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Background: The first evidence of micro- and nanoplastic (MNP) exposure in the human placenta is emerging. However, the toxicokinetics and toxicity of MNPs in the placenta, specifically environmentally relevant particles, remain unclear., Objectives: We examined the transport, uptake, and toxicity of pristine and experimentally weathered MNPs in nonsyncytialized and syncytialized BeWo b30 choriocarcinoma cells., Methods: We performed untargeted chemical characterization of pristine and weathered MNPs using liquid chromatography high-resolution mass spectrometry to evaluate compositional differences following particle weathering. We investigated cellular internalization of pristine and weathered polystyrene (PS; 0.05 - 10 μ m ) and high-density polyethylene (HDPE; 0 - 80 μ m ) particles using high-resolution confocal imaging and three-dimensional rendering. We investigated the influence of particle coating with human plasma on the cellular transport of PS particles using a transwell setup and examined the influence of acute MNP exposure on cell viability, damage to the plasma membrane, and expression of genes involved in steroidogenesis., Results: Chemical characterization of MNPs showed a significantly higher number of unique features in pristine particles in comparison with weathered particles. Size-dependent placental uptake of pristine and weathered MNPs was observed in both placental cell types after 24 h exposure. Cellular transport was limited and size-dependent and was not influenced by particle coating with human plasma. None of the MNPs affected cell viability. Damage to the plasma membrane was observed only for 0.05 μ m PS particles in the nonsyncytialized cells at the highest concentration tested ( 100 μ g / mL ). Modest down-regulation of hsd17b1 was observed in syncytialized cells exposed to pristine MNPs., Discussion: Our results suggest that pristine and weathered MNPs are internalized and translocated in placental cells in vitro . Effects on gene expression observed upon pristine PS and HDPE particle exposure warrant further examination. More in-depth investigations are needed to better understand the potential health risks of MNP and chemicals associated with them under environmentally relevant exposure scenarios. https://doi.org/10.1289/EHP10873.

Insulin secretion in pancreatic β-cells is regulated by cortical complexes that are enriched at the sites of adhesion to extracellular matrix facing the vasculature. Many components of these complexes, including bassoon, RIM, ELKS and liprins, are shared with neuronal synapses. Here, we show that insulin secretion sites also contain the non-neuronal proteins LL5β (also known as PHLDB2) and KANK1, which, in migrating cells, organize exocytotic machinery in the vicinity of integrin-based adhesions. Depletion of LL5β or focal adhesion disassembly triggered by myosin II inhibition perturbed the clustering of secretory complexes and attenuated the first wave of insulin release. Although previous analyses in vitro and in neurons have suggested that secretory machinery might assemble through liquid-liquid phase separation, analysis of endogenously labeled ELKS in pancreatic islets indicated that its dynamics is inconsistent with such a scenario. Instead, fluorescence recovery after photobleaching and single-molecule imaging showed that ELKS turnover is driven by binding and unbinding to low-mobility scaffolds. Both the scaffold movements and ELKS exchange were stimulated by glucose treatment. Our findings help to explain how integrin-based adhesions control spatial organization of glucose-stimulated insulin release., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Microtubules are dynamic cytoskeletal polymers that spontaneously switch between phases of growth and shrinkage. The probability of transitioning from growth to shrinkage, termed catastrophe, increases with microtubule age, but the underlying mechanisms are poorly understood. Here, we set out to test whether microtubule lattice defects formed during polymerization can affect growth at the plus end. To generate microtubules with lattice defects, we used microtubule-stabilizing agents that promote formation of polymers with different protofilament numbers. By employing different agents during nucleation of stable microtubule seeds and the subsequent polymerization phase, we could reproducibly induce switches in protofilament number and induce stable lattice defects. Such drug-induced defects led to frequent catastrophes, which were not observed when microtubules were grown in the same conditions but without a protofilament number mismatch. Microtubule severing at the site of the defect was sufficient to suppress catastrophes. We conclude that structural defects within the microtubule lattice can exert effects that can propagate over long distances and affect the dynamic state of the microtubule end., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Degradation of aggregates by selective autophagy is important as damaged proteins may impose a threat to cellular homeostasis. Although the core components of the autophagy machinery are well characterized, the spatiotemporal regulation of many selective autophagy processes, including aggrephagy, remains largely unexplored. Furthermore, because most live-cell imaging studies have so far focused on starvation-induced autophagy, little is known about the dynamics of aggrephagy. Here, we describe the development and application of the mKeima-PIM assay, which enables live-cell observation of autophagic turnover and degradation of inducible protein aggregates in conjunction with key autophagy players. This allowed us to quantify the relative timing and duration of different steps of aggrephagy in human cells and revealed the short-lived nature of the autophagosome. The assay furthermore showed the spatial distribution of omegasome formation, highlighting that autophagy initiation is directly instructed by the cargo. Moreover, we found that nascent autophagosomes mostly remain immobile until acidification occurs. Thus, our assay provides new insights into the spatiotemporal regulation and dynamics of aggrephagy. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

