Biology subjects

Kaverina, I.

Publications and source records attributed to Kaverina, I..

14 recordsLinked to original sources

bioRxiv2025

Cell cycle-regulated tug-of-war between microtubule motors positions major trafficking organelles

The morphology and integrity of the Golgi apparatus, which are critical for protein processing and sorting, undergo significant changes during interphase in proliferating mammalian cells. Initially compact during G1, the Golgi dissociates from the centrosomes in S-phase and transitions into an extended perinuclear ribbon in G2. The mechanisms underlying these changes remain unclear. Golgi positioning can be influenced by microtubule (MT)-dependent motors and membrane exchange with endoplasmic reticulum exit sites (ERES). We employ loss-of-function approaches and live-cell microscopy to demonstrate that CDK1-dependent signaling modulates the tug-of-war between plus- and minus-end-directed motors, resulting in differential positioning of the Golgi and ERES in G1 versus S/G2 phases. In G1, the Golgi and ERES are compacted by the minus-end-directed activity of dynein and KIFC3, respectively. In S/G2, organelle-specific combinations of kinesin-1 heavy and light chains overpower minus-end-directed motors. Our findings reveal a novel, cell cycle-regulated mechanism that coordinates organelle positioning via motor switching.

cell biology↗

bioRxiv2025

A Comprehensive Workflow for Imaging Live Insulin Secretion Events and Granules in Intact Islets

Accurate detection of insulin secretion from pancreatic beta cells is crucial for understanding normal physiological insulin secretion and its pathophysiological counterpart in diabetic states. Traditional methods using fluorescently labeled insulin granules or dye labeling often struggle to distinguish secretion from insulin granule dynamics. We present an optimized protocol using the cell-impermeable Zn2+-binding dye FluoZin-3, which fluoresces upon Zn2+ co-secretion with insulin outside of the islet, more accurately representing secretion. FluoZin-3 combined with intact islet attachment to vascular extracellular matrix and TIRF microscopy offers high spatial and temporal resolution as well as a high signal-to-noise ratio in a minimally perturbed system. Additionally, by integrating the cell-permeable Zn2+-binding dye ZIGIR, we can track insulin granule dynamics alongside secretion events. Our approach generates large datasets, which we efficiently analyze using ilastik machine learning software, enabling fast, accurate, and optionally supervised analysis. This technique builds on our groups previous protocols, detailing a streamlined workflow adaptable to high-resolution, live-cell microscopy for not just insulin but other secretory/granule systems as well. With this method, we investigated secretion behavior of different IG pools in real time during the first phase of insulin secretion: predocked, which appear before high glucose stimulation and are docked at the membrane; docked, which appear upon high glucose stimulation and dock at the membrane; and newcomer, which appear upon high glucose stimulation but dont dock at the membrane. The predocked and newcomer insulin granules are equally secreted and newcomer insulin granules dwell less than one second before secretion upon high glucose stimulation. The predocked and docked insulin granules, however, stay longer at the membrane before secretion. This method is useful for the investigation of functional beta cell heterogeneity of insulin granule secretion in space and time.

