Docking Of Secretory Vesicles Research Articles

Phospholipase D1 produces phosphatidic acid at sites of secretory vesicle docking and fusion.

Phospholipase D1 (PLD1) activity is essential for the stimulated exocytosis of secretory vesicles where it acts as a lipid-modifying enzyme to produces phosphatidic acid (PA). PLD1 localizes to the plasma membrane and secretory vesicles, and PLD1 inhibition or knockdowns reduce the rate of fusion. However, temporal data resolving when and where PLD1 and PA are required during exocytosis is lacking. In this work, PLD1 and production of PA are measured during the trafficking, docking, and fusion of secretory vesicles in PC12 cells. Using fluorescently tagged PLD1 and a PA-binding protein, cells were imaged using TIRF microscopy to monitor the presence of PLD1 and the formation of PA throughout the stages of exocytosis. Single docking and fusion events were imaged to measure the recruitment of PLD1 and the formation of PA. PLD1 is present on mobile, docking, and fusing vesicles and also colocalizes with Syx1a clusters. Treatment of cells with PLD inhibitors significantly reduces fusion, but not PLD1 localization to secretory vesicles. Inhibitors also alter the formation of PA; when PLD1 is active, PA slowly accumulates on docked vesicles. During fusion, PA is reduced in cells treated with PLD1 inhibitors, indicating that PLD1 produces PA during exocytosis.

A SNAP23-Dependent SNARE Complex Mediates Free Light Chain Secretion in AL Amyloidosis and Multiple Myeloma and Its Blockade Triggers a Terminal Unfolded Protein Response and Massive Apoptosis

Background AL amyloidosis (AL) is an incurable plasma cell (PC) disorder with only one FDA-approved therapy. Deposition of immunoglobulin free light chain (FLC) fibrils in target organs is the sole pathogenic mechanism in AL. However, therapeutic strategies directly targeting FLC secretion are not available. SNARE proteins, the specific targets of botulinum neurotoxin (BoNT), mediate the docking and fusion of secretory vesicles. We hypothesized that targeting specific sets of SNAREs via BoNT will block FLC exocytosis, causing retention of FLC-loaded vesicles and triggering a terminal unfolded protein response (UPR) and apoptosis in AL cells. We postulate that SNAREs are potential novel targets in AL and other plasma cell disorders. Materials and Methods We stably expressed 7 distinct, inducible BoNT serotypes (BoNT/A-F) in FLC-secreting AL (ALMC1 and ALMC2) and multiple myeloma (MM, KMS11) cell lines via tetracycline-inducible (Tet-On) bicistronic lentiviral vectors with a P2A-GFP which allowed us to use GFP as a biomarker of BoNT expression. We established monoclones of successfully transduced cells for each BoNT. We performed flow cytometry to assess the kinetics of GFP induction and assess for apoptosis via AnnexinV/DAPI staining. Western blot (WB) was used to assess the cleavage of SNAREs upon expression of each BoNT serotype. We used WB and ELISA to assess FLC secretion per cell upon expression of each BoNT serotype. We used TurboID proximity labeling followed by mass spectrometry to identify cytotoxic BoNT interacting partners and used co-IP for confirmation. We used RNP-based CRISPR-Cas9 gene editing to knock out (KO) putative BoNT targets. Results By querying the IFM170 gene expression database of newly diagnosed MM patients, we identified VAMP2, VAMP3, SNAP23, and SYNTAXIN4 as the top expressed SNAREs. Their high expression was retained in a large panel of AL and MM cell lines. Expression of all BoNT serotypes, except for BoNT B, led to rapid apoptosis of AL and MM cells, with BoNT F standing out for the extent and rapidity of cytotoxicity. Dual cleavage of SNAP23 and VAMP3 correlated with cytotoxicity of BoNT in AL and MM cell lines. Mechanistically, we showed that cytotoxic BoNTs, but not non-toxic BoNT B, led to decreased FLC secretion and increased FLC retention and triggered the PERK pathway of the UPR with increased expression of CHOP and GADD34, preceding the onset of apoptosis. We identified SNAP23 as an interacting partner of highly cytotoxic BoNT F, but not non-toxic BoNT B. KO experiment showed single SNAP23 KO to be partially toxic for AL and MM cell lines but did not recapitulate the effects of cytotoxic BoNTs entirely. Based on our preliminary data, we hypothesize VAMP3 and SYNTAXIN4 to be synthetic lethal with SNAP23. Conclusions Our data provide proof of concept that blocking FLC secretion in AL and MM cells is feasible and results in rapid cell apoptosis by triggering a terminal UPR, thus representing a promising, novel therapeutic approach. By using BoNT as a biological tool, we identified SNAP23 as a critical component of FLC exocytosisin AL cell lines, thus representing a novel, promising molecular target in AL amyloidosis and multiple myeloma.

