Vesicle and reaction-diffusion hybrid modeling with STEPS.

Vesicles carry out many essential functions within cells through the processes of endocytosis, exocytosis, and passive and active transport. This includes transporting and delivering molecules between different parts of the cell, and storing and releasing neurotransmitters in neurons. To date, computational simulation of these key biological players has been rather limited and has not advanced at the same pace as other aspects of cell modeling, restricting the realism of computational models. We describe a general vesicle modeling tool that has been designed for wide application to a variety of cell models, implemented within our software STochastic Engine for Pathway Simulation (STEPS), a stochastic reaction-diffusion simulator that supports realistic reconstructions of cell tissue in tetrahedral meshes. The implementation is validated in an extensive test suite, parallel performance is demonstrated in a realistic synaptic bouton model, and example models are visualized in a Blender extension module.

Patients and mice with deficiency in the SNARE protein SYNTAXIN-11 have a secondary B cell defect.

SYNTAXIN-11 (STX11) is a SNARE protein that mediates the fusion of cytotoxic granules with the plasma membrane at the immunological synapses of CD8 T or NK cells. Autosomal recessive inheritance of deleterious STX11 variants impairs cytotoxic granule exocytosis, causing familial hemophagocytic lymphohistiocytosis type 4 (FHL-4). In several FHL-4 patients, we also observed hypogammaglobulinemia, elevated frequencies of naive B cells, and increased double-negative DN2:DN1 B cell ratios, indicating a hitherto unrecognized role of STX11 in humoral immunity. Detailed analysis of Stx11-deficient mice revealed impaired CD4 T cell help for B cells, associated with disrupted germinal center formation, reduced isotype class switching, and low antibody avidity. Mechanistically, Stx11-/- CD4 T cells exhibit impaired membrane fusion leading to reduced CD107a and CD40L surface mobilization and diminished IL-2 and IL-10 secretion. Our findings highlight a critical role of STX11 in SNARE-mediated membrane trafficking and vesicle exocytosis in CD4 T cells, important for successful CD4 T cell-B cell interactions. Deficiency in STX11 impairs CD4 T cell-dependent B cell differentiation and humoral responses.

Parallel processing of quickly and slowly mobilized reserve vesicles in hippocampal synapses.

Vesicles within presynaptic terminals are thought to be segregated into a variety of readily releasable and reserve pools. The nature of the pools and trafficking between them is not well understood, but pools that are slow to mobilize when synapses are active are often assumed to feed pools that are mobilized more quickly, in a series. However, electrophysiological studies of synaptic transmission have suggested instead a parallel organization where vesicles within slowly and quickly mobilized reserve pools would separately feed independent reluctant- and fast-releasing subdivisions of the readily releasable pool. Here, we use FM-dyes to confirm the existence of multiple reserve pools at hippocampal synapses and a parallel organization that prevents intermixing between the pools, even when stimulation is intense enough to drive exocytosis at the maximum rate. The experiments additionally demonstrate extensive heterogeneity among synapses in the relative sizes of the slowly and quickly mobilized reserve pools, which suggests equivalent heterogeneity in the numbers of reluctant and fast-releasing readily releasable vesicles that may be relevant for understanding information processing and storage.

OCaR1 endows exocytic vesicles with autoregulatory competence by preventing uncontrolled Ca2+ release, exocytosis, and pancreatic tissue damage.

Regulated exocytosis is initiated by increased Ca2+ concentrations in close spatial proximity to secretory granules, which is effectively prevented when the cell is at rest. Here we showed that exocytosis of zymogen granules in acinar cells was driven by Ca2+ directly released from acidic Ca2+ stores including secretory granules through NAADP-activated two-pore channels (TPCs). We identified OCaR1 (encoded by Tmem63a) as an organellar Ca2+ regulator protein integral to the membrane of secretory granules that controlled Ca2+ release via inhibition of TPC1 and TPC2 currents. Deletion of OCaR1 led to extensive Ca2+ release from NAADP-responsive granules under basal conditions as well as upon stimulation of GPCR receptors. Moreover, OCaR1 deletion exacerbated the disease phenotype in murine models of severe and chronic pancreatitis. Our findings showed OCaR1 as a gatekeeper of Ca2+ release that endows NAADP-sensitive secretory granules with an autoregulatory mechanism preventing uncontrolled exocytosis and pancreatic tissue damage.

