Fusion Of Intracellular Vesicles Research Articles

Mechanical Strain Induces and Increases Vesicular Release Monitored by Microfabricated Stretchable Electrodes

AbstractExocytosis involving the fusion of intracellular vesicles with cell membrane, is thought to be modulated by the mechanical cues in the microenvironment. Single‐cell electrochemistry can offer unique information about the quantification and kinetics of exocytotic events; however, the effects of mechanical force on vesicular release have been poorly explored. Herein, we developed a stretchable microelectrode with excellent electrochemical stability under mechanical deformation by microfabrication of functionalized poly(3,4‐ethylenedioxythiophene) conductive ink, which achieved real‐time quantitation of strain‐induced vesicular exocytosis from a single cell for the first time. We found that mechanical strain could cause calcium influx via the activation of Piezo1 channels in chromaffin cell, initiating the vesicular exocytosis process. Interestingly, mechanical strain increases the amount of catecholamines released by accelerating the opening and prolonging the closing of fusion pore during exocytosis. This work is expected to provide revealing insights into the regulatory effects of mechanical stimuli on vesicular exocytosis.

Mechanical Strain Induces and Increases Vesicular Release Monitored by Microfabricated Stretchable Electrodes.

Exocytosis involving the fusion of intracellular vesicles with cell membrane, is thought to be modulated by the mechanical cues in the microenvironment. Single-cell electrochemistry can offer unique information about the quantification and kinetics of exocytotic events; however, the effects of mechanical force on vesicular release have been poorly explored. Herein, we developed a stretchable microelectrode with excellent electrochemical stability under mechanical deformation by microfabrication of functionalized poly(3,4-ethylenedioxythiophene) conductive ink, which achieved real-time quantitation of strain-induced vesicular exocytosis from a single cell for the first time. We found that mechanical strain could cause calcium influx via the activation of Piezo1 channels in chromaffin cell, initiating the vesicular exocytosis process. Interestingly, mechanical strain increases the amount of catecholamines released by accelerating the opening and prolonging the closing of fusion pore during exocytosis. This work is expected to provide revealing insights into the regulatory effects of mechanical stimuli on vesicular exocytosis.

Initiation of acute pancreatitis in mice is independent of fusion between lysosomes and zymogen granules

The co-localization of the lysosomal protease cathepsin B (CTSB) and the digestive zymogen trypsinogen is a prerequisite for the initiation of acute pancreatitis. However, the exact molecular mechanisms of co-localization are not fully understood. In this study, we investigated the role of lysosomes in the onset of acute pancreatitis by using two different experimental approaches. Using an acinar cell-specific genetic deletion of the ras-related protein Rab7, important for intracellular vesicle trafficking and fusion, we analyzed the subcellular distribution of lysosomal enzymes and the severity of pancreatitis in vivo and ex vivo. Lysosomal permeabilization was performed by the lysosomotropic agent Glycyl-L-phenylalanine 2-naphthylamide (GPN). Acinar cell-specific deletion of Rab7 increased endogenous CTSB activity and despite the lack of re-distribution of CTSB from lysosomes to the secretory vesicles, the activation of CTSB localized in the zymogen compartment still took place leading to trypsinogen activation and pancreatic injury. Disease severity was comparable to controls during the early phase but more severe at later time points. Similarly, GPN did not prevent CTSB activation inside the secretory compartment upon caerulein stimulation, while lysosomal CTSB shifted to the cytosol. Intracellular trypsinogen activation was maintained leading to acute pancreatitis similar to controls. Our results indicate that initiation of acute pancreatitis seems to be independent of the presence of lysosomes and that fusion of lysosomes and zymogen granules is dispensable for the disease onset. Intact lysosomes rather appear to have protective effects at later disease stages.

