Exophilin8 transiently clusters insulin granules at the actin-rich cell cortex prior to exocytosis

Exophilin8/MyRIP/Slac2-c is an effector protein of the small GTPase Rab27a and is specifically localized on retinal melanosomes and secretory granules. We investigated the role of exophilin8 in insulin granule trafficking. Exogenous expression of exophilin8 in pancreatic β cells or their cell line, MIN6, polarized (exophilin8-positive) insulin granules at the cell corners, where both cortical actin and the microtubule plus-end–binding protein, EB1, were present. Mutation analyses indicated that the ability of exophilin8 to act as a linker between Rab27a and myosin Va is essential for its granule-clustering activity. Moreover, exophilin8 and exophilin8-associated insulin granules were markedly stable and immobile. Total internal reflection fluorescence microscopy indicated that exophilin8 restricts the motion of insulin granules at a region deeper than that where another Rab27a effector, granuphilin, accumulates docked granules directly attached to the plasma membrane. However, the exophilin8-induced immobility of insulin granules was eliminated upon secretagogue stimulation and did not inhibit evoked exocytosis. Furthermore, exophilin8 depletion prevents insulin granules from being transported close to the plasma membrane and inhibits their fusion. These findings indicate that exophilin8 transiently traps insulin granules into the cortical actin network close to the microtubule plus-ends and supplies them for release during the stimulation.

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The Rab27a effector exophilin7 promotes fusion of secretory granules that have not been docked to the plasma membrane

Molecular Biology of the Cell ◽

10.1091/mbc.e12-04-0265 ◽

2013 ◽

Vol 24 (3) ◽

pp. 319-330 ◽

Cited By ~ 18

Author(s):

Hao Wang ◽

Ray Ishizaki ◽

Jun Xu ◽

Kazuo Kasai ◽

Eri Kobayashi ◽

...

Keyword(s):

Plasma Membrane ◽

Knockout Mice ◽

Secretory Granules ◽

Small Gtpase ◽

Membrane Phospholipids ◽

Β Cells ◽

Β Cell ◽

Pancreatic Β Cells ◽

Insulin Granules ◽

Insulin Exocytosis

Granuphilin, an effector of the small GTPase Rab27a, mediates the stable attachment (docking) of insulin granules to the plasma membrane and inhibits subsequent fusion of docked granules, possibly through interaction with a fusion-inhibitory Munc18-1/syntaxin complex. However, phenotypes of insulin exocytosis differ considerably between Rab27a- and granuphilin-deficient pancreatic β cells, suggesting that other Rab27a effectors function in those cells. We found that one of the putative Rab27a effector family proteins, exophilin7/JFC1/Slp1, is expressed in β cells; however, unlike granuphilin, exophilin7 overexpressed in the β-cell line MIN6 failed to show granule-docking or fusion-inhibitory activity. Furthermore, exophilin7 has no affinities to either Munc18-1 or Munc18-1–interacting syntaxin-1a, in contrast to granuphilin. Although β cells of exophilin7-knockout mice show no apparent abnormalities in intracellular distribution or in ordinary glucose-induced exocytosis of insulin granules, they do show impaired fusion in response to some stronger stimuli, specifically from granules that have not been docked to the plasma membrane. Exophilin7 appears to mediate the fusion of undocked granules through the affinity of its C2A domain toward the plasma membrane phospholipids. These findings indicate that the two Rab27a effectors, granuphilin and exophilin7, differentially regulate the exocytosis of either stably or minimally docked granules, respectively.

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An enzymatic cascade of Rab5 effectors regulates phosphoinositide turnover in the endocytic pathway

The Journal of Cell Biology ◽

10.1083/jcb.200505128 ◽

2005 ◽

Vol 170 (4) ◽

pp. 607-618 ◽

Cited By ~ 252

Author(s):

Hye-Won Shin ◽

Mitsuko Hayashi ◽

Savvas Christoforidis ◽

Sandra Lacas-Gervais ◽

Sebastian Hoepfner ◽

...

