Absence Of Microtubules Research Articles

The Role of Microtubules and Their Dynamics in Cell Migration

Although microtubules have long been implicated in cell locomotion, the mechanism of their involvement remains controversial. Most studies have concluded that microtubules play a positive role by regulating actin polymerization, transporting membrane vesicles to the leading edge, and/or facilitating the turnover of adhesion plaques. Here we used wild-type and mutant CHO cell lines with alterations in tubulin to demonstrate that microtubules can also act to restrain cell motility. Tubulin mutations or low concentrations of drugs that suppress microtubule dynamics without affecting the amount of microtubule polymer inhibited the rate of migration by preventing microtubule reorganization in the trailing portion of the cells where the more dynamic microtubules are normally found. Under these conditions, cells along the edge of a wound still extended lamellipodia and elongated toward the wound but were inhibited in their ability to retract their tails, thus retarding forward progress. The idea that microtubules normally act to restrain cell locomotion was confirmed by treating cells with high concentrations of nocodazole to depolymerize the microtubule network. In the absence of microtubules, wild-type CHO and HeLa cells could still move at near normal speeds, but the movement became more random. We conclude that microtubules act both to restrain cell movement and to establish directionality.

Abstract 2053: Aurora-A kinase is required for rapid establishment of the mitotic checkpoint

Abstract Aurora-A (AurA) is a conserved Serine/Threonine kinase and is mainly involved in multiple key steps in cell division. A high level of AurA is frequently detected in different human cancer types and is used as a poor prognostic biomarker. AurA inhibitors as potential anti-cancer drugs are widely being tested in clinical trials. To explore the mechanism(s) of possible efficacy, the roles of AurA in mitosis should be fully elucidated. AurA is known to be required for centrosome separation, spindle formation and kinetochore-microtubule attachment. However, the mechanism by which AurA is involved in regulation of the mitotic checkpoint is unclear. Most studies have used RNA interference, which does not abolish AurA activity, or inhibitors, which often target both AurA and AurB. In this study, we sought to dissect the role of AurA in checkpoint regulation by using cells derived from genetically engineered mice conditionally null for AurA, as well as a new selective AurA inhibitor. Upon deletion of AurA in mouse embryonic fibroblast (MEFs), 65% of cells formed monopolar spindles and exited mitosis without dividing. However, the average duration of mitosis in these cells was 88 minutes, significantly shorter than the 192 minutes in cells with monopolar spindles induced by Eg5 kinesin inhibition, in which checkpoint is fully functional. Previous work in our lab showed AurA is dispensable for checkpoint signaling in APC/C inhibition. Hence, we tested whether the truncated arrest in AurA-/- cells was due to defective checkpoint engagement and/or because of attenuated AurB mediated error correction, which reactivates checkpoint. Using live cell imaging followed by immunofluorescence, we found the recruitment of Mad2 to kinetochores at the start of mitosis was markedly delayed in AurA-/- MEFs. Mad2 levels at kinetochores were reduced to 28% of that in wild-type cells in the absence of microtubules. In addition, without AurA, AurB was not able to accumulate at the centromere at early prometa-phase. This observation may indicate that error correction is compromised, resulting in the abnormal acceleration of checkpoint satisfaction. Similar results were obtained using human immortalized RPE cells treated with a selective AurA inhibitor-MLN8237. Thus, preliminary data suggest AurA disruption causes delays in rapid establishment of a robust checkpoint and an effective error correction pathway at early mitosis. These defects accelerate mitotic exit in the presence of an aberrant spindle, leading to aneuploid progeny. Currently, we are assessing the dynamics of the checkpoint signaling on kinetochores throughout mitosis. We are also investigating whether AurA function in checkpoint regulation is directly through activation of AurB at centromere. This study is expected to further elucidate basic mechanisms of mitotic regulation and will provide important implications for the application of AurA kinase inhibitors in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2053. doi:1538-7445.AM2012-2053

Kif2C Minimal Functional Domain Has Unusual Nucleotide Binding Properties That Are Adapted to Microtubule Depolymerization

