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

.ROSSI, JUAN PABLO F.C.; M. VIGIL1, O. REY2, D. RINALDI1, M. V. ESPELT1, F. TEZANOS PINTO1, I. MANGIALAVORI1, R. C. ROSSI1, J. P. ROSSI1, M. FERREIRA-GOMES1

SAN LUIS

Congreso; 279. XLVIII Reunión Anual SAB, 27-29 Noviembre 2019. San Luis, Argentina; 2019

SOCIEDAD ARGENTINA DE BIOFÍSICA

Regulation of plasma membrane calcium pump (PMCA) by the cytoskeletonM. Vigil1, O. Rey2, D.Rinaldi1, M. V. Espelt1, F. Tezanos Pinto1, I.Mangialavori1, R. C. Rossi1, J. P. Rossi1, M.Ferreira-Gomes11Instituto de Química y Fisicoquímica Biológicas.?Prof. Paladini?. Departamento de Química Biológica, Facultad de Farmacia yBioquímica. Universidad de Buenos Aires. 2Instituto de Inmunología,Genética y Metabolismo (INIGEM). Cátedra de Genética. Facultad de Farmacia yBioquímica. Universidad de Buenos Aires.Wehave previously described a novel regulatory mechanism for PMCA activity thatinvolves the actin cytoskeleton. In HEK293 cells, PMCA activity wasregulated by the polymerization state of the cortical cytoskeleton: actindepolymerization with cytochalasin-D significantly increased PMCA activitywhile using jasplakinolide that stabilizes filamentous actin, decrease PMCAactivity. Reportssuggestthat the increase in cytosolic Ca2+ concentration ([Ca2+]CYT) induced transient cortical actindepolymerization. We hypothesize that after an increase in [Ca2+]CYT,PMCA will be activated by short actin oligomers formed at the cell cortex. Thismay lead PMCA to actively restore [Ca2+]CYT level. Bylowering the [Ca2+]CYT, actin may re-polymerize into longfilaments and the PMCA return to its inactive state. To test this hypothesis,we carried out experiments aimed at measuring [Ca2+]CYT and actindynamics in cells that overexpress different PMCA isoforms, both in normal andpathological cells. Thedynamics of [Ca2+]CYT were studied in HEK293T cells using the fluorescent probe Fluo-4 and following [Ca2+]CYTlevels generated by its release from the endoplasmic reticulum (ER), and by theextracellular uptake through store operated Ca2+ channels. Results show that fluorescence time courses measured inindividual cells are different from those obtained from the whole population ofcells, suggesting the presence of at least two different kinds of cellresponses. Overexpression of isoforms 2 and 4 of human PMCA contributed todecrease the [Ca2+]CYT arising from ER depletion and theextracellular medium. On the other hand, the use of different actin-markers andLife-act overexpression, that bio-mark actin, allowed us to measure differentpolymerization states of actin in the cells. The combination of thesedeterminations will be used to establish the role of PMCA in what might be aversatile feedback mechanism that contributes to the interaction between Ca2+and the cortical cytoskeleton.