Keyword(s):

Plasma Membrane ◽

Dual Role ◽

Small Gtpase ◽

Endocytic Pathway ◽

Cell Cortex ◽

Rab Gtpase ◽

Functional Importance ◽

Dual Mechanism ◽

Transferrin Uptake

Generation and turnover of phosphoinositides (PIs) must be coordinated in a spatial- and temporal-restricted manner. The small GTPase Rab5 interacts with two PI 3-kinases, Vps34 and PI3Kβ, suggesting that it regulates the production of 3-PIs at various stages of the early endocytic pathway. Here, we discovered that Rab5 also interacts directly with PI 5- and PI 4-phosphatases and stimulates their activity. Rab5 regulates the production of phosphatidylinositol 3-phosphate (PtdIns[3]P) through a dual mechanism, by directly phosphorylating phosphatidylinositol via Vps34 and by a hierarchical enzymatic cascade of phosphoinositide-3-kinaseβ (PI3Kβ), PI 5-, and PI 4-phosphatases. The functional importance of such an enzymatic pathway is demonstrated by the inhibition of transferrin uptake upon silencing of PI 4-phosphatase and studies in weeble mutant mice, where deficiency of PI 4-phosphatase causes an increase of PtdIns(3,4)P2 and a reduction in PtdIns(3)P. Activation of PI 3-kinase at the plasma membrane is accompanied by the recruitment of Rab5, PI 4-, and PI 5-phosphatases to the cell cortex. Our data provide the first evidence for a dual role of a Rab GTPase in regulating both generation and turnover of PIs via PI kinases and phosphatases to coordinate signaling functions with organelle homeostasis.

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Role of Dictyostelium racE in cytokinesis: mutational analysis and localization studies by use of green fluorescent protein.

Molecular Biology of the Cell ◽

10.1091/mbc.8.5.935 ◽

1997 ◽

Vol 8 (5) ◽

pp. 935-944 ◽

Cited By ~ 46

Author(s):

D A Larochelle ◽

K K Vithalani ◽

A De Lozanne

Keyword(s):

Plasma Membrane ◽

Fusion Protein ◽

Fluorescent Protein ◽

Mutational Analysis ◽

Small Gtpase ◽

Cell Cortex ◽

Entire Cell ◽

Exogenous Expression ◽

Green Fluorescent ◽

Constitutively Active

The small GTPase racE is essential for cytokinesis in Dictyostelium but its precise role in cell division is not known. To determine the molecular mechanism of racE function, we undertook a mutational analysis of racE. The exogenous expression of either wild-type racE or a constitutively active V20racE mutant effectively rescues the cytokinesis deficiency of racE null cells. In contrast, a constitutively inactive N25racE mutant fails to rescue the cytokinesis deficiency. Thus, cytokinesis requires only the activation of racE by GTP and not the inactivation of racE by hydrolysis of GTP. To determine the spatial distribution of racE, we created a fusion protein with GFP at the amino terminus of racE. Remarkably, GFP-racE fusion protein was fully competent to rescue the phenotype of racE null cells and, therefore, must reside in the same location as native racE. We found that GFP-racE localized to the plasma membrane of the cell throughout the entire cell cycle. Furthermore, constitutively active and inactive GFP-racE fusion proteins also localized to the plasma membrane. We mapped the domain required for plasma membrane localization to the carboxyl-terminal 40 amino acids of racE. This domain, however, is not sufficient to confer racE function onto a closely related GTPase. Taken together, these results suggest that racE functions at the cell cortex but it is not involved in determining the timing or placement of the contractile ring.

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Barrier role of actin filaments in regulated mucin secretion from airway goblet cells

AJP Cell Physiology ◽

10.1152/ajpcell.00397.2004 ◽

2005 ◽

Vol 288 (1) ◽

pp. C46-C56 ◽

Cited By ~ 50

Author(s):

Camille Ehre ◽

Andrea H. Rossi ◽

Lubna H. Abdullah ◽

Kathleen De Pestel ◽

Sandra Hill ◽

...

Keyword(s):

Plasma Membrane ◽

Actin Filaments ◽

Fluorescent Protein ◽

Secretory Granules ◽

Yellow Fluorescent Protein ◽

Goblet Cells ◽

Actin Binding ◽

Secretory Cells ◽

Cortical Actin ◽

Mucin Secretion

Airway goblet cells secrete mucin onto mucosal surfaces under the regulation of an apical, phospholipase C/Gq-coupled P2Y2receptor. We tested whether cortical actin filaments negatively regulate exocytosis in goblet cells by forming a barrier between secretory granules and plasma membrane docking sites as postulated for other secretory cells. Immunostaining of human lung tissues and SPOC1 cells (an epithelial, mucin-secreting cell line) revealed an apical distribution of β- and γ-actin in ciliated and goblet cells. In goblet cells, actin appeared as a prominent subplasmalemmal sheet lying between granules and the apical membrane, and it disappeared from SPOC1 cells activated by purinergic agonist. Disruption of actin filaments with latrunculin A stimulated SPOC1 cell mucin secretion under basal and agonist-activated conditions, whereas stabilization with jasplakinolide or overexpression of β- or γ-actin conjugated to yellow fluorescent protein (YFP) inhibited secretion. Myristoylated alanine-rich C kinase substrate, a PKC-activated actin-plasma membrane tethering protein, was phosphorylated after agonist stimulation, suggesting a translocation to the cytosol. Scinderin (or adseverin), a Ca2+-activated actin filament severing and capping protein was cloned from human airway and SPOC1 cells, and synthetic peptides corresponding to its actin-binding domains inhibited mucin secretion. We conclude that actin filaments negatively regulate mucin secretion basally in airway goblet cells and are dynamically remodeled in agonist-stimulated cells to promote exocytosis.