The neuronal microtubule cytoskeleton underlies the polarization and proper functioning of neurons, amongst others by providing tracks for motor proteins that drive intracellular transport. Different subsets of neuronal microtubules, varying in composition, stability, and motor preference, are known to exist, but the high density of microtubules has so far precluded mapping their relative abundance and three-dimensional organization. Here, we use different super-resolution techniques (STED, Expansion Microscopy) to explore the nanoscale organization of the neuronal microtubule network in rat hippocampal neurons. This revealed that in dendrites acetylated microtubules are enriched in the core of the dendritic shaft, while tyrosinated microtubules are enriched near the plasma membrane, thus forming a shell around the acetylated microtubules. Moreover, using a novel analysis pipeline we quantified the absolute number of acetylated and tyrosinated microtubules within dendrites and found that they account for 65-75% and ~20-30% of all microtubules, respectively, leaving only few microtubules that do not fall in either category. Because these different microtubule subtypes facilitate different motor proteins, these novel insights help to understand the spatial regulation of intracellular transport., Competing Interests: EK, DJ, DS, LK No competing interests declared, (© 2021, Katrukha et al.)

Axon formation critically relies on local microtubule remodeling and marks the first step in establishing neuronal polarity. However, the function of the microtubule-organizing centrosomes during the onset of axon formation is still under debate. Here, we demonstrate that centrosomes play an essential role in controlling axon formation in human-induced pluripotent stem cell (iPSC)-derived neurons. Depleting centrioles, the core components of centrosomes, in unpolarized human neuronal stem cells results in various axon developmental defects at later stages, including immature action potential firing, mislocalization of axonal microtubule-associated Trim46 proteins, suppressed expression of growth cone proteins, and affected growth cone morphologies. Live-cell imaging of microtubules reveals that centriole loss impairs axonal microtubule reorganization toward the unique parallel plus-end out microtubule bundles during early development. We propose that centrosomes mediate microtubule remodeling during early axon development in human iPSC-derived neurons, thereby laying the foundation for further axon development and function., (© 2021 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Bunyaviruses have a genome that is divided over multiple segments. Genome segmentation complicates the generation of progeny virus, since each newly formed virus particle should preferably contain a full set of genome segments in order to disseminate efficiently within and between hosts. Here, we combine immunofluorescence and fluorescence in situ hybridization techniques to simultaneously visualize bunyavirus progeny virions and their genomic content at single-molecule resolution in the context of singly infected cells. Using Rift Valley fever virus and Schmallenberg virus as prototype tri-segmented bunyaviruses, we show that bunyavirus genome packaging is influenced by the intracellular viral genome content of individual cells, which results in greatly variable packaging efficiencies within a cell population. We further show that bunyavirus genome packaging is more efficient in insect cells compared to mammalian cells and provide new insights on the possibility that incomplete particles may contribute to bunyavirus spread as well.

Intracellular transport relies on multiple kinesins, but it is poorly understood which kinesins are present on particular cargos, what their contributions are and whether they act simultaneously on the same cargo. Here, we show that Rab6-positive secretory vesicles are transported from the Golgi apparatus to the cell periphery by kinesin-1 KIF5B and kinesin-3 KIF13B, which determine the location of secretion events. KIF5B plays a dominant role, whereas KIF13B helps Rab6 vesicles to reach freshly polymerized microtubule ends, to which KIF5B binds poorly, likely because its cofactors, MAP7-family proteins, are slow in populating these ends. Sub-pixel localization demonstrated that during microtubule plus-end directed transport, both kinesins localize to the vesicle front and can be engaged on the same vesicle. When vesicles reverse direction, KIF13B relocates to the middle of the vesicle, while KIF5B shifts to the back, suggesting that KIF5B but not KIF13B undergoes a tug-of-war with a minus-end directed motor., Competing Interests: AS, MM, EK, IG, CP, QL, PH, YY, VS, IS, EM, LK No competing interests declared, LP Reviewing editor, eLife, AA Deputy editor, eLife, (© 2020, Serra-Marques et al.)

