreased affinity of the adenosine diphosphate (ADP)–inorganic phosphate- and ADP-bound forms of MCAK for the MT lattice. Detyrosination also impairs MT disassembly in neurons and inhibits the activity of the neuronal depolymerizing motor KIF2A in vitro. These results indicate that MT depolymerizing motors are directly inhibited by the detyrosination of tubulin, resulting in the stabilization of cellular MTs. Detyrosination of transiently stabilized MTs may give rise to persistent subpopulations of disassembly-resistant polymers to sustain subcellular cytoskeletal differentiation.     

Establishment of a stable, acetylated microtubule bundle during neuronal commitment

Two posttranslational modifications of alpha-tubulin, acetylation and detyrosination, are associated with stable microtubule (MT) populations, including those of neuronal processes. We have used a pluripotent embryonal carcinoma cell line, P19, to investigate changes in MT isotype and stability found in MT arrays during neurogenesis. This cell line has an advantage in that both commitment- and differentiation-related events can be observed. Uncommitted P19 cells have minimal arrays of acetylated and detyrosinated MTs. Following neuronal induction with retinoic acid (RA), indirect immunofluorescence microscopy shows that the first MT modifications occur during commitment and before any morphological change is observed. RA-induced cells initially polymerize a temporarily enlarged population of MTs. Included in this population is a new array of acetylated MTs arranged in a bundle of parallel MTs. This bundle is colchicine-stable, although no MT-associated proteins (MAPs) are detectable using a battery of anti-MAP antibodies. Observation of MT arrays with patterns that are intermediate between the early bundles and short neurites suggests that the acetylated MT bundle subsequently extends to form a neurite. MAP 2 is first detected at about the time of neurite extension. However, at this early stage of differentiation, MAP 2 is not yet limited to dendritic processes. This report provides the first evidence that the stable MTs of mature neurons may be initiated during neuronal commitment.     

Process outgrowth in oligodendrocytes is mediated by CNP, a novel microtubule assembly myelin protein

Oligodendrocytes (OLs) extend arborized processes that are supported by microtubules (MTs) and microfilaments. Little is known about proteins that modulate and interact with the cytoskeleton during myelination. Several lines of evidence suggest a role for 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in mediating process formation in OLs. In this study, we report that tubulin is a major CNP-interacting protein. In vitro, CNP binds preferentially to tubulin heterodimers compared with MTs and induces MT assembly by copolymerizing with tubulin. CNP overexpression induces dramatic morphology changes in both glial and nonglial cells, resulting in MT and F-actin reorganization and formation of branched processes. These morphological effects are attributed to CNP MT assembly activity; branched process formation is either substantially reduced or abolished with the expression of loss-of-function mutants. Accordingly, cultured OLs from CNP-deficient mice extend smaller outgrowths with less arborized processes. We propose that CNP is an important component of the cytoskeletal machinery that directs process outgrowth in OLs.     

The microtubule-destabilizing kinesin XKCM1 regulates microtubule dynamic instability in cells

The dynamic activities of cellular microtubules (MTs) are tightly regulated by a balance between MT-stabilizing and -destabilizing proteins. Studies in Xenopus egg extracts have shown that the major MT destabilizer during interphase and mitosis is the kinesin-related protein XKCM1, which depolymerizes MT ends in an ATP-dependent manner. Herein, we examine the effects of both overexpression and inhibition of XKCM1 on the regulation of MT dynamics in vertebrate somatic cells. We found that XKCM1 is a MT-destabilizing enzyme in PtK2 cells and that XKCM1 modulates cellular MT dynamics. Our results indicate that perturbation of XKCM1 levels alters the catastrophe frequency and the rescue frequency of cellular MTs. In addition, we found that overexpression of XKCM1 or inhibition of KCM1 during mitosis leads to the formation of aberrant spindles and a mitotic delay. The predominant spindle defects from excess XKCM1 included monoastral and monopolar spindles, as well as small prometaphase-like spindles with improper chromosomal attachments. Inhibition of KCM1 during mitosis led to prometaphase spindles with excessively long MTs and spindles with partially separated poles and a radial MT array. These results show that KCM1 plays a critical role in regulating both interphase and mitotic MT dynamics in mammalian cells.     