The kinesin-13 Kif2C hydrolyzes ATP and uses the energy released to disassemble microtubules. The mechanism by which this is achieved remains elusive. Here we show that Kif2C-(sN+M), a monomeric construct consisting of the motor domain with the proximal part of the N-terminal Neck extension but devoid of its more distal, unstructured, and highly basic part, has a robust depolymerase activity. When detached from microtubules, the Kif2C-(sN+M) nucleotide-binding site is occupied by ATP at physiological concentrations of adenine nucleotides. As a consequence, Kif2C-(sN+M) starts its interaction with microtubules in that state, which differentiates kinesin-13s from motile kinesins. Moreover, in this ATP-bound conformational state, Kif2C-(sN+M) has a higher affinity for soluble tubulin compared with microtubules. We propose a mechanism in which, in the first step, the specificity of ATP-bound Kif2C for soluble tubulin causes it to stabilize a curved conformation of tubulin heterodimers at the ends of microtubules. Data from an ATPase-deficient Kif2C mutant suggest that, then, ATP hydrolysis precedes and is required for tubulin release to take place. Finally, comparison with Kif2C-Motor indicates that the binding specificity for curved tubulin and, accordingly, the microtubule depolymerase activity are conferred to the motor domain by its N-terminal Neck extension.

Interactions between Leishmania braziliensis and Macrophages Are Dependent on the Cytoskeleton and Myosin Va.

Leishmaniasis is a neglected tropical disease with no effective vaccines. Actin, microtubules and the actin-based molecular motor myosin Va were investigated for their involvement in Leishmania braziliensis macrophage interactions. Results showed a decrease in the association index when macrophages were without F-actin or microtubules regardless of the activation state of the macrophage. In the absence of F-actin, the production of NO in non-activated cells increased, while in activated cells, the production of NO was reduced independent of parasites. The opposite effect of an increased NO production was observed in the absence of microtubules. In activated cells, the loss of cytoskeletal components inhibited the release of IL-10 during parasite interactions. The production of IL-10 also decreased in the absence of actin or microtubules in non-activated macrophages. Only the disruption of actin altered the production of TNF-α in activated macrophages. The expression of myosin Va tail resulted in an acute decrease in the association index between transfected macrophages and L. braziliensis promastigotes. These data reveal the importance of F-actin, microtubules, and myosin-Va suggesting that modulation of the cytoskeleton may be a mechanism used by L. braziliensis to overcome the natural responses of macrophages to establish infections.

Aspergillus Myosin-V Supports Polarized Growth in the Absence of Microtubule-Based Transport

In the filamentous fungus Aspergillus nidulans, both microtubules and actin filaments are important for polarized growth at the hyphal tip. Less clear is how different microtubule-based and actin-based motors work together to support this growth. Here we examined the role of myosin-V (MYOV) in hyphal growth. MYOV-depleted cells form elongated hyphae, but the rate of hyphal elongation is significantly reduced. In addition, although wild type cells without microtubules still undergo polarized growth, microtubule disassembly abolishes polarized growth in MYOV-depleted cells. Thus, MYOV is essential for polarized growth in the absence of microtubules. Moreover, while a triple kinesin null mutant lacking kinesin-1 (KINA) and two kinesin-3s (UNCA and UNCB) undergoes hyphal elongation and forms a colony, depleting MYOV in this triple mutant results in lethality due to a severe defect in polarized growth. These results argue that MYOV, through its ability to transport secretory cargo, can support a significant amount of polarized hyphal tip growth in the absence of any microtubule-based transport. Finally, our genetic analyses also indicate that KINA (kinesin-1) rather than UNCA (kinesin-3) is the major kinesin motor that supports polarized growth in the absence of MYOV.

The characean internodal cell as a model system for studying wound healing.