cell biology↗

bioRxiv2025

CLASP1/2 REGULATE IMMUNE SYNAPSE MATURATION IN NATURAL KILLER CELLS

Natural killer (NK) cells are the first line of defense against viral infections and tumors. Their cytotoxic activity relies on the formation of an immune synapse (IS) with target cells. The lymphocyte function-associated antigen (LFA)-1 plays a central role in NK cell cytotoxicity by modulating NK-IS assembly and maturation. LFA-1 organization at the IS involves a Golgi-dependent mechanism, which has not been completely elucidated. CLIP-associating proteins (CLASP) 1/2 are microtubule plus-tip interacting proteins that control the dynamics of Golgi derived microtubules (GDMTs). In the present study, we found that CLASP1/2 depletion impaired LFA-1 organization at the IS and inhibited the polarization of the centrosome and the lytic granules towards the target cell. Our results also revealed the role of the Golgi apparatus as a microtubule organizing center (MTOC) in these cells. Furthermore, we found that, similarly to what was described in other cell types, NK cells require CLASP1/2 and AKAP350 for efficient nucleation of microtubules at the Golgi. Overall, this study uncovers the role of CLASP1/2 in the maturation of the lytic IS in NK cells, and presents evidence supporting the contribution of GDMTs in this process. Summary sentenceThe Golgi apparatus (GA) functions as a microtubule-organizing center (MTOC) in NK cells. During the recognition of tumoral cells by NK cells, CLASP1/2-mediated stabilization of GA-derived microtubules (GDMTs) facilitates vesicular LFA-1 (LFA-1v) trafficking toward the interaction surface, thereby promoting the immune synapse (IS) maturation.

immunology↗

bioRxiv2024

Directed insulin secretion occurs at precise cortical regions with optimal ELKS content that are devoid of microtubules

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 LL5{beta}. However, using TIRF microscopy of intact mouse islets to precisely localize secretion events within ELKS/LL5{beta} patches, we now show that secretion is restricted to only 5% of ELKS/LL5{beta} 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. Significance StatementO_LIInsulin has to be secreted directly into the bloodstream for efficient regulation of glucose metabolism. Molecular requirements for precise secretion location and microtubule-mediated regulation in directed secretion are uncharacterized. C_LIO_LIUsing intact mouse islets and a live insulin secretion assay, we demonstrate that cortical patches containing ELKS and LL5{beta} display secretory heterogeneity and secretion hot spots are precisely localized to patch edges that are devoid of microtubules. C_LIO_LIThese findings suggest that microtubule absence is critical for secretion and secretion occurs away from presumed sites of secretory granule delivery. This expands the current knowledge of regulation and spatial characteristics of insulin secretion. C_LI

cell biology↗

bioRxiv2024

Pancreatic islet alpha cells regulate microtubule stability in neighboring beta cells to tune insulin secretion and induce functional heterogeneity in individual mouse and human islets

We have reported that the microtubule (MT) network in {beta} 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 -cell secreted hormones, Gcg and/or Glp1, regulate the MT stability in {beta} cells. Activating the receptors of Gcg or Glp1 (GcgR or Glp1R) with chemical agonists induces MT destabilization in {beta} 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 {beta} cells of islets with higher /{beta} cell ratio are less stable than those with lower /{beta} cell ratio. Within each individual islet, {beta} cells that are located close to cells show faster MTs remodeling upon glucose stimulation than those away. Consequently, islets with higher /{beta} 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 cells, using Gcg and or Glp1-mediated paracrine signaling, tune {beta}-cell secretion. In addition, the different -{beta} cell ratios in individual islets lead to their heterogeneous secretory responses, which may be important for handling secretory function needs under different physiological conditions. HighlightsO_LIGcg sensitizes glucose-induced MT remodeling in mouse and human {beta} cells C_LIO_LIMT density in single islets anti-correlates with /{beta} cell ratio C_LIO_LIGSIS levels in single islets positively correlate with /{beta} cell ratio C_LIO_LIDifferent /{beta} cell ratio contributes to heterogeneity of single islet GSIS C_LI

cell biology↗

bioRxiv2023

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. The {beta}-cell microtubule network has a complex architecture and is non-directional, which provides 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 the 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.

cell biology↗

bioRxiv2022

CLASP2 facilitates dynamic actin filament organization along the microtubule lattice

