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Maintenance of active longevity, preservation of physical activity, and prevention of decreased mobility associated with injury or age of patients are among the most urgent tasks for modern healthcare. The suppression of pathological processes and activation of defense systems at the cellular and organismal levels are the main routes for solving these problems. Several initial anti-aging therapy approaches are detoxification, rheocorrection, and immunocorrection. In these areas, methods of extracorporeal hemocorrection, in particular, therapeutic plasmapheresis, are effective. This study aimed to evaluate the effectiveness of hardware plasmapheresis with albumin compensation by assessing the dynamics of circulating age-related biomarker levels in randomly selected patients. Twenty human subjects of both sexes aged 40–55 years with an increase in one or more aging-related biomarkers participated in this study. The patients were randomly divided into two groups with ten people each. Patients from Group 1 underwent therapeutic plasmapheresis with albumin replacement (four procedures with a 2-day interval). Patients from Group 2 were offered plasmapheresis treatment with saline replacement. The levels of aging-related biomarkers were determined in the blood of patients before and 30 days after starting treatment. Preliminary data showed that plasmapheresis with albumin replacement in randomly selected male and female patients was accompanied by normalization of the selected aging biomarkers. Thirty days after the start of the plasmapheresis treatment, a decrease in both biological and phenotypic age was determined. Further studies are needed to investigate the effects of nutritional factors on aging biomarkers with and without plasmapheresis treatment. Based on the obtained results, recommendations will be made on the use of plasmapheresis in preventive and sports medicine. The use of this method will help reduce the biological age of patients and, as a result, reduce the risks of developing age-related diseases and disabilities and contribute to prolonging life and improving its quality.

To maintain normal blood glucose levels, pancreatic beta cells secrete insulin into the bloodstream at specialized regions at the cell periphery, often called secretion hot spots. While many secretory machinery components are located all over the cell membrane, directed secretion relies on distinct cortical patches of the scaffolding protein ELKS and the microtubule (MT)-anchoring protein LL5β. However, using TIRF microscopy of intact mouse islets to precisely localize secretion events within ELKS/LL5β patches, we now show that secretion is restricted to only 5% of ELKS/LL5β patch area. Moreover, the majority of secretion occurs at the margins of ELKS patches. This suggests that additional factor(s) must be responsible for hot spot definition. Because the MT cytoskeleton plays a regulatory role in the insulin secretion process via both delivery and removal of secretory granules from the secretion sites, we test whether local MT organization defines secretory activity at hot spots. We find that the majority of secretion events occur at regions devoid of MTs. Based on our findings, we present a model in which local MT disassembly and optimal ELKS content are strong predictors of directed insulin secretion.

We have reported that the microtubule (MT) network in β cells attenuates this function by withdrawing insulin secretory granules (ISGs) away from the plasma membrane. Thus, high glucose-induced MT remodeling is required for robust glucose-stimulated insulin secretion (GSIS). We now show that α-cell secreted hormones, Gcg and/or Glp1, regulate the MT stability in β cells. Activating the receptors of Gcg or Glp1 (GcgR or Glp1R) with chemical agonists induces MT destabilization in β ells in the absence of high glucose. In contrast, inhibiting these receptors with antagonists attenuates high glucose-induced MT destabilization. Supporting the significance of this regulation, the MT networks in β cells of islets with higher α/β cell ratio are less stable than those with lower α/β cell ratio. Within each individual islet, β cells that are located close to α cells show faster MTs remodeling upon glucose stimulation than those away. Consequently, islets with higher α/β cell ratio secrete more insulin in response to high glucose and plasma membrane depolarization, which is recapitulated by direct Gcg stimulation. These combined results reveal a new MT-dependent pathway by which α cells, using Gcg and or Glp1-mediated paracrine signaling, tune β-cell secretion. In addition, the different α-β cell ratios in individual islets lead to their heterogeneous secretory responses, which may be important for handling secretory function needs under different physiological conditions., Highlights: Gcg sensitizes glucose-induced MT remodeling in mouse and human β cellsMT density in single islets anti-correlates with α/β cell ratioGSIS levels in single islets positively correlate with α/β cell ratioDifferent α/β cell ratio contributes to heterogeneity of single islet GSIS.

