‘Neurosecretion’ by aminergic synaptic terminals In vivo—a study of secretory granule exocytosis in the corpus cardiacum of the flying locust

A single transcription factor, MIST1 (BHLHA15), maximizes secretory function in diverse secretory cells (like pancreatic acinar cells) by transcriptionally upregulating genes that elaborate secretory architecture. Here, we show that the scantly-studied MIST1 target, ELAPOR1, is an evolutionarily conserved, novel Mannose-6-phosphate receptor (M6PR) domain-containing protein. ELAPOR1 expression was specific to zymogenic cells (ZCs, the MIST1-expressing population in the stomach). ELAPOR1 expression was lost as tissue injury caused ZCs to undergo paligenosis (ie, to become metaplastic and reenter the cell cycle). In cultured cells, ELAPOR1 trafficked with cis-Golgi resident proteins and with the trans-Golgi and late endosome protein: cation-independent M6PR. Secretory vesicle trafficking was disrupted by expression of ELAPOR1 truncation mutants. Mass spectrometric analysis of co-immunoprecipitated proteins showed ELAPOR1 and CI-M6PR shared many binding partners. However, CI-M6PR and ELAPOR1 must function differently, as CI-M6PR co-immunoprecipitated more lysosomal proteins and was not decreased during paligenosis in vivo. We generated Elapor1−/− mice to determine ELAPOR1 function in vivo. Consistent with in vitro findings, secretory granule maturation was defective in Elapor1−/− ZCs. Our results identify a role for ELAPOR1 in secretory granule maturation and help clarify how a single transcription factor maintains mature exocrine cell architecture in homeostasis and helps dismantles it during paligenosis.

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CONCOMITANT SYNTHESIS OF MEMBRANE PROTEIN AND EXPORTABLE PROTEIN OF THE SECRETORY GRANULE IN RAT PAROTID GLAND

The Journal of Cell Biology ◽

10.1083/jcb.50.1.187 ◽

1971 ◽

Vol 50 (1) ◽

pp. 187-200 ◽

Cited By ~ 86

Author(s):

Abraham Amsterdam ◽

Michael Schramm ◽

Itzhak Ohad ◽

Yoram Salomon ◽

Zvi Selinger

Keyword(s):

Amino Acids ◽

Secretory Granule ◽

De Novo ◽

Secretory Granules ◽

Specific Radioactivity ◽

Staining Intensity ◽

Granule Membrane ◽

Rat Parotid ◽

Cell Fractions

After enzyme secretion the membrane of the secretory granule, which had been fused to the cell membrane, was resorbed into the cell. Experiments were therefore carried out to test whether formation of new secretory granules involves reutilization of the resorbed membrane or synthesis of a new membrane, de novo, from amino acids. Incorporation of amino acids-14C into proteins of various cell fractions was measured in vivo, 30, 120, and. 300 min after labeling. At all times the specific radioactivity of the secretory granule membrane was about equal to that of the granule's exportable content. At 120 and 300 min the specific radioactivity of the granule membrane and of the granule content was much higher than that of any other subcellular fraction. It is therefore concluded that the protein of the membrane is synthesized de novo concomitantly with the exportable protein. The proteins of the granule membrane could be distinguished from those of the granule content by gel electrophoresis. All major bands were labeled proportionately to their staining intensity. The amino acid composition of the secretory granule membrane was markedly different from that of the granule's content and also from that of the mitochondrial membrane. The granule membrane showed a high proline content, 30 moles/100 moles amino acids. The analyses show that the radioactivity of the granule membrane is indeed inherent in its proteins and is not due to contamination by other fractions. The possibility is considered that the exportable protein leaves the endoplasmic reticulum already enveloped by the newly synthesized membrane.

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Protease Nexin-1 Promotes Secretory Granule Biogenesis by Preventing Granule Protein Degradation

Molecular Biology of the Cell ◽

10.1091/mbc.e05-08-0755 ◽

2006 ◽

Vol 17 (2) ◽

pp. 789-798 ◽

Cited By ~ 34

Author(s):

Taeyoon Kim ◽

Y. Peng Loh

Keyword(s):

