Massive calcium-activated endocytosis without involvement of classical endocytic proteins

Vincenzo Lariccia, Michael Fine, Simona Magi, Mei Jung Lin, Alp Yaradanakul, Marc C. Llaguno, Donald W. Hilgemann

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Abstract

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 (PIP2) synthesis: PIP2 substitutes for ATP to induce MEND. ATP-activated MEND is blocked by an inositol 5-phosphatase and by guanosine 5?-[?-thio]triphosphate (GTPγS). Block by GTPγS is overcome by the phospholipase C inhibitor, U73122, and PIP2 induces MEND in the presence of GTP?S. MEND can occur in the absence of ATP and PIP2 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 PIP2 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 A2 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.

Original languageEnglish (US)
Pages (from-to)111-132
Number of pages22
JournalJournal of General Physiology
Volume137
Issue number1
DOIs
StatePublished - Jan 2011

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Endocytosis
Calcium
Adenosine Triphosphate
Proteins
Exocytosis
Sphingomyelin Phosphodiesterase
Polyamines
Membranes
Cholesterol
Dynamins
Clathrin
Spermidine
Guanosine
Calcineurin
Ceramides
HEK293 Cells
Type C Phospholipases
Phosphatidylinositols
Guanosine Triphosphate
Actin Cytoskeleton

ASJC Scopus subject areas

  • Physiology

Cite this

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.

In: Journal of General Physiology, Vol. 137, No. 1, 01.2011, p. 111-132.

Research output: Contribution to journalArticle

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. In: Journal of General Physiology. 2011 ; Vol. 137, No. 1. pp. 111-132.
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