Pancreatic islet β cells preferentially secrete insulin toward the plasma membrane, making contact with the capillary extracellular matrix (ECM). Isolated islets separated from the exocrine acinar cells are the best system for cell biology studies of primary β cells, whereas isolated islets lose their capillary network during ex vivo culture. Providing the appropriate extracellular signaling by attaching islets to vascular ECM-coated surfaces can restore the polarized insulin secretion toward the ECM. The guided secretion toward ECM-coated glass coverslips provides a good model for recording insulin secretion in real time to study its regulation. Additionally, β cells attached to the ECM-coated coverslips are suitable for confocal live imaging of subcellular components including adhesion molecules, cytoskeleton, and ion channels. This procedure is also compatible for total internal reflection fluorescence (TIRF) microscopy, which provides optimal signal-to-noise ratio and high spatial precision of structures close to the plasma membrane. In this article, we describe the optimized protocol for vascular ECM-coating of glass coverslips and the process of attachment of isolated mouse islets on the coverslip. This preparation is compatible with any high-resolution microscopy of live primary β cells. Key features • Optimized coating procedure to attach isolated islets, compatible for both confocal and TIRF microscopy. • The ECM-coated glass coverslip functions as the artificial capillary surface to guide secretion toward the coated surface for optimal imaging of secretion events. • Shows the process of islets attachment to the ECM-coated surface in a 6-day ex vivo culture., Competing Interests: Competing interestsThe authors declare no competing interests., (©Copyright : © 2023 The Authors; This is an open access article under the CC BY license.)

Obesity is a major public health problem that requires new approaches. Despite all interventions, the behavioural and therapeutic interventions developed have demonstrated limited effectiveness in curbing the obesity epidemic. Findings from imaging studies of the brain suggest the existence of neural vulnerabilities and structural changes that are associated with the development of obesity and eating disorders. This review highlights the clinical relevance of brain neuroimaging research in obese individuals to prevent risky behaviour, early diagnosis, and the development of new safer and more effective treatments.

Pancreatic β cell secretion of insulin is crucial to the maintenance of glucose homeostasis and prevention of diseases related to glucose regulation, including diabetes. Pancreatic β cells accomplish efficient insulin secretion by clustering secretion events at the cell membrane facing the vasculature. Regions at the cell periphery characterized by clustered secretion are currently termed insulin secretion hot spots. Several proteins, many associated with the microtubule and actin cytoskeletons, are known to localize to and serve specific functions at hot spots. Among these proteins are the scaffolding protein ELKS, the membrane-associated proteins LL5β and liprins, the focal adhesion-associated protein KANK1, and other factors typically associated with the presynaptic active zone in neurons. These hot spot proteins have been shown to contribute to insulin secretion, but many questions remain regarding their organization and dynamics at hot spots. Current studies suggest microtubule- and F-actin are involved in regulation of hot spot proteins and their function in secretion. The hot spot protein association with the cytoskeleton networks also suggests a potential role for mechanical regulation of these proteins and hot spots in general. This perspective summarizes the existing knowledge of known hot spot proteins, their cytoskeletal-mediated regulation, and discuss questions remaining regarding mechanical regulation of pancreatic beta cell hot spots., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fye and Kaverina.)

Glucose stimulation induces the remodeling of microtubules, which potentiates insulin secretion in pancreatic β-cells. CAMSAP2 binds to microtubule minus ends to stabilize microtubules in several cultured clonal cells. Here, we report that the knockdown of CAMSAP2 in primary β-cells reduces total insulin content and attenuates GSIS without affecting the releasability of insulin vesicles. Surprisingly, CAMSAP2 knockdown does not change microtubule stability. Unlike in cultured insulinoma cells, CAMSAP2 in primary β-cells predominantly localizes to the Golgi apparatus instead of microtubule minus ends. This novel localization is specific to primary β- but not α-cells and is independent of microtubule binding. Consistent with its specific localization at the Golgi, CAMSAP2 promotes efficient Golgi-ER trafficking in primary β-cells. Moreover, primary β-cells and insulinoma cells likely express different CAMSAP2 isoforms. We propose that a novel CAMSAP2 isoform in primary β-cells has a non-canonical function, which promotes Golgi-ER trafficking to support efficient production of insulin and secretion., Competing Interests: The authors declare no conflicts of interest., (© 2023 The Authors.)