Abstract 141: Targeting protein secretion as a novel therapeutic strategy in AL amyloidosis

Abstract AL amyloidosis (AL) is an incurable plasma cell disorder. The solely pathogenic mechanism in AL is deposition of immunoglobulin free light chains (FLC) organized in fibrils in target organs. Surprisingly, therapeutic strategies directly targeting FLC secretion are not available. SNARE proteins, which are the specific target of botulinum neurotoxin (BoNT), are involved in the docking and fusion of secretory vesicles. We hypothesized that certain BoNT serotypes may block FLC secretion and trigger cytotoxicity triggering a terminal UPR. Gene expression profiling in a cohort of 170 newly diagnosed multiple myeloma patients (IFM170) was used to interrogate SNAREs expression in malignant plasma cells. We developed tetracycline inducible, bicistronic vectors expressing distinct BoNT serotypes (BoNT/A-F), T2A and GFP. Lentivirally transduced cells would express BoNT in a 1:1 stochiometric ratio with GFP, upon doxycycline (dox) administration. We transduced AL cell lines stably expressing rRTA with Tet-On lentivirus expressing 7 distinct BoNTs and performed two sets of experiments. First, we performed time-course viability assays on polyclonally transduced cells and compared relative proportion of GFP+ cells over time. Then, we single-cell sorted transduced cells, triggered BoNT expression and assessed GFP kinetic and apoptosis at 24, 48 and 72 hours post dox. SNAREs cleavage following induction of BoNT expression was evaluated via WB in GFP+ clones. To assess if BoNT cytotoxicity correlated with cessation of FLC secretion, we performed a secretion assay in monoclones expressing distinct BoNTs. IFM170 GEP and AL/MM cell lines analysis showed VAMP2, VAMP3 and SNAP23 as the top expressed SNAREs. By using polyclonally transduced cells, we show that GFP+ (transduced) cells are rapidly depleted over time after dox, across all serotypes, except BoNT/B, consistent with cytotoxic effect. We noted an association between SNAP23 and VAMP3 cleavage and BoNT toxicity, suggesting that dual targeting of SNAP23/VAMP3 may be necessary to mediate BoNT cytotoxicity. We next show that only BoNTs causing early cytotoxicity (A, D, E and F) significantly inhibited FLC secretion and induced UPR activation, presumably through FLC retention. Indeed, cytotoxic BoNTs, activated PERK pathway with eIF2a phosphorylation (p-eIF2a); ATF4, CHOP and GADD34 upregulation. Importantly preliminary data in CHOP KO AL cells show rescue of BoNT_induced death, confirming that a terminal UPR is the mechanism of cytotoxicity of BoNT in AL. We show that in all seven BoNT serotypes examined, except for BoNT/B, simultaneous cleavage of SNAP23 and VAMP3 predicts BoNT cytotoxicity and correlates with cessation of FLC secretion and terminal UPR. Overall, our preliminary data provide proof of concept that targeting FLC secretion is feasible and of therapeutic efficacy in preclinical AL models, suggesting potential clinical translatability of this innovative approach. Citation Format: Maria Moscvin, Peter Gregor Czarnecki, Tianzeng Chen, Annamaria Gullá, Kenneth Carl Anderson, Giada Bianchi. Targeting protein secretion as a novel therapeutic strategy in AL amyloidosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 141.

