Localized Aurora B activity spatially controls non-kinetochore microtubules during spindle assembly

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Microtubules (MTs) in the axon have a uniform polarity orientation that is recapitulated during recovery from episodes of MT depolymerization (Heidemann, S. R., M. A. Hamborg, S. J. Thomas, B. Song, S. Lindley, and D. Chu. 1984. J. Cell Biol. 99:1289-1295). This tight regulation of their organization indicates that axonal MTs are spatially regulated by discrete nucleating structures comparable in function to the centrosome. Several authors have proposed that an especially stable class of MTs in the axon may serve as these nucleating structures. In a previous report (Baas, P. W., and M. M. Black. 1990. J. Cell Biol. 111:495-509), we determined that the axons of cultured sympathetic neurons contain two classes of MT polymer, stable and labile, that differ in their sensitivity to nocodazole by roughly 35-fold. The stable and labile polymer represent long-lived and recently assembled polymer, respectively. We also determined that these two classes of polymer can be visually distinguished at the immunoelectron microscopic level based on their content of tyrosinated alpha-tubulin: the labile polymer stains densely, while the stable polymer does not stain. In the present study, we have taken advantage of these observations to directly identify MT nucleating structures in the axon. Neuron cultures were treated with nocodazole for 6 h to completely depolymerize the labile polymer in the axon, and substantially shorten the stable polymer. The cultures were then rinsed free of the drug, permitted to reassemble polymer for various periods of time, and prepared for immunoelectron microscopic localization of tyrosinated alpha-tubulin. Serial reconstruction of consecutive thin sections was undertaken to determine the spatial relationship between the stable MTs and the newly assembled polymer. All of the new polymer assembled in direct continuity with the plus ends of stable MTs, indicating that these ends are assembly competent, and hence capable of acting as nucleating structures. Our results further indicate that no self-assembly of MTs occurs in the axon, nor do any MT nucleating structures exist in the axon other than the plus ends of stable MTs. Thus the plus ends of stable MTs are the exclusive nucleating structures for MTs in the axon.

The cytoplasmic microtubule array in Neurospora crassa depends on microtubule-organizing centers at spindle pole bodies and microtubule +end-depending pseudo-MTOCs at septa

An Essential Function of the C. elegans Ortholog of TPX2 Is to Localize Activated Aurora A Kinase to Mitotic Spindles

Dimeric Centromere Protein E (CENP-E) Promotes Microtubule-elongation At The Plus-ends Of Microtubules

Kinesin-8s are plus-end-directed motors that negatively regulate microtubule (MT) length. Well-characterized members of this subfamily (Kip3, Kif18A) exhibit two important properties: (i) They are "ultraprocessive," a feature enabled by a second MT-binding site that tethers the motors to a MT track, and (ii) they dissociate infrequently from the plus end. Together, these characteristics combined with their plus-end motility cause Kip3 and Kif18A to enrich preferentially at the plus ends of long MTs, promoting MT catastrophes or pausing. Kif18B, an understudied human kinesin-8, also limits MT growth during mitosis. In contrast to Kif18A and Kip3, localization of Kif18B to plus ends relies on binding to the plus-end tracking protein EB1, making the relationship between its potential plus-end-directed motility and plus-end accumulation unclear. Using single-molecule assays, we show that Kif18B is only modestly processive and that the motor switches frequently between directed and diffusive modes of motility. Diffusion is promoted by the tail domain, which also contains a second MT-binding site that decreases the off rate of the motor from the MT lattice. In cells, Kif18B concentrates at the extreme tip of a subset of MTs, superseding EB1. Our data demonstrate that kinesin-8 motors use diverse design principles to target MT plus ends, which likely target them to the plus ends of distinct MT subpopulations in the mitotic spindle.

Cells with blocked microtubule polymerization are delayed in mitosis, but eventually manage to proliferate despite substantial chromosome missegregation. While several studies have analyzed the first cell division after microtubule depolymerization, we have asked how cells cope long-term with microtubule impairment. We allowed 24 clonal populations of yeast cells with beta-tubulin mutations preventing proper microtubule polymerization, to evolve for ˜150 generations. At the end of the laboratory evolution experiment, cells had regained the ability to form microtubules and were less sensitive to microtubule-depolymerizing drugs. Whole-genome sequencing identified recurrently mutated genes, in particular for tubulins and kinesins, as well as pervasive duplication of chromosome VIII. Recreating these mutations and chromosome VIII disomy prior to evolution confirmed that they allow cells to compensate for the original mutation in beta-tubulin. Most of the identified mutations did not abolish function, but rather restored microtubule functionality. Analysis of the temporal order of resistance development in independent populations repeatedly revealed the same series of events: disomy of chromosome VIII followed by a single additional adaptive mutation in either tubulins or kinesins. Since tubulins are highly conserved among eukaryotes, our results have implications for understanding resistance to microtubule-targeting drugs widely used in cancer therapy.