The vertebrate Golgi complex is a large dynamic organelle which undergoes morphological changes and fragmentation both as a part of normal physiological dynamics and under disease conditions. The Golgi is known to have a functionally important relationship with the centrosome. The extent of the spatial association between these two organelles varies in a dynamic and regulated manner. It is essential to have a reliable unbiased approach to evaluate Golgi volume, Golgi extension/scattering in the 3D cell space, and spatial association of the Golgi with the centrosome. It is also important that each of these features is evaluated by a simple metric, one measurement per cell, so that the variability and deviations in the cell population can be easily assessed. Here, we present an approach to analyze confocal microscopy image stacks to easily measure Golgi volume, scattering, and association with the centrosome. The approach is based on a custom MATLAB script, provided here as a supplement, and also uses widely available software (ImageJ and/or Imaris). The output of the script is a table with the following parameters: Golgi volume in voxels, Golgi volume in μm 3 , "Golgi-Golgi" distance (averaged distance between all Golgi voxels), Golgi-centrosome distance (averaged distance between each Golgi voxel and the nearest mother centriole), and centrosome-centrosome distance (for cells with duplicated centrosome, the distance between the mother centrioles). The approach can also be applied to analyze distribution of any fluorescently- labeled structure within a cell and its association with the centrosome or any single point within the cell volume., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Objectives: Pancreatic beta cells secrete insulin postprandially and during fasting to maintain glucose homeostasis. Although glucose-stimulated insulin secretion (GSIS) has been extensively studied, much less is known about basal insulin secretion. Here, we performed a genome-wide CRISPR/Cas9 knockout screen to identify novel regulators of insulin secretion., Methods: To identify genes that cell autonomously regulate insulin secretion, we engineered a Cas9-expressing MIN6 subclone that permits irreversible fluorescence labeling of exocytic insulin granules. Using a fluorescence-activated cell sorting assay of exocytosis in low glucose and high glucose conditions in individual cells, we performed a genome-wide CRISPR/Cas9 knockout screen., Results: We identified several members of the COMMD family, a conserved family of proteins with central roles in intracellular membrane trafficking, as positive regulators of basal insulin secretion, but not GSIS. Mechanistically, we show that the Commander complex promotes insulin granules docking in basal state. This is mediated, at least in part, by its function in ITGB1 recycling. Defective ITGB1 recycling reduces its membrane distribution, the number of focal adhesions and cortical ELKS-containing complexes., Conclusions: We demonstrated a previously unknown function of the Commander complex in basal insulin secretion. We showed that by ITGB1 recycling, Commander complex increases cortical adhesions, which enhances the assembly of the ELKS-containing complexes. The resulting increase in the number of insulin granules near the plasma membrane strengthens basal insulin secretion., (Copyright © 2022 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Ventral stress fibers (VSFs) are contractile actin fibers dynamically attached to cell-matrix focal adhesions. VSFs are critical in cellular traction force production and migration. VSFs vary from randomly oriented short, thinner fibers to long, thick fibers that span along the whole long axis of a cell. De novo VSF formation was shown to occur by cortical actin mesh condensation or by crosslinking of dorsal stress fibers and transverse arcs at the cell front. However, the formation of long VSFs that extend across the whole cell axis is not well understood. Here, we report a novel phenomenon of VSF merging in migratory fibroblast cells, which is guided by mechanical force balance and contributes to VSF alignment along the long cell axis. The mechanism of VSF merging involves two steps: connection of two ventral fibers by an emerging myosin II bridge at an intervening adhesion and intervening adhesion dissolution. Our data indicate that these two steps are interdependent: slow adhesion disassembly leads to the slowing of the myosin bridge formation. Cellular data and computational modeling show that the contact angle between merging fibers decides successful merging, with shallow angles leading to merge failure. Our data and modeling further show that merging increases the share of uniformly aligned long VSFs, likely contributing to directional traction force production. Thus, we characterize merging as a process for dynamic reorganization of VSFs with functional significance for directional cell migration., (© 2022 Wiley Periodicals LLC.)

Pancreatic islet β cells regulate glucose homeostasis via glucose-stimulated insulin secretion (GSIS). Cytoskeletal polymers microtubules (MTs) serve as tracks for the transport and positioning of secretory insulin granules. MT network in β cells has unique morphology with several distinct features, which support granule biogenesis ( via Golgi-derived MT array), net non-directional transport ( via interlocked MT mesh), and control availability of granules at secretion sites ( via submembrane MT bundle). The submembrane MT array, which is parallel to the plasma membrane and serves to withdraw excessive granules from the secretion hot spots, is destabilized and fragmented downstream of high glucose stimulation, allowing for regulated secretion. The origin of such an unusual MT network, the features that define its functionality, and metabolic pathways that regulate it are still to a large extent elusive and are a matter of active investigation and debate. Besides the MT network itself, it is important to consider the interplay of molecular motors that drive and fine-tune insulin granule transport. Importantly, activity of kinesin-1, which is the major MT-dependent motor in β cells, transports insulin granules, and has a capacity to remodel MT network, is also regulated by glucose. We discuss yet unknown potential avenues toward understanding how MT network and motor proteins provide control for secretion in coordination with other GSIS-regulating mechanisms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bracey, Gu and Kaverina.)