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

Current Protein and Peptide Science ◽

10.2174/1389203722666210325110231 ◽

2021 ◽

Vol 22 ◽

Author(s):

Najeeb Ullah ◽

Ezzouhra El Maaiden ◽

Md. Sahab Uddin ◽

Ghulam Md Ashraf

Keyword(s):

Plasma Membrane ◽

Secretory Granules ◽

Secretory Vesicle ◽

Neuroendocrine Cells ◽

Snare Complex ◽

Secretory Vesicles ◽

Functional Protein ◽

Vesicle Docking ◽

Synaptotagmin 1

: 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 promotes vesicle fusion. This review assesses the role of the Syt-1 protein involved in the secretory vesicle docking, priming, and fusion.

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Recycling domains in plant cell morphogenesis: small GTPase effectors, plasma membrane signalling and the exocyst

Biochemical Society Transactions ◽

10.1042/bst0380723 ◽

2010 ◽

Vol 38 (2) ◽

pp. 723-728 ◽

Cited By ~ 15

Author(s):

Viktor Žárský ◽

Martin Potocký

Keyword(s):

Plasma Membrane ◽

Plant Cell ◽

Secretory Pathway ◽

Root Hairs ◽

Small Gtpases ◽

Small Gtpase ◽

Cell Cortex ◽

Membrane Phospholipids ◽

Recycling Endosome ◽

Regulatory Module

The Rho/Rop small GTPase regulatory module is central for initiating exocytotically ACDs (active cortical domains) in plant cell cortex, and a growing array of Rop regulators and effectors are being discovered in plants. Structural membrane phospholipids are important constituents of cells as well as signals, and phospholipid-modifying enzymes are well known effectors of small GTPases. We have shown that PLDs (phospholipases D) and their product, PA (phosphatidic acid), belong to the regulators of the secretory pathway in plants. We have also shown that specific NOXs (NADPH oxidases) producing ROS (reactive oxygen species) are involved in cell growth as exemplified by pollen tubes and root hairs. Most plant cells exhibit several distinct plasma membrane domains (ACDs), established and maintained by endocytosis/exocytosis-driven membrane protein recycling. We proposed recently the concept of a ‘recycling domain’ (RD), uniting the ACD and the connected endosomal recycling compartment (endosome), as a dynamic spatiotemporal entity. We have described a putative GTPase–effector complex exocyst involved in exocytic vesicle tethering in plants. Owing to the multiplicity of its Exo70 subunits, this complex, along with many RabA GTPases (putative recycling endosome organizers), may belong to core regulators of RD organization in plants.

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Dynamics of Immature Secretory Granules: Role of Cytoskeletal Elements during Transport, Cortical Restriction, and F-Actin-dependent Tethering

Molecular Biology of the Cell ◽

10.1091/mbc.12.5.1353 ◽

2001 ◽

Vol 12 (5) ◽

pp. 1353-1365 ◽

Cited By ~ 77

Author(s):

Rüdiger Rudolf ◽

Thorsten Salm ◽

Amin Rustom ◽

Hans-Hermann Gerdes

Keyword(s):

Secretory Granules ◽

Late Phase ◽

Cell Cortex ◽

Cell Periphery ◽

Dependent Manner ◽

Chromogranin B ◽

Cooperative Action ◽

Cytoskeletal Elements ◽

Golgi Network

Secretory granules store neuropeptides and hormones and exhibit regulated exocytosis upon appropriate cellular stimulation. They are generated in the trans-Golgi network as immature secretory granules, short-lived vesicular intermediates, which undergo a complex and poorly understood maturation process. Due to their short half-life and low abundance, real-time studies of immature secretory granules have not been previously possible. We describe here a pulse/chase-like system based on the expression of a human chromogranin B-GFP fusion protein in neuroendocrine PC12 cells, which permits direct visualization of the budding of immature secretory granules and their dynamics during maturation. Live cell imaging revealed that newly formed immature secretory granules are transported in a direct and microtubule-dependent manner within a few seconds to the cell periphery. Our data suggest that the cooperative action of microtubules and actin filaments restricts immature secretory granules to the F-actin-rich cell cortex, where they move randomly and mature completely within a few hours. During this maturation period, secretory granules segregate into pools of different motility. In a late phase of maturation, 60% of secretory granules were found to be immobile and about half of these underwent F-actin-dependent tethering.