Targeting Free Light Chain Secretion Via Botulinum Neurotoxin Is a Novel Therapeutic Strategy in AL Amyloidosis By Inducing a Terminal Unfolded Protein Response

BackgroundAL amyloidosis (AL) is an incurable plasma cell (PC) disorder. The solely pathogenic mechanism in AL is deposition of immunoglobulin free light chains (FLC) organized in fibrils in target organs. Surprisingly, therapeutic strategies directly targeting FLC secretion are not available. SNARE proteins, which are the specific target of botulinum neurotoxin (BoNT), are involved in the docking and fusion of secretory vesicles. We hypothesized that certain BoNT serotypes may block FLC exocytosis, causing retention of FLC-loaded vesicles and triggering a terminal unfolded protein response (UPR).Materials and MethodsGene expression profiling in a cohort of 170 newly diagnosed multiple myeloma (MM) patients (IFM170) was used to interrogate SNAREs expression in malignant plasma cells. Western blotting (WB) was used to assess SNARE expression across MM and AL cell lines. We developed tetracycline inducible, lentiviral vectors expressing distinct BoNT serotypes (BoNT/A-F), T2A and GFP. Lentivirally transduced cells would express BoNT in a 1:1 stochiometric ratio with GFP, upon doxycycline (dox) administration, allowing for flow cytometry-based analysis. A vector comprising solely T2A and GFP was used as negative control. We transduced AL cell lines with Tet-On lentivirus expressing 7 distinct BoNTs and performed two sets of experiments. First, we performed time-course viability assays on polyclonally transduced cells and compared relative proportion of GFP+ cells over time. Then, we single-cell sorted transduced cells, triggered BoNT expression and assessed GFP kinetic and apoptosis via AnnexinV/DAPI flow cytometry at 24, 48 and 72 hours post dox. SNAREs cleavage following induction of BoNT expression was evaluated via WB in GFP+ clones. To assess if BoNT cytotoxicity correlated with cessation of FLC secretion, we performed a secretion assay in monoclones expressing distinct BoNTs.ResultsIFM170 GEP analysis showed VAMP2, VAMP3 and SNAP23 as the top expressed SNAREs. This was further confirmed in AL/MM cell lines. By using polyclonally transduced cells, we show that GFP+ cells are rapidly depleted over time after dox, across all serotypes, except BoNT/B, consistent with cytotoxic effect. Similarly, we observed rapid apoptosis in monoclones expressing any BoNT serotypes, except BoNT/B. We noted an association between SNAP23 and VAMP3 cleavage and BoNT toxicity, suggesting that dual targeting of SNAP23/VAMP3 may be necessary to mediate BoNT cytotoxicity. We next show that only BoNTs causing early cytotoxicity significantly inhibited FLC secretion. Cytotoxic BoNTs, activated PERK pathway with eIF2a phosphorylation (p-eIF2a); CHOP and GADD34 upregulation, presumably through FLC retention.ConclusionsWe show that cytotoxic BoNTs block FLC secretion, trigger a terminal UPR and induce apoptosis in AL and MM models. We provide proof of concept that targeting FLC secretion has a potential clinical translatability. DisclosuresCzarnecki: Clearview: Consultancy. Anderson: Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees. Bianchi: Karyopharm: Consultancy, Honoraria; MJH: Honoraria; Jacob D. Fuchsberg Law Firm: Consultancy; Pfizer: Consultancy, Honoraria.

OAB-042: Targeting free light chain secretion via Botulinum Neurotoxin is a novel therapeutic strategy in AL amyloidosis by inducing a terminal unfolded protein response