Alpha herpesvirus exocytosis from neuron cell bodies uses constitutive secretory mechanisms, and egress and spread from axons is independent of neuronal firing activity.

Alpha herpesviruses naturally infect the peripheral nervous system, and can spread to the central nervous system, causing severe debilitating or deadly disease. Because alpha herpesviruses spread along synaptic circuits, and infected neurons exhibit altered electrophysiology and increased spontaneous activity, we hypothesized that alpha herpesviruses use activity-dependent synaptic vesicle-like regulated secretory mechanisms for egress and spread from neurons. Using live-cell fluorescence microscopy, we show that Pseudorabies Virus (PRV) particles use the constitutive Rab6 post-Golgi secretory pathway to exit from the cell body of primary neurons, independent of local calcium signaling. Some PRV particles colocalize with Rab6 in the proximal axon, but we did not detect colocalization/co-transport in the distal axon. Thus, the specific secretory mechanisms used for viral egress from axons remains unclear. To address the role of neuronal activity more generally, we used a compartmentalized neuron culture system to measure the egress and spread of PRV from axons, and pharmacological and optogenetics approaches to modulate neuronal activity. Using tetrodotoxin to silence neuronal activity, we observed no inhibition, and using potassium chloride or optogenetics to elevate neuronal activity, we also show no increase in virus spread from axons. We conclude that PRV egress from neurons uses constitutive secretory mechanisms: generally, activity-independent mechanisms in axons, and specifically, the constitutive Rab6 post-Golgi secretory pathway in cell bodies.

Simultaneous TIRF imaging of subplasmalemmal Ca2+ dynamics and granule fusions in insulin-secreting INS-1 cells reveals coexistent synchronized and asynchronous release.

The basal and glucose-induced insulin secretion from pancreatic beta cells is a tightly regulated process that is triggered in a Ca2+-dependent fashion and further positively modulated by substances that raise intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) or by certain antidiabetic drugs. In a previous study, we have temporally resolved the subplasmalemmal [Ca2+]i dynamics in beta cells that are characterized by trains of sharply delimited spikes, reaching peak values up to 5 µM. Applying total internal reflection fluorescence (TIRF) microscopy and synaptopHluorin to visualize fusion events of individual granules, we found that several fusion events can coincide within 50 to 150 ms. To test whether subplasmalemmal [Ca2+]i microdomains around single or clustered Ca2+ channels may cause a synchronized release of insulin-containing vesicles, we applied simultaneous dual-color TIRF microscopy and monitored Ca2+ fluctuations and exocytotic events in INS-1 cells at high frame rates. The results indicate that fusions can be triggered by subplasmalemmal Ca2+ spiking. This, however, does account for a minority of fusion events. About 90 %-95 % of fusion events either happen between Ca2+ spikes or incidentally overlap with subplasmalemmal Ca2+ spikes. We conclude that only a fraction of exocytotic events in glucose-induced and tolbutamide- or forskolin-enhanced insulin release from INS-1 cells is tightly coupled to Ca2+ microdomains around voltage-gated Ca2+ channels.

Endothelial PTP1B Deletion Promotes VWF Exocytosis and Venous Thromboinflammation.