ADP release is the rate-limiting step of the MT activated ATPase of non-claret disjunctional and kinesin

Efficient assembly of a mitotic spindle and stable attachment of microtubules (k-fibers) to kinetochores are essential for the high fidelity of chromosome segregation. Both spindle assembly and 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 acts 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.k-fiber formation require robust nucleation and polymerization of microtubules mediated by the γ-tubulin ring complex (γ TuRC). It has been well established that centrosomes and chromatin are the two centers for microtubule nucleation. We recently demonstrate a third mechanism for microtubule nucleation and polymerization, in which the existing microtubules in the spindle act as templates to promote the formation of new microtubules. We showed that a novel spindle-associated protein, FAM29A, plays a critical role in this microtubule-dependent microtubule amplification. FAM29A associates with spindle microtubules and directly interacts with and recruits NEDD1, the targeting subunit of γTuRC. Spindle-associated γTuRC then promotes microtubule nucleation required for spindle assembly and k-fiber formation. This novel microtubule amplification pathway provides a powerful mechanism to control the local cytoskeleton structures independent of centrosomes and chromatin. We speculate that microtubule amplification not only functions in mitosis, but may also act in other physiological processes to re-enforce existing cytoskeleton structures.

Genome-wide Analysis Reveals Novel and Discrete Functions for Tubulin Carboxy-Terminal Tails

The structure and function of microtubules (MTs) are regulated by post-translational modifications of tubulin subunits, such as acetylation of the Lys40 residue of α-tubulin. Regulation of the organization and dynamics of MTs is essential for the precise formation of the mitotic spindle. Spindle MTs are highly acetylated, but the mechanism regulating this acetylation is largely unknown. Furry (Fry) is an evolutionarily conserved protein that binds to MTs and colocalizes with acetylated MTs in the mitotic spindle. In this study, we examined the role of Fry in the acetylation of MTs in the mitotic spindle. Depletion of Fry significantly reduced the level of MT acetylation in the mitotic spindle. Expression of the N-terminal fragment of Fry induced hyperacetylation of MTs in both mitotic and interphase cells. These results indicate that Fry promotes MT acetylation in the mitotic spindle. We also found that Fry binds to the tubulin deacetylase SIRT2, preferentially in mitotic cells. Cell-free experiments revealed that the N-terminal region of Fry is the domain responsible for binding to and inhibiting the tubulin-deacetylase activity of SIRT2. AGK2, a specific inhibitor of SIRT2, increased the level of MT acetylation in the mitotic spindle, indicating that SIRT2 is involved in the deacetylation of spindle MTs. Furthermore, AGK2 reversed the decrease in MT acetylation induced by Fry depletion. In summary, these results suggest that Fry plays a crucial role in promoting the level of MT acetylation in the mitotic spindle by inhibiting the tubulin-deacetylase activity of SIRT2.

In the fungus Ustilago maydis, early endosomes move bidirectionally along microtubules (MTs) and facilitate growth by local membrane recycling at the tip of the infectious hypha. Here, we set out to elucidate the molecular mechanism of this process. We show that endosomes travel by Kinesin-3 activity into the hyphal apex, where they reverse direction and move backwards in a dynein-dependent manner. Our data demonstrate that dynein, dynactin and Lis1 accumulate at MT plus-ends within the hyphal tip, where they provide a reservoir of inactive motors for retrograde endosome transport. Consistently, endosome traffic is abolished after depletion of the dynein activator Lis1 and in Kinesin-1 null mutants, which was due to a defect in targeting of dynein and dynactin to the apical MT plus-ends. Furthermore, biologically active GFP-dynein travels on endosomes in retrograde and not in anterograde direction. Surprisingly, a CLIP170 homologue was neither needed for dynein localization nor for endosome transport. These results suggest an apical dynein loading zone in the hyphal tip, which ensure that endosomes reach the expanding growth region before they reverse direction.