Shiga toxin facilitates its retrograde transport by modifying microtubule dynamics

The bacterial exotoxin Shiga toxin is endocytosed by mammalian host cells and transported retrogradely through the secretory pathway before entering the cytosol. Shiga toxin also increases the levels of microfilaments and microtubules (MTs) upon binding to the cell surface. The purpose for this alteration in cytoskeletal dynamics is unknown. We have investigated whether Shiga toxin-induced changes in MT levels facilitate its intracellular transport. We have tested the effects of the Shiga toxin B subunit (STB) on MT-dependent and -independent transport steps. STB increases the rate of MT-dependent Golgi stack repositioning after nocodazole treatment. It also enhances the MT-dependent accumulation of transferrin in a perinuclear recycling compartment. By contrast, the rate of MT-independent transferrin recycling is not significantly different when STB is present. We found that STB normally requires MTs and dynein for its retrograde transport to the juxtanuclear Golgi complex and that STB increases MT assembly. Furthermore, we find that MT polymerization is limiting for STB transport in cells. These results show that STB-induced changes in cytoskeletal dynamics influence intracellular transport. We conclude that the increased rate of MT assembly upon Shiga toxin binding facilitates the retrograde transport of the toxin through the secretory pathway.     

Live cell imaging reveals structural associations between the actin and microtubule cytoskeleton in Arabidopsis

In eukaryotic cells, the actin and microtubule (MT) cytoskeletal networks are dynamic structures that organize intracellular processes and facilitate their rapid reorganization. In plant cells, actin filaments (AFs) and MTs are essential for cell growth and morphogenesis. However, dynamic interactions between these two essential components in live cells have not been explored. Here, we use spinning-disc confocal microscopy to dissect interaction and cooperation between cortical AFs and MTs in Arabidopsis thaliana, utilizing fluorescent reporter constructs for both components. Quantitative analyses revealed altered AF dynamics associated with the positions and orientations of cortical MTs. Reorganization and reassembly of the AF array was dependent on the MTs following drug-induced depolymerization, whereby short AFs initially appeared colocalized with MTs, and displayed motility along MTs. We also observed that light-induced reorganization of MTs occurred in concert with changes in AF behavior. Our results indicate dynamic interaction between the cortical actin and MT cytoskeletons in interphase plant cells.     

Toxicity and interaction of titanium dioxide nanoparticles with microtubule protein

Titanium dioxide (TiO2) nanoparticles (NPs) are widely used in several manufactured products. The small size of NPs facilitates their uptake into cells as well as transcytosis across epithelial cells into blood and lymph circulation to reach different sites, such as the central nervous system. Different studies have shown the risks that TiO2 NPs in the neuronal system and other organs present. As membrane-bound layer aggregates or single particles, TiO2 NPs can enter not only cells, but also mitochondria and nuclei. Therefore these particles can interact with cytoplasmic proteins such as microtubules (MTs). MTs are cytoskeletal proteins that are essential in eukaryotic cells for a variety of functions, such as cellular transport, cell motility and mitosis. MTs in neurons are used to transport substances such as neurotransmitters. Single TiO2 NPs in cytoplasm can interact with these proteins and affect their crucial functions in different tissues. In this study, we showed the effects of TiO2 NPs on MT polymerization and structure using ultraviolet spectrophotometer and fluorometry. The fluorescent spectroscopy showed a significant tubulin conformational change in the presence of TiO2 NPs and the ultraviolet spectroscopy results showed that TiO2 NPs affect tubulin polymerization and decrease it. The aim of this study was to find the potential risks that TiO2 NPs pose to human organs and cells.     

Tau-induced traffic jams reflect organelles accumulation at points of microtubule polar mismatching

It is currently accepted that tau overexpression leads to impaired organelle transport and thus to neuronal degeneration. Nevertheless, the underlying mechanisms that lead to impaired organelle transport are not entirely clear. Using cultured Aplysia neurons and online confocal imaging of human tau, microtubules (MTs), the plus-end tracking protein – end-binding protein 3, retrogradely and anterogradely transported organelles, we found that overexpression of tau generates the hallmarks of human tau pathogenesis. Nevertheless, in contrast to earlier reports, we found that the tau-induced impairment of organelle transport is because of polar reorientation of the MTs along the axon or their displacement to submembrane domains. 'Traffic jams' reflect the accumulation of organelles at points of MT polar discontinuations or polar mismatching rather than because of MT depolymerization. Our findings offer a new mechanistic explanation for earlier observations, which established that tau overexpression leads to impaired retrograde and anterograde organelle transport, while the MT skeleton appeared intact.     

