The kinesin-4 member KIF7 plays critical roles in Hedgehog signaling in vertebrate cells. KIF7 is an atypical kinesin as it binds to microtubules but is immotile. We demonstrate that, like conventional kinesins, KIF7 is regulated by autoinhibition as the full-length protein is inactive for microtubule binding in cells. We identify a segment, the inhibitory coiled coil (inhCC), that is required for autoinhibition of KIF7 whereas the adjacent regulatory coiled coil (rCC) that contributes to autoinhibition of the motile kinesin-4's KIF21A and KIF21B, is not sufficient for KIF7 autoinhibition. Disease-associated mutations in the inhCC relieve autoinhibition and result in strong microtubule binding. Surprisingly, uninhibited KIF7 proteins did not bind preferentially to or track the plus ends of growing microtubules in cells, as suggested by previous in vitro work, but rather bound along cytosolic and axonemal microtubules. Localization to the tip of the primary cilium also required the inhCC and could be increased by disease-associated mutations regardless of the protein's autoinhibition state. These findings suggest that loss of KIF7 autoinhibition and/or altered cilium tip localization can contribute to pathogenesis of human disease.

ABSTRACT Axons and dendrites are distinguished by microtubule polarity. In Drosophila , dendrites are dominated by minus-end-out microtubules, whereas axons contain plus-end-out microtubules. Local nucleation in dendrites generates microtubules in both orientations. To understand why dendritic nucleation does not disrupt polarity, we used live imaging to analyze the fate of microtubules generated at branch points. We found that they had different rates of success exiting the branch based on orientation: correctly oriented minus-end-out microtubules succeeded in leaving about twice as often as incorrectly oriented microtubules. Increased success relied on other microtubules in a parallel orientation. From a candidate screen, we identified Trim9 and kinesin-5 (Klp61F) as machinery that promoted growth of new microtubules. In S2 cells, Eb1 recruited Trim9 to microtubules. Klp61F promoted microtubule growth in vitro and in vivo , and could recruit Trim9 in S2 cells. In summary, the data argue that Trim9 and kinesin-5 act together at microtubule plus ends to help polymerizing microtubules parallel to pre-existing ones resist catastrophe.

ABSTRACT Radial microtubule (MT) arrays or asters determine cell geometry in animal cells. Multiple asters interacting with motors, such as those in syncytia, form intracellular patterns, but the mechanical principles behind this are not clear. Here, we report that oocytes of the marine ascidian Phallusia mammillata treated with the drug BI-D1870 spontaneously form cytoplasmic MT asters, or cytasters. These asters form steady state segregation patterns in a shell just under the membrane. Cytaster centers tessellate the oocyte cytoplasm, that is divide it into polygonal structures, dominated by hexagons, in a kinesin-5-dependent manner, while inter-aster MTs form ‘mini-spindles’. A computational model of multiple asters interacting with kinesin-5 can reproduce both tessellation patterns and mini-spindles in a manner specific to the number of MTs per aster, MT lengths and kinesin-5 density. Simulations predict that the hexagonal tessellation patterns scale with increasing cell size, when the packing fraction of asters in cells is ∼1.6. This self-organized in vivo tessellation by cytasters is comparable to the ‘circle packing problem’, suggesting that there is an intrinsic mechanical pattern-forming module that is potentially relevant to understanding the role of collective mechanics of cytoskeletal elements in embryogenesis. This article has an associated First Person interview with the first author of the paper.

ABSTRACT During anaphase, a microtubule-containing structure called the midzone forms between the segregating chromosomes. The midzone is composed of an antiparallel array of microtubules and numerous microtubule-associated proteins that contribute to midzone formation and function. In many cells, the midzone is an important source of signals that specify the location of contractile ring assembly and constriction. The midzone also contributes to the events of anaphase by generating forces that impact chromosome segregation and spindle elongation; some midzone components contribute to both processes. The results of recent experiments have increased our understanding of the importance of the midzone, a microtubule array that has often been overlooked. This Journal of Cell Science at a Glance article will review, and illustrate on the accompanying poster, the organization, formation and dynamics of the midzone, and discuss open questions for future research.