<h3>Background</h3> AL amyloidosis (AL) is an incurable plasma cell (PC) disorder. The solely pathogenic mechanism in AL is deposition of immunoglobulin free light chains (FLC) organized in fibrils in target organs. Surprisingly, therapeutic strategies directly targeting FLC secretion are not available. SNARE proteins, which are the specific target of botulinum neurotoxin (BoNT), are involved in the docking and fusion of secretory vesicles. We hypothesized that certain BoNT serotypes may block FLC exocytosis, causing retention of FLC-loaded vesicles and triggering a terminal unfolded protein response (UPR). <h3>Methods</h3> Gene expression profiling in a cohort of 170 newly diagnosed multiple myeloma (MM) patients (IFM170) was used to interrogate SNAREs expression in malignant plasma cells. Western blotting (WB) was used to assess SNARE expression across MM and AL cell lines. We developed tetracycline inducible, lentiviral vectors expressing distinct BoNT serotypes (BoNT/A-F), T2A and GFP. Lentivirally transduced cells would express BoNT in a 1:1 stochiometric ratio with GFP, upon doxycycline (dox) administration, allowing for flow cytometry-based analysis. A vector comprising solely T2A and GFP was used as negative control. We transduced AL cell lines with Tet-On lentivirus expressing 7 distinct BoNTs and performed two sets of experiments. First, we performed time-course viability assays on polyclonally transduced cells and compared relative proportion of GFP+ cells over time. Then, we single-cell sorted transduced cells, triggered BoNT expression and assessed GFP kinetic and apoptosis via AnnexinV/DAPI flow cytometry at 24, 48 and 72 hours post dox. SNAREs cleavage following induction of BoNT expression was evaluated via WB in GFP+ clones. To assess if BoNT cytotoxicity correlated with cessation of FLC secretion, we performed a secretion assay in monoclones expressing distinct BoNTs. <h3>Results</h3> IFM170 GEP analysis showed VAMP2, VAMP3 and SNAP23 as the top expressed SNAREs. This was further confirmed in AL/MM cell lines. By using polyclonally transduced cells, we show that GFP+ cells are rapidly depleted over time after dox, across all serotypes, except BoNT/B, consistent with cytotoxic effect. Similarly, we observed rapid apoptosis in monoclones expressing any BoNT serotypes, except BoNT/B. We noted an association between SNAP23 and VAMP3 cleavage and BoNT toxicity, suggesting that dual targeting of SNAP23/VAMP3 may be necessary to mediate BoNT cytotoxicity. We next show that only BoNTs causing early cytotoxicity significantly inhibited FLC secretion. Cytotoxic BoNTs, activated PERK pathway with eIF2a phosphorylation (p-eIF2a); CHOP and GADD34 upregulation, presumably through FLC retention. <h3>Conclusions</h3> We show that cytotoxic BoNTs block FLC secretion, trigger a terminal UPR and induce apoptosis in AL and MM models. We provide proof of concept that targeting FLC secretion has a potential clinical translatability.

A comprehensive review of the influence of Epigallocatechin gallate on Sjögren's syndrome associated molecular regulators of exocytosis (Review)

Sjögren's syndrome (SS) is an autoimmune disorder that affects the salivary glands, leading to reduced secretory functions and oral and ocular dryness. The salivary glands are composed of acinar cells that are responsible for the secretion and production of secretory granules, which contain salivary components, such as amylase, mucins and immunoglobulins. This secretion process involves secretory vesicle trafficking, docking, priming and membrane fusion. A failure during any of the steps in exocytosis in the salivary glands results in the altered secretion of saliva. Soluble N-ethylmaleimide-sensitive-factor attachment protein receptors, actin, tight junctions and aquaporin 5 all serve an important role in the trafficking regulation of secretory vesicles in the secretion of saliva via exocytosis. Alterations in the expression and distribution of these selected proteins leads to salivary gland dysfunction, including SS. Several studies have demonstrated that green tea polyphenols, most notably Epigallocatechin gallate (EGCG), possess both anti-inflammatory and anti-apoptotic properties in normal human cells. Molecular, cellular and animal studies have indicated that EGCG can provide protective effects against autoimmune and inflammatory reactions in salivary glands in diseases such as SS. The aim of the present article is to provide a comprehensive and up-to-date review on the possible therapeutic interactions between EGCG and the selected molecular mechanisms associated with SS.

The nanoscale molecular morphology of docked exocytic dense-core vesicles in neuroendocrine cells

Rab-GTPases and their interacting partners are key regulators of secretory vesicle trafficking, docking, and fusion to the plasma membrane in neurons and neuroendocrine cells. Where and how these proteins are positioned and organized with respect to the vesicle and plasma membrane are unknown. Here, we use correlative super-resolution light and platinum replica electron microscopy to map Rab-GTPases (Rab27a and Rab3a) and their effectors (Granuphilin-a, Rabphilin3a, and Rim2) at the nanoscale in 2D. Next, we apply a targetable genetically-encoded electron microscopy labeling method that uses histidine based affinity-tags and metal-binding gold-nanoparticles to determine the 3D axial location of these exocytic proteins and two SNARE proteins (Syntaxin1A and SNAP25) using electron tomography. Rab proteins are distributed across the entire surface and t-SNARE proteins at the base of docked vesicles. We propose that the circumferential distribution of Rabs and Rab-effectors could aid in the efficient transport, capture, docking, and rapid fusion of calcium-triggered exocytic vesicles in excitable cells.

