Massive Endocytosis (MEND) Activated by Ca and Polyamines in Fibroblasts and Cardiac Myoyctes: Dependencies on nucleotides, PIP2, cholesterol, clathrin, and other factors

We describe four protocols that result in internalization of ~50% of the surface membrane of BHK fibroblasts and cardiac myocytes within <1 min. To do so, we use patch clamp with large pipette tips for cell dialysis and Na/Ca exchangers to evoke cytoplasmic Ca transients (5 to 200 µM Ca2+ ) for 1-5 s. Endocytosis is monitored via capacitance and/or optically by standard membrane dyes. In the first protocol, ATP is depleted from the cytoplasm, a Ca transient is evoked, and MEND is then activated by replenishment of ATP and GTP. GTP alone is not sufficient, Ca transients are required, and AMPPNP does not subsitute for ATP. Second, when membrane cytoskeleton is stabilized with phalloidin, MEND is made 'available' for 1 to 3 min, and it occurs within 5 s during a Ca transient without ATP depletion. Third, high ATP concentrations (4 to 8 mM) promote MEND to occur within 20 to 60 s after (but not during) a Ca transient. Fourth, polyamines, spermine or spermidine, at physiological concentrations (1 mM) cause MEND to occur within <5 s during Ca transients without ATP depletion. MEND is not blocked by protein domains and other interventions that block chathrin-dependent endocytosis or by tyrosine kinase inhibitors. MEND is blocked by cholesterol depletion, GTPgS, and PIP2 phosphatases, and MEND is promoted by perfusion of PIP2 into cells when ATP and GTP are depleted. In neonatal myocytes, transient GTPgS perfusion substitutes for Ca transients in permissive steps leading to MEND activation upon ATP perfusion. We conclude that MEND is a regulated and massive cell stress response that can remove large fractions of the cell surface of multiple cell types by clathrin-independent mechanisms.