Munc18c accelerates SNARE-dependent membrane fusion in the presence of regulatory proteins α-SNAP and NSF

Intracellular vesicle fusion is driven by the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and their cofactors, including Sec1/Munc18 (SM), α-SNAP, and NSF. α-SNAP and NSF play multiple layers of regulatory roles in the SNARE assembly, disassembling the cis-SNARE complex and the prefusion SNARE complex. How SM proteins coupled with NSF and α-SNAP regulate SNARE-dependent membrane fusion remains incompletely understood. Munc18c, an SM protein involved in the exocytosis of the glucose transporter GLUT4, binds and activates target (t-) SNAREs to accelerate the fusion reaction through a SNARE-like peptide (SLP). Here, using an in vitro reconstituted system, we discovered that α-SNAP blocks the GLUT4 SNAREs-mediated membrane fusion. Munc18c interacts with t-SNAREs to displace α-SNAP, which overcomes the fusion inhibition. Furthermore, Munc18c shields the trans-SNARE complex from α-SNAP/NSF-mediated disassembly and accelerates SNARE-dependent fusion kinetics in the presence of NSF and α-SNAP. The SLP in domain 3a is indispensable in Munc18c-assisted resistance to NSF and α-SNAP. Together, our findings demonstrate that Munc18c protects the prefusion SNARE complex from α-SNAP and NSF, promoting SNARE-dependent membrane fusion through its SLP.

Rab2 and Rab6 are Implicated in Acrosome Formation during Spermatogenesis in Eriocheir sinensis: Based on Sperm Proteome.

Rab proteins are GTP-dependent small proteins that function as regulators of intracellular vesicle transport, fusion, and localization. However, few studies have investigated their function in Decapoda reproduction. The Eriocheir sinensis sperm has no tail and the nuclei are uncondensed. With the acrosome forming the majority of the sperm mass, it provides an ideal model for studying acrosome formation. We firstly analyzed the sperm proteome using LC-MS/MS. To study the functions of Rab2 and Rab6, related to the Golgi apparatus, in the acrosome formation during spermatogenesis, the genes of Rab2 and Rab6 were cloned based on the testis transcriptome of E.sinensis and poly-clonal antibodies were prepared. The presence of 2 Rab proteins was confirmed in the testis and sperm by western blot. We further observed the characteristics of target 2 Rab proteins using immunofluorescence (IF). A total of 1247 proteins including 7 Rab proteins, Rab1, Rab2, Rab5, Rab6, Rab11, Rab14, and Rab18 were identified in the sperm proteome. The IF results showed that Rab2 co-localizes with GM130, a cis-Golgi matrix protein, in the spermatagonia and spermatocytes. In the early spermatids, Rab2 and Rab6 participate in the formation of pre-acrosomal vesicles. In maturing spermatids, both Rab2 and Rab6 settle on the acrosomal membrane but present different characteristics wrapping the pre-acrosome. In the mature sperm, Rab2 localizes in the perinuclear theca surrounding the nuclei cup, while Rab6 remains on the acrosomal membrane. Our research found 7 Rab proteins based on the analysis of the sperm proteome in E.sinensis, and confirmed the involvement of Rab2 and Rab6 in acrosome formation. These findings provide a foundation for studying the functions of Rab proteins during spermatogenesis in Decapoda animals.

Impact of mesenchymal stromal cell-derived vesicular cargo on B-cell acute lymphoblastic leukemia progression.

Leukemia cells reciprocally interact with their surrounding bone marrow microenvironment (BMM), rendering it hospitable to leukemia cell survival, for instance through the release of small extracellular vesicles (sEVs). In contrast, we show here that BMM deficiency of pleckstrin homology domain family M member 1 (PLEKHM1), which serves as a hub between fusion and secretion of intracellular vesicles and is important for vesicular secretion in osteoclasts, accelerates murine BCR-ABL1+ B-cell acute lymphoblastic leukemia (B-ALL) via regulation of the cargo of sEVs released by BMM-derived mesenchymal stromal cells (MSCs). PLEKHM1-deficient MSCs and their sEVs carry increased amounts of syntenin and syndecan-1, resulting in a more immature B-cell phenotype and an increased number/function of leukemia-initiating cells (LICs) via focal adhesion kinase and AKT signaling in B-ALL cells. Exvivo pretreatment of LICs with sEVs derived from PLEKHM1-deficient MSCs led to a strong trend toward acceleration of murine and human BCR-ABL1+ B-ALL. In turn, inflammatory mediators such as recombinant or B-ALL cell-derived tumor necrosis factor α or interleukin-1β condition murine and human MSCs invitro, decreasing PLEKHM1, while increasing syntenin and syndecan-1 in MSCs, thereby perpetuating the sEV-associated circuit. Consistently, human trephine biopsies of patients with B-ALL showed a reduced percentage of PLEKHM1+ MSCs. In summary, our data reveal an important role of BMM-derived sEVs for driving specifically BCR-ABL1+ B-ALL, possibly contributing to its worse prognosis compared with BCR-ABL1- B-ALL, and suggest that secretion of inflammatory cytokines by cancer cells in general may similarly modulate the tumor microenvironment.