ABSTRACT Cell and tissue functions rely on the genetic programmes and cascades of biochemical signals. It has become evident during the past decade that the physical properties of soft material that govern the mechanics of cells and tissues play an important role in cellular function and morphology. The biophysical properties of cells and tissues are determined by the cytoskeleton, consisting of dynamic networks of F-actin and microtubules, molecular motors, crosslinkers and other associated proteins, among other factors such as cell–cell interactions. The Drosophila syncytial embryo represents a simple pseudo-tissue, with its nuclei orderly embedded in a structured cytoskeletal matrix at the embryonic cortex with no physical separation by cellular membranes. Here, we review the stereotypic dynamics and regulation of the cytoskeleton in Drosophila syncytial embryos and how cytoskeletal dynamics underlies biophysical properties and the emergence of collective features. We highlight the specific features and processes of syncytial embryos and discuss the applicability of biophysical approaches.

ABSTRACT Tubulin enters the cilium by diffusion and motor-based intraflagellar transport (IFT). However, the respective contribution of each route in providing tubulin for axonemal assembly remains unknown. Using Chlamydomonas , we attenuated IFT-based tubulin transport of GFP–β-tubulin by altering the IFT74N–IFT81N tubulin-binding module and the C-terminal E-hook of tubulin. E-hook-deficient GFP–β-tubulin was incorporated into the axonemal microtubules, but its transport frequency by IFT was reduced by ∼90% in control cells and essentially abolished when the tubulin-binding site of IFT81 was incapacitated. Despite the strong reduction in IFT, the proportion of E-hook-deficient GFP–β-tubulin in the axoneme was only moderately reduced. In vivo imaging showed more GFP–β-tubulin particles entering cilia by diffusion than by IFT. Extrapolated to endogenous tubulin, the data indicate that diffusion provides most of the tubulin required for axonemal assembly. We propose that IFT of tubulin is nevertheless needed for ciliogenesis, because it augments the tubulin pool supplied to the ciliary tip by diffusion, thus ensuring that free tubulin there is maintained at the critical concentration for plus-end microtubule assembly during rapid ciliary growth.

ABSTRACT Giardia lamblia , a widespread parasitic protozoan, attaches to the host gastrointestinal epithelium by using the ventral disc, a complex microtubule (MT) organelle. The ‘cup-like’ disc is formed by a spiral MT array that scaffolds numerous disc-associated proteins (DAPs) and higher-order protein complexes. In interphase, the disc is hyperstable and has limited MT dynamics; however, it remains unclear how DAPs confer these properties. To investigate mechanisms of hyperstability, we confirmed the disc-specific localization of over 50 new DAPs identified by using both a disc proteome and an ongoing GFP localization screen. DAPs localize to specific disc regions and many lack similarity to known proteins. By screening 14 CRISPRi-mediated DAP knockdown (KD) strains for defects in hyperstability and MT dynamics, we identified two strains – DAP5188KD and DAP6751KD –with discs that dissociate following high-salt fractionation. Discs in the DAP5188KD strain were also sensitive to treatment with the MT-polymerization inhibitor nocodazole. Thus, we confirm here that at least two of the 87 known DAPs confer hyperstable properties to the disc MTs, and we anticipate that other DAPs contribute to disc MT stability, nucleation and assembly.

ABSTRACT During cell division, misaligned chromosomes are captured and aligned by motors before their segregation. The CENP-E motor is recruited to polar unattached kinetochores to facilitate chromosome alignment. The spindle checkpoint protein BubR1 (also known as BUB1B) has been reported as a CENP-E interacting partner, but the extent to which BubR1 contributes to CENP-E localization at kinetochores has remained controversial. Here we define the molecular determinants that specify the interaction between BubR1 and CENP-E. The basic C-terminal helix of BubR1 is necessary but not sufficient for CENP-E interaction, and a minimal key acidic patch on the kinetochore-targeting domain of CENP-E is also essential. We then demonstrate that BubR1 is required for the recruitment of CENP-E to kinetochores to facilitate chromosome alignment. This BubR1–CENP-E axis is critical for alignment of chromosomes that have failed to congress through other pathways and recapitulates the major known function of CENP-E. Overall, our studies define the molecular basis and the function for CENP-E recruitment to BubR1 at kinetochores during mammalian mitosis. This article has an associated First Person interview with the first author of the paper.