Coordination between the microtubule and actin networks is essential for cell motility, neuronal growth cone guidance, and wound healing. Members of the CLASP (Cytoplasmic Linker-Associated Protein) family of proteins have been implicated in the cytoskeletal crosstalk between microtubules and actin networks, however, the molecular mechanisms underlying CLASPs role in cytoskeletal coordination are unclear. Here, we investigate CLASP2s crosslinking function with microtubules and F-actin. Our results demonstrate that CLASP2 crosslinks F-actin to the microtubule lattice in vitro. We find that the crosslinking ability is retained by L-TOG2-S, a minimal construct containing the TOG2 domain and serine-arginine rich region of CLASP2. Furthermore, CLASP2 promotes the accumulation of multiple actin filaments along the microtubule, supporting up to 11 F-actin landing events on a single microtubule lattice region. CLASP2 also facilitates dynamic organization of polymerizing actin filaments templated by the microtubule network, with F-actin forming bridges between individual microtubules. Finally, we find that depletion of CLASPs in vascular smooth muscle cells results in disorganized actin fibers and reduced co-alignment of actin fibers with microtubules, suggesting that CLASP and microtubules contribute to higher-order actin structures. Taken together, our results indicate that CLASP2 can directly crosslink F-actin to microtubules, and that this microtubule-CLASP-actin interaction may influence overall cytoskeletal organization in cells.

cell biology↗

bioRxiv2022

The microtubule minus end-binding protein CAMSAP2 does not regulate microtubule dynamics in primary pancreatic β-cells but facilitates insulin secretion

Glucose stimulation induces the remodeling of microtubules in Islet {beta}-cells to potentiate glucose-stimulated insulin secretion. CAMSAP2 is a microtubule minus-end binding protein and is reported to stabilize and position microtubules in several non-{beta}-cells, such as human retinal pigment epithelium cells. In immortalized insulinoma MIN6 cells, CAMSAP2 binds to and forms short stretches at microtubule minus ends in the cytoplasm, which is consistent with the reported subcellular localization and functions of CAMSAP2 in non-{beta}-cells. Surprisingly, we found that CAMSAP2 expressed in primary islet {beta}-cells does not form short stretches in the cytoplasm, but instead is localized to the Golgi apparatus. This novel localization is specific to {beta}-but not -cells in islets and it is independent of MT-binding. Knockdown of CAMSAP2 by shRNA impairs Golgi-ER trafficking, reduces total insulin content, and attenuates GSIS without affecting the MT dynamics or releasability of insulin granules in islet {beta}-cells. Corresponding to these results, we found that primary islets and MIN6 cells express different CAMSAP2 isoforms. We propose that primary islet {beta} cells use a novel CAMSAP2 isoform for a MT-independent non-canonical function, which is to promote Golgi-ER trafficking that supports efficient production of insulin secretory granules.

cell biology↗

bioRxiv2022

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

Ventral stress fibers (VSFs) are contractile actin fibers present in the ventral plane of the cell and existing in a dynamic attachment with cell-matrix focal adhesions. VSFs are critical in cellular mechanobiological functions such as traction force production, cell polarization, and migration. VSF within their intracellular network vary from short, thinner fibers that are randomly oriented to long, thick fibers that span along the whole long axis of a cell. De novo VSF formation was shown to occur by condensation from the cortical actin mesh or by crosslinking of other stress fiber subtypes (dorsal stress fibers and transverse arcs) at the cell front. However, 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 to form a cohesive, contractile VSF. Our data indicate that these two steps are interdependent, since under conditions where adhesion disassembly is slowed, formation of the myosin bridge is slowed as well. Cellular data and computational modeling show that the angle of contact between merging fibers decides successful merging, with angles closer to 180 yielding merging events and shallower angles leading to merge failure. Our data and modeling further show that merging increases the share of uniformly aligned long VSFs, which would contribute to directional traction force production. Thus, we thoroughly characterize merging as process for dynamic reorganization of VSFs in steady state, investigating the steps and variants of the process as well as its functional significance in migratory cells.

cell biology↗

bioRxiv2021

A Focal Adhesion Filament Cross-correlation Kit for fast, automated segmentation and correlation of focal adhesions and actin stress fibers in cells