BACKGROUND: Endothelial activation promotes the release of procoagulant extracellular vesicles and inflammatory mediators from specialized storage granules. Endothelial membrane exocytosis is controlled by phosphorylation. We hypothesized that the absence of PTP1B (protein tyrosine phosphatase 1B) in endothelial cells promotes venous thromboinflammation by triggering endothelial membrane fusion and exocytosis. METHODS: Mice with inducible endothelial deletion of PTP1B (End.PTP1B-KO) underwent inferior vena cava ligation to induce stenosis and venous thrombosis. Primary endothelial cells from transgenic mice and human umbilical vein endothelial cells were used for mechanistic studies. RESULTS: Vascular ultrasound and histology showed significantly larger venous thrombi containing higher numbers of Ly6G (lymphocyte antigen 6 family member G)-positive neutrophils in mice with endothelial PTP1B deletion, and intravital microscopy confirmed the more pronounced neutrophil recruitment following inferior vena cava ligation. RT2 PCR profiler array and immunocytochemistry analysis revealed increased endothelial activation and adhesion molecule expression in primary End.PTP1B-KO endothelial cells, including CD62P (P-selectin) and VWF (von Willebrand factor). Pretreatment with the NF-κB (nuclear factor kappa B) kinase inhibitor BAY11-7082, antibodies neutralizing CD162 (P-selectin glycoprotein ligand-1) or VWF, or arginylglycylaspartic acid integrin-blocking peptides abolished the neutrophil adhesion to End.PTP1B-KO endothelial cells in vitro. Circulating levels of annexin V+ procoagulant endothelial CD62E+ (E-selectin) and neutrophil (Ly6G+) extracellular vesicles were also elevated in End.PTP1B-KO mice after inferior vena cava ligation. Higher plasma MPO (myeloperoxidase) and Cit-H3 (citrullinated histone-3) levels and neutrophil elastase activity indicated neutrophil activation and extracellular trap formation. Infusion of End.PTP1B-KO extracellular vesicles into C57BL/6J wild-type mice most prominently enhanced the recruitment of endogenous neutrophils, and this response was blunted in VWF-deficient mice or by VWF-blocking antibodies. Reduced PTP1B binding and tyrosine dephosphorylation of SNAP23 (synaptosome-associated protein 23) resulting in increased VWF exocytosis and neutrophil adhesion were identified as mechanisms, all of which could be restored by NF-κB kinase inhibition using BAY11-7082. CONCLUSIONS: Our findings show that endothelial PTP1B deletion promotes venous thromboinflammation by enhancing SNAP23 phosphorylation, endothelial VWF exocytosis, and neutrophil recruitment.

Escape sites from the endo-lysosomal trafficking route manipulate exocytosis of nanoparticles in polar epithelium.

The endo-lysosomal pathway is a major barrier for the trans-epithelial transport of nanoparticles (NPs), but escape strategies could facilitate trans-epithelial delivery. Based on the polarization properties of the epithelium, different escape compartments may result in different exocytosis fates of NPs and further affect the delivery efficiency. Therefore, optimizing the escape sites is critical for trans-epithelial delivery. Here, commonly used PEG-coated-poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles were fabricated as model nanoparticles (MNPs) and the intestinal epithelium was chosen as the polarized epithelium. The MNPs were incubated with different endosomolytic agents for early endosomal escape, late endosomal escape and lysosomal escape, respectively. According to in vitro and in vivo studies, MNPs escaping from early endosomes and late endosomes exhibited stronger capacity for trans-epithelial transport than those escaping from lysosomes. By further probing into the mechanism, we surprisingly found that although MNPs escaping from early endosomes quickly egressed from the apical side of epithelia, they were subsequently followed by "reuptake" via caveolae and trafficked through the endoplasmic reticulum-Golgi apparatus (ER/GA) secretory pathway, achieving efficient trans-epithelial transport; MNPs escaping from late endosomes, which were located near the nucleus, were prone to enter the ER/GA for efficient basolateral exocytosis. However, MNPs escaping from lysosomes were detained within cells by autophagosomes. Collectively, our research suggested that early endosomes and late endosomes were ideal escape sites for trans-epithelial delivery.

A hydrophobic groove in secretagogin allows for alternate interactions with SNAP-25 and syntaxin-4 in endocrine tissues.