Protein Degradation ◽

Secretory Granule ◽

Endocrine Cells ◽

Underlying Mechanism ◽

Genetic Profiling ◽

Regulated Secretion ◽

Granule Protein ◽

Protease Nexin

Dense-core secretory granule (DCG) biogenesis is a prerequisite step for the sorting, processing, and secretion of neuropeptides and hormones in (neuro)endocrine cells. Previously, chromogranin A (CgA) has been shown to play a key role in the regulation of DCG biogenesis in vitro and in vivo. However, the underlying mechanism of CgA-mediated DCG biogenesis has not been explored. In this study, we have uncovered a novel mechanism for the regulation of CgA-mediated DCG biogenesis. Transfection of CgA into endocrine 6T3 cells lacking CgA and DCGs not only recovered DCG formation and regulated secretion but also prevented granule protein degradation. Genetic profiling of CgA-expressing 6T3 versus CgA- and DCG-deficient 6T3 cells, followed by real-time reverse transcription-polymerase chain reaction and Western blotting analyses, revealed that a serine protease inhibitor, protease nexin-1 (PN-1), was significantly up-regulated in CgA-expressing 6T3 cells. Overexpression of PN-1 in CgA-deficient 6T3 cells prevented degradation of DCG proteins at the Golgi apparatus, enhanced DCG biogenesis, and recovered regulated secretion. Moreover, depletion of PN-1 by antisense RNAs in CgA-expressing 6T3 cells resulted in the specific degradation of DCG proteins. We conclude that CgA increases DCG biogenesis in endocrine cells by up-regulating PN-1 expression to stabilize granule proteins against degradation.

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SM protein Munc18-2 facilitates transition of Syntaxin 11-mediated lipid mixing to complete fusion for T-lymphocyte cytotoxicity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1617981114 ◽

2017 ◽

Vol 114 (11) ◽

pp. E2176-E2185 ◽

Cited By ~ 20

Author(s):

Waldo A. Spessott ◽

Maria L. Sanmillan ◽

Margaret E. McCormick ◽

Vineet V. Kulkarni ◽

Claudio G. Giraudo

Keyword(s):

Transmembrane Domain ◽

Snare Complex ◽

Complete Fusion ◽

Sm Proteins ◽

Granule Exocytosis ◽

Cytolytic Granule ◽

Syntaxin 11 ◽

Sm Protein ◽

Lytic Granule

The atypical lipid-anchored Syntaxin 11 (STX11) and its binding partner, the Sec/Munc (SM) protein Munc18-2, facilitate cytolytic granule release by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Patients carrying mutations in these genes develop familial hemophagocytic lymphohistiocytosis, a primary immunodeficiency characterized by impaired lytic granule exocytosis. However, whether a SNARE such as STX11, which lacks a transmembrane domain, can support membrane fusion in vivo is uncertain, as is the precise role of Munc18-2 during lytic granule exocytosis. Here, using a reconstituted “flipped” cell–cell fusion assay, we show that lipid-anchored STX11 and its cognate SNARE proteins mainly support exchange of lipids but not cytoplasmic content between cells, resembling hemifusion. Strikingly, complete fusion is stimulated by addition of wild-type Munc18-2 to the assay, but not of Munc18-2 mutants with abnormal STX11 binding. Our data reveal that Munc18-2 is not just a chaperone of STX11 but also directly contributes to complete membrane merging by promoting SNARE complex assembly. These results further support the concept that SM proteins in general are part of the core fusion machinery. This fusion mechanism likely contributes to other cell-type–specific exocytic processes such as platelet secretion.

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Melanophilin accelerates insulin granule fusion without predocking to the plasma membrane

10.2337/figshare.12990773.v1 ◽

2020 ◽

Author(s):

Ada Admin ◽

Hao Wang ◽

Kouichi Mizuno ◽

Noriko Takahashi ◽

Eri Kobayashi ◽

...

Keyword(s):

Plasma Membrane ◽

Secretory Granule ◽

Β Cells ◽

Pancreatic Β Cells ◽

Granule Exocytosis ◽

Synaptic Vesicle Exocytosis ◽

Granule Membrane ◽

Myosin Va ◽

Vesicle Exocytosis ◽

Glucose Stimulated Insulin Secretion

Direct observation of fluorescence-labeled secretory granule exocytosis in living pancreatic β cells has revealed heterogeneous prefusion behaviors: some granules dwell beneath the plasma membrane before fusion, while others fuse immediately once they are recruited to the plasma membrane. Although the former mode seems to follow sequential docking-priming-fusion steps as found in synaptic vesicle exocytosis, the latter mode, which is unique to secretory granule exocytosis, has not been explored well. Here, we show that melanophilin, one of the effectors of the monomeric GTPase Rab27 on the granule membrane, is involved in such an accelerated mode of exocytosis. Both melanophilin-mutated leaden mouse and melanophilin-downregulated human pancreatic β cells exhibit impaired glucose-stimulated insulin secretion, with a specific reduction in fusion events that bypass stable docking to the plasma membrane. Upon stimulus-induced [Ca2+]i rise, melanophilin mediates this type of fusion by dissociating granules from myosin-Va and actin in the actin cortex and by associating them with a fusion-competent, open form of syntaxin-4 on the plasma membrane. These findings provide the hitherto unknown mechanism to support sustainable exocytosis by which granules are recruited from the cell interior and fuse promptly without stable predocking to the plasma membrane.

