Subcellular distribution of acetylcholinesterase forms in chromaffin cells

Bon, Suzanne; Bader, Marie‐France; Aunis, Dominique; Massoulié, Jean; Henry, Jean‐Pierre

doi:10.1111/j.1432-1033.1990.tb15567.x

Cited by 23 publications

(17 citation statements)

References 67 publications

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“…In both PC12 and adrenal chromaffin cells, PIKfyve was present in membrane and cytosol fractions. We also ran an equal amount of purified chromaffin granule membranes prepared as previously described (12,38,39). Importantly, PIKfyve immunoreactivity was also detected in the granule fraction, suggesting that PIKfyve may be recruited to large dense core vesicles and could play a role in secretory granule function.…”

Section: Resultsmentioning

confidence: 99%

PIKfyve Negatively Regulates Exocytosis in Neurosecretory Cells

Osborne

Wen

Boucheron

et al. 2008

Journal of Biological Chemistry

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Regulated secretion depends upon a highly coordinated series of protein-protein and protein-lipid interactions. Two phosphoinositides, phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3-phosphate, are important for the ATP-dependent priming of the secretory apparatus prior to Ca 2؉ -dependent exocytosis. Mechanisms that control phosphoinositide levels are likely to play an important role in priming fine tuning. Here we have investigated the involvement of PIKfyve, a phosphoinositide 5-kinase that can phosphorylate phosphatidylinositol 3-phosphate to produce phosphatidylinositol 3,5-bisphosphate on large dense core vesicle exocytosis from neuroendocrine cells. PIKfyve localizes to a subpopulation of secretory granules in chromaffin and PC12 cells. Nicotine stimulation promoted recruitment of PIKfyve-EGFP onto secretory vesicles in PC12 cells. YM-201636, a selective inhibitor of PIKfyve activity, and PIKfyve knockdown by small interfering RNA potentiated secretory granule exocytosis. Overexpression of PIKfyve or its yeast orthologue Fab1p inhibited regulated secretion in PC12 cells, whereas a catalytically inactive PIKfyve mutant had no effect. These results demonstrate a novel inhibitory role for PIKfyve catalytic activity in regulated secretion and provide further evidence for a fine tuning of exocytosis by 3-phosphorylated phosphoinositides.Neurotransmitters and hormones are released from their storage vesicles into the extracellular space via calciumdependent exocytosis. Much work has been directed toward identifying the proteins involved in and regulating the exocytic process. These include members of the SNARE family of proteins, synaptotagmins (1, 2, 4, 5, 13), and a host of regulatory proteins, such as Munc18a (6, 7). In addition to the critical role played by membrane proteins, there is an increasing amount of literature pointing to the importance of lipids, in particular phosphoinositides, in the regulation of membrane trafficking events, including regulated exocytosis (8 -13).Phosphoinositides are generated by the reversible phosphorylation of the inositol head group of phosphatidylinositol (PtdIns)2 by an array of kinases and phosphatases at one or more of the 3-, 4-, and 5-OH positions (10). Previous studies have highlighted the importance of a plasma membrane pool of phosphatidylinositol 4,5-bisphosphate for regulated exocytosis of large dense core vesicles (LDCV) (14 -17).We recently demonstrated an involvement of the type II PI3K, PI3K-C2␣, and its product PtdIns(3)P in priming, an ATPdependent step during which LDCV acquire the ability to fuse with the plasma membrane (12). This novel role played by PtdIns(3)P in promoting the priming stage of exocytosis has opened a number of new research avenues regarding the mechanism(s) regulating the concentration of PtdIns(3)P and the potential role played by other 3-phosphorylated phosphoinositides in neurosecretory cells.Priming is a reversible step and must be tightly controlled to prevent too many vesicles from reaching the primed state. Pt...