Synaptotagmin-1: A Multi-Functional Protein that Mediates Vesicle Docking, Priming, and Fusion

The fusion of secretory vesicles with the plasma membrane depends on the assembly of v-SNAREs (VAMP2/synaptobrevin2) and t-SNAREs (SNAP25/syntaxin1) into the SNARE complex. Vesicles go through several upstream steps, referred to as docking and priming, to gain fusion competence. The vesicular protein synaptotagmin-1 (Syt-1) is the principal Ca2+ sensor for fusion in several central nervous system neurons and neuroendocrine cells and part of the docking complex for secretory granules. Syt-1 binds to the acceptor complex such as synaxin1, SNAP-25 on the plasma membrane to facilitate secretory vesicle docking, and upon Ca2+-influx, it promotes vesicle fusion. This review assesses the role of the Syt-1 protein involved in the secretory vesicle docking, priming, and fusion.

The Nanoscale Anatomy of Exocytic Dense-Core Vesicles in Neuroendocrine Cells

Rab-GTPases and their binding partners are key regulators of secretory vesicle trafficking, docking, and fusion to the plasma membrane in neurons and neuroendocrine cells. Where and how these proteins are located and organized with respect to the vesicle and plasma membrane are unknown. Here, we use correlative super-resolution light and platinum replica electron microscopy to map Rab-GTPases (Rab27a and Rab3a) and their effectors (Granuphilin-a, Rabphilin3a, and Rim2) at the nanoscale in 2D. Next, we develop a targetable genetically-encoded electron microscopy labeling method that uses histidine based affinity-tags and metal-binding gold-nanoparticles to determine the axial location of exocytic proteins using electron tomography.

Structure Prediction and Molecular Phylogenetic Analysis of Membrane Interactions in Synaptotagmin-Like Proteins

Membrane trafficking and exocytosis are evolutionarily conserved throughout eukaryotes and involve many interactions between proteins and membrane surfaces. In vertebrates, synaptotagmin-like proteins (Slp) play key roles in trafficking of large dense-core secretory vesicles. Slp family proteins promote secretory vesicle docking to the plasma membrane through their N-terminal Slp homology domains, which bind to vesicular Rab proteins, and their tandem C-terminal C2 domains (C2A and C2B), which bind plasma membrane lipids independently of calcium. Our previous experimental and computational studies showed that the C2A domain of Slp-4 (also called granuphilin) binds with high affinity to membrane surfaces containing phosphatidylinositol-(4,5)-bisphosphate (PIP2) and phosphatidylserine (PS). A conserved PIP2 binding site centered on three lysine residues in the beta3-beta4 region specifically anchors the domain to PIP2 lipids, while many other basic residues surrounding the Lys-cluster enhance interactions with PS. We hypothesize that the positive electrostatic potential of the membrane binding surface is a key determinant of its membrane affinity and function, and therefore the net charge on the protein surface is likely to be evolutionarily conserved. To test this hypothesis, the C2A domain sequences for Slp-4 and Slp-2 in vertebrates were compared to assess the evolution of their respective polybasic surfaces. Using a molecular phylogenetic approach, clades were identified and the net charges of individual and consensus sequences were calculated. Structural homology models were generated using a crystal structure of Slp-4 C2A, and Poisson-Boltzmann calculations were carried out to quantify the positive electrostatic surfaces. This approach may help characterize electrostatic interactions with charged membrane surfaces and identify conserved differences that govern affinity and function of membrane-binding proteins.