Heterogeneity of glucose-stimulated insulin secretion (GSIS) in pancreatic islets is physiologically important but poorly understood. Here, we utilize mouse islets to determine how microtubules (MTs) affect secretion toward the vascular extracellular matrix at single cell and subcellular levels. Our data indicate that MT stability in the β-cell population is heterogenous, and that GSIS is suppressed in cells with highly stable MTs. Consistently, MT hyper-stabilization prevents, and MT depolymerization promotes the capacity of single β-cell for GSIS. Analysis of spatiotemporal patterns of secretion events shows that MT depolymerization activates otherwise dormant β-cells via initiation of secretion clusters (hot spots). MT depolymerization also enhances secretion from individual cells, introducing both additional clusters and scattered events. Interestingly, without MTs, the timing of clustered secretion is dysregulated, extending the first phase of GSIS and causing oversecretion. In contrast, glucose-induced Ca 2+ influx was not affected by MT depolymerization yet required for secretion under these conditions, indicating that MT-dependent regulation of secretion hot spots acts in parallel with Ca 2+ signaling. Our findings uncover a novel MT function in tuning insulin secretion hot spots, which leads to accurately measured and timed response to glucose stimuli and promotes functional β-cell heterogeneity., Competing Interests: KT, JL, KB, KH, HM, XZ, GA, TF, AO, MM, MZ, GG, WH, IK No competing interests declared, (© 2021, Trogden et al.)

Focal adhesions (FAs) and associated actin stress fibers (SFs) form a complex mechanical system that mediates bidirectional interactions between cells and their environment. This linked network is essential for mechanosensing, force production and force transduction, thus directly governing cellular processes like polarization, migration and extracellular matrix remodeling. We introduce a tool for fast and robust coupled analysis of both FAs and SFs named the Focal Adhesion Filament Cross-correlation Kit (FAFCK). Our software can detect and record location, axes lengths, area, orientation, and aspect ratio of focal adhesion structures as well as the location, length, width and orientation of actin stress fibers. This enables users to automate analysis of the correlation of FAs and SFs and study the stress fiber system in a higher degree, pivotal to accurately evaluate transmission of mechanocellular forces between a cell and its surroundings. The FAFCK is particularly suited for unbiased and systematic quantitative analysis of FAs and SFs necessary for novel approaches of traction force microscopy that uses the additional data from the cellular side to calculate the stress distribution in the substrate. For validation and comparison with other tools, we provide datasets of cells of varying quality that are labelled by a human expert. Datasets and FAFCK are freely available as open source under the GNU General Public License., Competing Interests: The authors have declared that no competing interests exist.

For sustainable function, each pancreatic islet β cell maintains thousands of insulin secretory granules (SGs) at all times. Glucose stimulation induces the secretion of a small portion of these SGs and simultaneously boosts SG biosynthesis to sustain this stock. The failure of these processes, often induced by sustained high-insulin output, results in type 2 diabetes. Intriguingly, young insulin SGs are more likely secreted during glucose-stimulated insulin secretion (GSIS) for unknown reasons, while older SGs tend to lose releasability and be degraded. Here, we examine the roles of microtubule (MT) and Gαo-signaling in regulating the preferential secretion of young versus old SGs. We show that both MT-destabilization and Gαo inactivation results in more SGs localization near plasma membrane (PM) despite higher levels of GSIS and reduced SG biosynthesis. Intriguingly, MT-destabilization or Gαo-inactivation results in higher secretion probabilities of older SGs, while combining both having additive effects on boosting GSIS. Lastly, Gαo inactivation does not detectably destabilize the β-cell MT network. These findings suggest that Gαo and MT can modulate the preferential release of younger insulin SGs via largely parallel pathways., Competing Interests: One of the co-authors (Dr. Ho) has received a fellowship from Eli Lilly and Company. However, this does not alter our adherence to PLOS ONE policies on sharing data and materials.