Assembly-promoting protein Munc18c stimulates SNARE-dependent membrane fusion through its SNARE-like peptide

Intracellular vesicle fusion requires the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and their cognate Sec1/Munc18 (SM) proteins. How SM proteins act in concert with trans-SNARE complexes to promote membrane fusion remains incompletely understood. Munc18c, a broadly distributed SM protein, selectively regulates multiple exocytotic pathways, including GLUT4 exocytosis. Here, using an in vitro reconstituted system, we discovered a SNARE-like peptide (SLP), conserved in Munc18-1 of synaptic exocytosis, is crucial to the stimulatory activity of Munc18c in vesicle fusion. The direct stimulation of the SNARE-mediated fusion reaction by SLP further supported the essential role of this fragment. Interestingly, we found SLP strongly accelerates the membrane fusion rate when anchored to the target membrane but not the vesicle membrane, suggesting it primarily interacts with t-SNAREs in cis to drive fusion. Furthermore, we determined the SLP fragment is competitive with the full-length Munc18c protein and specific to the cognate v-SNARE isoforms, supporting how it could resemble Munc18c’s activity in membrane fusion. Together, our findings demonstrate that Munc18c facilitates SNARE-dependent membrane fusion through SLP, revealing that the t-SNARE-SLP binding mode might be a conserved mechanism for the stimulatory function of SM proteins in vesicle fusion.

HDL as Bidirectional Lipid Vectors: Time for New Paradigms.

The anti-atherogenic properties of high-density lipoproteins (HDL) have been explained mainly by reverse cholesterol transport (RCT) from peripheral tissues to the liver. The RCT seems to agree with most of the negative epidemiological correlations between HDL cholesterol levels and coronary artery disease. However, therapies designed to increase HDL cholesterol failed to reduce cardiovascular risk, despite their capacity to improve cholesterol efflux, the first stage of RCT. Therefore, the cardioprotective role of HDL may not be explained by RCT, and it is time for new paradigms about the physiological function of these lipoproteins. It should be considered that the main HDL apolipoprotein, apo AI, has been highly conserved throughout evolution. Consequently, these lipoproteins play an essential physiological role beyond their capacity to protect against atherosclerosis. We propose HDL as bidirectional lipid vectors carrying lipids from and to tissues according to their local context. Lipid influx mediated by HDL appears to be particularly important for tissue repair right on site where the damage occurs, including arteries during the first stages of atherosclerosis. In contrast, the HDL-lipid efflux is relevant for secretory cells where the fusion of intracellular vesicles drastically enlarges the cytoplasmic membrane with the potential consequence of impairment of cell function. In such circumstances, HDL could deliver some functional lipids and pick up not only cholesterol but an integral part of the membrane in excess, restoring the viability of the secretory cells. This hypothesis is congruent with the beneficial effects of HDL against atherosclerosis as well as with their capacity to induce insulin secretion and merits experimental exploration.

Vesicle Impact Electrochemical Cytometry to Determine Carbon Nanotube-Induced Fusion of Intracellular Vesicles.