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Sequential in vivo labeling of insulin secretory granule pools in INS-SNAP transgenic pigs

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2107665118 ◽

2021 ◽

Vol 118 (37) ◽

pp. e2107665118

Author(s):

Elisabeth Kemter ◽

Andreas Müller ◽

Martin Neukam ◽

Anna Ivanova ◽

Nikolai Klymiuk ◽

...

Keyword(s):

Secretory Granule ◽

Cell Function ◽

Ex Vivo ◽

Secretory Granules ◽

Pig Model ◽

Transgenic Pigs ◽

Glucose Levels ◽

Molecular Features ◽

Insulin Secretory Granule

β cells produce, store, and secrete insulin upon elevated blood glucose levels. Insulin secretion is a highly regulated process. The probability for insulin secretory granules to undergo fusion with the plasma membrane or being degraded is correlated with their age. However, the molecular features and stimuli connected to this behavior have not yet been fully understood. Furthermore, our understanding of β cell function is mostly derived from studies of ex vivo isolated islets in rodent models. To overcome this translational gap and study insulin secretory granule turnover in vivo, we have generated a transgenic pig model with the SNAP-tag fused to insulin. We demonstrate the correct targeting and processing of the tagged insulin and normal glycemic control of the pig model. Furthermore, we show specific single- and dual-color granular labeling of in vivo–labeled pig pancreas. This model may provide unprecedented insights into the in vivo insulin secretory granule behavior in an animal close to humans.

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Sparse force-bearing bridges between neighboring synaptic vesicles

Brain Structure and Function ◽

10.1007/s00429-019-01966-x ◽

2019 ◽

Vol 224 (9) ◽

pp. 3263-3276 ◽

Cited By ~ 1

Author(s):

John F. Wesseling ◽

Sébastien Phan ◽

Eric A. Bushong ◽

Léa Siksou ◽

Serge Marty ◽

...

Keyword(s):

Plasma Membrane ◽

Active Zone ◽

Synaptic Vesicles ◽

Electron Tomography ◽

Centrifugal Forces ◽

Small Minority ◽

Synaptic Terminals ◽

Stable Structures

Abstract Most vesicles in the interior of synaptic terminals are clustered in clouds close to active zone regions of the plasma membrane where exocytosis occurs. Electron-dense structures, termed bridges, have been reported between a small minority of pairs of neighboring vesicles within the clouds. Synapsin proteins have been implicated previously, but the existence of the bridges as stable structures in vivo has been questioned. Here we use electron tomography to show that the bridges are present but less frequent in synapsin knockouts compared to wildtype. An analysis of distances between neighbors in wildtype tomograms indicated that the bridges are strong enough to resist centrifugal forces likely induced by fixation with aldehydes. The results confirm that the bridges are stable structures and that synapsin proteins are involved in formation or stabilization.

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Secretory Granule Exocytosis

Physiological Reviews ◽

10.1152/physrev.00031.2002 ◽

2003 ◽

Vol 83 (2) ◽

pp. 581-632 ◽

Cited By ~ 562

Author(s):

Robert D. Burgoyne ◽

Alan Morgan

Keyword(s):

Secretory Granule ◽

Secretory Granules ◽

Physiological Role ◽

Cell Types ◽

Control Mechanisms ◽

Granule Exocytosis ◽

Synaptic Vesicle Exocytosis ◽

Wide Range ◽

Vesicle Exocytosis ◽

Protein Components

Regulated exocytosis of secretory granules or dense-core granules has been examined in many well-characterized cell types including neurons, neuroendocrine, endocrine, exocrine, and hemopoietic cells and also in other less well-studied cell types. Secretory granule exocytosis occurs through mechanisms with many aspects in common with synaptic vesicle exocytosis and most likely uses the same basic protein components. Despite the widespread expression and conservation of a core exocytotic machinery, many variations occur in the control of secretory granule exocytosis that are related to the specialized physiological role of particular cell types. In this review we describe the wide range of cell types in which regulated secretory granule exocytosis occurs and assess the evidence for the expression of the conserved fusion machinery in these cells. The signals that trigger and regulate exocytosis are reviewed. Aspects of the control of exocytosis that are specific for secretory granules compared with synaptic vesicles or for particular cell types are described and compared to define the range of accessory control mechanisms that exert their effects on the core exocytotic machinery.

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