The microtubule cytoskeleton of pancreatic islet β-cells regulates glucose-stimulated insulin secretion (GSIS). We have reported that the microtubule-mediated movement of insulin vesicles away from the plasma membrane limits insulin secretion. High glucose-induced remodeling of microtubule network facilitates robust GSIS. This remodeling involves disassembly of old microtubules and nucleation of new microtubules. Here, we examine the mechanisms whereby glucose stimulation decreases microtubule lifetimes in β-cells. Using real-time imaging of photoconverted microtubules, we demonstrate that high levels of glucose induce rapid microtubule disassembly preferentially in the periphery of individual β-cells, and this process is mediated by the phosphorylation of microtubule-associated protein tau. Specifically, high glucose induces tau hyper-phosphorylation via glucose-responsive kinases GSK3, PKA, PKC, and CDK5. This causes dissociation of tau from and subsequent destabilization of microtubules. Consequently, tau knockdown in mouse islet β-cells facilitates microtubule turnover, causing increased basal insulin secretion, depleting insulin vesicles from the cytoplasm, and impairing GSIS. More importantly, tau knockdown uncouples microtubule destabilization from glucose stimulation. These findings suggest that tau suppresses peripheral microtubules turning over to restrict insulin oversecretion in basal conditions and preserve the insulin pool that can be released following stimulation; high glucose promotes tau phosphorylation to enhance microtubule disassembly to acutely enhance GSIS., (© 2020 by the American Diabetes Association.)

Swi-independent 3a and 3b (Sin3a and Sin3b) are paralogous transcriptional coregulators that direct cellular differentiation, survival, and function. Here, we report that mouse Sin3a and Sin3b are coproduced in most pancreatic cells during embryogenesis but become much more enriched in endocrine cells in adults, implying continued essential roles in mature endocrine cell function. Mice with loss of Sin3a in endocrine progenitors were normal during early postnatal stages but gradually developed diabetes before weaning. These physiological defects were preceded by the compromised survival, insulin-vesicle packaging, insulin secretion, and nutrient-induced Ca 2+ influx of Sin3a -deficient β-cells. RNA sequencing coupled with candidate chromatin immunoprecipitation assays revealed several genes that could be directly regulated by Sin3a in β-cells, which modulate Ca 2+ /ion transport, cell survival, vesicle/membrane trafficking, glucose metabolism, and stress responses. Finally, mice with loss of both Sin3a and Sin3b in multipotent embryonic pancreatic progenitors had significantly reduced islet cell mass at birth, caused by decreased endocrine progenitor production and increased β-cell death. These findings highlight the stage-specific requirements for the presumed "general" coregulators Sin3a and Sin3b in islet β-cells, with Sin3a being dispensable for differentiation but required for postnatal function and survival., (© 2020 by the American Diabetes Association.)

Here, we characterize spatial distribution of the Golgi complex in human cells. In contrast to the prevailing view that the Golgi compactly surrounds the centrosome throughout interphase, we observe characteristic differences in the morphology of Golgi ribbons and their association with the centrosome during various periods of the cell cycle. The compact Golgi complex is typical in G1; during S-phase, Golgi ribbons lose their association with the centrosome and extend along the nuclear envelope to largely encircle the nucleus in G2. Interestingly, pre-mitotic separation of duplicated centrosomes always occurs after dissociation from the Golgi. Shortly before the nuclear envelope breakdown, scattered Golgi ribbons reassociate with the separated centrosomes restoring two compact Golgi complexes. Transitions between the compact and distributed Golgi morphologies are microtubule-dependent. However, they occur even in the absence of centrosomes, which implies that Golgi reorganization is not driven by the centrosomal microtubule asters. Cells with different Golgi morphology exhibit distinct differences in the directional persistence and velocity of migration. These data suggest that changes in the radial distribution of the Golgi around the nucleus define the extent of cell polarization and regulate cell motility in a cell cycle-dependent manner.