Carbon nanotube (CNT)-modified electrodes are used to obtain new measurements of vesicle content via amperometry. We have investigated the interaction between CNTs and isolated adrenal chromaffin vesicles (as a model) by vesicle impact electrochemical cytometry. Our data show that the presence of CNTs not only significantly increased the vesicular catecholamine number from 2,250,000 ± 112,766 molecules on a bare electrode to 3,880,000 ± 686,573 molecules on CNT/carbon fiber electrodes but also caused an enhancement in the maximum intensity of the current, which implies the existence of strong interactions between vesicle biolayers and CNTs and an altered electroporation process. We suggest that CNTs might perturb and destabilize the membrane structure of intracellular vesicles and cause the aggregation or fusion of vesicles into new vesicles with larger size and higher content. Our findings are consistent with previous computational and experimental results and support the hypothesis that CNTs as a mediator can rearrange the phospholipid bilayer membrane and trigger homotypic fusion of intracellular vesicles.

NKG7 - regulating endosomal pathways?

Ng et al. have identified NKG7 as a regulator of inflammation in response to diverse immunological challenges. While NKG7 was required for the degranulation of cytotoxic cells, additional defects including reduced expansion and trafficking of CD8 T cells, and altered antigen presentation, were noted in NKG7-deficient mice. The precise mechanism by which NKG7 mediates its effects has not been resolved but may involve regulation of endosomal vesicle trafficking.

The Sec1/Munc18 protein Vps45 holds the Qa-SNARE Tlg2 in an open conformation.

Fusion of intracellular trafficking vesicles is mediated by the assembly of SNARE proteins into membrane-bridging complexes. SNARE-mediated membrane fusion requires Sec1/Munc18-family (SM) proteins, SNARE chaperones that can function as templates to catalyze SNARE complex assembly. Paradoxically, the SM protein Munc18-1 traps the Qa-SNARE protein syntaxin-1 in an autoinhibited closed conformation. Here we present the structure of a second SM-Qa-SNARE complex, Vps45-Tlg2. Strikingly, Vps45 holds Tlg2 in an open conformation, with its SNARE motif disengaged from its Habc domain and its linker region unfolded. The domain 3a helical hairpin of Vps45 is unfurled, exposing the presumptive R-SNARE binding site to allow template complex formation. Although Tlg2 has a pronounced tendency to form homo-tetramers, Vps45 can rescue Tlg2 tetramers into stoichiometric Vps45-Tlg2 complexes. Our findings demonstrate that SM proteins can engage Qa-SNAREs using at least two different modes, one in which the SNARE is closed and one in which it is open.

Molecular Mechanisms of Intercellular Dissemination of Bacterial Pathogens.

Several intracellular bacterial pathogens, including Listeria monocytogenes, Shigella flexerni, and Rickettsia spp. use an actin-based motility process to spread in mammalian cell monolayers. Cell-to-cell spread is mediated by protrusive structures that contain bacteria encased in the host cell plasma membrane. These protrusions, which form in infected host cells, are internalized by neighboring cells. In this review, we summarize key findings on cell-to-cell spread, focusing on recent work on mechanisms of protrusion formation and internalization. We also discuss the dynamic behavior of bacterial populations during spread, and highlight recent findings showing that intercellular spread by an extracellular bacterial pathogen.

Regulation of human cerebral cortical development by EXOC7 and EXOC8, components of the exocyst complex, and roles in neural progenitor cell proliferation and survival

PurposeThe exocyst complex is a conserved protein complex that mediates fusion of intracellular vesicles to the plasma membrane and is implicated in processes including cell polarity, cell migration, ciliogenesis, cytokinesis, autophagy, and fusion of secretory vesicles. The essential role of these genes in human genetic disorders, however, is unknown. MethodsWe performed homozygosity mapping and exome sequencing of consanguineous families with recessively inherited brain development disorders. We modeled an EXOC7 splice variant in vitro and examined EXOC7 messenger RNA (mRNA) expression in developing mouse and human cortex. We modeled exoc7 loss-of-function in a zebrafish knockout. ResultsWe report variants in exocyst complex members, EXOC7 and EXOC8, in a novel disorder of cerebral cortex development. In EXOC7, we identified four independent partial loss-of-function (LOF) variants in a recessively inherited disorder characterized by brain atrophy, seizures, and developmental delay, and in severe cases, microcephaly and infantile death. In EXOC8, we found a homozygous truncating variant in a family with a similar clinical disorder. We modeled exoc7 deficiency in zebrafish and found the absence of exoc7 causes microcephaly. ConclusionOur results highlight the essential role of the exocyst pathway in normal cortical development and how its perturbation causes complex brain disorders.