Microtubule-dependent organization of membranous organelles occurs through motor-based pulling and by coupling microtubule dynamics to membrane remodeling. For example, tubules of endoplasmic reticulum (ER) can be extended by kinesin- and dynein-mediated transport and through the association with the tips of dynamic microtubules. The binding between ER and growing microtubule plus ends requires End Binding (EB) proteins and the transmembrane protein STIM1, which form a tip-attachment complex (TAC), but it is unknown whether these proteins are sufficient for membrane remodeling. Furthermore, EBs and their partners undergo rapid turnover at microtubule ends, and it is unclear how highly transient protein-protein interactions can induce load-bearing processive motion. Here, we reconstituted membrane tubulation in a minimal system with giant unilamellar vesicles, dynamic microtubules, an EB protein, and a membrane-bound protein that can interact with EBs and microtubules. We showed that these components are sufficient to drive membrane remodeling by three mechanisms: membrane tubulation induced by growing microtubule ends, motor-independent membrane sliding along microtubule shafts, and membrane pulling by shrinking microtubules. Experiments and modeling demonstrated that the first two mechanisms can be explained by adhesion-driven biased membrane spreading on microtubules. Optical trapping revealed that growing and shrinking microtubule ends can exert forces of ∼0.5 and ∼5 pN, respectively, through attached proteins. Rapidly exchanging molecules that connect membranes to dynamic microtubules can thus bear a sufficient load to induce membrane deformation and motility. Furthermore, combining TAC components and a membrane-attached kinesin in the same in vitro assays demonstrated that they can cooperate in promoting membrane tubule extension., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Single-molecule localization microscopy (SMLM) enables fluorescent microscopy with nanometric resolution. While localizing molecules close to the coverslip is relatively straightforward using high numerical aperture (NA) oil immersion (OI) objectives, optical aberrations impede SMLM deeper in watery samples. Adaptive optics (AO) with a deformable mirror (DM) can be used to correct such aberrations and to induce precise levels of astigmatism to encode the z- position of molecules. Alternatively, the use of water immersion (WI) objectives might be sufficient to limit the most dominant aberrations. Here we compare SMLM at various depths using either WI or OI with or without AO. In addition, we compare the performance of a cylindrical lens and a DM for astigmatism-based z- encoding. We find that OI combined with adaptive optics improves localization precision beyond the performance of WI-based imaging and enables deep (>10 µm) 3D localization., Competing Interests: The authors declare that there are no conflicts of interest related to this article., (© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement.)

The axon initial segment (AIS) is a unique neuronal compartment that plays a crucial role in the generation of action potential and neuronal polarity. The assembly of the AIS requires membrane, scaffolding, and cytoskeletal proteins, including Ankyrin-G and TRIM46. How these components cooperate in AIS formation is currently poorly understood. Here, we show that Ankyrin-G acts as a scaffold interacting with End-Binding (EB) proteins and membrane proteins such as Neurofascin-186 to recruit TRIM46-positive microtubules to the plasma membrane. Using in vitro reconstitution and cellular assays, we demonstrate that TRIM46 forms parallel microtubule bundles and stabilizes them by acting as a rescue factor. TRIM46-labeled microtubules drive retrograde transport of Neurofascin-186 to the proximal axon, where Ankyrin-G prevents its endocytosis, resulting in stable accumulation of Neurofascin-186 at the AIS. Neurofascin-186 enrichment in turn reinforces membrane anchoring of Ankyrin-G and subsequent recruitment of TRIM46-decorated microtubules. Our study reveals feedback-based mechanisms driving AIS assembly., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

In neurons, the continuous and dynamic endoplasmic reticulum (ER) network extends throughout the axon, and its dysfunction causes various axonopathies. However, it remains largely unknown how ER integrity and remodeling modulate presynaptic function in mammalian neurons. Here, we demonstrated that ER membrane receptors VAPA and VAPB are involved in modulating the synaptic vesicle (SV) cycle. VAP interacts with secernin-1 (SCRN1) at the ER membrane via a single FFAT-like motif. Similar to VAP, loss of SCRN1 or SCRN1-VAP interactions resulted in impaired SV cycling. Consistently, SCRN1 or VAP depletion was accompanied by decreased action potential-evoked Ca 2+ responses. Additionally, we found that VAP-SCRN1 interactions play an important role in maintaining ER continuity and dynamics, as well as presynaptic Ca 2+ homeostasis. Based on these findings, we propose a model where the ER-localized VAP-SCRN1 interactions provide a novel control mechanism to tune ER remodeling and thereby modulate Ca 2+ dynamics and SV cycling at presynaptic sites. These data provide new insights into the molecular mechanisms controlling ER structure and dynamics, and highlight the relevance of ER function for SV cycling., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Kinesin-1 is responsible for microtubule-based transport of numerous cellular cargoes. Here, we explored the regulation of kinesin-1 by MAP7 proteins. We found that all four mammalian MAP7 family members bind to kinesin-1. In HeLa cells, MAP7, MAP7D1, and MAP7D3 act redundantly to enable kinesin-1-dependent transport and microtubule recruitment of the truncated kinesin-1 KIF5B-560, which contains the stalk but not the cargo-binding and autoregulatory regions. In vitro, purified MAP7 and MAP7D3 increase microtubule landing rate and processivity of kinesin-1 through transient association with the motor. MAP7 proteins promote binding of kinesin-1 to microtubules both directly, through the N-terminal microtubule-binding domain and unstructured linker region, and indirectly, through an allosteric effect exerted by the kinesin-binding C-terminal domain. Compared with MAP7, MAP7D3 has a higher affinity for kinesin-1 and a lower affinity for microtubules and, unlike MAP7, can be cotransported with the motor. We propose that MAP7 proteins are microtubule-tethered kinesin-1 activators, with which the motor transiently interacts as it moves along microtubules., (© 2019 Hooikaas et al.)