Two key prerequisites for glucose-stimulated insulin secretion (GSIS) in β cells are the proximity of insulin granules to the plasma membrane and their anchoring or docking to the plasma membrane (PM). Although recent evidence has indicated that both of these factors are altered in the context of diabetes, it is unclear what regulates localization of insulin granules and their interactions with the PM within single cells. Here, we demonstrate that microtubule (MT)-motor-mediated transport dynamics have a critical role in regulating both factors. Super-resolution imaging shows that whereas the MT cytoskeleton resembles a random meshwork in the cells' interior, MTs near the cell surface are preferentially aligned with the PM. Computational modeling suggests two consequences of this alignment. First, this structured MT network preferentially withdraws granules from the PM. Second, the binding and transport of insulin granules by MT motors prevents their stable anchoring to the PM. These findings suggest the MT cytoskeleton may negatively regulate GSIS by both limiting the amount of insulin proximal to the PM and preventing or breaking interactions between the PM and the remaining nearby insulin granules. These results predict that altering MT network structure in β cells can be used to tune GSIS. Thus, our study points to the potential of an alternative therapeutic strategy for diabetes by targeting specific MT regulators., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Cell polarization is important for multiple physiological processes. In motile cells, microtubules (MTs) are organized as a polarized array, which is to a large extent comprised of Golgi-derived MTs (GDMTs), which asymmetrically extend toward the cell front. We have recently found that GDMT asymmetry is based on a nonrandom positioning of spatially restricted nucleation hotspots, where MTs form in a cooperative manner. Here, we summarize methods used for GDMT identification including microtubule regrowth after complete drug-induced depolymerization and tracking of growing microtubules using fluorescent MT plus-end-tracking proteins (+TIPs) in living cells, and subsequent detection of those GDMTs that originate from the nucleation hotspots. These approaches can be used for quantification of the spatial distribution of MT nucleation events associated with the Golgi or another large structure.

The microtubule (MT) network is an essential regulator of insulin secretion from pancreatic β cells, which is central to blood-sugar homeostasis. We find that when glucose metabolism induces insulin secretion, it also increases formation of Golgi-derived microtubules (GDMTs), notably with the same biphasic kinetics as insulin exocytosis. Furthermore, GDMT nucleation is controlled by a glucose signal-transduction pathway through cAMP and its effector EPAC2. Preventing new GDMT nucleation dramatically affects the pipeline of insulin production, storage, and release. There is an overall reduction of β-cell insulin content, and remaining insulin becomes retained within the Golgi, likely because of stalling of insulin-granule budding. While not preventing glucose-induced insulin exocytosis, the diminished granule availability substantially blunts the amount secreted. Constant dynamic maintenance of the GDMT network is therefore critical for normal β-cell physiology. Our study demonstrates that the biogenesis of post-Golgi carriers, particularly large secretory granules, requires ongoing nucleation and replenishment of the GDMT network., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The kinesin-3 KIF1C is a fast organelle transporter implicated in the transport of dense core vesicles in neurons and the delivery of integrins to cell adhesions. Here we report the mechanisms of autoinhibition and release that control the activity of KIF1C. We show that the microtubule binding surface of KIF1C motor domain interacts with its stalk and that these autoinhibitory interactions are released upon binding of protein tyrosine phosphatase PTPN21. The FERM domain of PTPN21 stimulates dense core vesicle transport in primary hippocampal neurons and rescues integrin trafficking in KIF1C-depleted cells. In vitro, human full-length KIF1C is a processive, plus-end directed motor. Its landing rate onto microtubules increases in the presence of either PTPN21 FERM domain or the cargo adapter Hook3 that binds the same region of KIF1C tail. This autoinhibition release mechanism allows cargo-activated transport and might enable motors to participate in bidirectional cargo transport without undertaking a tug-of-war.

Most epithelial cells contain apical membrane structures associated to bundles of actin filaments, which constitute the brush border. Whereas microtubule participation in the maintenance of the brush border identity has been characterized, their contribution to de novo microvilli organization remained elusive. Hereby, using a cell model of individual enterocyte polarization, we found that nocodazole induced microtubule depolymerization prevented the de novo brush border formation. Microtubule participation in brush border actin organization was confirmed in polarized kidney tubule MDCK cells. We also found that centrosome, but not Golgi derived microtubules, were essential for the initial stages of brush border development. During this process, microtubule plus ends acquired an early asymmetric orientation toward the apical membrane, which clearly differs from their predominant basal orientation in mature epithelia. In addition, overexpression of the microtubule plus ends associated protein CLIP170, which regulate actin nucleation in different cell contexts, facilitated brush border formation. In combination, the present results support the participation of centrosomal microtubule plus ends in the activation of the polarized actin organization associated to brush border formation, unveiling a novel mechanism of microtubule regulation of epithelial polarity., (© 2017 Wiley Periodicals, Inc.)

