Microtubules Regulate Localization and Availability of Insulin Granules in Pancreatic Beta Cells.

Two key prerequisites for glucose-stimulated insulin secretion (GSIS) in ß 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 ß 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.

Microtubules regulate brush border formation.

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.

Centrosomal AKAP350 and CIP4 act in concert to define the polarized localization of the centrosome and Golgi in migratory cells.

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.

Podosome-regulating kinesin KIF1C translocates to the cell periphery in a CLASP-dependent manner.

The kinesin KIF1C is known to regulate podosomes, actin-rich adhesion structures that remodel the extracellular matrix during physiological processes. Here, we show that KIF1C is a player in the podosome-inducing signaling cascade. Upon induction of podosome formation by protein kinase C (PKC), KIF1C translocation to the cell periphery intensifies and KIF1C accumulates both in the proximity of peripheral microtubules that show enrichment for the plus-tip-associated proteins CLASPs and around podosomes. Importantly, without CLASPs, both KIF1C trafficking and podosome formation are suppressed. Moreover, chimeric mitochondrially targeted CLASP2 recruits KIF1C, suggesting a transient CLASP-KIF1C association. We propose that CLASPs create preferred microtubule tracks for KIF1C to promote podosome induction downstream of PKC.

Asymmetric focal adhesion disassembly in motile cells.

Cell migration requires the integration and coordination of specific focal adhesion dynamics at the cell front, center and rear. In this review, we will present our understanding of the regulation of adhesion turnover and disassembly in various regions of the cell. Adhesion turnover involves a number of tyrosine kinases and phosphatases, most of which are engaged in FAK signaling pathways. Additionally, adhesions are regulated by tensile forces that depend on dynamic coupling with the actin cytoskeleton. The distribution of adhesion disassembly throughout a motile cell is likely coordinated by the asymmetry of the microtubule network. We present a model that suggests two stages of microtubule-driven adhesion disassembly: destabilization and detachment.

Regulation of cell migration by dynamic microtubules.

Microtubules define the architecture and internal organization of cells by positioning organelles and activities, as well as by supporting cell shape and mechanics. One of the major functions of microtubules is the control of polarized cell motility. In order to support the asymmetry of polarized cells, microtubules have to be organized asymmetrically themselves. Asymmetry in microtubule distribution and stability is regulated by multiple molecular factors, most of which are microtubule-associated proteins that locally control microtubule nucleation and dynamics. At the same time, the dynamic state of microtubules is key to the regulatory mechanisms by which microtubules regulate cell polarity, modulate cell adhesion and control force-production by the actin cytoskeleton. Here, we propose that even small alterations in microtubule dynamics can influence cell migration via several different microtubule-dependent pathways. We discuss regulatory factors, potential feedback mechanisms due to functional microtubule-actin crosstalk and implications for cancer cell motility.

The leading role of microtubules in endothelial barrier dysfunction: disassembly of peripheral microtubules leaves behind the cytoskeletal reorganization.

Disturbance of the endothelial barrier is characterized by dramatic cytoskeleton reorganization, activation of actomyosin contraction and, finally, leads to intercellular gap formation. Here we demonstrate that the edemagenic agent, thrombin, causes a rapid increase in the human pulmonary artery endothelial cell (EC) barrier permeability accompanied by fast decreasing in the peripheral microtubules quantity and reorganization of the microtubule system in the internal cytoplasm of the EC within 5 min of the treatment. The actin stress-fibers formation occurs gradually and the maximal effect is observed relatively later, 30 min of the thrombin treatment. Thus, microtubules reaction develops faster than the reorganization of the actin filaments system responsible for the subsequent changes of the cell shape during barrier dysfunction development. Direct microtubules depolymerization by nocodazole initiates the cascade of barrier dysfunction reactions. Nocodazole-induced barrier disruption is connected directly with the degree of peripheral microtubules depolymerization. Short-term loss of endothelial barrier function occurs at the minimal destruction of peripheral microtubules, when actin filament system is still intact. Specifically, we demonstrate that the EC microtubule dynamics examined by time-lapse imaging of EB3-GFP comets movement has changed under these conditions: microtubule plus ends growth rate significantly decreased near the cell periphery. The microtubules, apparently, are the first target in the circuit of reactions leading to the pulmonary EC barrier compromise. Our results show that dynamic microtubules play an essential role in the barrier function in vitro; peripheral microtubules depolymerization is necessary and sufficient condition for initiation of endothelial barrier dysfunction.