Intracellular Vesicle Fusion Requires a Membrane-Destabilizing Peptide Located at the Juxtamembrane Region of the v-SNARE

SUMMARYIntracellular vesicle fusion is mediated by soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) and Sec1/Munc18 (SM) proteins. It is generally accepted that membrane fusion occurs when the vesicle and target membranes are brought into close proximity by SNAREs and SM proteins. In this work, we demonstrate that, for fusion to occur, membrane bilayers must be destabilized by a conserved membrane-embedded motif located at the juxtamembrane region of the vesicle-anchored v-SNARE. Comprised of basic and hydrophobic residues, the juxtamembrane motif perturbs the lipid bilayer structure and promotes SNARE-SM-mediated membrane fusion. The juxtamembrane motif can be functionally substituted with an unrelated membrane-disrupting peptide in the membrane fusion reaction. These findings establish the juxtamembrane motif of the v-SNARE as a membrane-destabilizing peptide. Requirement of membrane-destabilizing peptides is likely a common feature of biological membrane fusion.

Pannexin-1 limits the production of proinflammatory cytokines during necroptosis.

The activation of mixed lineage kinase-like (MLKL) by receptor-interacting protein kinase-3 (RIPK3) controls the execution of necroptosis, a regulated form of necrosis that occurs in apoptosis-deficient conditions. Active oligomerized MLKL triggers the exposure of phosphatidylserine residues on the cell surface and disrupts the plasma membrane integrity by forming lytic pores. MLKL also governs endosomal trafficking and biogenesis of small extracellular vesicles as well as the production of proinflammatory cytokines during the early steps of necroptosis; however, the molecular basis continues to be elucidated. Here, we find that MLKL oligomers activate Pannexin-1 (PANX1) channels, concomitantly to the loss of phosphatidylserine asymmetry. This plasma membrane "leakiness" requires the small GTPase RAB27A and RAB27B isoforms, which regulate intracellular vesicle trafficking, docking, and fusion with the plasma membrane. Although cells in which PANX1 is silenced or inhibited normally undergo necroptotic death, they display enhanced production of cytokines such as interleukin-8, indicating that PANX1 may tamper with inflammation. These data identify a novel signaling nexus between MLKL, RAB27, and PANX1 and propose ways to interfere with inflammation associated with necroptosis.

Synaptotagmin 17 controls neurite outgrowth and synaptic physiology via distinct cellular pathways

The synaptotagmin (syt) proteins have been widely studied for their role in regulating fusion of intracellular vesicles with the plasma membrane. Here we report that syt-17, an unusual isoform of unknown function, plays no role in exocytosis, and instead plays multiple roles in intracellular membrane trafficking. Syt-17 is localized to the Golgi complex in hippocampal neurons, where it coordinates import of vesicles from the endoplasmic reticulum to support neurite outgrowth and facilitate axon regrowth after injury. Further, we discovered a second pool of syt-17 on early endosomes in neurites. Loss of syt-17 disrupts endocytic trafficking, resulting in the accumulation of excess postsynaptic AMPA receptors and defective synaptic plasticity. Two distinct pools of syt-17 thus control two crucial, independent membrane trafficking pathways in neurons. Function of syt-17 appears to be one mechanism by which neurons have specialized their secretory and endosomal systems to support the demands of synaptic communication over sprawling neurite arbors.

Faster regeneration associated to high expression of Fam65b and Hdac6 in dysferlin-deficient mouse

