A triggered mechanism retrieves membrane in seconds after Ca(2+)-stimulated exocytosis in single pituitary cells

In neuroendocrine cells, cytosolic Ca2+ triggers exocytosis in tens of milliseconds, yet known pathways of endocytic membrane retrieval take minutes. To test for faster retrieval mechanisms, we have triggered short bursts of exocytosis by flash photolysis of caged Ca2+, and have tracked subsequent retrieval by measuring the plasma membrane capacitance. We find that a limited amount of membrane can be retrieved with a time constant of 4 s at 21-26 degrees C, and that this occurs partially via structures larger than coated vesicles. This novel mechanism may be arrested at a late step. Incomplete retrieval structures then remain on the cell surface for minutes until the consequences of a renewed increase in cytosolic [Ca2+] disconnect them from the cell surface in < 1 s. Our results provide evidence for a rapid, triggered membrane retrieval pathway in excitable cells.

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alpha 2 Macroglobulin binding to the plasma membrane of cultured fibroblasts. Diffuse binding followed by clustering in coated regions.

The Journal of Cell Biology ◽

10.1083/jcb.82.3.614 ◽

1979 ◽

Vol 82 (3) ◽

pp. 614-625 ◽

Cited By ~ 140

Author(s):

M C Willingham ◽

F R Maxfield ◽

I H Pastan

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Coated Vesicles ◽

The Other ◽

Con A ◽

Coated Pits ◽

Cultured Fibroblasts ◽

Receptor Complexes ◽

Transmission Electron ◽

Epidermal Growth

Using transmission electron microscopy, we have studied the interaction of alpha 2 macroglobulin (alpha 2 M) with the surface of cultured fibroblasts. When cells were incubated for 2 h at 4 degrees C with ferritin-conjugated alpha 2 M, approximately 90% of the alpha 2 M was diffusely distributed on the cell surface, and the other 10% was concentrated in "coated" pits. A pattern of diffuse labeling with some clustering in "coated" pits was also obtained when cells were incubated for 5 min at 4 degrees C with alpha 2 M, fixed with glutaraldehyde, and the alpha 2 M was localized with affinity-purified, peroxidase-labeled antibody to alpha 2 M. Experiments in which cells were fixed with 0.2% paraformaldehyde before incubation with alpha 2 M showed that the native distribution of alpha 2 M receptors was entirely diffuse without significant clustering in "coated" pits. This indicates that some redistribution of the alpha 2 M-receptor complexes into clusters occurred even at 4 degrees C. In experiments with concanavalin A(Con A), we found that some of the Con A clustered in coated regions of the membrane and was internalized in coated vesicles, but much of the Con A was directly internalized in uncoated vesicles or pinosomes. We conclude that unoccupied alpha 2 M receptors are diffusely distributed on the cell surface. When alpha 2 M-receptor complexes are formed, they rapidly cluster in coated regions or pits in the plasma membrane and subsequently are internalized in coated vesicles. Because insulin and epidermal growth factor are internalized in the same structures as alpha 2 M (Maxfield, F.R., J. Schlessinger, Y. Schechter, I. Pastan, and M.C. Willingham. 1978. Cell, 14: 805--810.), we suggest that all peptide hormones, as well as other proteins that enter the cell by receptor-mediated endocytosis, follow this same pathway.

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Vps10p Cycles between the TGN and the Late Endosome via the Plasma Membrane in Clathrin Mutants

Molecular Biology of the Cell ◽

10.1091/mbc.02-07-0105 ◽

2002 ◽

Vol 13 (12) ◽

pp. 4296-4307 ◽

Cited By ~ 17

Author(s):

Olivier Deloche ◽

Randy W. Schekman

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Fluorescent Protein ◽

Chimeric Protein ◽

Late Endosome ◽

Coated Vesicles ◽

Cargo Protein ◽

Endosomal Membranes ◽

Green Fluorescent ◽

Vacuolar Protein

Clathrin-coated vesicles mediate the transport of the soluble vacuolar protein CPY from the TGN to the endosomal/prevacuolar compartment. Surprisingly, CPY sorting is not affected in clathrin deletion mutant cells. Here, we have investigated the clathrin-independent pathway that allows CPY transport to the vacuole. We find that CPY transport is mediated by the endosome and requires normal trafficking of its sorting receptor, Vps10p, the steady state distribution of which is not altered in chc1 cells. In contrast, Vps10p accumulates at the cell surface in achc1/end3 double mutant, suggesting that Vps10p is rerouted to the cell surface in the absence of clathrin. We used a chimeric protein containing the first 50 amino acids of CPY fused to a green fluorescent protein (CPY-GFP) to mimic CPY transport inchc1. In the absence of clathrin, CPY-GFP resides in the lumen of the vacuole as in wild-type cells. However, inchc1/sec6 double mutants, CPY-GFP is present in internal structures, possibly endosomal membranes, that do not colocalize with the vacuole. We propose that Vps10p must be transported to and retrieved from the plasma membrane to mediate CPY sorting to the vacuole in the absence of clathrin-coated vesicles. In this circumstance, precursor CPY may be captured by retrieved Vps10p in an early or late endosome, rather than as it normally is in the trans-Golgi, and delivered to the vacuole by the normalVPS gene-dependent process. Once relieved of cargo protein, Vps10p would be recycled to the trans-Golgi and then to the cell surface for further rounds of sorting.