Golgi as an MTOC: making microtubules for its own good.

In cells, microtubules (MTs) are nucleated at MT-organizing centers (MTOCs). The centrosome-based MTOCs organize radial MT arrays, which are often not optimal for polarized trafficking. A recently discovered subset of non-centrosomal MTs nucleated at the Golgi has proven to be indispensable for the Golgi organization, post-Golgi trafficking and cell polarity. Here, we summarize the history of this discovery, known molecular prerequisites of MT nucleation at the Golgi and unique functions of Golgi-derived MTs.

Targeted mutation of Cyln2 in the Williams syndrome critical region links CLIP-115 haploinsufficiency to neurodevelopmental abnormalities in mice.

Williams syndrome is a neurodevelopmental disorder caused by the hemizygous deletion of 1.6 Mb on human chromosome 7q11.23. This region comprises the gene CYLN2, encoding CLIP-115, a microtubule-binding protein of 115 kD. Using a gene-targeting approach, we provide evidence that mice with haploinsufficiency for Cyln2 have features reminiscent of Williams syndrome, including mild growth deficiency, brain abnormalities, hippocampal dysfunction and particular deficits in motor coordination. Absence of CLIP-115 also leads to increased levels of CLIP-170 (a closely related cytoplasmic linker protein) and dynactin at the tips of growing microtubules. This protein redistribution may affect dynein motor regulation and, together with the loss of CLIP-115-specific functions, underlie neurological alterations in Williams syndrome.

Insulin secretion hot spots in pancreatic ß cells as secreting adhesions.

Pancreatic ß cell secretion of insulin is crucial to the maintenance of glucose homeostasis and prevention of diseases related to glucose regulation, including diabetes. Pancreatic ß 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 LL5ß 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.

Unbiased Quantification of Golgi Scattering and Golgi-Centrosome Association.

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 µm3, "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.

Preparation of Whole-mount Mouse Islets on Vascular Extracellular Matrix for Live Islet Cell Microscopy.

Pancreatic islet ß 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 ß 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, ß 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 ß 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.

Glucose-stimulated KIF5B-driven microtubule sliding organizes microtubule networks in pancreatic beta cells.

In pancreatic islet beta cells, molecular motors use cytoskeletal polymers microtubules as tracks for intracellular transport of insulin secretory granules. Beta-cell microtubule network has a complex architecture and is non-directional, which provide insulin granules at the cell periphery for rapid secretion response, yet to avoid over-secretion and subsequent hypoglycemia. We have previously characterized a peripheral sub-membrane microtubule array, which is critical for withdrawal of excessive insulin granules from the secretion sites. Microtubules in beta cells originate at the Golgi in the cell interior, and how the peripheral array is formed is unknown. Using real-time imaging and photo-kinetics approaches in clonal mouse pancreatic beta cells MIN6, we now demonstrate that kinesin KIF5B, a motor protein with a capacity to transport microtubules as cargos, slides existing microtubules to the cell periphery and aligns them to each other along the plasma membrane. Moreover, like many physiological beta-cell features, microtubule sliding is facilitated by a high glucose stimulus. These new data, together with our previous report that in high glucose sub-membrane MT array is destabilized to allow for robust secretion, indicate that MT sliding is another integral part of glucose-triggered microtubule remodeling, likely replacing destabilized peripheral microtubules to prevent their loss over time and beta-cell malfunction.

CAMSAP2 localizes to the Golgi in islet ß-cells and facilitates Golgi-ER trafficking.