Selective types of autophagy mediate the clearance of specific cellular components and are essential to maintain cellular homeostasis. However, tools to directly induce and monitor such pathways are limited. Here we introduce the PIM (particles induced by multimerization) assay as a tool for the study of aggrephagy, the autophagic clearance of aggregates. The assay uses an inducible multimerization module to assemble protein clusters, which upon induction recruit ubiquitin, p62, and LC3 before being delivered to lysosomes. Moreover, use of a dual fluorescent tag allows for the direct observation of cluster delivery to the lysosome. Using flow cytometry and fluorescence microscopy, we show that delivery to the lysosome is partially dependent on p62 and ATG7. This assay will help in elucidating the spatiotemporal dynamics and control mechanisms underlying aggregate clearance by the autophagy-lysosomal system.

In neurons, microtubules form dense bundles and run along the length of axons and dendrites. Occasionally, dendritic microtubules can grow from the shaft directly into dendritic spines. Microtubules target dendritic spines that are undergoing activity-dependent changes, but the mechanism by which microtubules enter spines has remained poorly understood. Using live-cell imaging, high-resolution microscopy, and local glutamate uncaging, we show that local actin remodeling at the base of a spine promotes microtubule spine targeting. Microtubule spine entry is triggered by activation of N-Methyl-D-aspartic acid (NMDA) receptors and calcium influx and requires dynamic actin remodeling. Activity-dependent translocation of the actin remodeling protein cortactin out of the spine correlates with increased microtubule targeting at a single spine level. Our data show that the structural changes in the actin cytoskeleton at the base of the spine are directly involved in microtubule entry and emphasize the importance of actin-microtubule crosstalk in orchestrating synapse function and plasticity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The dynamic instability of microtubules plays a key role in controlling their organization and function, but the cellular mechanisms regulating this process are poorly understood. Here, we show that cytoplasmic linker-associated proteins (CLASPs) suppress transitions from microtubule growth to shortening, termed catastrophes, including those induced by microtubule-destabilizing agents and physical barriers. Mammalian CLASPs encompass three TOG-like domains, TOG1, TOG2, and TOG3, none of which bind to free tubulin. TOG2 is essential for catastrophe suppression, whereas TOG3 mildly enhances rescues but cannot suppress catastrophes. These functions are inhibited by the C-terminal domain of CLASP2, while the TOG1 domain can release this auto-inhibition. TOG2 fused to a positively charged microtubule-binding peptide autonomously accumulates at growing but not shrinking ends, suppresses catastrophes, and stimulates rescues. CLASPs suppress catastrophes by stabilizing growing microtubule ends, including incomplete ones, preventing their depolymerization and promoting their recovery into complete tubes. TOG2 domain is the key determinant of these activities., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Microtubules control different aspects of cell polarization. In cells with a radial microtubule system, a pivotal role in setting up asymmetry is attributed to the relative positioning of the centrosome and the nucleus. Here, we show that centrosome loss had no effect on the ability of endothelial cells to polarize and move in 2D and 3D environments. In contrast, non-centrosomal microtubules stabilized by the microtubule minus-end-binding protein CAMSAP2 were required for directional migration on 2D substrates and for the establishment of polarized cell morphology in soft 3D matrices. CAMSAP2 was also important for persistent endothelial cell sprouting during in vivo zebrafish vessel development. In the absence of CAMSAP2, cell polarization in 3D could be partly rescued by centrosome depletion, indicating that in these conditions the centrosome inhibited cell polarity. We propose that CAMSAP2-protected non-centrosomal microtubules are needed for establishing cell asymmetry by enabling microtubule enrichment in a single-cell protrusion., Competing Interests: MM, AV, JW, EK No competing interests declared, AA Senior editor, eLife, (© 2018, Martin et al.)

