Modulation of plasma membrane calcium ATPase (PMCA) by cortical actin cytoskeleton in living cells

MAXIMILIANO VIGIL; SANTIAGO FARAJ ; PICCO M; DEBORA RINALDI; IRENE C MANGIALAVORI; ROLANDO ROSSI; OSVALDO REY; JUAN PABLO ROSSI; MARIELA FERREIRA-GOMES

Congreso; L Reunión Anual de la Sociedad Argentina de Biofísica; 2022

The associations between the cortical cytoskeleton and the elements of the plasma membrane are currently not only considered to be structural and mechanical in nature but are now recognized as dynamic interrelationships that modulate cellular responses. We recently explained a new regulatory mechanism of plasma membrane Ca2+-ATPase (PMCA) in which the actin cytoskeleton could participate in the regulation of cytosolic Ca2+ ([Ca2+]cyt) homeostasis.To assess whether this regulatory mechanism may have physiological relevance, we decided to further characterize it in a living cell by monitoring changes in actin and [Ca2+]cyt polymerization. We used HEK293T cells that expressed PMCA transiently. The dynamics of [Ca2+]cyt were performed using the Fluo-4 fluorescent probe, whereas the actin dynamic in cells was visualized by transiently expressing LifeAct-Ruby. The transient height [Ca2+]cyt was generated by the release of Ca2+ from the endoplasmic reticulum (ER) or by the influx of extracellular Ca2+ via storage-operated Ca2+ channels (SOC).Results show that the increase of [Ca2+]cyt resulting from ER and extracellular media was significantly attenuated in cells that overexpressed the hPMCA2 or hPMCA4 isoforms. Furthermore, the increase of [Ca2+]cyt induced actin polymerization in the nuclear periphery and depolymerization in cortical regions only when Ca2+ enters through the SOC. This actin reorganization also occurred when PMCA was overexpressed, although in these cells the height of [Ca2+]cyt was lower. To explore the results, we design a kinetic model that explains PMCA activity and its activation in the [Ca2+]cyt functionality. The model was fitted to the experimental data and shows that PMCA activity increases while actin depolymerizes in cortical regions.These results suggest that actin reorganization is triggered by the rise of [Ca2+]cyt and that PMCA could be activated by the depolymerization of cortical actin in cells to restore the basal level of [Ca2+]cyt.With grants from ANPCYT, CONICET, UBA