ABSTRACT The radial spoke is a key element in a transducer apparatus controlling the motility of eukaryotic cilia. The transduction biomechanics is a long-standing question in cilia biology. The radial spoke has three regions – a spoke head, a bifurcated neck and a stalk. Although the neck and the stalk are asymmetric, twofold symmetry of the head has remained controversial. In this work we used single particle cryo-electron microscopy (cryo-EM) analysis to generate a 3D structure of the whole radial spoke at unprecedented resolution. We show the head region at 15 Å (1.5 nm) resolution and confirm twofold symmetry. Using distance constraints generated by cross-linking mass spectrometry, we locate two components, RSP2 and RSP4, at the head and neck regions. Our biophysical analysis of isolated RSP4, RSP9, and RSP10 affirmed their oligomeric state. Our results enable us to redefine the boundaries of the regions and propose a model of organization of the radial spoke component proteins.

ABSTRACT Osteoclasts are giant multinucleated myeloid cells specialized for bone resorption, which is essential for the preservation of bone health throughout life. The activity of osteoclasts relies on the typical organization of osteoclast cytoskeleton components into a highly complex structure comprising actin, microtubules and other cytoskeletal proteins that constitutes the backbone of the bone resorption apparatus. The development of methods to differentiate osteoclasts in culture and manipulate them genetically, as well as improvements in cell imaging technologies, has shed light onto the molecular mechanisms that control the structure and dynamics of the osteoclast cytoskeleton, and thus the mechanism of bone resorption. Although essential for normal bone physiology, abnormal osteoclast activity can cause bone defects, in particular their hyper-activation is commonly associated with many pathologies, hormonal imbalance and medical treatments. Increased bone degradation by osteoclasts provokes progressive bone loss, leading to osteoporosis, with the resulting bone frailty leading to fractures, loss of autonomy and premature death. In this context, the osteoclast cytoskeleton has recently proven to be a relevant therapeutic target for controlling pathological bone resorption levels. Here, we review the present knowledge on the regulatory mechanisms of the osteoclast cytoskeleton that control their bone resorption activity in normal and pathological conditions.

ABSTRACT Glucocorticoids (GCs) act through the glucocorticoid receptor (GR, also known as NR3C1) to regulate immunity, energy metabolism and tissue repair. Upon ligand binding, activated GR mediates cellular effects by regulating gene expression, but some GR effects can occur rapidly without new transcription. Here, we show that GCs rapidly inhibit cell migration, in response to both GR agonist and antagonist ligand binding. The inhibitory effect on migration is prevented by GR knockdown with siRNA, confirming GR specificity, but not by actinomycin D treatment, suggesting a non-transcriptional mechanism. We identified a rapid onset increase in microtubule polymerisation following GC treatment, identifying cytoskeletal stabilisation as the likely mechanism of action. HDAC6 overexpression, but not knockdown of αTAT1, rescued the GC effect, implicating HDAC6 as the GR effector. Consistent with this hypothesis, ligand-dependent cytoplasmic interaction between GR and HDAC6 was demonstrated by quantitative imaging. Taken together, we propose that activated GR inhibits HDAC6 function, and thereby increases the stability of the microtubule network to reduce cell motility. We therefore report a novel, non-transcriptional mechanism whereby GCs impair cell motility through inhibition of HDAC6 and rapid reorganization of the cell architecture. This article has an associated First Person interview with the first author of the paper.

ABSTRACT To segregate the chromosomes faithfully during cell division, cells assemble a spindle that captures the kinetochores and pulls them towards opposite poles. Proper spindle function requires correct interplay between microtubule motors and non-motor proteins. Defects in spindle assembly or changes in spindle dynamics are associated with diseases, such as cancer or developmental disorders. Here, we compared mitotic and meiotic spindles in fission yeast. We show that, even though mitotic and meiotic spindles underwent the typical three phases of spindle elongation, they have distinct features. We found that the relative concentration of the kinesin-14 family protein Pkl1 is decreased in meiosis I compared to mitosis, while the concentration of the kinesin-5 family protein Cut7 remains constant. We identified the second kinesin-14 family protein Klp2 and microtubule dynamics as factors necessary for proper meiotic spindle assembly. This work defines the differences between mitotic and meiotic spindles in fission yeast Schizosaccharomyces pombe , and provides prospect for future comparative studies. This article has an associated First Person interview with the first author of the paper.