The position of the mitotic spindle is tightly controlled in animal cells as it determines the plane and orientation of cell division. Contacts between cytoplasmic dynein and astral microtubules (MTs) at the cell cortex generate pulling forces that position the spindle. An evolutionarily conserved Gα-GPR-1/2 Pins/LGN -LIN-5 Mud/NuMA cortical complex interacts with dynein and is required for pulling force generation, but the dynamics of this process remain unclear. In this study, by fluorescently labeling endogenous proteins in Caenorhabditis elegans embryos, we show that dynein exists in two distinct cortical populations. One population directly depends on LIN-5, whereas the other is concentrated at MT plus ends and depends on end-binding (EB) proteins. Knockout mutants lacking all EBs are viable and fertile and display normal pulling forces and spindle positioning. However, EB protein-dependent dynein plus end tracking was found to contribute to force generation in embryos with a partially perturbed dynein function, indicating the existence of two mechanisms that together create a highly robust force-generating system., (© 2017 Schmidt et al.)

Polarized cargo transport is essential for neuronal function. However, the minimal basic components required for selective cargo sorting and distribution in neurons remain elusive. We found that in sensory neurons the axon initial segment is largely absent and that microtubule-associated protein 2 (MAP2) defines the cargo-filtering zone in the proximal axon. Here, MAP2 directs axonal cargo entry by coordinating the activities of molecular motors. We show that distinct kinesins differentially regulate cargo velocity: kinesin-3 drives fast axonal cargo trafficking, while kinesin-1 slows down axonal cargo transport. MAP2 inhibits "slow" kinesin-1 motor activity and allows kinesin-3 to drive robust cargo transport from the soma into the axon. In the distal axon, the inhibitory action of MAP2 decreases, leading to regained kinesin-1 activity and vesicle distribution. We propose that selective axonal cargo trafficking requires the MAP2-defined pre-axonal filtering zone and the ability of cargos to switch between distinct kinesin motor activities., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The cytoplasm is a highly complex and heterogeneous medium that is structured by the cytoskeleton. How local transport depends on the heterogeneous organization and dynamics of F-actin and microtubules is poorly understood. Here we use a novel delivery and functionalization strategy to utilize quantum dots (QDs) as probes for active and passive intracellular transport. Rapid imaging of non-functionalized QDs reveals two populations with a 100-fold difference in diffusion constant, with the faster fraction increasing upon actin depolymerization. When nanobody-functionalized QDs are targeted to different kinesin motor proteins, their trajectories do not display strong actin-induced transverse displacements, as suggested previously. Only kinesin-1 displays subtle directional fluctuations, because the subset of microtubules used by this motor undergoes prominent undulations. Using actin-targeting agents reveals that F-actin suppresses most microtubule shape remodelling, rather than promoting it. These results demonstrate how the spatial heterogeneity of the cytoskeleton imposes large variations in non-equilibrium intracellular dynamics.

Microtubules are dynamic polymers that in cells can grow, shrink or pause, but the factors that promote pausing are poorly understood. Here, we show that the mammalian kinesin-4 KIF21B is a processive motor that can accumulate at microtubule plus ends and induce pausing. A few KIF21B molecules are sufficient to induce strong growth inhibition of a microtubule plus end in vitro. This property depends on non-motor microtubule-binding domains located in the stalk region and the C-terminal WD40 domain. The WD40-containing KIF21B tail displays preference for a GTP-type over a GDP-type microtubule lattice and contributes to the interaction of KIF21B with microtubule plus ends. KIF21B also contains a motor-inhibiting domain that does not fully block the interaction of the protein with microtubules, but rather enhances its pause-inducing activity by preventing KIF21B detachment from microtubule tips. Thus, KIF21B combines microtubule-binding and regulatory activities that together constitute an autonomous microtubule pausing factor.