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Dynasore inhibits rapid endocytosis in bovine chromaffin cells

AJP Cell Physiology ◽

10.1152/ajpcell.00562.2008 ◽

2009 ◽

Vol 297 (2) ◽

pp. C397-C406 ◽

Cited By ~ 12

Author(s):

Chia-Chang Tsai ◽

Chih-Lung Lin ◽

Tzu-Lun Wang ◽

Ai-Chuan Chou ◽

Min-Yi Chou ◽

...

Keyword(s):

Plasma Membrane ◽

Chromaffin Cells ◽

Membrane Capacitance ◽

Neuroendocrine Cells ◽

Maximal Increase ◽

Force Microscopy ◽

Vesicle Recycling ◽

Atomic Force ◽

Bovine Chromaffin Cells ◽

Patch Configuration

Vesicle recycling is vital for maintaining membrane homeostasis and neurotransmitter release. Multiple pathways for retrieving vesicles fused to the plasma membrane have been reported in neuroendocrine cells. Dynasore, a dynamin GTPase inhibitor, has been shown to specifically inhibit endocytosis and vesicle recycling in nerve terminals. To characterize its effects in modulating vesicle recycling and repetitive exocytosis, changes in the whole cell membrane capacitance of bovine chromaffin cells were recorded in the perforated-patch configuration. Constitutive endocytosis was blocked by dynasore treatment, as shown by an increase in membrane capacitance. The membrane capacitance was increased during strong depolarizations and declined within 30 s to a value lower than the prestimulus level. The amplitude, but not the time constant, of the rapid exponential decay was significantly decreased by dynasore treatment. Although the maximal increase in capacitance induced by stimulation was significantly increased by dynasore treatment, the intercepts at time 0 of the curve fitted to the decay phase were all ∼110% of the membrane capacitance before stimulation, regardless of the dynasore concentration used. Membrane depolarization caused clathrin aggregation and F-actin continuity disruption at the cell boundary, whereas dynasore treatment induced clathrin aggregation without affecting F-actin continuity. The number of invagination pits on the surface of the plasma membrane determined using atomic force microscopy was increased and the pore was wider in dynasore-treated cells. Our data indicate that dynamin-mediated endocytosis is the main pathway responsible for rapid compensatory endocytosis.

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Transport of the membrane glycoprotein of vesicular stomatitis virus to the cell surface in two stages by clathrin-coated vesicles.

The Journal of Cell Biology ◽

10.1083/jcb.86.1.162 ◽

1980 ◽

Vol 86 (1) ◽

pp. 162-171 ◽

Cited By ~ 99

Author(s):

J E Rothman ◽

H Bursztyn-Pettegrew ◽

R E Fine

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Golgi Apparatus ◽

Cell Surface ◽

G Protein ◽

Vesicular Stomatitis Virus ◽

Coated Vesicles ◽

Transmembrane Glycoprotein ◽

Vesicular Stomatitis ◽

Two Stages

The G protein of vesicular stomatitis virus is a transmembrane glycoprotein that is transported from its site of synthesis in the rough endoplasmic reticulum to the plasma membrane via the Golgi apparatus. Pulse-chase experiments suggest that G is transported to the cell surface in two successive waves of clathrin-coated vesicles. The oligosaccharides of G protein carried in the early wave are of the "high-mannose" (G1) form, whereas the oligosaccharides in the second, later wave are of the mature "complex" (G2) form. the early wave is therefore proposed to correspond to transport of G in coated vesicles from the endoplasmic reticulum to the Golgi apparatus, and the succeeding wave to transport from the Golgi apparatus to the plasma membrane. The G1- and G2-containing coated vesicles appear to be structurally distinct, as judged by their differential precipitation by anticoated vesicle serum.

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Receptor-mediated endocytosis in cultured fibroblasts: cryptic coated pits and the formation of receptosomes.

Journal of Histochemistry & Cytochemistry ◽

10.1177/29.9.6169759 ◽

1981 ◽

Vol 29 (9) ◽

pp. 1003-1013 ◽

Cited By ~ 53

Author(s):

M C Willingham ◽

A V Rutherford ◽

M G Gallo ◽

J Wehland ◽

R B Dickson ◽

...