Vesicular release of neurotransmitters and hormones relies on the dynamic assembly of the exocytosis/trans-SNARE complex through sequential interactions of synaptobrevins, syntaxins, and SNAP-25. Despite SNARE-mediated release being fundamental for intercellular communication in all excitable tissues, the role of auxiliary proteins modulating the import of reserve vesicles to the active zone, and thus, scaling repetitive exocytosis remains less explored. Secretagogin is a Ca2+-sensor protein with SNAP-25 being its only known interacting partner. SNAP-25 anchors readily releasable vesicles within the active zone, thus being instrumental for 1st phase release. However, genetic deletion of secretagogin impedes 2nd phase release instead, calling for the existence of alternative protein-protein interactions. Here, we screened the secretagogin interactome in the brain and pancreas, and found syntaxin-4 grossly overrepresented. Ca2+-loaded secretagogin interacted with syntaxin-4 at nanomolar affinity and 1:1 stoichiometry. Crystal structures of the protein complexes revealed a hydrophobic groove in secretagogin for the binding of syntaxin-4. This groove was also used to bind SNAP-25. In mixtures of equimolar recombinant proteins, SNAP-25 was sequestered by secretagogin in competition with syntaxin-4. Kd differences suggested that secretagogin could shape unidirectional vesicle movement by sequential interactions, a hypothesis supported by in vitro biological data. This mechanism could facilitate the movement of transport vesicles toward release sites, particularly in the endocrine pancreas where secretagogin, SNAP-25, and syntaxin-4 coexist in both α- and ß-cells. Thus, secretagogin could modulate the pace and fidelity of vesicular hormone release by differential protein interactions.

Autophagy and Exocytosis of Lipofuscin Into the Basolateral Extracellular Space of Human Retinal Pigment Epithelium From Fetal Development to Adolescence.

Purpose: To undertake the first ultrastructural characterization of human retinal pigment epithelial (RPE) differentiation from fetal development to adolescence. Methods: Ten fetal eyes and three eyes aged six, nine, and 17 years were examined in the temporal retina adjacent to the optic nerve head by transmission electron microscopy. The area, number, and distribution of RPE organelles were quantified and interpreted within the context of adjacent photoreceptors, Bruch's membrane, and choriocapillaris maturation. Results: Between eight to 12 weeks' gestation (WG), pseudostratified columnar epithelia with apical tight junctions differentiate to a simple cuboidal epithelium with random distribution of melanosomes and mitochondria. Between 12 to 26 WG, cells enlarge and show long apical microvilli and apicolateral junctional complexes. Coinciding with eye opening at 26 WG, melanosomes migrate apically whereas mitochondria distribute to perinuclear regions, with the first appearance of phagosomes, complex granules, and basolateral extracellular space (BES) formation. Significantly, autophagy and heterophagy, as evidenced by organelle recycling, and the gold standard of ultrastructural evidence for autophagy of double-membrane autophagosomes and mitophagosomes were evident from 32 WG, followed by basal infoldings of RPE cell membrane at 36 WG. Lipofuscin formation and deposition into the BES evident at six years increased at 17 years. Conclusions: We provide compelling ultrastructural evidence that heterophagy and autophagy begins in the third trimester of human fetal development and that deposition of cellular byproducts into the extracellular space of RPE takes place via exocytosis. Transplanted RPE cells must also demonstrate the capacity to subserve autophagic and heterophagic functions for effective disease mitigation.

Phospholipids Differentially Regulate Ca2+ Binding to Synaptotagmin-1.

Synaptotagmin-1 (Syt-1) is a calcium sensing protein that is resident in synaptic vesicles. It is well established that Syt-1 is essential for fast and synchronous neurotransmitter release. However, the role of Ca2+ and phospholipid binding in the function of Syt-1, and ultimately in neurotransmitter release, is unclear. Here, we investigate the binding of Ca2+ to Syt-1, first in the absence of lipids, using native mass spectrometry to evaluate individual binding affinities. Syt-1 binds to one Ca2+ with a KD ∼ 45 µM. Each subsequent binding affinity (n ≥ 2) is successively unfavorable. Given that Syt-1 has been reported to bind anionic phospholipids to modulate the Ca2+ binding affinity, we explored the extent that Ca2+ binding was mediated by selected anionic phospholipid binding. We found that phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and dioleoylphosphatidylserine (DOPS) positively modulated Ca2+ binding. However, the extent of Syt-1 binding to phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) was reduced with increasing [Ca2+]. Overall, we find that specific lipids differentially modulate Ca2+ binding. Given that these lipids are enriched in different subcellular compartments and therefore may interact with Syt-1 at different stages of the synaptic vesicle cycle, we propose a regulatory mechanism involving Syt-1, Ca2+, and anionic phospholipids that may also control some aspects of vesicular exocytosis.