The Ncd kinesin-14 motor is required for meiotic spindle assembly in Drosophila oocytes and produces force in mitotic spindles that opposes other motors. Despite extensive studies, the way the motor binds to the spindle to perform its functions is not well understood. By analyzing Ncd deleted for the conserved head or the positively charged tail, we found that the tail is essential for binding to spindles and centrosomes, but both the head and tail are needed for normal spindle assembly and function. Fluorescence photobleaching assays to analyze binding interactions with the spindle yielded data for headless and full-length Ncd that did not fit well to previous recovery models. We report a new model that accounts for Ncd transport towards the equator revealed by fluorescence flow analysis of early mitotic spindles and gives rate constants that confirm the dominant role the Ncd tail plays in binding to the spindle. By contrast, the head binds weakly to spindles based on analysis of the tailless fluorescence recovery data. Minus-end Ncd thus binds tightly to spindles and is transported in early metaphase towards microtubule plus-ends, the opposite direction to that in which the motor moves, to produce force in the spindle later in mitosis.

The plus-end microtubule (MT) motor kinesin-1 is essential for normal development, with key roles in the nervous system. Kinesin-1 drives axonal transport of membrane cargoes to fulfill the metabolic needs of neurons and maintain synapses. We have previously demonstrated that kinesin-1, in addition to its well-established role in organelle transport, can drive MT-MT sliding by transporting "cargo" MTs along "track" MTs, resulting in dramatic cell shape changes. The mechanism and physiological relevance of this MT sliding are unclear. In addition to its motor domain, kinesin-1 contains a second MT-binding site, located at the C terminus of the heavy chain. Here, we mutated this C-terminal MT-binding site such that the ability of kinesin-1 to slide MTs is significantly compromised, whereas cargo transport is unaffected. We introduced this mutation into the genomic locus of kinesin-1 heavy chain (KHC), generating the Khc(mutA) allele. Khc(mutA) neurons displayed significant MT sliding defects while maintaining normal transport of many cargoes. Using this mutant, we demonstrated that MT sliding is required for axon and dendrite outgrowth in vivo. Consistent with these results, Khc(mutA) flies displayed severe locomotion and viability defects. To test the role of MT sliding further, we engineered a chimeric motor that actively slides MTs but cannot transport organelles. Activation of MT sliding in Khc(mutA) neurons using this chimeric motor rescued axon outgrowth in cultured neurons and in vivo, firmly establishing the role of sliding in axon outgrowth. These results demonstrate that MT sliding by kinesin-1 is an essential biological phenomenon required for neuronal morphogenesis and normal nervous system development.

Microtubules are polarized polymers that exhibit dynamic instability, with alternating phases of elongation and shortening, particularly at the more dynamic plus-end. Microtubule plus-end tracking proteins (+TIPs) localize to and track with growing microtubule plus-ends in the cell. +TIPs regulate microtubule dynamics and mediate interactions with other cellular components. The molecular mechanisms responsible for the +TIP tracking activity are not well understood, however. We reconstituted the +TIP tracking of mammalian proteins EB1 and CLIP-170 in vitro at single-molecule resolution using time-lapse total internal reflection fluorescence microscopy. We found that EB1 is capable of dynamically tracking growing microtubule plus-ends. Our single-molecule studies demonstrate that EB1 exchanges rapidly at microtubule plus-ends with a dwell time of <1 s, indicating that single EB1 molecules go through multiple rounds of binding and dissociation during microtubule polymerization. CLIP-170 exhibits lattice diffusion and fails to selectively track microtubule ends in the absence of EB1; the addition of EB1 is both necessary and sufficient to mediate plus-end tracking by CLIP-170. Single-molecule analysis of the CLIP-170-EB1 complex also indicates a short dwell time at growing plus-ends, an observation inconsistent with the copolymerization of this complex with tubulin for plus-end-specific localization. GTP hydrolysis is required for +TIP tracking, because end-specificity is lost when tubulin is polymerized in the presence of guanosine 5'-[alpha,beta-methylene]triphosphate (GMPCPP). Together, our data provide insight into the mechanisms driving plus-end tracking by mammalian +TIPs and suggest that EB1 specifically recognizes the distinct lattice structure at the growing microtubule end.