This work describes the characean internodal cell as a model system for the study of wound healing and compares wounds induced by certain chemicals and UV irradiation with wounds occurring in the natural environment. We review the existing literature and define three types of wound response: (1) cortical window formation characterised by disassembly of microtubules, transient inhibition of actin-dependent cytoplasmic streaming and chloroplast detachment, (2) fibrillar wound walls characterised by exocytosis of vesicles carrying wall polysaccharides and membrane-bound cellulose synthase complexes coupled with endocytosis of surplus membrane and (3) amorphous, callose- and membrane-containing wound walls characterised by exocytosis of vesicles and endoplasmic reticulum cisternae in the absence of membrane recycling. We hypothesize that these three wound responses reflect the extent of damage, probably Ca(2+) influx, and that the secretion of Ca(2+) -loaded endoplasmic reticulum cisternae is an emergency reaction in case of severe Ca(2+) load. Microtubules are not required for wound healing but their disassembly could have a signalling function. Transient reorganisation of the actin cytoskeleton into a meshwork of randomly oriented filaments is required for the migration of wound wall forming organelles, just as occurs in tip-growing plant cells. New data presented in this study show that during the deposition of an amorphous wound wall numerous actin rings are present, which may indicate specific ion fluxes and/or a storage form for actin. In addition, we present new evidence for the exocytosis of FM1-43-stained organelles, putative endosomes, required for plasma membrane repair during wound healing. Finally, we show that quickly growing fibrillar wound walls, even when deposited in the absence of microtubules, have a highly ordered helical structure of consistent handedness comprised of cellulose microfibrils.

WAVE2 regulates meiotic spindle stability, peripheral positioning and polar body emission in mouse oocytes

During oocyte meiotic maturation, meiotic spindles form in the central cytoplasm and then migrate to the cortex to extrude a small polar body, forming a highly polarized cell through a process involving actin and actin-related molecules. The mechanisms underlying oocyte polarization are still unclear. The Arp2/3 complex regulates oocyte polarization but it is not known whether the WASP family of proteins, a known regulator of the Arp2/3 complex, is involved in this context. In the present study, the role of WASP family member WAVE2 in mouse oocyte asymmetric division was investigated. (1) WAVE2 mRNA and protein were detected during mouse oocyte meiosis. (2) siRNA-mediated and antibody-mediated disruption of WAVE2 resulted in the failure of chromosome congression, spindle formation, spindle positioning and polar body extrusion. (3) WAVE2 regulated actin-driven chromosome migration since chromosomes were arrested in the central cytoplasm by WAVE2 RNAi in the absence of microtubules. (4) Localization of γ-tubulin and MAPK was disrupted after RNAi, confirming the effect of WAVE2 on spindle formation. (5) Actin cap and cortical granule-free domain (CGFD) formation was also disrupted, further confirming the failure of oocyte polarization. Our data suggest that WAVE2 regulates oocyte polarization by regulating meiotic spindle, peripheral positioning, probably via an actin-mediated pathway, and is involved in polar body emission during mouse oocyte meiotic maturation.

KNL1/Spc105 Recruits PP1 to Silence the Spindle Assembly Checkpoint

The spindle assembly checkpoint (SAC) delays anaphase onset until kinetochores accomplish bioriented microtubule attachments [1]. Although several centromeric and kinetochore kinases, including Aurora B, regulate kinetochore-microtubule attachment and/or SAC activation [2-4], the molecular mechanism that translates bioriented attachment into SAC silencing remains unclear [5]. Employing a method to rapidly induce exact gene replacement in budding yeast [6], we show here that the binding of protein phosphatase 1(PP1/Glc7) to the evolutionarily conserved RVSF motif of the kinetochore protein Spc105 (KNL1/Blinkin/CASC5) is essential for viability by silencing the SAC, while it plays an auxiliary nonessential role for physical chromosome segregation. Although Aurora B may inhibit this binding, persistent PP1-Spc105 interaction does not affect chromosome segregation and is insufficient to silence the SAC in the absence of microtubules, indicating that dynamic regulation of thisinteraction is dispensable. However, the amount of PP1 targeted to kinetochores must be finely tuned, because recruitment of either no or one extra copy of PP1 to Spc105 is detrimental, illustrating the vital impact of targeting an exiguous fraction of PP1 to the kinetochore. We propose that the PP1-Spc105 interaction enables local regulation of dynamic phosphorylation and dephosphorylation at the kinetochore to couple microtubule attachment and SAC silencing.

Nuclear Fission without Microtubules—a Return to the Past?

