Regulation of plasma membrane calcium pumps (PMCA) by cytoskeleton.

VIGIL, M.; REY, O; RINALDI, D.E; PICCO, ME; ESPELT, M.V.; DE TEZANOS PINTO, F; MANGIALAVORI, I.; ROSSI, R.C; ROSSI, J.P.; FERREIRA GOMES, M

San Luis

Congreso; XLVIII Reunión Anual de la SAB; 2019

We have previously described a novel regulatory mechanism for PMCA activity thatinvolves the actin cytoskeleton. In HEK293 cells, PMCA activity was regulated by thepolymerization state of the cortical cytoskeleton: actin depolymerization withcytochalasin-D significantly increased PMCA activity while using jasplakinolide thatstabilizes filamentous actin, decrease PMCA activity. Reports suggest that the increase incytosolic Ca2+ concentration ([Ca 2+]CYT) induced transient cortical actindepolymerization. We hypothesize that after an increase in [Ca2+]CYT, PMCA will beactivated by short actin oligomers formed at the cell cortex. This may lead PMCA toactively restore [Ca2+]CYT level. By lowering the [Ca2+]CYT, actin may re-polymerize intolong filaments and the PMCA return to its inactive state. To test this hypothesis, wecarried out experiments aimed at measuring [Ca2+]CYT and actin dynamics in cells thatoverexpress different PMCA isoforms, both in normal and pathological cells.The dynamics of [Ca2+]CYT were studied in HEK293T cells using the fluorescent probeFluo-4 and following [Ca2+]CYT levels generated by its release from the endoplasmicreticulum (ER), and by the extracellular uptake through store-operated Ca2+ channels.Results show that fluorescence time courses measured in individual cells are differentfrom those obtained from the whole population of cells, suggesting the presence of atleast two different kinds of cell responses. Overexpression of isoforms 2 and 4 of humanPMCA contributed to decrease the [Ca2+]CYT arising from ER depletion and theextracellular medium. On the other hand, the use of different actin-markers and Life-actoverexpression, that bio-mark actin, allowed us to measure different polymerizationstates of actin in the cells. The combination of these determinations will be used toestablish the role of PMCA in what might be a versatile feedback mechanism thatcontributes to the interaction between Ca2+ and the cortical cytoskeleton.