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.
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.
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.
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.
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.
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
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
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.
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.
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.
► Microtubule-dependent regulation of cell migration is particularly important in large cells. ► Microtubules transport post-Golgi carriers, mRNA and recycling endosomes to cortical locations. ► ...Microtubules regulate actin assembly kinetics and focal adhesion turnover. ► Microtubule functions in cell migration require proper regulation of microtubule dynamics. ► Changes in microtubule dynamics affect cell migration through multiple pathways.
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.
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