Efficient formation of bipolar microtubule bundles requires microtubule-bound gamma-tubulin complexes

The mechanism for forming linear microtubule (MT) arrays in cells such as neurons, polarized epithelial cells, and myotubes is not well understood. A simpler bipolar linear array is the fission yeast interphase MT bundle, which in its basic form contains two MTs that are bundled at their minus ends. Here, we characterize mto2p as a novel fission yeast protein required for MT nucleation from noncentrosomal gamma-tubulin complexes (gamma-TuCs). In interphase mto2Delta cells, MT nucleation was strongly inhibited, and MT bundling occurred infrequently and only when two MTs met by chance in the cytoplasm. In wild-type 2, we observed MT nucleation from gamma-TuCs bound along the length of existing MTs. We propose a model on how these nucleation events can more efficiently drive the formation of bipolar MT bundles in interphase. Key to the model is our observation of selective antiparallel binding of MTs, which can both explain the generation and spatial separation of multiple bipolar bundles.     

Mechanism,function and regulation of microtubule-dependent microtubule amplification in mitosis

Mitotic spindle mediates the segregation of chromosomes in the cell cycle and the proper function of the spindle is crucial to the high fidelity of chromosome segregation and to the stability of the genome. Nucleation of microtubules (MTs) from centrosomes and chromatin represents two well-characterized pathways essential for the assembly of a dynamic spindle in mitosis. Recently, we identified a third MT nucleation pathway, in which existing MTs in the spindle act as a template to promote the nucleation and polymerization of MTs, thereby efficiently amplifying MTs in the spindle. We will review here our current understanding on the molecular mechanism, the physiological function and the cell-cycle regulation of MT amplification.     

A Highlights from MBoC Selection: MAP65/Ase1 promote microtubule flexibility

Microtubules (MTs) are dynamic cytoskeletal elements involved in numerous cellular processes. Although they are highly rigid polymers with a persistence length of 1–8 mm, they may exhibit a curved shape at a scale of few micrometers within cells, depending on their biological functions. However, how MT flexural rigidity in cells is regulated remains poorly understood. Here we ask whether MT-associated proteins (MAPs) could locally control the mechanical properties of MTs. We show that two major cross-linkers of the conserved MAP65/PRC1/Ase1 family drastically decrease MT rigidity. Their MT-binding domain mediates this effect. Remarkably, the softening effect of MAP65 observed on single MTs is maintained when MTs are cross-linked. By reconstituting physical collisions between growing MTs/MT bundles, we further show that the decrease in MT stiffness induced by MAP65 proteins is responsible for the sharp bending deformations observed in cells when they coalign at a steep angle to create bundles. Taken together, these data provide new insights into how MAP65, by modifying MT mechanical properties, may regulate the formation of complex MT arrays.     

Septin GTPases spatially guide microtubule organization and plus end dynamics in polarizing epithelia

Establishment of epithelial polarity requires the reorganization of the microtubule (MT) cytoskeleton from a radial array into a network positioned along the apicobasal axis of the cell. Little is known about the mechanisms that spatially guide the remodeling of MTs during epithelial polarization. Septins are filamentous guanine triphosphatases (GTPases) that associate with MTs, but the function of septins in MT organization and dynamics is poorly understood. In this paper, we show that in polarizing epithelia, septins guide the directionality of MT plus end movement by suppressing MT catastrophe. By enabling persistent MT growth, two spatially distinct populations of septins, perinuclear and peripheral filaments, steer the growth and capture of MT plus ends. This navigation mechanism is essential for the maintenance of perinuclear MT bundles and for the orientation of peripheral MTs as well as for the apicobasal positioning of MTs. Our results suggest that septins provide the directional guidance cues necessary for polarizing the epithelial MT network.     

