Regulation of plasma membrane calcium pump (PMCA) by the cytoskeleton

VIGIL MA; REY O; RINALDI D; PICCO ME; ESPELT MV; DE TEZANOS PINTO F; MANGIALAVORI I; ROSSI RC; ROSSI JPFC; FERREIRA-GOMES MS

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Biofísica; 2019

We have previously described a novel regulatory mechanism for PMCA activity that involves the actin cytoskeleton. In HEK293 cells, PMCA activity was regulated by the polymerization state of the cortical cytoskeleton: actin depolymerization with cytochalasin-D significantly increased PMCA activity while using jasplakinolide that stabilizes filamentous actin, decrease PMCA activity. Reports suggest that the increase in cytosolic Ca2+ concentration ([Ca 2+]CYT) induced transient cortical actin depolymerization. We hypothesize that after an increase in [Ca2+]CYT, PMCA will be activated by short actin oligomers formed at the cell cortex. This may lead PMCA to actively restore [Ca2+]CYT level. By lowering the [Ca2+]CYT, actin may re-polymerize into long filaments and the PMCA return to its inactive state. To test this hypothesis, we carried out experiments aimed at measuring [Ca2+]CYT and actin dynamics in cells that overexpress different PMCA isoforms, both in normal and pathological cells. The dynamics of [Ca2+]CYT were studied in HEK293T cells using the fluorescent probe Fluo-4 and following [Ca2+]CYT levels generated by its release from the endoplasmic reticulum (ER), and by the extracellular uptake through store-operated Ca2+ channels. Results show that fluorescence time courses measured in individual cells are different from those obtained from the whole population of cells, suggesting the presence of at least two different kinds of cell responses. Overexpression of isoforms 2 and 4 of human PMCA contributed to decrease the [Ca2+]CYT arising from ER depletion and the extracellular medium. On the other hand, the use of different actin-markers and Life-act overexpression, that bio-mark actin, allowed us to measure different polymerization states of actin in the cells. The combination of these determinations will be used to establish the role of PMCA in what might be a versatile feedback mechanism that contributes to the interaction between Ca2+ and the cortical cytoskeleton.