Noncentrosomal microtubule (MT) nucleation at the Golgi generates MT network asymmetry in motile vertebrate cells. Investigating the Golgi-derived MT (GDMT) distribution, we find that MT asymmetry arises from nonrandom nucleation sites at the Golgi (hotspots). Using computational simulations, we propose two plausible mechanistic models of GDMT nucleation leading to this phenotype. In the "cooperativity" model, formation of a single GDMT promotes further nucleation at the same site. In the "heterogeneous Golgi" model, MT nucleation is dramatically up-regulated at discrete and sparse locations within the Golgi. While MT clustering in hotspots is equally well described by both models, simulating MT length distributions within the cooperativity model fits the data better. Investigating the molecular mechanism underlying hotspot formation, we have found that hotspots are significantly smaller than a Golgi subdomain positive for scaffolding protein AKAP450, which is thought to recruit GDMT nucleation factors. We have further probed potential roles of known GDMT-promoting molecules, including γ-TuRC-mediated nucleation activator (γ-TuNA) domain-containing proteins and MT stabilizer CLASPs. While both γ-TuNA inhibition and lack of CLASPs resulted in drastically decreased GDMT nucleation, computational modeling revealed that only γ-TuNA inhibition suppressed hotspot formation. We conclude that hotspots require γ-TuNA activity, which facilitates clustered GDMT nucleation at distinct Golgi sites., (© 2017 Sanders et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Extracellular matrix (ECM) remodeling during physiological processes is mediated by invasive protrusions called podosomes. Positioning and dynamics of podosomes define the extent of ECM degradation. Microtubules are known to be involved in podosome regulation, but the role of microtubule (MT) network configuration in podosome dynamics and positioning is not well understood. Here, we show that the arrangement of the microtubule network defines the pattern of podosome formation and relocation in vascular smooth muscle cells (VSMCs). We show that microtubule plus-end targeting facilitates de novo formation of podosomes, in addition to podosome remodeling. Moreover, specialized bent microtubules with plus ends reversed towards the cell center promote relocation of podosomes from the cell edge to the cell center, resulting in an evenly distributed podosome pattern. Microtubule bending is induced downstream of protein kinase C (PKC) activation and requires microtubule-stabilizing proteins known as cytoplasmic linker associated proteins (CLASPs) and retrograde actin flow. Similar to microtubule depolymerization, CLASP depletion by siRNA blocks microtubule bending and eliminates centripetal relocation of podosomes. Podosome relocation also coincides with translocation of podosome-stimulating kinesin KIF1C, which is known to move preferentially along CLASP-associated microtubules. These findings indicate that CLASP-dependent microtubule network configuration is critical to the cellular location and distribution of KIF1C-dependent podosomes. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The proto-oncogene c-Src is involved in a variety of signaling processes. Therefore, c-Src spatiotemporal localization is critical for interaction with downstream targets. However, the mechanisms regulating this localization have remained elusive. Previous studies have shown that c-Src trafficking is a microtubule-dependent process that facilitates c-Src turnover in neuronal growth cones. As such, microtubule depolymerization lead to the inhibition of c-Src recycling. Alternatively, c-Src trafficking was also shown to be regulated by RhoB-dependent actin polymerization. Our results show that c-Src vesicles primarily exhibit microtubule-dependent trafficking; however, microtubule depolymerization does not inhibit vesicle movement. Instead, vesicular movement becomes both faster and less directional. This movement was associated with actin polymerization directly at c-Src vesicle membranes. Interestingly, it has been shown previously that c-Src delivery is an actin polymerization-dependent process that relies on small GTPase RhoB at c-Src vesicles. In agreement with this finding, microtubule depolymerization induced significant activation of RhoB, together with actin comet tail formation. These effects occurred downstream of GTP-exchange factor, GEF-H1, which was released from depolymerizing MTs. Accordingly, GEF-H1 activity was necessary for actin comet tail formation at the Src vesicles. Our results indicate that regulation of c-Src trafficking requires both microtubules and actin polymerization, and that GEF-H1 coordinates c-Src trafficking, acting as a molecular switch between these two mechanisms.