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. Author summaryOur novel Focal Adhesion Filament Cross-correlation Kit (FAFCK) allows for fast, reliable, unbiased, and systematic detection of focal adhesions and actin stress fibers in cells and their mutual correlation. Detailed analysis of these structures which are both key elements in mechano-sensing and force transduction will help tremendously to improve quantitative analysis of mechanocellular experiments, key to understanding the complex interplay between cells and the extracellular matrix. In particular, sophisticated analysis methods such as model-based traction force microscopy will benefit from correlating the detailed datasets of stress fibers and focal adhesions.

biophysics↗

bioRxiv2020

Microtubules and Gαo-signaling independently regulate the preferential secretion of newly synthesized insulin granules in pancreatic islet β cells

For sustainable function, each pancreatic islet {beta} cell maintains thousands of insulin granules (IGs) at all times. Glucose stimulation induces the secretion of a small portion of these IGs and simultaneously triggers IG biosynthesis to sustain this stock. The failure of these processes, often induced by sustained high-insulin output, results in type 2 diabetes. Intriguingly, newly synthesized IGs are more likely secreted during glucose-stimulated insulin secretion. The older IGs tend to lose releasability and be degraded, which represents a futile metabolic load that can sensitize {beta} cells to workload-induced dysfunction and even death. Here, we examine the factor(s) that allows the preferential secretion of younger IGs. We show that {beta} cells without either microtubules (MTs) or Go signaling secrete a bigger portion of older IGs, which is associated with increased IG docking on plasma membrane. Yet Go inactivation does not alter the {beta}-cell MT network. These findings suggest that Go and MT regulate the preferential release of newer IGs via parallel pathways and provide two potential models to further explore the underlying mechanisms and physiological significance of this regulation in functional {beta} cells.

cell biology↗

bioRxiv2020

Microtubules regulate pancreatic beta cell heterogeneity via spatiotemporal control of insulin secretion hot spots

Heterogeneity of glucose-stimulated insulin secretion (GSIS) in pancreatic islets is physiologically important but poorly understood. Here, we utilize whole mouse islets to determine how microtubules affect secretion toward the vascular extracellular matrix. Our data indicate that microtubule stability in the {beta}-cell population is heterogenous, and that cells with more stable microtubules secrete less in response to a stimulus. Consistently, microtubule hyper-stabilization prevents, and microtubule depolymerization promotes {beta}-cell activation. Analysis of spatiotemporal patterns of secretion events shows that microtubule depolymerization activates otherwise dormant {beta}-cells via initiation of secretion clusters (hot spots). Microtubule depolymerization also enhances secretion from individual cells, introducing both additional clusters and scattered events. Interestingly, without microtubules, the timing of clustered secretion is dysregulated, extending the first phase of GSIS. Our findings uncover a novel microtubule function in tuning insulin secretion hot spots, which leads to accurately measured and timed response to glucose stimuli and promotes functional {beta}-cell heterogeneity.

cell biology↗

bioRxiv2019

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

Two key prerequisites for glucose stimulated insulin secretion (GSIS) in Beta cells are the proximity of insulin granules to the plasma membrane and their anchoring or docking to the plasma membrane (PM). While recent evidence has indicated that both of these factors are altered in the context of diabetes, it is unclear what regulates localization of insulin and its 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 while the MT cytoskeleton resembles a random meshwork in the cells interior, MTs near the cells surface are preferentially aligned with the PM. Computational modeling demonstrates 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. The MT cytoskeleton thus negatively regulates GSIS by both limiting the amount of insulin proximal to the PM and preventing/breaking interactions between the PM and the remaining nearby insulin. These results predict that altering MT structure in beta cells can be used to tune GSIS. Thus, our study points to a potential of an alternative therapeutic strategy for diabetes by targeting specific MT regulators.

cell biology↗

bioRxiv2018

Activation of intracellular transport by relieving KIF1C autoinhibition

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.

cell biology↗