​OneOne of the most familiar images in biology, even to high school students taking an introductory course, is that of newly duplicated chromosomes lined up along the center of a cell at the stage of cell division called metaphase. As the cell enters anaphase, microtubules, rod-like filaments that comprise the mitotic spindle, pull the chromosomes away from one another to opposite sides of the cell. The process is then completed when the mother cell pinches apart, producing two daughter cells, each with a full chromosomal complement. Cell division is subject to rigorous quality control in the form of checkpoints. One such checkpoint occurs during metaphase and ensures that all is well with the mitotic spindle, and that the microtubules are securely attached at one end to the kinetochore, an assemblage of proteins stuck to the center of each chromosome, and at the other to one of the two microtubule-organizing structures (“spindle pole bodies” in yeast) on opposite ends of the cell. Though microtubule-based mitosis is a fundamental feature of cell division, new work in fission yeast provides evidence for a microtubule-independent nuclear division process in a eukaryotic cell, potentially uncovering an intermediate stage in the evolution ... Not surprisingly then, spindle microtubules play a fundamental role in chromosome segregation; cells that lack microtubules are unable to pass the mitotic checkpoint and continue on to cell division. However, Stefania Castagnetti, Snezhana Oiferendo, and Paul Nurse (this issue of PLoS Biology) have made an unexpected observation in the fission yeast Schizosaccharomyces pombe, which undergoes a special form of mitosis, called closed mitosis, during which the nuclear membrane remains intact and the mitotic spindle elongates between two spindle pole bodies embedded in the nuclear envelope. Using a microtubule-depolymerizing drug (microtubules are made of protein subunits called tubulin) to prevent spindle formation and S. pombe mutants incapable of forming microtubules, the authors found an alternative form of nuclear division occurring in the absence of microtubules. This variation, which they call nuclear fission, could produce two clusters of segregated chromosomes, each encapsulated within a nuclear membrane. To further investigate the conditions in which nuclear fission can occur, the authors used a variety of genetic tools and protein markers. They found that, unlike normal mitosis, nuclear fission was not under spindle checkpoint control; even though the absence of microtubules activated the spindle checkpoint, mitosis was only slightly delayed, and the nucleus proceeded to divide. They also determined that separation of sister chromatids (identical strands of a replicated chromosome connected by a structure called the centromere) was required for nuclear fission to occur, and that the centromeres of chromosomes remained clustered with the spindle pole bodies during nuclear fission. Using a drug that depolymerizes the cytoskeletal protein actin, they found that nuclear fission requires filamentous actin. Actin structures may be involved in the nuclear membrane redistribution that occurs in anaphase, and rapid redistribution facilitated by actin could be responsible for the ruffled appearance that the nuclear membrane acquires during fission. Taking all of their results into consideration, the authors suggest a possible mechanism for this microtubule-independent cell division process: at the beginning of mitosis, rather than lining up at the cell center, the sister chromatids, connected by their centromeres, remain associated with the two spindle pole bodies. The spindle pole bodies then slowly move apart within the nuclear membrane, and as the sister chromatids lose cohesion with one another, they are pulled apart by their connection to the spindle pole bodies. Subsequent membrane expansion associated with lipid biosynthesis during mitosis and deformation of the nuclear envelope around the separated chromosomes then leads to the formation of distinct nuclear bodies. Although not as gracefully orchestrated or efficient as conventional mitosis in fission yeast or other eukaryotes, this alternative form of nuclear division may have evolutionary implications. Unlike in eukaryotes, bacterial and archaeal cells segregate their DNA without a mitotic spindle. Instead, bacterial chromosome segregation is driven by polymerization of an actin-like protein. By showing that S. pombe can undergo nuclear fission without microtubules, Nurse and his colleagues may have peeled back the mitotic spindle overlay to provide a glimpse of an evolutionarily distant intermediate process in which cell division occurred when replicated sister chromosomes remained attached to centrosomes or spindle pole bodies at the nuclear envelope and were segregated as these structures moved apart. Further studies will reveal whether processes similar to nuclear fission in S. pombe occur in other eukaryotes, indicating that it could be an ancestral means of cell division prior to the evolution of the mitotic spindle. Castagnetti S, Oliferenko S, Nurse P (2010) Fission yeast cells undergo nuclear division in the absence of spindle microtubules. doi10.1371/journal.pbio.1000512

Modulation of the Kinesin ATPase Cycle by Neck Linker Docking and Microtubule Binding