Regulation of leading edge microtubule and actin dynamics downstream of Rac1

Actin in migrating cells is regulated by Rho GTPases. However, Rho proteins might also affect microtubules (MTs). Here, we used time-lapse microscopy of PtK1 cells to examine MT regulation downstream of Rac1. In these cells, "pioneer" MTs growing into leading-edge protrusions exhibited a decreased catastrophe frequency and an increased time in growth as compared with MTs further from the leading edge. Constitutively active Rac1(Q61L) promoted pioneer behavior in most MTs, whereas dominant-negative Rac1(T17N) eliminated pioneer MTs, indicating that Rac1 is a regulator of MT dynamics in vivo. Rac1(Q61L) also enhanced MT turnover through stimulation of MT retrograde flow and breakage. Inhibition of p21-activated kinases (Paks), downstream effectors of Rac1, inhibited Rac1(Q61L)-induced MT growth and retrograde flow. In addition, Rac1(Q61L) promoted lamellipodial actin polymerization and Pak-dependent retrograde flow. Together, these results indicate coordinated regulation of the two cytoskeletal systems in the leading edge of migrating cells.     

Single-molecule analysis of the microtubule cross-linking protein MAP65-1 reveals a molecular mechanism for contact-angle-dependent microtubule bundling

Bundling of microtubules (MTs) is critical for the formation of complex MT arrays. In land plants, the interphase cortical MTs form bundles specifically following shallow-angle encounters between them. To investigate how cells select particular MT contact angles for bundling, we used an in vitro reconstitution approach consisting of dynamic MTs and the MT-cross-linking protein MAP65-1. We found that MAP65-1 binds to MTs as monomers and inherently targets antiparallel MTs for bundling. Dwell-time analysis showed that the affinity of MAP65-1 for antiparallel overlapping MTs is about three times higher than its affinity for single MTs and parallel overlapping MTs. We also found that purified MAP65-1 exclusively selects shallow-angle MT encounters for bundling, indicating that this activity is an intrinsic property of MAP65-1. Reconstitution experiments with mutant MAP65-1 proteins with different numbers of spectrin repeats within the N-terminal rod domain showed that the length of the rod domain is a major determinant of the range of MT bundling angles. The length of the rod domain also determined the distance between MTs within a bundle. Together, our data show that the rod domain of MAP65-1 acts both as a spacer and as a structural element that specifies the MT encounter angles that are conducive for bundling.     

Myosin II activity facilitates microtubule bundling in the neuronal growth cone neck

The cell biological processes underlying axon growth and guidance are still not well understood. An outstanding question is how a new segment of the axon shaft is formed in the wake of neuronal growth cone advance. For this to occur, the highly dynamic, splayed-out microtubule (MT) arrays characteristic of the growth cone must be consolidated (bundled together) to form the core of the axon shaft. MT-associated proteins stabilize bundled MTs, but how individual MTs are brought together for initial bundling is unknown. Here, we show that laterally moving actin arcs, which are myosin II-driven contractile structures, interact with growing MTs and transport them from the sides of the growth cone into the central domain. Upon Myosin II inhibition, the movement of actin filaments and MTs immediately stopped and MTs unbundled. Thus, Myosin II-dependent compressive force is necessary for normal MT bundling in the growth cone neck.     

Septin 6 localizes to microtubules in neuronal dendrites

In neuronal dendrites, septins localize to the base of the spine, a unique position which is sandwiched between the microtubule (MT)-rich dendritic shaft and the actin filament-rich spine. Here, we provide evidence for the association of SEPT6 with MTs in cultured rat hippocampal neurons. In normal cultures, SEPT6 clusters localized to MTs, but not to actin clusters. Only MT-disrupting agents (vincristine and nocodazole), but not microfilament-disrupting one (latrunculin A), induced the redistribution of SEPT6 to the disrupted MTs. The nascent MT fibers that were recovered from vincristine or nocodazole treatments also accompanied SEPT6. Blocking MT disruption by Taxol prevented such phenomena, proving that the redistribution of SEPT6 was due to the MT disruption. Our results indicate that SEPT6 complexes at the base of the dendritic spine are associated with MTs.