Tracing the STING exocytosis pathway during herpes viruses infection.

The STimulator of INterferon Genes (STING) constitutes a major DNA-sensing pathway that restricts HSV-1 infection in different models by activating type I interferon and pro-inflammatory responses. To counteract STING, HSV-1 has evolved numerous strategies including mechanisms to interfere with its oligomerization, post-translational modifications, and downstream signaling. Previously, we demonstrated that STING is packaged in extracellular vesicles (EVs) produced from HSV-1-infected cells. These EVs activated antiviral responses in uninfected recipient cells and suppressed a subsequent HSV-1 infection in a STING-dependent manner. Here, we provide information on the packaging of STING in EVs and its exocytosis. We found that STING exocytosis did not occur in CD63 knockdown cells supporting that STING follows the CD63 exocytosis pathway. Consistently, we found that STING co-localized with CD63 in cytoplasmic globular structures and exosomal STING and CD63 co-fractionated. Both golgicide A and brefeldin A prevented STING exocytosis during HSV-1 infection suggesting that STING trafficking through the Golgi is required. A STING ligand was insufficient for STING exocytosis, and downstream signaling through TBK1 was not required. However, STING palmitoylation and tethering to the ER by STIM1 were required for STING exocytosis. Finally, we found that HSV-1 replication/late gene expression triggered CD63 exocytosis that was required for STING exocytosis. Surprisingly, HSV-2 strain G did not trigger CD63 or STING exocytosis as opposed to VZV and HCMV. Also, EVs from HSV-1(F)- and HSV-2(G)-infected cells displayed differences in their ability to restrict these viruses. Overall, STING exocytosis is induced by certain viruses and shapes the microenvironment of infection.IMPORTANCEExtracellular vesicles (EVs) are released by all types of cells as they constitute a major mechanism of intercellular communication. The packaging of specific cargo in EVs and the pathway of exocytosis are not fully understood. STING is a sensor of a broad spectrum of pathogens and a key component of innate immunity. STING exocytosis during HSV-1 infection has been an intriguing observation, raising questions of whether this is a virus-induced process, the purpose it serves, and whether it is observed after infection with other viruses. Here, we have provided insights into the pathway of STING exocytosis and determined factors involved. STING exocytosis is a virus-induced process and not a response of the host to the infection. Besides HSV-1, other herpes viruses triggered STING exocytosis, but HSV-2(G) did not. HSV-1 EVs displayed different restriction capabilities compared with HSV-2(G) EVs. Overall, STING exocytosis is triggered by viruses to shape the microenvironment of infection.

Synaptotagmin 7 docks synaptic vesicles to support facilitation and Doc2α-triggered asynchronous release.

Despite decades of intense study, the molecular basis of asynchronous neurotransmitter release remains enigmatic. Synaptotagmin (syt) 7 and Doc2 have both been proposed as Ca2+ sensors that trigger this mode of exocytosis, but conflicting findings have led to controversy. Here, we demonstrate that at excitatory mouse hippocampal synapses, Doc2α is the major Ca2+ sensor for asynchronous release, while syt7 supports this process through activity-dependent docking of synaptic vesicles. In synapses lacking Doc2α, asynchronous release after single action potentials is strongly reduced, while deleting syt7 has no effect. However, in the absence of syt7, docked vesicles cannot be replenished on millisecond timescales. Consequently, both synchronous and asynchronous release depress from the second pulse onward during repetitive activity. By contrast, synapses lacking Doc2α have normal activity-dependent docking, but continue to exhibit decreased asynchronous release after multiple stimuli. Moreover, disruption of both Ca2+ sensors is non-additive. These findings result in a new model whereby syt7 drives activity-dependent docking, thus providing synaptic vesicles for synchronous (syt1) and asynchronous (Doc2 and other unidentified sensors) release during ongoing transmission.

MADD regulates natural killer cell degranulation through Rab27a activation.