Kinesin motor proteins use an ATP hydrolysis cycle to perform various functions in eukaryotic cells. Many questions remain about how the kinesin mechanochemical ATPase cycle is fine-tuned for specific work outputs. In this study, we use isothermal titration calorimetry and stopped-flow fluorometry to determine and analyze the thermodynamics of the human kinesin-5 (Eg5/KSP) ATPase cycle. In the absence of microtubules, the binding interactions of kinesin-5 with both ADP product and ATP substrate involve significant enthalpic gains coupled to smaller entropic penalties. However, when the wild-type enzyme is titrated with a non-hydrolyzable ATP analog or the enzyme is mutated such that it is able to bind but not hydrolyze ATP, substrate binding is 10-fold weaker than ADP binding because of a greater entropic penalty due to the structural rearrangements of switch 1, switch 2, and loop L5 on ATP binding. We propose that these rearrangements are reversed upon ATP hydrolysis and phosphate release. In addition, experiments on a truncated kinesin-5 construct reveal that upon nucleotide binding, both the N-terminal cover strand and the neck linker interact to modulate kinesin-5 nucleotide affinity. Moreover, interactions with microtubules significantly weaken the affinity of kinesin-5 for ADP without altering the affinity of the enzyme for ATP in the absence of ATP hydrolysis. Together, these results define the energy landscape of a kinesin ATPase cycle in the absence and presence of microtubules and shed light on the role of molecular motor mechanochemistry in cellular microtubule dynamics.

Microtubule-assisted mechanism for functional metabolic macromolecular complex formation

Evidence has been presented for a metabolic multienzyme complex, the purinosome, that participates in de novo purine biosynthesis to form clusters in the cytoplasm of living cells under purine-depleted conditions. Here we identified, using fluorescent live cell imaging, that a microtubule network appears to physically control the spatial distribution of purinosomes in the cytoplasm. Application of a cell-based assay measuring the rate of de novo purine biosynthesis confirmed that the metabolic activity of purinosomes was significantly suppressed in the absence of microtubules. Collectively, we propose a microtubule-assisted mechanism for functional purinosome formation in HeLa cells.

Centrosome biogenesis continues in the absence of microtubules during prolonged S‐phase arrest

When CHO cells are arrested in S-phase, they undergo repeated rounds of centrosome duplication without cell-cycle progression. While the increase is slow and asynchronous, the number of centrosomes in these cells does rise with time. To investigate mechanisms controlling this duplication, we have arrested CHO cells in S-phase for up to 72 h, and coordinately inhibited new centriole formation by treatment with the microtubule poison colcemid. We find that in such cells, the pre-existing centrosomes remain, and a variable number of foci--containing alpha/gamma-tubulin and centrin 2--assemble at the nuclear periphery. When the colcemid is washed out, the nuclear-associated foci disappear, and cells assemble new centriole-containing centrosomes, which accumulate the centriole scaffold protein SAS-6, nucleate microtubule asters, and form functional mitotic spindle poles. The number of centrosomes that assemble following colcemid washout increases with duration of S-phase arrest, even though the number of nuclear-associated foci or pre-existing centrosomes does not increase. This suggests that during S-phase, a cryptic generative event occurs repeatedly, even in the absence of new triplet microtubule assembly. When triplet microtubule assembly is restored, these cryptic generative events become realized, and multiple centriole-containing centrosomes assemble.

A Developmentally Regulated Two-Step Process Generates a Noncentrosomal Microtubule Network in Drosophila Tracheal Cells

Microtubules (MTs) are essential for many cell features, such as polarity, motility, shape, and vesicle trafficking. Therefore, in a multicellular organism, their organization differs between cell types and during development; however, the control of this process remains elusive. Here, we show that during Drosophila tracheal morphogenesis, MT reorganization is coupled to relocalization of the microtubule organizing centers (MTOC) components from the centrosome to the apical cell domain from where MTs then grow. We reveal that this process is controlled by the trachealess patterning gene in a two-step mechanism. MTOC components are first released from the centrosome by the activity of the MT-severing protein Spastin, and then anchored apically through the transmembrane protein Piopio. We further show that these changes are essential for tracheal development, thus stressing the functional relevance of MT reorganization for morphogenesis.