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Structure and development of cortical microtubule arrays in plant cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100082182 ◽

1978 ◽

Vol 36 (2) ◽

pp. 264-265

Author(s):

A.R. Hardham ◽

B.E.S. Gunning

Keyword(s):

Cell Wall ◽

Plasma Membrane ◽

Cortical Microtubule ◽

Plant Cells ◽

Original Description ◽

Cell Cortex ◽

Cortical Microtubules ◽

Fundamental Part ◽

Cellulose Component

Microtubules in the plant cell cortex are usually aligned parallel to microfibrils of cellulose that are being deposited in the cell wall, and are considered to function in guiding or orienting cellulose synthetase complexes that lie in or on the plasma membrane. The cellulose component is largely responsible for the mechanical reaction of the wall to turgor forces, thereby determining cell size and shape, and therefore the role of the cortical microtubules is a fundamental part of the overall morphogenetic process in plants. It is important to determine the structure of cortical arrays of microtubules and to learn how the cell regulates their development, neither of these aspects having been investigated adequately since the original description likened the microtubules to “hundreds of hoops around the cell”.

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Regulation of Stimulated and Basal Release of Weibel-Palade Bodies By Syntaxin-3 Containing SNARE-Complexes

Blood ◽

10.1182/blood.v124.21.1440.1440 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1440-1440

Author(s):

Dorothee Van Breevoort ◽

Maryam Wahedi ◽

Maaike Schillemans ◽

Tom Carter ◽

Stephan Huveneers ◽

...

Keyword(s):

Endothelial Cells ◽

Conflicts Of Interest ◽

Vascular Endothelial Cells ◽

Secretory Granules ◽

Subcellular Location ◽

Small Gtpase ◽

Snare Complex ◽

Interaction Partners ◽

Syntaxin 3

Abstract Vascular endothelial cells contain unique rod-shaped secretory granules, called Weibel-Palade bodies (WPBs), which contain a number of haemostatic, angiogenic and inflammatory mediators. Several components that are critical for regulated WPB exocytosis have been identified, including the small GTPase Rab27A and its effector synaptotagmin-like protein 4-a (Slp4-a), but the mechanism remains unclear. We have previously identified syntaxin binding protein 1 (STXBP1) as an endogenous Slp4-a binding partner involved in WPB release, along with the SNARE proteins syntaxin-2 and -3. In this study we investigated the possible role of syntaxin-2 and -3 in WPB exocytosis. We characterized the subcellular location of these syntaxins in endothelial cells using immunocytochemistry. Syntaxin-2 was primarily associated with the plasma membrane where it localized at VE-caderin-based adherens junctions and at integrin-based adhesions to the extracellular matrix. Interestingly, the t-SNARE syntaxin-3 was primarily associated with WPBs. To further explore its role in WPB biology we mapped the endothelial interaction partners of syntaxin-3 through an unbiased mass spectrometry approach using pull downs of lentivirally-expressed mEGFP-syntaxin-3 with anti-GFP nanobeads. Among its interaction partners are various SNAREs and associated proteins such as syntaxin binding proteins 2 and 5 (STXBP2/5), N-ethylmaleimide-sensitive factor (NSF), SNAP23 and α-SNAP, suggesting we successfully pulled down a SNARE complex and its regulatory machinery that are involved in exocytosis. We further addressed the role of syntaxin-3 in stimulated WPB exocytosis using siRNA-mediated knockdowns and found that depletion of syntaxin-3 led to a significant potentiation of Ca2+- as well as cAMP-mediated VWF secretion. In contrast, we observed a decrease in basal (unstimulated) VWF secretion after silencing of syntaxin-3, which amounts to a significantly increased intracellular VWF content. The potentiation of VWF secretion after depletion of syntaxin-3 was almost completely attributable to an increased WPB pool size; when corrected for increased WPB content the probability of stimulated release of a WPB was unaltered in the absence of syntaxin-3. Our data position syntaxin-3 as a WPB-linked SNARE-protein that regulates basal secretion of VWF. Disclosures No relevant conflicts of interest to declare.

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