Integrity of the Golgi apparatus requires the microtubule (MT) network. A subset of MTs originates at the Golgi itself, which in this case functions as a MT-organizing center (MTOC). Golgi-derived MTs serve important roles in post-Golgi trafficking, maintenance of Golgi integrity, cell polarity and motility, as well as cell type-specific functions, including neurite outgrowth/branching. Here, we discuss possible models describing the formation and dynamics of Golgi-derived MTs. How Golgi-derived MTs are formed is not fully understood. A widely discussed model implicates that the critical step of the process is recruitment of molecular factors, which drive MT nucleation (γ-tubulin ring complex, or γ-TuRC), to the Golgi membrane via specific scaffolding interactions. Based on recent findings, we propose to introduce an additional level of regulation, whereby MT-binding proteins and/or local tubulin dimer concentration at the Golgi helps to overcome kinetic barriers at the initial nucleation step. According to our model, emerging MTs are subsequently stabilized by Golgi-associated MT-stabilizing proteins. We discuss molecular factors potentially involved in all three steps of MT formation. To preserve proper cell functioning, a balance must be maintained between MT subsets at the centrosome and the Golgi. Recent work has shown that certain centrosomal factors are important in maintaining this balance, suggesting a close connection between regulation of centrosomal and Golgi-derived MTs. Finally, we will discuss potential functions of Golgi-derived MTs based on their nucleation site location within a Golgi stack.

For glucose-stimulated insulin secretion (GSIS), insulin granules have to be localized close to the plasma membrane. The role of microtubule-dependent transport in granule positioning and GSIS has been debated. Here, we report that microtubules, counterintuitively, restrict granule availability for secretion. In β cells, microtubules originate at the Golgi and form a dense non-radial meshwork. Non-directional transport along these microtubules limits granule dwelling at the cell periphery, restricting granule availability for secretion. High glucose destabilizes microtubules, decreasing their density; such local microtubule depolymerization is necessary for GSIS, likely because granule withdrawal from the cell periphery becomes inefficient. Consistently, microtubule depolymerization by nocodazole blocks granule withdrawal, increases their concentration at exocytic sites, and dramatically enhances GSIS in vitro and in mice. Furthermore, glucose-driven MT destabilization is balanced by new microtubule formation, which likely prevents over-secretion. Importantly, microtubule density is greater in dysfunctional β cells of diabetic mice., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The acquisition of a migratory phenotype is central in processes as diverse as embryo differentiation and tumor metastasis. An early event in this phenomenon is the generation of a nucleus-centrosome-Golgi back-to-front axis. AKAP350 (also known as AKAP9) is a Golgi and centrosome scaffold protein that is involved in microtubule nucleation. AKAP350 interacts with CIP4 (also known as TRIP10), a cdc42 effector that regulates actin dynamics. The present study aimed to characterize the participation of centrosomal AKAP350 in the acquisition of migratory polarity, and the involvement of CIP4 in the pathway. The decrease in total or in centrosomal AKAP350 led to decreased formation of the nucleus-centrosome-Golgi axis and defective cell migration. CIP4 localized at the centrosome, which was enhanced in migratory cells, but inhibited in cells with decreased centrosomal AKAP350. A decrease in the CIP4 expression or inhibition of the CIP4-AKAP350 interaction also led to defective cell polarization. Centrosome positioning, but not nuclear movement, was affected by loss of CIP4 or AKAP350 function. Our results support a model in which AKAP350 recruits CIP4 to the centrosome, providing a centrosomal scaffold to integrate microtubule and actin dynamics, thus enabling centrosome polarization and ensuring cell migration directionality., (© 2015. Published by The Company of Biologists Ltd.)

CLIP-associated proteins CLASPs are mammalian microtubule (MT) plus-end tracking proteins (+TIPs) that promote MT rescue in vivo. Their plus-end localization is dependent on other +TIPs, EB1 and CLIP-170, but in the leading edge of the cell, CLASPs display lattice-binding activity. MT association of CLASPs is suggested to be regulated by multiple TOG (tumor overexpressed gene) domains and by the serine-arginine (SR)-rich region, which contains binding sites for EB1. Here, we report the crystal structures of the two TOG domains of CLASP2. Both domains consist of six HEAT repeats, which are similar to the canonical paddle-like tubulin-binding TOG domains, but have arched conformations. The degrees and directions of curvature are different between the two TOG domains, implying that they have distinct roles in MT binding. Using biochemical, molecular modeling and cell biological analyses, we have investigated the interactions between the TOG domains and αβ-tubulin and found that each domain associates differently with αβ-tubulin. Our findings suggest that, by varying the degrees of domain curvature, the TOG domains may distinguish the structural conformation of the tubulin dimer, discriminate between different states of MT dynamic instability and thereby function differentially as stabilizers of MTs., (Copyright © 2015 Elsevier Ltd. All rights reserved.)