Keyword(s):

Plasma Membrane ◽

Horseradish Peroxidase ◽

Cell Surface ◽

Concanavalin A ◽

Ruthenium Red ◽

Coated Vesicles ◽

Surface Markers ◽

Coated Pits ◽

Cultured Fibroblasts ◽

Receptor Mediated Endocytosis

Concentrative receptor-mediated endocytosis of many specific ligands by cultured fibroblasts occurs through the coated pit-receptosome pathway. The formation of receptosomes was studied using two impermeant electron-dense labels for the cell surface, ruthenium red and concanavalin A-horseradish peroxidase. These studies show that at 4 degrees C, virtually all coated structures near the plasma membrane are in communication with the cell surface, and are not isolated coated vesicles. On warming cells to 37 degrees C for only 1 minute, a major portion of these structures become cryptic, that is, not labeled by these surface markers. However, on cooling cells immediately back to 4 degrees C, virtually all of these structures are again in communication with the surface. Many images showed that membrane of these cryptic pits to be continuous with the cell surface when caught in the appropriate plane of section; often there was a very narrow entrance that excluded extracellular label. At 37 degrees C, receptosomes could be occasionally seen forming as an invagination of membrane adjacent to the coated region. Mechanisms by which receptosomes may form and other evidence demonstrating the failure of coated pits to pinch off to form isolated coated vesicles during endocytosis are discussed.

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COOH-terminal signals mediate the trafficking of a peptide processing enzyme in endocrine cells.

The Journal of Cell Biology ◽

10.1083/jcb.121.1.23 ◽

1993 ◽

Vol 121 (1) ◽

pp. 23-36 ◽

Cited By ~ 50

Author(s):

S L Milgram ◽

R E Mains ◽

B A Eipper

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Endocrine Cells ◽

Secretory Pathway ◽

Bioactive Peptides ◽

Full Length ◽

Neuroendocrine Cells ◽

Subcellular Compartment ◽

Peptide Processing ◽

Regulated Secretory Pathway

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the COOH-terminal amidation of bioactive peptides through a two step reaction catalyzed by separate enzymes contained within the PAM precursor. To characterize the trafficking of integral membrane PAM proteins in neuroendocrine cells, we have generated stable AtT-20 cell lines expressing full length and COOH-terminally truncated integral membrane PAM proteins. Full length integral membrane PAM was present on the cell surface in low but detectable amounts and PAM proteins which reached the cell surface were rapidly internalized but not immediately degraded in lysosomes. Internalized PAM complexed with PAM antibody was found in a subcellular compartment which overlapped with internalized transferrin and with structures binding WGA. Thus the punctate juxtanuclear staining of full length PAM represents PAM in endosomes. Endoproteolytic processing of full length PAM-1 and PAM-2 resulted in the secretion of soluble PAM proteins; the secretion of these soluble PAM proteins was stimulus dependent. Although some of the truncated PAM protein was also processed and stored in AtT-20 cells, much of the expressed protein was redistributed to the plasma membrane. Soluble proteins not observed in large amounts in cells expressing full length PAM were released from the surface of cells expressing truncated PAM and little internalization of truncated integral membrane PAM was observed. Thus, the COOH-terminal domain of PAM contains information important for its trafficking within the regulated secretory pathway as well as information necessary for its retrieval from the cell surface.

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Dynamics of clathrin and adaptor proteins during endocytosis

AJP Cell Physiology ◽

10.1152/ajpcell.00160.2006 ◽

2006 ◽

Vol 291 (5) ◽

pp. C1072-C1081 ◽

Cited By ~ 46

Author(s):

Joshua Z. Rappoport ◽

Shahrnaz Kemal ◽

Alexandre Benmerah ◽

Sanford M. Simon

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Time Course ◽

Adaptor Proteins ◽

Nucleation Site ◽

Heterogeneous Population ◽

Coated Vesicles ◽

Subunit Exchange ◽

Dead End ◽

Endocytic Vesicles

The endocytic adaptor complex AP-2 colocalizes with the majority of clathrin-positive spots at the cell surface. However, we previously observed that AP-2 is excluded from internalizing clathrin-coated vesicles (CCVs). The present studies quantitatively demonstrate that AP-2 disengages from sites of endocytosis seconds before internalization of the nascent CCV. In contrast, epsin, an alternate adaptor for clathrin at the plasma membrane, disappeared, along with clathrin. This suggests that epsin remains an integral part of the CCV throughout endocytosis. Clathrin spots at the cell surface represent a heterogeneous population: a majority (70%) of the spots disappeared with a time course of 4 min, whereas a minority (22%) remained static for ≥30 min. The static clathrin spots undergo constant subunit exchange, suggesting that although they are static structures, these spots comprise functional clathrin molecules, rather than dead-end aggregates. These results support a model where AP-2 serves a cargo-sorting function before endocytosis, whereas alternate adaptors, such as epsin, actually link cargo to the clathrin coat surrounding nascent endocytic vesicles. These data also support a role for static clathrin, providing a nucleation site for endocytosis.