Dysferlin is a sarcolemmal muscle protein associated with the processes of membrane repair, trafficking, and fusion of intracellular vesicles and muscle regeneration.Mutations in the DYSF gene cause clinically distinct forms of muscular dystrophies. The dysferlin-deficient SJL/J mouse model presents a reduction of 85% of the protein but shows mild weakness and discrete histopathological alterations. To study the effect of dysferlin deficiency in the muscle regenerative process, we used a model of electrical injury by electroporation to induce muscle degeneration/regeneration in the SJL/J mouse. The relative expression of the genes Pax7, MyoD, Myf5, and Myog was accompanied by the histopathological evaluation during muscle recovery at different time points after injury. We also investigated the effects of dysferlin deficiency in the expression of genes encoding FAM65B and HDAC6 proteins, recently described as forming a tricomplex with dysferlin at the beginning of myoblast differentiation. We observed an altered time course through the process of degeneration and regeneration in dysferlin-deficient mice, with remarkable regenerative capacity characterized by a faster and effective response in the first days after injury, as compared to the WT mice. Also, dysferlin deficiency seems to significantly alter the gene expression of Fam65b and Hdac6 during regeneration, since higher levels of expression of both genes were observed in dysferlin-deficient mice. These results need further attention to define their relevance in the disease mechanism.

The N-peptide–binding mode is critical to Munc18-1 function in synaptic exocytosis

Sec1/Munc18 (SM) proteins promote intracellular vesicle fusion by binding to N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). A key SNARE-binding mode of SM proteins involves the N-terminal peptide (N-peptide) motif of syntaxin, a SNARE subunit localized to the target membrane. In in vitro membrane fusion assays, inhibition of N-peptide motif binding previously has been shown to abrogate the stimulatory function of Munc18-1, a SM protein involved in synaptic exocytosis in neurons. The physiological role of the N-peptide-binding mode, however, remains unclear. In this work, we addressed this key question using a "clogged" Munc18-1 protein, in which an ectopic copy of the syntaxin N-peptide motif was directly fused to Munc18-1. We found that the ectopic N-peptide motif blocks the N-peptide-binding pocket of Munc18-1, preventing the latter from binding to the native N-peptide motif on syntaxin-1. In a reconstituted system, we observed that clogged Munc18-1 is defective in promoting SNARE zippering. When introduced into induced neuronal cells (iN cells) derived from human pluripotent stem cells, clogged Munc18-1 failed to mediate synaptic exocytosis. As a result, both spontaneous and evoked synaptic transmission was abolished. These genetic findings provide direct evidence for the crucial role of the N-peptide-binding mode of Munc18-1 in synaptic exocytosis. We suggest that clogged SM proteins will also be instrumental in defining the physiological roles of the N-peptide-binding mode in other vesicle-fusion pathways.

Functional Reconstitution of Intracellular Vesicle Fusion Using Purified SNAREs and Sec1/Munc18 (SM) Proteins.

The fusion of intracellular vesicles with target membranes is mediated by two classes of conserved molecules-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAP receptors or SNAREs) and Sec1/Munc18 (SM) proteins. A conserved function of SM proteins is to recognize their cognate trans-SNARE complexes and accelerate fusion kinetics. Here, we describe a physiologically relevant reconstitution system in which macromolecular crowding agents are included to recapitulate the crowded intracellular environment. Through this system, we elucidate the molecular mechanisms by which SNAREs and SM proteins drive vesicle fusion.

SNAP23 regulates BAX-dependent adipocyte programmed cell death independently of canonical macroautophagy.

The t-SNARE protein SNAP23 conventionally functions as a component of the cellular machinery required for intracellular transport vesicle fusion with target membranes and has been implicated in the regulation of fasting glucose levels, BMI, and type 2 diabetes. Surprisingly, we observed that adipocyte-specific KO of SNAP23 in mice resulted in a temporal development of severe generalized lipodystrophy associated with adipose tissue inflammation, insulin resistance, hyperglycemia, liver steatosis, and early death. This resulted from adipocyte cell death associated with an inhibition of macroautophagy and lysosomal degradation of the proapoptotic regulator BAX, with increased BAX activation. BAX colocalized with LC3-positive autophagic vacuoles and was increased upon treatment with lysosome inhibitors. Moreover, BAX deficiency suppressed the lipodystrophic phenotype in the adipocyte-specific SNAP23-KO mice and prevented cell death. In addition, ATG9 deficiency phenocopied SNAP23 deficiency, whereas ATG7 deficiency had no effect on BAX protein levels, BAX activation, or apoptotic cell death. These data demonstrate a role for SNAP23 in the control of macroautophagy and programmed cell death through an ATG9-dependent, but ATG7-independent, pathway regulating BAX protein levels and BAX activation.