ABSTRACT In cells, microtubule dynamics are regulated by plus-end tracking proteins (+TIPs). End-binding protein 1 (EB1, also known as MAPRE1) acts as a master regulator of +TIP networks by targeting the growing ends of microtubules and recruiting other factors. However, the molecular mechanism underlying high-affinity binding of EB1 to microtubule ends remains an open area of research. Using single-molecule imaging, we show that the end-binding kinetics of EB1 change when the polymerization and hydrolysis rates of tubulin dimers are altered, confirming that EB1 binds to GTP–tubulin and/or GDP– P i –tubulin at microtubule growing ends. The affinity of wild-type EB1 to these sites is higher than that of monomeric EB1 mutants, suggesting that both calponin homology domains present in the EB1 dimer contribute to end binding. Introduction of phosphomimetic mutations into the EB1 linker domain weakens the end-binding affinity and confers a more curved conformation on the EB1 dimer without compromising dimerization, suggesting that the overall architecture of EB1 is important for its end-binding affinity. Taken together, our results provide insights into how the high-affinity end-binding of EB1 is achieved and how this activity may be regulated in cells.

ABSTRACT Previously, we have shown that bulk microtubule (MT) movement correlates with neurite elongation, and blocking either dynein activity or MT assembly inhibits both processes. However, whether the contributions of MT dynamics and dynein activity to neurite elongation are separate or interdependent is unclear. Here, we investigated the underlying mechanism by testing the roles of dynein and MT assembly in neurite elongation of Aplysia and chick neurites using time-lapse imaging, fluorescent speckle microscopy, super-resolution imaging and biophysical analysis. Pharmacologically inhibiting either dynein activity or MT assembly reduced neurite elongation rates as well as bulk and individual MT anterograde translocation. Simultaneously suppressing both processes did not have additive effects, suggesting a shared mechanism of action. Single-molecule switching nanoscopy revealed that inhibition of MT assembly decreased the association of dynein with MTs. Finally, inhibiting MT assembly prevented the rise in tension induced by dynein inhibition. Taken together, our results suggest that MT assembly is required for dynein-driven MT translocation and neurite outgrowth.

ABSTRACT Formins are actin regulators critical for diverse processes across eukaryotes. With many formins in plants and animals, it has been challenging to determine formin function in vivo . We found that the phylogenetically distinct class I integral membrane formins (denoted For1) from the moss P. patens enrich at sites of membrane turnover, with For1D more tightly associated with the plasma membrane than For1A. To probe formin function, we generated formin-null lines with greatly reduced formin complexity. We found that For1A and For1D help to anchor actin near the cell apex, with For1A contributing to formation of cytosolic actin, while For1D contributes to plasma membrane-associated actin. At the cortex, For1A and For1D localized to motile puncta and differentially impacted actin dynamics. We found that class I cortical formin mobility depended on microtubules and only moderately on actin, whereas class II formin (denoted For2) mobility solely depended on actin. Moreover, cortical For2A tightly correlated with the puncta labeled by the endocytic membrane dye FM4-64, and null mutants in class I formins did not affect uptake of a similar dye, FM1-43, suggesting that class I and II formins are involved in distinct membrane trafficking pathways.

ABSTRACT Motile cilia generate directed hydrodynamic flow that is important for the motility of cells and extracellular fluids. To optimize directed hydrodynamic flow, motile cilia are organized and oriented into a polarized array. Basal bodies (BBs) nucleate and position motile cilia at the cell cortex. Cytoplasmic BB-associated microtubules are conserved structures that extend from BBs. By using the ciliate, Tetrahymena thermophila , combined with EM-tomography and light microscopy, we show that BB-appendage microtubules assemble coincidently with new BB assembly and that they are attached to the cell cortex. These BB-appendage microtubules are specifically marked by post translational modifications of tubulin, including glycylation. Mutations that prevent glycylation shorten BB-appendage microtubules and disrupt BB positioning and cortical attachment. Consistent with the attachment of BB-appendage microtubules to the cell cortex to position BBs, mutations that disrupt the cellular cortical cytoskeleton disrupt the cortical attachment and positioning of BBs. In summary, BB-appendage microtubules promote the organization of ciliary arrays through attachment to the cell cortex.