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A glycolipid-anchored prion protein is endocytosed via clathrin-coated pits.

The Journal of Cell Biology ◽

10.1083/jcb.125.6.1239 ◽

1994 ◽

Vol 125 (6) ◽

pp. 1239-1250 ◽

Cited By ~ 169

Author(s):

S L Shyng ◽

J E Heuser ◽

D A Harris

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Prion Protein ◽

Transmembrane Protein ◽

Primary Cultures ◽

Neuroblastoma Cells ◽

Cellular Prion Protein ◽

Coated Vesicles ◽

Coated Pits ◽

Hypertonic Medium

The cellular prion protein (PrPc) is a glycolipid-anchored, cell surface protein of unknown function, a posttranslationally modified isoform of which PrPSc is involved in the pathogenesis of Creutzfeldt-Jakob disease, scrapie, and other spongiform encephalopathies. We have shown previously that chPrP, a chicken homologue of mammalian PrPC, constitutively cycles between the cell surface and an endocytic compartment, with a transit time of approximately 60 min in cultured neuroblastoma cells. We now report that endocytosis of chPrP is mediated by clathrin-coated pits. Immunogold labeling of neuroblastoma cells demonstrates that the concentration of chPrP within 0.05 microns of coated pits is 3-5 times higher than over other areas of the plasma membrane. Moreover, gold particles can be seen within coated vesicles and deeply invaginated coated pits that are in the process of pinching off from the plasma membrane. ChPrP is also localized to coated pits in primary cultures of neurons and glia, and is found in coated vesicles purified from chicken brain. Finally, internalization of chPrP is reduced by 70% after neuroblastoma cells are incubated in hypertonic medium, a treatment that inhibits endocytosis by disrupting clathrin lattices. Caveolae, plasmalemmal invaginations in which several other glycolipid-anchored proteins are concentrated, are not seen in neuroblastoma cells analyzed by thin-section or deep-etch electron microscopy. Moreover, these cells do not express detectable levels of caveolin, a caveolar coat protein. Since chPrP lacks a cytoplasmic domain that could interact directly with the intracellular components of clathrin-coated pits, we propose that the polypeptide chain of chPrP associates with the extracellular domain of a transmembrane protein that contains a coated pit internalization signal.

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Syntaxin 1A inhibits regulated CFTR trafficking inXenopus oocytes

AJP Cell Physiology ◽

10.1152/ajpcell.1999.277.1.c174 ◽

1999 ◽

Vol 277 (1) ◽

pp. C174-C180 ◽

Cited By ~ 59

Author(s):

Kathryn W. Peters ◽

Juanjuan Qi ◽

Simon C. Watkins ◽

Raymond A. Frizzell

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Membrane Trafficking ◽

Glucose Transporter ◽

Functional Expression ◽

Membrane Capacitance ◽

K Channel ◽

Syntaxin 1A ◽

Viral Hemagglutinin ◽

Hemagglutinin Protein

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial cell Cl channel, whose gating activity and membrane trafficking are controlled by cAMP/protein kinase A (PKA)-mediated phosphorylation. CFTR Cl currents are regulated also by syntaxin 1A (A. P. Naren, D. J. Nelson, W. W. Xie, B. Jovov, J. Pevsner, M. K. Bennett, D. J. Benos, M. W. Quick, and K. L. Kirk. Nature 390: 302–305, 1997), a protein best known for its role in membrane trafficking and neurosecretion. To examine the mechanism of syntaxin 1A inhibition, we expressed these proteins in Xenopusoocytes and monitored agonist-induced changes in plasma membrane capacitance and cell surface fluorescence of CFTR that contains an external epitope tag. cAMP stimulation elicited large increases in membrane capacitance and in cell surface labeling of flag-tagged CFTR. Coexpression of CFTR with syntaxin 1A, but not syntaxin 3, inhibited cAMP-induced increases in membrane capacitance and plasma membrane CFTR content. Injection of botulinum toxin/C1 rapidly reversed syntaxin’s effects on current and capacitance, indicating that they cannot be explained by an effect on CFTR synthesis. Functional expression of other integral membrane proteins, including Na-coupled glucose transporter hSGLT1, inwardly rectified K channel hIK1, P2Y2 nucleotide receptor, and viral hemagglutinin protein, was not affected by syntaxin 1A coexpression. These findings indicate that acute regulation of the number of CFTR Cl channels in plasma membrane is one mechanism by which cAMP/PKA regulates Cl currents. Inhibition of plasma membrane CFTR content by syntaxin 1A is consistent with the concept that syntaxin and other components of the SNARE machinery are involved in regulated trafficking of CFTR.

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