Microtubules regulate signaling, trafficking, and cell mechanics, but the respective contribution of these functions to cell morphogenesis and migration in 3D matrices is unclear. Here, we report that the microtubule plus-end tracking protein (+TIP) SLAIN2, which suppresses catastrophes, is not required for 2D cell migration but is essential for mesenchymal cell invasion in 3D culture and in a mouse cancer model. We show that SLAIN2 inactivation does not affect Rho GTPase activity, trafficking, and focal adhesion formation. However, SLAIN2-dependent catastrophe inhibition determines microtubule resistance to compression and pseudopod elongation. Another +TIP, CLASP1, is also needed to form invasive pseudopods because it prevents catastrophes specifically at their tips. When microtubule growth persistence is reduced, inhibition of depolymerization is sufficient for pseudopod maintenance but not remodeling. We propose that catastrophe inhibition by SLAIN2 and CLASP1 supports mesenchymal cell shape in soft 3D matrices by enabling microtubules to perform a load-bearing function., Competing Interests: The authors declare no competing financial interests., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The epidermal growth factor receptor (EGFR) represents one of the most common target proteins in anti-cancer therapy. To directly examine the structural and dynamical properties of EGFR activation by the epidermal growth factor (EGF) in native membranes, we have developed a solid-state nuclear magnetic resonance (ssNMR)-based approach supported by dynamic nuclear polarization (DNP). In contrast to previous crystallographic results, our experiments show that the ligand-free state of the extracellular domain (ECD) is highly dynamic, while the intracellular kinase domain (KD) is rigid. Ligand binding restricts the overall and local motion of EGFR domains, including the ECD and the C-terminal region. We propose that the reduction in conformational entropy of the ECD by ligand binding favors the cooperative binding required for receptor dimerization, causing allosteric activation of the intracellular tyrosine kinase., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The Golgi apparatus controls the formation of non-centrosomal microtubule arrays important for Golgi organization, polarized transport, cell motility, and cell differentiation. Here, we show that CAMSAP2 stabilizes and attaches microtubule minus ends to the Golgi through a complex of AKAP450 and myomegalin. CLASPs stabilize CAMSAP2-decorated microtubules but are not required for their Golgi tethering. AKAP450 is also essential for Golgi microtubule nucleation, and myomegalin and CDK5RAP2 but not CAMSAP2 contribute to this function. In the absence of centrosomes, AKAP450- and CAMSAP2-dependent pathways of microtubule minus-end organization become dominant, and the presence of at least one of them is needed to maintain microtubule density. Strikingly, a compact Golgi can be assembled in the absence of both centrosomal and Golgi microtubules. However, CAMSAP2- and AKAP450-dependent Golgi microtubules facilitate Golgi reorientation and cell invasion in a 3D matrix. We propose that Golgi-anchored microtubules are important for polarized cell movement but not for coalescence of Golgi membranes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Tight regulation of kinesin activity is crucial and malfunction is linked to neurological diseases. Point mutations in the KIF21A gene cause congenital fibrosis of the extraocular muscles type 1 (CFEOM1) by disrupting the autoinhibitory interaction between the motor domain and a regulatory region in the stalk. However, the molecular mechanism underlying the misregulation of KIF21A activity in CFEOM1 is not understood. Here, we show that the KIF21A regulatory domain containing all disease-associated substitutions in the stalk forms an intramolecular antiparallel coiled coil that inhibits the kinesin. CFEOM1 mutations lead to KIF21A hyperactivation by affecting either the structural integrity of the antiparallel coiled coil or the autoinhibitory binding interface, thereby reducing its affinity for the motor domain. Interaction of the KIF21A regulatory domain with the KIF21B motor domain and sequence similarities to KIF7 and KIF27 strongly suggest a conservation of this regulatory mechanism in other kinesin-4 family members.

Microtubules are dynamic polymers built of tubulin dimers that attach in a head-to-tail fashion to form protofilaments, which further associate laterally to form a tube. Asynchronous elongation of individual protofilaments can potentially lead to an altered microtubule-end structure that promotes sudden depolymerization, termed catastrophe [1-4]. However, how the dynamics of individual protofilaments relates to overall growth persistence has remained unclear. Here, we used the microtubule targeting anti-cancer drug Eribulin [5-7] to explore the consequences of stalled protofilament elongation on microtubule growth. Using X-ray crystallography, we first revealed that Eribulin binds to a site on β-tubulin that is required for protofilament plus-end elongation. Based on the structural information, we engineered a fluorescent Eribulin molecule. We demonstrate that single Eribulin molecules specifically interact with microtubule plus ends and are sufficient to either trigger a catastrophe or induce slow and erratic microtubule growth in the presence of EB3. Interestingly, we found that Eribulin increases the frequency of EB3 comet "splitting," transient events where a slow and erratically progressing comet is followed by a faster comet. This observation possibly reflects the "healing" of a microtubule lattice. Because EB3 comet splitting was also observed in control microtubules in the absence of any drugs, we propose that Eribulin amplifies a natural pathway toward catastrophe by promoting the arrest of protofilament elongation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Centrioles are fundamental and evolutionarily conserved microtubule-based organelles whose assembly is characterized by microtubule growth rates that are orders of magnitude slower than those of cytoplasmic microtubules. Several centriolar proteins can interact with tubulin or microtubules, but how they ensure the exceptionally slow growth of centriolar microtubules has remained mysterious. Here, we bring together crystallographic, biophysical, and reconstitution assays to demonstrate that the human centriolar protein CPAP (SAS-4 in worms and flies) binds and "caps" microtubule plus ends by associating with a site of β-tubulin engaged in longitudinal tubulin-tubulin interactions. Strikingly, we uncover that CPAP activity dampens microtubule growth and stabilizes microtubules by inhibiting catastrophes and promoting rescues. We further establish that the capping function of CPAP is important to limit growth of centriolar microtubules in cells. Our results suggest that CPAP acts as a molecular lid that ensures slow assembly of centriolar microtubules and, thereby, contributes to organelle length control., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Axon formation, the initial step in establishing neuronal polarity, critically depends on local microtubule reorganization and is characterized by the formation of parallel microtubule bundles. How uniform microtubule polarity is achieved during axonal development remains an outstanding question. Here, we show that the tripartite motif containing (TRIM) protein TRIM46 plays an instructive role in the initial polarization of neuronal cells. TRIM46 is specifically localized to the newly specified axon and, at later stages, partly overlaps with the axon initial segment (AIS). TRIM46 specifically forms closely spaced parallel microtubule bundles oriented with their plus-end out. Without TRIM46, all neurites have a dendrite-like mixed microtubule organization resulting in Tau missorting and altered cargo trafficking. By forming uniform microtubule bundles in the axon, TRIM46 is required for neuronal polarity and axon specification in vitro and in vivo. Thus, TRIM46 defines a unique axonal cytoskeletal compartment for regulating microtubule organization during neuronal development., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Microtubules are hollow biopolymers of 25-nm diameter and are key constituents of the cytoskeleton. In neurons, microtubules are organized differently between axons and dendrites, but their precise organization in different compartments is not completely understood. Super-resolution microscopy techniques can detect specific structures at an increased resolution, but the narrow spacing between neuronal microtubules poses challenges because most existing labelling strategies increase the effective microtubule diameter by 20-40 nm and will thereby blend neighbouring microtubules into one structure. Here we develop single-chain antibody fragments (nanobodies) against tubulin to achieve super-resolution imaging of microtubules with a decreased apparent diameter. To test the resolving power of these novel probes, we generate microtubule bundles with a known spacing of 50-70 nm and successfully resolve individual microtubules. Individual bundled microtubules can also be resolved in different mammalian cells, including hippocampal neurons, allowing novel insights into fundamental mechanisms of microtubule organization in cell- and neurobiology.