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Section: Resultsmentioning

confidence: 99%

PIKfyve Negatively Regulates Exocytosis in Neurosecretory Cells

Osborne

Wen

Boucheron

et al. 2008

Journal of Biological Chemistry

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“…Subcellular Fractionation and Immunoisolation-Bovine adrenal medulla were isolated, homogenized, and subjected to differential centrifugation, and purified chromaffin granules were obtained as described previously (27,28). Protein concentrations were determined by Bradford protein assay (Bio-Rad).…”

Section: Methodsmentioning

confidence: 99%

Identification of Secretory Granule Phosphatidylinositol 4,5-Bisphosphate-interacting Proteins Using an Affinity Pulldown Strategy

Osborne

Wallis

Jiménez

et al. 2007

Molecular & Cellular Proteomics

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Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) synthesis is required for calcium-dependent exocytosis in neurosecretory cells. We developed a PtdIns(4,5)P 2 bead pulldown strategy combined with subcellular fractionation to identify endogenous chromaffin granule proteins that interact with PtdIns(4,5)P 2 . We identified two synaptotagmin isoforms, synaptotagmins 1 and 7; spectrin; ␣-adaptin; and synaptotagmin-like protein 4 (granuphilin) by mass spectrometry and Western blotting. The interaction between synaptotagmin 7 and PtdIns(4,5)P 2 and its functional relevance was investigated. The 45-kDa isoform of synaptotagmin 7 was found to be highly expressed in adrenal chromaffin cells compared with PC12 cells and to mainly localize to secretory granules by subcellular fractionation, immunoisolation, and immunocytochemistry. We demonstrated that synaptotagmin 7 binds PtdIns(4,5)P 2 via the C2B domain in the absence of calcium and via both the C2A and C2B domains in the presence of calcium. We mutated the polylysine stretch in synaptotagmin 7 C2B and demonstrated that this mutant domain lacks the calcium-independent PtdIns(4,5)P 2 binding. Synaptotagmin 7 C2B domain inhibited catecholamine release from digitonin-permeabilized chromaffin cells, and this inhibition was abrogated with the C2B polylysine mutant. These data indicate that synaptotagmin 7 C2B-effector interactions, which occur via the polylysine stretch, including calcium-independent PtdIns(4,5)P 2 binding, are important for chromaffin granule exocytosis. Molecular & Cellular Proteomics 6:1158 -1169, 2007.Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) 1 plays a critical role in neurosecretory cells for both the exocytosis and endocytosis of secretory vesicles (1-3). The PtdIns(4,5)P 2 required for secretion is synthesized on the plasma membrane (4), and the binding of vesicle-associated proteins to PtdIns(4,5)P 2 in trans is thought to be important for bringing the vesicle and plasma membranes together prior to exocytosis to ensure rapid and efficient fusion upon calcium influx (5). Putative PtdIns(4,5)P 2 effectors include synaptotagmin 1 (Syt1) (6, 7), calcium-activated protein for secretion (CAPS) (8, 9), and rabphilin (10).CAPS binds to PtdIns(4,5)P 2 and is a potential PtdIns(4,5)P 2 effector for priming of secretory granule exocytosis (9). However, recent work on the CAPS1 knock-out mouse highlighted an involvement of CAPS1 in the refilling or storage of catecholamine in mature secretory granules therefore suggesting an alternative role for CAPS (11).Syts play a fundamental role in triggering membrane fusion in a variety of systems, and much work has focused on Syt1 as the main candidate for calcium sensor in exocytosis (12)(13)(14). Syt1 binds to PtdIns(4,5)P 2 , and this interaction is believed to play a critical role in bridging the secretory vesicle and plasma membranes during exocytosis (5, 6). However, multiple Syt isoforms are expressed in the central nervous system and neurosecretory cells, suggesting that different isoforms ma...

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“…In chromaffin cells, AChE has been reported to exist in the cell membrane and in different subcellular organelles (7,49). Bovine chromaffin cells also secrete AChE upon depolarizing stimulation and in the presence of Ca 2ϩ , suggesting an exocytotic mechanism, and thus the presence of the enzyme in some kind of secretory vesicle or endosome (51,58).…”

Section: Discussionmentioning

confidence: 99%