Multivalent lipid targeting by the calcium-independent C2A domain of synaptotagmin-like protein 4/granuphilin

Synaptotagmin-like protein 4 (Slp-4), also known as granuphilin, is a Rab effector responsible for docking secretory vesicles to the plasma membrane before exocytosis. Slp-4 binds vesicular Rab proteins via an N-terminal Slp homology domain, interacts with plasma membrane SNARE complex proteins via a central linker region, and contains tandem C-terminal C2 domains (C2A and C2B) with affinity for phosphatidylinositol-(4,5)-bisphosphate (PIP2). The Slp-4 C2A domain binds with low nanomolar apparent affinity to PIP2 in lipid vesicles that also contain background anionic lipids such as phosphatidylserine but much weaker when either the background anionic lipids or PIP2 is removed. Through computational and experimental approaches, we show that this high-affinity membrane binding arises from concerted interaction at multiple sites on the C2A domain. In addition to a conserved PIP2-selective lysine cluster, a larger cationic surface surrounding the cluster contributes substantially to the affinity for physiologically relevant lipid compositions. Although the K398A mutation in the lysine cluster blocks PIP2 binding, this mutated protein domain retains the ability to bind physiological membranes in both a liposome-binding assay and MIN6 cells. Molecular dynamics simulations indicate several conformationally flexible loops that contribute to the nonspecific cationic surface. We also identify and characterize a covalently modified variant that arises through reactivity of the PIP2-binding lysine cluster with endogenous bacterial compounds and binds weakly to membranes. Overall, multivalent lipid binding by the Slp-4 C2A domain provides selective recognition and high-affinity docking of large dense core secretory vesicles to the plasma membrane.

1897-P: BECN1, a Key Autophagy Protein, Regulates Adiponectin Secretion in Adipocytes by Interacting with the Exocyst Components

Autophagy is a lysosomal degradation pathway that plays pivotal roles in maintaining cellular homeostasis. Beclin1/BECN1 is a key autophagy protein that regulates the stability and kinase activity of the class III phosphatidylinositol 3-kinase complex. Dysregulation of autophagy results in the pathogenesis of a variety of diseases, including type 2 diabetes. However, the molecular mechanism of the autophagy machinery in metabolic regulation is poorly understood. To elucidate how autophagy and BECN1 are involved in energy homeostasis and insulin sensitivity, we generated a unique knock-in mouse model showing hyperactive autophagy. This mouse model carries a single amino acid substitution (F121A) in the BECN1 protein (BECN1F121A), which disrupts an inhibitory binding between BECN1 and BCL2, leading to constitutive activation of autophagy. Using this moue model, we discovered that in addition to a role in autophagic regulation, BECN1 also executes a non-degradative function. We found that in response to high-fat diet feeding, BECN1F121A mice show improved systemic insulin sensitivity, elevated adiponectin levels in the circulation, and enhanced adiponectin signaling in metabolic tissues, compared to wild type (WT) mice, despite that body weight and white adipose tissue weight are similar between the two genotypes. Using adipocyte-differentiated stromal vascular fraction cells, we further found that compared to WT BECN1, the BECN1F121A mutant interacts more strongly with components of the exocyst, a complex required for secretory vesicle docking at the plasma membrane. Finally, we demonstrated that the BECN1-exocyst interaction is mechanistically mediated through the coiled-coil domain of BECN1. Altogether, our findings suggest that BECN1 regulates adiponectin secretion through interacting with the exocyst in white adipose tissues, and highlight that autophagy proteins may play important non-degradative roles in energy metabolism. Disclosure K. Kuramoto: None. C. He: None.

Photolysis of Caged Inositol Pyrophosphate InsP8 Directly Modulates Intracellular Ca2+ Oscillations and Controls C2AB Domain Localization.

Inositol pyrophosphates constitute a family of hyperphosphorylated signaling molecules involved in the regulation of glucose uptake and insulin sensitivity. While our understanding of the biological roles of inositol heptaphosphates (PP-InsP5) has greatly improved, the functions of the inositol octaphosphates ((PP)2-InsP4) have remained unclear. Here we present the synthesis of two enantiomeric cell-permeant and photocaged (PP)2-InsP4 derivatives and apply them to study the functions in living β-cells. Photorelease of the naturally occurring isomer 1,5-(PP)2-InsP4 led to an immediate and concentration-dependent reduction of intracellular calcium oscillations, while other caged inositol pyrophosphates (3,5-(PP)2-InsP4, 5-PP-InsP5, 1-PP-InsP5, 3-PP-InsP5) showed no immediate effect. Furthermore, uncaging of 1,5-(PP)2-InsP4 but not 3,5-(PP)2-InsP4 induced translocation of the C2AB domain of granuphilin from the plasma membrane to the cytosol. Granuphilin is involved in membrane docking of secretory vesicles. This suggests that 1,5-(PP)2-InsP4 impacts β-cell activity by regulating granule localization and/or priming and calcium signaling in concert.