In neurons, most microtubules are not associated with a central microtubule-organizing center (MTOC), and therefore, both the minus and plus-ends of these non-centrosomal microtubules are found throughout the cell. Microtubule plus-ends are well established as dynamic regulatory sites in numerous processes, but the role of microtubule minus-ends has remained poorly understood. Using live-cell imaging, high-resolution microscopy, and laser-based microsurgery techniques, we show that the CAMSAP/Nezha/Patronin family protein CAMSAP2 specifically localizes to non-centrosomal microtubule minus-ends and is required for proper microtubule organization in neurons. CAMSAP2 stabilizes non-centrosomal microtubules and is required for neuronal polarity, axon specification, and dendritic branch formation in vitro and in vivo. Furthermore, we found that non-centrosomal microtubules in dendrites are largely generated by γ-Tubulin-dependent nucleation. We propose a two-step model in which γ-Tubulin initiates the formation of non-centrosomal microtubules and CAMSAP2 stabilizes the free microtubule minus-ends in order to control neuronal polarity and development., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Microtubules are cytoskeletal polymers with two structurally and functionally distinct ends, the plus- and the minus-end. Here, we focus on the mechanisms underlying the regulation of microtubule minus-ends by the CAMSAP/Nezha/Patronin protein family. We show that CAMSAP2 is required for the proper organization and stabilization of interphase microtubules and directional cell migration. By combining live-cell imaging and in vitro reconstitution of microtubule assembly from purified components with laser microsurgery, we demonstrate that CAMSAPs regulate microtubule minus-end growth and are specifically deposited on the lattice formed by microtubule minus-end polymerization. This process leads to the formation of CAMSAP-decorated microtubule stretches, which are stabilized from both ends and serve as sites of noncentrosomal microtubule outgrowth. The length of the stretches is regulated by the microtubule-severing protein katanin, which interacts with CAMSAPs. Our data thus indicate that microtubule minus-end assembly drives the stabilization of noncentrosomal microtubules and that katanin regulates this process., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Proper organization of microtubule networks depends on microtubule-associated proteins and motors that use different spatial cues to guide microtubule growth [1-3]. For example, it has been proposed that the uniform minus-end-out microtubule organization in dendrites of Drosophila neurons is maintained by steering of polymerizing microtubules along the stable ones by kinesin-2 motors bound to growing microtubule plus ends [4]. To explore the mechanics of kinesin-guided microtubule growth, we reconstituted this process in vitro. In the presence of microtubule plus-end tracking EB proteins, a constitutively active kinesin linked to the EB-interacting motif SxIP effectively guided polymerizing microtubules along other microtubules both in cells and in vitro. Experiments combined with modeling revealed that at angles larger than 90°, guidance efficiency is determined by the force needed for microtubule bending. At angles smaller than 90°, guidance requires microtubule growth, and guidance efficiency depends on the ability of kinesins to maintain contact between the two microtubules despite the geometrical constraints imposed by microtubule length and growth rate. Our findings provide a conceptual framework for understanding microtubule guidance during the generation of different types of microtubule arrays., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Mechanisms controlling microtubule dynamics at the cell cortex play a crucial role in cell morphogenesis and neuronal development. Here, we identified kinesin-4 KIF21A as an inhibitor of microtubule growth at the cell cortex. In vitro, KIF21A suppresses microtubule growth and inhibits catastrophes. In cells, KIF21A restricts microtubule growth and participates in organizing microtubule arrays at the cell edge. KIF21A is recruited to the cortex by KANK1, which coclusters with liprin-α1/β1 and the components of the LL5β-containing cortical microtubule attachment complexes. Mutations in KIF21A have been linked to congenital fibrosis of the extraocular muscles type 1 (CFEOM1), a dominant disorder associated with neurodevelopmental defects. CFEOM1-associated mutations relieve autoinhibition of the KIF21A motor, and this results in enhanced KIF21A accumulation in axonal growth cones, aberrant axon morphology, and reduced responsiveness to inhibitory cues. Our study provides mechanistic insight into cortical microtubule regulation and suggests that altered microtubule dynamics contribute to CFEOM1 pathogenesis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Axon regeneration after injury requires the extensive reconstruction, reorganization, and stabilization of the microtubule cytoskeleton in the growth cones. Here, we identify KIF3C as a key regulator of axonal growth and regeneration by controlling microtubule dynamics and organization in the growth cone. KIF3C is developmentally regulated. Rat embryonic sensory axons and growth cones contain undetectable levels of KIF3C protein that is locally translated immediately after injury. In adult neurons, KIF3C is axonally transported from the cell body and is enriched at the growth cone where it preferentially binds to tyrosinated microtubules. Functionally, the interaction of KIF3C with EB3 is necessary for its localization at the microtubule plus-ends in the growth cone. Depletion of KIF3C in adult neurons leads to an increase in stable, overgrown and looped microtubules because of a strong decrease in the microtubule frequency of catastrophes, suggesting that KIF3C functions as a microtubule-destabilizing factor. Adult axons lacking KIF3C, by RNA interference or KIF3C gene knock-out, display an impaired axonal outgrowth in vitro and a delayed regeneration after injury both in vitro and in vivo. Murine KIF3C knock-out embryonic axons grow normally but do not regenerate after injury because they are unable to locally translate KIF3C. These data show that KIF3C is an injury-specific kinesin that contributes to axon growth and regeneration by regulating and organizing the microtubule cytoskeleton in the growth cone.