Natural killer (NK) cells have the ability to lyse other cells through the release of lytic granules (LGs). This is in part mediated by the small GTPase Rab27a, which was first identified to play a crucial role in degranulation through the study of individuals harboring mutations in the gene encoding Rab27a. However, the guanine nucleotide exchange factor (GEF) regulating the activation of Rab27a in cytotoxic lymphocytes was unknown. Here, we show that knockout of MADD significantly decreased the levels of GTP-bound Rab27a in both resting and stimulated NK cells, and MADD-deficient NK cells and CD8+ T cells displayed severely reduced degranulation and cytolytic ability, similar to that seen with Rab27a deficiency. Although MADD colocalized with Rab27a on LGs and was enriched at the cytolytic synapse, the loss of MADD did not impact Rab27a association with LGs nor their recruitment to the cytolytic synapse. Together, our results demonstrate an important role for MADD in cytotoxic lymphocyte killing.

Quantitative Proteomics Reveal That CB2R Agonist JWH-133 Downregulates NF-κB Activation, Oxidative Stress, and Lysosomal Exocytosis from HIV-Infected Macrophages.

HIV-associated neurocognitive disorders (HAND) affect 15-55% of HIV-positive patients and effective therapies are unavailable. HIV-infected monocyte-derived macrophages (MDM) invade the brain of these individuals, promoting neurotoxicity. We demonstrated an increased expression of cathepsin B (CATB), a lysosomal protease, in monocytes and post-mortem brain tissues of women with HAND. Increased CATB release from HIV-infected MDM leads to neurotoxicity, and their secretion is associated with NF-κB activation, oxidative stress, and lysosomal exocytosis. Cannabinoid receptor 2 (CB2R) agonist, JWH-133, decreases HIV-1 replication, CATB secretion, and neurotoxicity from HIV-infected MDM, but the mechanisms are not entirely understood. We hypothesized that HIV-1 infection upregulates the expression of proteins associated with oxidative stress and that a CB2R agonist could reverse these effects. MDM were isolated from healthy women donors (n = 3), infected with HIV-1ADA, and treated with JWH-133. After 13 days post-infection, cell lysates were labeled by Tandem Mass Tag (TMT) and analyzed by LC/MS/MS quantitative proteomics bioinformatics. While HIV-1 infection upregulated CATB, NF-κB signaling, Nrf2-mediated oxidative stress response, and lysosomal exocytosis, JWH-133 treatment downregulated the expression of the proteins involved in these pathways. Our results suggest that JWH-133 is a potential alternative therapy against HIV-induced neurotoxicity and warrant in vivo studies to test its potential against HAND.

A new look at an old body: molecular determinants of Weibel-Palade body composition and von Willebrand factor exocytosis.

Endothelial cells, forming a monolayer along blood vessels, intricately regulate vascular hemostasis, inflammatory responses, and angiogenesis. A key determinant of these functions is the controlled secretion of Weibel-Palade bodies (WPBs), which are specialized endothelial storage organelles housing a presynthesized pool of the hemostatic protein von Willebrand factor and various other hemostatic, inflammatory, angiogenic, and vasoactive mediators. This review delves into recent mechanistic insights into WPB biology, including the biogenesis that results in their unique morphology, the acquisition of intraluminal vesicles and other cargo, and the contribution of proton pumps to organelle acidification. Additionally, in light of a number of proteomic approaches to unravel the regulatory networks that control WPB formation and secretion, we provide a comprehensive overview of the WPB exocytotic machinery, including their molecular and cellular mechanisms.

Exosomal circRNAs: Novel biomarkers and therapeutic targets for urinary tumors.

Exosomal circRNAs have emerged as promising biomarkers and therapeutic targets for urinary tumors. In this review, we explored the intricate role of exosomal circRNAs in urological cancers, focusing on their biological functions, dysregulation in tumors, and potential clinical applications. The review delves into the mechanisms by which exosomal circRNAs contribute to tumor progression and highlights their diagnostic and therapeutic implications. By synthesizing current research findings, we present a compelling case for the significance of exosomal circRNAs in the context of urinary tumors. Furthermore, the review discusses the challenges and opportunities associated with utilizing exosomal circRNAs as diagnostic tools and targeted therapeutic agents. There is a need for further research to elucidate the specific mechanisms of exosomal circRNA secretion and delivery, as well as to enhance the detection methods for clinical translational applications. Overall, this comprehensive review underscores the pivotal role of exosomal circRNAs in urinary tumors and underscores their potential as valuable biomarkers and therapeutic tools in the management of urological cancers.