MUNC18-1 regulates the submembrane F-actin network, independently of syntaxin1 targeting, via hydrophobicity in β-sheet 10.

MUNC18-1 (also known as STXBP1) is an essential protein for docking and fusion of secretory vesicles. Mouse chromaffin cells (MCCs) lacking MUNC18-1 show impaired secretory vesicle docking, but also mistargeting of SNARE protein syntaxin1 and an abnormally dense submembrane F-actin network. Here, we tested the contribution of both these phenomena to docking and secretion defects in MUNC18-1-deficient MCCs. We show that an abnormal F-actin network and syntaxin1 targeting defects are not observed in Snap25- or Syt1-knockout (KO) MCCs, which are also secretion deficient. We identified a MUNC18-1 mutant (V263T in β-sheet 10) that fully restores syntaxin1 targeting but not F-actin abnormalities in Munc18-1-KO cells. MUNC18-2 and -3 (also known as STXBP2 and STXBP3, respectively), which lack the hydrophobic residue at position 263, also did not restore a normal F-actin network in Munc18-1-KO cells. However, these proteins did restore the normal F-actin network when a hydrophobic residue was introduced at the corresponding position. Munc18-1-KO MCCs expressing MUNC18-1(V263T) showed normal vesicle docking and exocytosis. These results demonstrate that MUNC18-1 regulates the F-actin network independently of syntaxin1 targeting via hydrophobicity in β-sheet 10. The abnormally dense F-actin network in Munc18-1-deficient cells is not a rate-limiting barrier in secretory vesicle docking or fusion.This article has an associated First Person interview with the first author of the paper.

Sphingolipids modulate docking, Ca2+ sensitivity and membrane fusion of native cortical vesicles.

Docking, priming, and membrane fusion of secretory vesicles (i.e. regulated exocytosis) requires lipids and proteins. Sphingolipids, in particular, sphingosine and sphingosine-1-phosphate, have been implicated in the modulation of exocytosis. However, the specific exocytotic steps that sphingolipids modulate and the enzymes that regulate sphingolipid concentrations on native secretory vesicle membranes remain unknown. Here we use tightly coupled functional and molecular analyses of fusion-ready cell surface complexes and cortical vesicles isolated from oocytes to assess the role of sphingolipids in the late, Ca2+-triggered steps of exocytosis. The molecular changes resulting from treatments with sphingolipid modifying compounds coupled with immunoblotting analysis revealed the presence of sphingosine kinase on native vesicles; the presence of a sphingosine-1-phosphate phosphatase is also indicated. Changes in sphingolipid concentrations on vesicles altered their docking/priming, Ca2+-sensitivity, and ability to fuse, indicating that sphingolipid concentrations are tightly regulated and maintained at optimal levels and ratios to ensure efficient exocytosis.

Molecules involved in acrosomal exocytosis and cortical granule exocytosis

At fertilization, the acrosome reaction and cortical reaction are crucial process to block polyspermy and the prevention of triploidy. Although molecules involved in trigger secretion are various in different exocytosis events, the two processes may share the similar exocytosis mechanism. Exocytosis is an accurate regulated process that consists of multiple stages such as recruitment, targeting, tethering and docking of secretory vesicles with the plasma membrane, priming of the fusion machinery and calcium-triggered membrane fusion. After fusion, the membrane around the secretory vesicle is incorporated into the plasma membrane and the granule releases its contents. The proteins involved in these processes belong to several highly conserved families: Rab GTPases, SNAREs (soluble NSF-attachment protein receptors), α-SNAP (α-NSF attachment protein), NSF (N-ethylmaleimide-sensitive factor), Munc13, Munc18, complexins and synaptotagmins. This review discussed that molecules believed to participate in the secretory function and their involvement in acrosome and cortical granule exocytosis.