Glucose stimulation induces the remodeling of microtubules, which potentiates insulin secretion in pancreatic ß-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 ß-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 ß-cells predominantly localizes to the Golgi apparatus instead of microtubule minus ends. This novel localization is specific to primary ß- 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 ß-cells. Moreover, primary ß-cells and insulinoma cells likely express different CAMSAP2 isoforms. We propose that a novel CAMSAP2 isoform in primary ß-cells has a non-canonical function, which promotes Golgi-ER trafficking to support efficient production of insulin and secretion.

Asymmetric CLASP-dependent nucleation of noncentrosomal microtubules at the trans-Golgi network.

Proper organization of microtubule arrays is essential for intracellular trafficking and cell motility. It is generally assumed that most if not all microtubules in vertebrate somatic cells are formed by the centrosome. Here we demonstrate that a large number of microtubules in untreated human cells originate from the Golgi apparatus in a centrosome-independent manner. Both centrosomal and Golgi-emanating microtubules need gamma-tubulin for nucleation. Additionally, formation of microtubules at the Golgi requires CLASPs, microtubule-binding proteins that selectively coat noncentrosomal microtubule seeds. We show that CLASPs are recruited to the trans-Golgi network (TGN) at the Golgi periphery by the TGN protein GCC185. In sharp contrast to radial centrosomal arrays, microtubules nucleated at the peripheral Golgi compartment are preferentially oriented toward the leading edge in motile cells. We propose that Golgi-emanating microtubules contribute to the asymmetric microtubule networks in polarized cells and support diverse processes including post-Golgi transport to the cell front.

Proper regulation of Cdc42 activity is required for tight actin concentration at the equator during cytokinesis in adherent mammalian cells.

Cytokinesis in mammalian cells requires actin assembly at the equatorial region. Although functions of RhoA in this process have been well established, additional mechanisms are likely involved. We have examined if Cdc42 is involved in actin assembly during cytokinesis. Depletion of Cdc42 had no apparent effects on the duration of cytokinesis, while overexpression of constitutively active Cdc42 (CACdc42) caused cytokinesis failure in normal rat kidney epithelial cells. Cells depleted of Cdc42 displayed abnormal cell morphology and caused a failure of tight accumulation of actin and RhoA at the equator. In contrast, in cells overexpressing CACdc42, actin formed abnormal bundles and RhoA was largely eliminated from the equator. Our results suggest that accurate regulation of Cdc42 activity is crucial for proper equatorial actin assembly and RhoA localization during cytokinesis. Notably, our observations also suggest that tight actin concentration is not essential for cytokinesis in adherent mammalian cells.

Microtubules in Pancreatic ß Cells: Convoluted Roadways Toward Precision.

Pancreatic islet ß 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 ß 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 ß 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.

Merging of ventral fibers at adhesions drives the remodeling of cellular contractile systems in fibroblasts.

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.

Genome-wide CRISPR screen identified a role for commander complex mediated ITGB1 recycling in basal insulin secretion.

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.

Dynamics and mechanism of p130Cas localization to focal adhesions.

The docking protein p130Cas is a major Src substrate involved in integrin signaling and mechanotransduction. Tyrosine phosphorylation of p130Cas in focal adhesions (FAs) has been linked to enhanced cell migration, invasion, proliferation, and survival. However, the mechanism of p130Cas targeting to FAs is uncertain, and dynamic aspects of its localization have not been explored. Using live cell microscopy, we show that fluorophore-tagged p130Cas is a component of FAs throughout the FA assembly and disassembly stages, although it resides transiently in FAs with a high mobile fraction. Deletion of either the N-terminal Src homology 3 (SH3) domain or the Cas-family C-terminal homology (CCH) domain significantly impaired p130Cas FA localization, and deletion of both domains resulted in full exclusion. Focal adhesion kinase was implicated in the FA targeting function of the p130Cas SH3 domain. Consistent with their roles in FA targeting, both the SH3 and CCH domains were found necessary for p130Cas to fully undergo tyrosine phosphorylation and promote cell migration. By revealing the capacity of p130Cas to function in FAs throughout their lifetime, clarifying FA targeting mechanism, and demonstrating the functional importance of the highly conserved CCH domain, our results advance the understanding of an important aspect of integrin signaling.