Microtubule-targeting agents (MTAs) are widely used for treatment of cancer and other diseases, and a detailed understanding of the mechanism of their action is important for the development of improved microtubule-directed therapies. Although there is a large body of data on the interactions of different MTAs with purified tubulin and microtubules, much less is known about how the effects of MTAs are modulated by microtubule-associated proteins. Among the regulatory factors with a potential to have a strong impact on MTA activity are the microtubule plus end-tracking proteins, which control multiple aspects of microtubule dynamic instability. Here, we reconstituted microtubule dynamics in vitro to investigate the influence of end-binding proteins (EBs), the core components of the microtubule plus end-tracking protein machinery, on the effects that MTAs exert on microtubule plus-end growth. We found that EBs promote microtubule catastrophe induction in the presence of all MTAs tested. Analysis of microtubule growth times supported the view that catastrophes are microtubule age dependent. This analysis indicated that MTAs affect microtubule aging in multiple ways: destabilizing MTAs, such as colchicine and vinblastine, accelerate aging in an EB-dependent manner, whereas stabilizing MTAs, such as paclitaxel and peloruside A, induce not only catastrophes but also rescues and can reverse the aging process.

Intracellular transport is driven by motor proteins that either use microtubules or actin filaments as their tracks, but the interplay between these transport pathways is poorly understood. Whereas many microtubule-based motors are known to drive long-range transport, several actin-based motors have been proposed to function predominantly in cargo tethering. How these opposing activities are integrated on cargoes that contain both types of motors is unknown. Here we use inducible intracellular transport assays to show that acute recruitment of myosin-V to kinesin-propelled cargo reduces their motility near the cell periphery and enhances their localization at the actin-rich cell cortex. Myosin-V arrests rapid microtubule-based transport without the need for regulated auto- or other inhibition of kinesin motors. In addition, myosin-V, despite being an ineffective long-range transporter, can drive slow, medium-range (1-5 μm), point-to-point transport in cortical cell regions. Altogether, these data support a model in which myosin-V establishes local cortical delivery of kinesin-bound cargos through a combination of tethering and active transport., (Copyright © 2013 Elsevier Ltd. All rights reserved.)