Routing of the RAB6 secretory pathway towards the lysosome related organelle of melanocytes

Exocytic carriers convey neo-synthesized components from the Golgi apparatus to the cell surface. While the release and anterograde movement of Golgi-derived vesicles require the small GTPase RAB6, its effector ELKS promotes the targeting and docking of secretory vesicles to particular areas of the plasma membrane. Here, we show that specialized cell types exploit and divert the secretory pathway towards lysosome related organelles. In cultured melanocytes, the secretory route relies on RAB6 and ELKS to directly transport and dock Golgi-derived carriers to melanosomes. By delivering specific cargos, such as MART-1 and TYRP2/ DCT, the RAB6/ELKS-dependent secretory pathway controls the formation and maturation of melanosomes but also pigment synthesis. In addition, pigmentation defects are observed in RAB6 KO mice. Our data together reveal for the first time that the secretory pathway can be directed towards intracellular organelles of endosomal origin to ensure their biogenesis and function.

In vivo single-molecule imaging of syntaxin1A reveals polyphosphoinositide- and activity-dependent trapping in presynaptic nanoclusters.

Syntaxin1A is organized in nanoclusters that are critical for the docking and priming of secretory vesicles from neurosecretory cells. Whether and how these nanoclusters are affected by neurotransmitter release in nerve terminals from a living organism is unknown. Here we imaged photoconvertible syntaxin1A-mEos2 in the motor nerve terminal of Drosophila larvae by single-particle tracking photoactivation localization microscopy. Opto- and thermo-genetic neuronal stimulation increased syntaxin1A-mEos2 mobility, and reduced the size and molecular density of nanoclusters, suggesting an activity-dependent release of syntaxin1A from the confinement of nanoclusters. Syntaxin1A mobility was increased by mutating its polyphosphoinositide-binding site or preventing SNARE complex assembly via co-expression of tetanus toxin light chain. In contrast, syntaxin1A mobility was reduced by preventing SNARE complex disassembly. Our data demonstrate that polyphosphoinositide favours syntaxin1A trapping, and show that SNARE complex disassembly leads to syntaxin1A dissociation from nanoclusters. Lateral diffusion and trapping of syntaxin1A in nanoclusters therefore dynamically regulate neurotransmitter release.

Multivalent Membrane Lipid Targeting by the Calcium-Independent C2 Domains of Granuphilin: Evidence from Computation and Experiment

Granuphilin, also known as synaptotagmin-like protein 4 (Slp4), is a Rab effector responsible for docking insulin secretory vesicles to the plasma membrane prior to exocytosis. Granuphilin binds vesicular Rab proteins via an N-terminal Slp homology (SHD) domain, binds plasma membrane SNARE complex proteins via a central linker region, and contains tandem C-terminal C2 domains (C2A and C2B) with affinity for phosphatidylinositol-(4,5)-bisphosphate (PIP2). Granuphilin's C2A domain has previously been shown to bind PIP2 or its soluble analogues with low micromolar affinity; however, the domain docks with low nanomolar apparent affinity to PIP2 in lipid vesicles that also contain background anionic lipids such as phosphatidylserine (PS). Here we show using a combination of computational and experimental approaches that this high-affinity membrane interaction arises from concerted binding at two or more sites on the C2A domain. First, docking calculations predict at least two possible binding pockets for anionic ligands. Second, liposome binding measurements indicate that affinity depends on concentrations of PIP2 as well as PS and phosphatidic acid (PA) in the membrane. Third, fluorescence measurements indicate that different regions of the protein surface are responsible for binding membranes containing or lacking PIP2. Fourth, single-molecule lateral diffusion measurements indicate distinct membrane-bound states depending on the available target lipids. Mutational analysis is currently underway to confirm locations of these lipid binding sites. Overall, multivalent membrane binding by granuphilin likely contributes to selective recognition and high affinity docking of large dense-core secretory vesicles to the plasma membrane.

A Post-Docking Role of Synaptotagmin 1-C2B Domain Bottom Residues R398/399 in Mouse Chromaffin Cells.

This study provides new insights in how the two opposite sides of the C2B domain of Synaptotagmin-1 participate in secretory vesicle fusion, and in more upstream steps, especially vesicle docking. We show that the "bottom" surface of the C2B domain is required for triggering fusion, but not for docking. Synaptotagmin-1 promotes docking by multiple, PI(4,5)P2-dependent and PI(4,5)P2-independent mechanisms. Mutations in the C2B bottom surface (R398/399) selectively disrupt the latter. These mutations help to discriminate protein regions involved in different aspects of Synaptotagmin-1 function in docking and fusion.