The mechanics behind plant development

Morphogenesis in living organisms relies on the integration of both biochemical and mechanical signals. During the last decade, attention has been mainly focused on the role of biochemical signals in patterning and morphogenesis, leaving the contribution of mechanics largely unexplored. Fortunately, the development of new tools and approaches has made it possible to re-examine these processes. In plants, shape is defined by two local variables: growth rate and growth direction. At the level of the cell, these variables depend on both the cell wall and turgor pressure. Multidisciplinary approaches have been used to understand how these cellular processes are integrated in the growing tissues. These include quantitative live imaging to measure growth rate and direction in tissues with cellular resolution. In parallel, stress patterns have been artificially modified and their impact on strain and cell behavior been analysed. Importantly, computational models based on analogies with continuum mechanics systems have been useful in interpreting the results. In this review, we will discuss these issues focusing on the shoot apical meristem, a population of stem cells that is responsible for the initiation of the aerial organs of the plant.

New spindle morphogenesis model by Dynein, Nudel, and the spindle matrix

New spindle morphogenesis model by Dynein, Nudel, and the spindle matrix

RANBP2 is an allosteric activator of the conventional kinesin‐1 motor protein, KIF5B, in a minimal cell‐free system

The association of cargoes to kinesins is thought to promote kinesin activation, yet the validation of such a model with native cargoes is lacking because none is known to activate kinesins directly in an in vitro system of purified components. The RAN-binding protein 2 (RANBP2), through its kinesin-binding domain (KBD), associates in vivo with kinesin-1, KIF5B/KIF5C. Here, we show that KBD and its flanking domains, RAN GTPase-binding domains 2 and 3 (RBD2/RBD3), activate the ATPase activity of KIF5B approximately 30-fold in the presence of microtubules and ATP. The activation kinetics of KIF5B by RANBP2 is biphasic and highly cooperative. Deletion of one of its RBDs lowers the activation of KIF5B threefold and abolishes cooperativity. Remarkably, RBD2-KBD-RBD3 induces unfolding and modest activation of KIF5B in the absence of microtubules. Hence, RANBP2 is the first native and positive allosteric activator known to jump-start and boost directly the activity of a kinesin.

EPR Spectroscopy Shows That There Are Multiple Closed Nucleotide Pocket States When Kinesin-family Motors Bind To Microtubules With A Triphosphate At The Motor Nucleotide Site

Identifying the conformational changes in kinesin-family motors associated with the binding of nucleotide and microtubules is essential to determining an atomic-level model for the production of force and motion by these molecular motors. We have previously used EPR spectroscopy and diphosphate-state nucleotide-analog spin probes to show that the open nucleotide site closes when kinesin-family motors bind to microtubules. We have extended these studies to kinesin and ncd motors in ATP-analog states. Upon binding to microtubules, probe mobility decreases implying a closing of the nucleotide site. Comparison with the microtubule•motor•diphosphate state shows two differences: 1. In the triphosphate state, there are two spectral components implying two different closed conformations of the nucleotide pocket. The equilibrium between the conformations is independent of temperature. 2. The probe mobilities in the closed triphosphate states are less than those seen in the diphosphate state implying a further closing of the nucleotide pocket in the triphosphate state. 3. To correlate better spectroscopy and structure, molecular dynamics simulations were done on the motor-probe complex. We find the simulated probe mobility for the open x-ray structure is comparable to that observed experimentally in the absence of microtubules. Minehardt et al (2001) proposed an ncd structure with a closed phosphate tube based on myosin x-ray structures. Simulated probe mobility from this structure is comparable to the most immobile of the microtubule•kinesin-motor•triphosphate states. We conclude that the nucleotide pocket is in a closed conformation when kinesin-family motors are bound to nucleotide and microtubules, with the triphosphate states more tightly closed. The most tightly closed state has a phosphate-tube structure apparently similar to myosin.