ABSTRACT Microtubules are cytoskeletal filaments essential for numerous aspects of cell physiology. They are polarized polymeric tubes with a fast growing plus end and a slow growing minus end. In this Cell Science at a Glance article and the accompanying poster, we review the current knowledge on the dynamics and organization of microtubule minus ends. Several factors, including the γ-tubulin ring complex, CAMSAP/Patronin, ASPM/Asp, SPIRAL2 (in plants) and the KANSL complex recognize microtubule minus ends and regulate their nucleation, stability and interactions with partners, such as microtubule severing enzymes, microtubule depolymerases and protein scaffolds. Together with minus-end-directed motors, these microtubule minus-end targeting proteins (−TIPs) also control the formation of microtubule-organizing centers, such as centrosomes and spindle poles, and mediate microtubule attachment to cellular membrane structures, including the cell cortex, Golgi complex and the cell nucleus. Structural and functional studies are starting to reveal the molecular mechanisms by which dynamic −TIP networks control microtubule minus ends.

ABSTRACT During mitosis, the cell sequentially constructs two microtubule-based spindles to ensure faithful segregation of chromosomes. A bipolar spindle first pulls apart the sister chromatids, then a central spindle further separates them away. Although the assembly of the first spindle is well described, the assembly of the second remains poorly understood. We report here that the inhibition of Aurora A leads to an absence of the central spindle resulting from a lack of nucleation of microtubules in the midzone. In the absence of Aurora A, the HURP (also known as DLGAP5) and NEDD1 proteins that are involved in nucleation of microtubules fail to concentrate in the midzone. HURP is an effector of RanGTP, whereas NEDD1 serves as an anchor for the γ-tubulin ring complex (γTURC). Interestingly, Aurora A phosphorylates HURP and NEDD1 during assembly of the initial bipolar spindle. We show here that the expression of a NEDD1 isoform mimicking phosphorylation by Aurora A is sufficient to restore microtubule nucleation in the midzone under conditions of Aurora A inhibition. These results reveal a new control mechanism of microtubule nucleation by Aurora A during assembly of the central spindle.

ABSTRACT During plant cytokinesis a radially expanding membrane-enclosed cell plate is formed from fusing vesicles that compartmentalizes the cell in two. How fusion is spatially restricted to the site of cell plate formation is unknown. Aggregation of cell-plate membrane starts near regions of microtubule overlap within the bipolar phragmoplast apparatus of the moss Physcomitrella patens . Since vesicle fusion generally requires coordination of vesicle tethering and subsequent fusion activity, we analyzed the subcellular localization of several subunits of the exocyst, a tethering complex active during plant cytokinesis. We found that the exocyst complex subunit Sec6 but not the Sec3 or Sec5 subunits localized to microtubule overlap regions in advance of cell plate construction in moss. Moreover, Sec6 exhibited a conserved physical interaction with an ortholog of the Sec1/Munc18 protein KEULE, an important regulator for cell-plate membrane vesicle fusion in Arabidopsis . Recruitment of the P. patens protein KEULE and vesicles to the early cell plate was delayed upon Sec6 gene silencing. Our findings, thus, suggest that vesicle-vesicle fusion is, in part, enabled by a pool of exocyst subunits at microtubule overlaps, which is recruited independently of vesicle delivery.

ABSTRACT The specific organization of the neuronal microtubule cytoskeleton in axons and dendrites is an evolutionarily conserved determinant of neuronal polarity that allows for selective cargo sorting. However, how dendritic microtubules are organized and whether local differences influence cargo transport remains largely unknown. Here, we use live-cell imaging to systematically probe the microtubule organization in Caenorhabditis elegans neurons, and demonstrate the contribution of distinct mechanisms in the organization of dendritic microtubules. We found that most non-ciliated neurons depend on unc-116 (kinesin-1), unc-33 (CRMP) and unc-44 (ankyrin) for correct microtubule organization and polarized cargo transport, as previously reported. Ciliated neurons and the URX neuron, however, use an additional pathway to nucleate microtubules at the tip of the dendrite, from the base of the cilium in ciliated neurons. Since inhibition of distal microtubule nucleation affects distal dendritic transport, we propose a model in which the presence of a microtubule-organizing center at the dendrite tip ensures correct dendritic cargo transport.