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Massive calcium–activated endocytosis without involvement of classical endocytic proteins
We describe rapid massive endocytosis (MEND) of >50% of the plasmalemma in baby hamster kidney (BHK) and HEK293 cells in response to large Ca transients. Constitutively expressed Na/Ca exchangers (NCX1) are used to generate Ca transients, whereas capacitance recording and a membrane tracer dye, F...
Autores principales: | , , , , , , |
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Formato: | Texto |
Lenguaje: | English |
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The Rockefeller University Press
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3010057/ https://www.ncbi.nlm.nih.gov/pubmed/21187336 http://dx.doi.org/10.1085/jgp.201010468 |
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author | Lariccia, Vincenzo Fine, Michael Magi, Simona Lin, Mei-Jung Yaradanakul, Alp Llaguno, Marc C. Hilgemann, Donald W. |
author_facet | Lariccia, Vincenzo Fine, Michael Magi, Simona Lin, Mei-Jung Yaradanakul, Alp Llaguno, Marc C. Hilgemann, Donald W. |
author_sort | Lariccia, Vincenzo |
collection | PubMed |
description | We describe rapid massive endocytosis (MEND) of >50% of the plasmalemma in baby hamster kidney (BHK) and HEK293 cells in response to large Ca transients. Constitutively expressed Na/Ca exchangers (NCX1) are used to generate Ca transients, whereas capacitance recording and a membrane tracer dye, FM 4–64, are used to monitor endocytosis. With high cytoplasmic adenosine triphosphate (ATP; >5 mM), Ca influx causes exocytosis followed by MEND. Without ATP, Ca transients cause only exocytosis. MEND can then be initiated by pipette perfusion of ATP, and multiple results indicate that ATP acts via phosphatidylinositol-bis 4,5-phosphate (PIP(2)) synthesis: PIP(2) substitutes for ATP to induce MEND. ATP-activated MEND is blocked by an inositol 5-phosphatase and by guanosine 5′-[γ-thio]triphosphate (GTPγS). Block by GTPγS is overcome by the phospholipase C inhibitor, U73122, and PIP(2) induces MEND in the presence of GTPγS. MEND can occur in the absence of ATP and PIP(2) when cytoplasmic free Ca is clamped to 10 µM or more by Ca-buffered solutions. ATP-independent MEND occurs within seconds during Ca transients when cytoplasmic solutions contain polyamines (e.g., spermidine) or the membrane is enriched in cholesterol. Although PIP(2) and cholesterol can induce MEND minutes after Ca transients have subsided, polyamines must be present during Ca transients. MEND can reverse over minutes in an ATP-dependent fashion. It is blocked by brief β-methylcyclodextrin treatments, and tests for involvement of clathrin, dynamins, calcineurin, and actin cytoskeleton were negative. Therefore, we turned to the roles of lipids. Bacterial sphingomyelinases (SMases) cause similar MEND responses within seconds, suggesting that ceramide may be important. However, Ca-activated MEND is not blocked by reagents that inhibit SMases. MEND is abolished by the alkylating phospholipase A(2) inhibitor, bromoenol lactone, whereas exocytosis remains robust, and Ca influx causes MEND in cardiac myocytes without preceding exocytosis. Thus, exocytosis is not prerequisite for MEND. From these results and two companion studies, we suggest that Ca promotes the formation of membrane domains that spontaneously vesiculate to the cytoplasmic side. |
format | Text |
id | pubmed-3010057 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | The Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-30100572011-07-01 Massive calcium–activated endocytosis without involvement of classical endocytic proteins Lariccia, Vincenzo Fine, Michael Magi, Simona Lin, Mei-Jung Yaradanakul, Alp Llaguno, Marc C. Hilgemann, Donald W. J Gen Physiol Article We describe rapid massive endocytosis (MEND) of >50% of the plasmalemma in baby hamster kidney (BHK) and HEK293 cells in response to large Ca transients. Constitutively expressed Na/Ca exchangers (NCX1) are used to generate Ca transients, whereas capacitance recording and a membrane tracer dye, FM 4–64, are used to monitor endocytosis. With high cytoplasmic adenosine triphosphate (ATP; >5 mM), Ca influx causes exocytosis followed by MEND. Without ATP, Ca transients cause only exocytosis. MEND can then be initiated by pipette perfusion of ATP, and multiple results indicate that ATP acts via phosphatidylinositol-bis 4,5-phosphate (PIP(2)) synthesis: PIP(2) substitutes for ATP to induce MEND. ATP-activated MEND is blocked by an inositol 5-phosphatase and by guanosine 5′-[γ-thio]triphosphate (GTPγS). Block by GTPγS is overcome by the phospholipase C inhibitor, U73122, and PIP(2) induces MEND in the presence of GTPγS. MEND can occur in the absence of ATP and PIP(2) when cytoplasmic free Ca is clamped to 10 µM or more by Ca-buffered solutions. ATP-independent MEND occurs within seconds during Ca transients when cytoplasmic solutions contain polyamines (e.g., spermidine) or the membrane is enriched in cholesterol. Although PIP(2) and cholesterol can induce MEND minutes after Ca transients have subsided, polyamines must be present during Ca transients. MEND can reverse over minutes in an ATP-dependent fashion. It is blocked by brief β-methylcyclodextrin treatments, and tests for involvement of clathrin, dynamins, calcineurin, and actin cytoskeleton were negative. Therefore, we turned to the roles of lipids. Bacterial sphingomyelinases (SMases) cause similar MEND responses within seconds, suggesting that ceramide may be important. However, Ca-activated MEND is not blocked by reagents that inhibit SMases. MEND is abolished by the alkylating phospholipase A(2) inhibitor, bromoenol lactone, whereas exocytosis remains robust, and Ca influx causes MEND in cardiac myocytes without preceding exocytosis. Thus, exocytosis is not prerequisite for MEND. From these results and two companion studies, we suggest that Ca promotes the formation of membrane domains that spontaneously vesiculate to the cytoplasmic side. The Rockefeller University Press 2011-01 /pmc/articles/PMC3010057/ /pubmed/21187336 http://dx.doi.org/10.1085/jgp.201010468 Text en © 2010 Lariccia et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). |
spellingShingle | Article Lariccia, Vincenzo Fine, Michael Magi, Simona Lin, Mei-Jung Yaradanakul, Alp Llaguno, Marc C. Hilgemann, Donald W. Massive calcium–activated endocytosis without involvement of classical endocytic proteins |
title | Massive calcium–activated endocytosis without involvement of classical endocytic proteins |
title_full | Massive calcium–activated endocytosis without involvement of classical endocytic proteins |
title_fullStr | Massive calcium–activated endocytosis without involvement of classical endocytic proteins |
title_full_unstemmed | Massive calcium–activated endocytosis without involvement of classical endocytic proteins |
title_short | Massive calcium–activated endocytosis without involvement of classical endocytic proteins |
title_sort | massive calcium–activated endocytosis without involvement of classical endocytic proteins |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3010057/ https://www.ncbi.nlm.nih.gov/pubmed/21187336 http://dx.doi.org/10.1085/jgp.201010468 |
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