Quantifying Neurite Growth Mediated by Interactions among Secretory Vesicles, Microtubules, and Actin Networks

Neurite growth is a fundamental process of neuronal development, which requires both membrane expansions by exocytosis and cytoskeletal dynamics. However, the specific contribution of these processes has not been yet assessed quantitatively. To study and quantify the growth process, we construct a biophysical model in which we relate the overall neurite outgrowth rate to the vesicle dynamics. By considering the complex motion of vesicles in the cell soma, we demonstrate from biophysical consideration that the main step of finding the neurite initiation site relies mainly on a two-dimensional diffusion/sequestration/fusion at the cell surface and we obtain a novel formula for the flux of vesicles at the neurite base. In the absence of microtubules, we show that a nascent neurite initiated by vesicular delivery can only reach a small length. By adding the microtubule dynamics to the secretory pathway and using stochastic analysis and simulations, we study the complex dynamics of neurite growth. Within this model, depending on the coupling parameter between the microtubules and the neurite, we find different regimes of growth, which describe dendritic and axonal growth. To validate one aspect of our model, we demonstrate that the experimental flux of TI-VAMP but not Synaptobrevin 2 vesicles contributes to the neurite growth. We conclude that although vesicles can be generated randomly in the cell body, the search for the neurite position using the microtubule network and diffusion is quite fast. Furthermore, when the TI-VAMP vesicular flow is large enough, the interactions between the microtubule bundle and the neurite control the growth process. In addition, all of these processes intimately cooperate to mediate the various modes of neurite growth: the model predicts three different growing modes including, in addition to the stable axonal growth and the stochastic dendritic growth, a fast oscillatory regime. Finally our study demonstrates that cytoskeletal dynamics is necessary to generate long protrusion, while vesicular delivery alone can only generate small neurite.

Mitotic exit in the absence of separase activity.

In budding yeast, three interdigitated pathways regulate mitotic exit (ME): mitotic cyclin-cyclin-dependent kinase (Cdk) inactivation; the Cdc14 early anaphase release (FEAR) network, including a nonproteolytic function of separase (Esp1); and the mitotic exit network (MEN) driven by interaction between the spindle pole body and the bud cortex. Here, we evaluate the contributions of these pathways to ME kinetics. Reducing Cdk activity is critical for ME, and the MEN contributes strongly to ME efficiency. Esp1 contributes to ME kinetics mainly through cohesin cleavage: the Esp1 requirement can be largely bypassed if cells are provided Esp1-independent means of separating sister chromatids. In the absence of Esp1 activity, we observed only a minor ME delay consistent with a FEAR defect. Esp1 overexpression drives ME in Cdc20-depleted cells arrested in metaphase. We have found that this activity of overexpressed Esp1 depended on spindle integrity and the MEN. We defined the first quantitative measure for Cdc14 release based on colocalization with the Net1 nucleolar anchor. This measure indicates efficient Cdc14 release upon MEN activation; release driven by Esp1 in the absence of microtubules was inefficient and incapable of driving ME. We also found a novel role for the MEN: activating Cdc14 nuclear export, even in the absence of Net1.

Nucleotide-Induced Flexibility Change in Neck Linkers of Dimeric Kinesin as Detected by Distance Measurements Using Spin-Labeling EPR

Using dipolar continuous-wave and pulsed electron paramagnetic resonance methods, we have determined the distribution of the distances between two spin labels placed on the middle of each of the neck linkers of dimeric kinesin. In the absence of microtubules, the distance was centered at 3.3 nm, but displayed a broad distribution with a width of 2.7 nm. This broad distribution implies that the linkers are random coils and extend well beyond the 2.5-nm distance expected of crystal structures. In the presence of microtubules, two linker populations were found: one similar to that observed in the absence of microtubules (a broad distribution centered at 3.3 nm), and the second population with a narrower distribution centered at 1.3–2.5 nm. In the absence of nucleotide but in the presence of microtubules, ∼ 40% of the linkers were at a distance centered at 1.9 nm with a 1.2-nm width; the remaining fraction was at 3.3 nm, as before. This suggests that neck linkers exhibit dynamics covering a wide distance range between 1.0 and 5.0 nm. In the presence of ATP analogs adenosine 5′-(β,γ-imido)triphosphate and adenosine 5′-(γ-thio)triphosphate, 40–50% of the spins showed a very narrow distribution centered at 1.6 nm, with a width of 0.4–0.5 nm. The remaining population displayed the broad 3.3-nm distribution. Under these conditions, a large fraction of linkers are docked firmly onto a motor core or microtubule, while the remainder is disordered. We propose that large nucleotide-dependent flexibility changes in the linkers contribute to the directional bias of the kinesin molecule stepping 8 nm along the microtubule.