Purification of time-resolved insulin granules reveals proteomic and lipidomic changes during granule aging.

Endocrine cells employ regulated exocytosis of secretory granules to secrete hormones and neurotransmitters. Secretory granule exocytosis depends on spatiotemporal variables such as proximity to the plasma membrane and age, with newly generated granules being preferentially released. Despite recent advances, we lack a comprehensive view of the molecular composition of insulin granules and associated changes over their lifetime. Here, we report a strategy for the purification of insulin secretory granules of distinct age from insulinoma INS-1 cells. Tagging the granule-resident protein phogrin with a cleavable CLIP tag, we obtain intact fractions of age-distinct granules for proteomic and lipidomic analyses. We find that the lipid composition changes over time, along with the physical properties of the membrane, and that kinesin-1 heavy chain (KIF5b) as well as Ras-related protein 3a (RAB3a) associate preferentially with younger granules. Further, we identify the Rho GTPase-activating protein (ARHGAP1) as a cytosolic factor associated with insulin granules.

Platelet C3G: a key player in vesicle exocytosis, spreading and clot retraction.

C3G is a Rap1 GEF that plays a pivotal role in platelet-mediated processes such as angiogenesis, tumor growth, and metastasis by modulating the platelet secretome. Here, we explore the mechanisms through which C3G governs platelet secretion. For this, we utilized animal models featuring either overexpression or deletion of C3G in platelets, as well as PC12 cell clones expressing C3G mutants. We found that C3G specifically regulates α-granule secretion via PKCδ, but it does not affect δ-granules or lysosomes. C3G activated RalA through a GEF-dependent mechanism, facilitating vesicle docking, while interfering with the formation of the trans-SNARE complex, thereby restricting vesicle fusion. Furthermore, C3G promotes the formation of lamellipodia during platelet spreading on specific substrates by enhancing actin polymerization via Src and Rac1-Arp2/3 pathways, but not Rap1. Consequently, C3G deletion in platelets favored kiss-and-run exocytosis. C3G also controlled granule secretion in PC12 cells, including pore formation. Additionally, C3G-deficient platelets exhibited reduced phosphatidylserine exposure, resulting in decreased thrombin generation, which along with defective actin polymerization and spreading, led to impaired clot retraction. In summary, platelet C3G plays a dual role by facilitating platelet spreading and clot retraction through the promotion of outside-in signaling while concurrently downregulating α-granule secretion by restricting granule fusion.

Pre-fusion motion state determines the heterogeneity of membrane fusion dynamics for large dense-core vesicles.

AIM: In neuroendocrine cells, large dense-core vesicles (LDCVs) undergo highly regulated pre-fusion processes before releasing hormones via membrane fusion. Significant heterogeneity has been found for LDCV population based on the dynamics of membrane fusion. However, how the pre-fusion status impacts the heterogeneity of LDCVs still remains unclear. Hence, we explored pre-fusion determinants of heterogeneous membrane fusion procedure of LDCV subpopulations. METHODS: We assessed the pre-fusion motion of two LDCV subpopulations with distinct membrane fusion dynamics individually, using total internal reflection fluorescence microscopy. These two subpopulations were isolated by blocking Rho GTPase-dependent actin reorganization using Clostridium difficile toxin B (ToxB), which selectively targets the fast fusion vesicle pool. RESULTS: We found that the fast fusion subpopulation was in an active motion mode prior to release, termed "active" LDCV pool, while vesicles from the slow fusion subpopulation were also moving but in a significantly more confined status, forming an "inert" pool. The depletion of the active pool by ToxB also eliminated fast fusion vesicles and was not rescued by pre-treatment with phorbol ester. A mild actin reorganization blocker, latrunculin A, that partially disrupted the active pool, only slightly attenuated the fast fusion subpopulation. CONCLUSION: The pre-fusion motion state of LDCVs also exhibits heterogeneity and dictates the heterogeneous fusion pore dynamics. Rearrangement of F-actin network mediates vesicle pre-fusion motion and subsequently determines the membrane fusion kinetics.