Cortical cytoskeleton dynamics regulates plasma membrane calcium ATPase isoform-2 (PMCA2) activity

DALGHI, M; FERREIRA GOMES, MARIELA; MONTALBETTI N; SIMONIN A; STREHLER EE; HEDIGER MA; ROSSI JPFC

BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2017

0167-4889

We have previously shown that purified actin can directly bind to human plasma membrane Ca2 + ATPase 4b (hPMCA4b) and exert a dual modulation on its Ca2 +-ATPase activity: F-actin inhibits PMCA while short actin oligomers may contribute to PMCA activation. These studies had to be performed with purified proteins given the nature of the biophysical and biochemical approaches used. To assess whether a functional interaction between the PMCAs and the cortical cytoskeleton is of physiological relevance, we characterized this phenomenon in the context of a living cell by monitoring in real-time the changes in the cytosolic calcium levels ([Ca2 +]CYT). In this study, we tested the influence of drugs that change the actin and microtubule polymerization state on the activity and membrane expression of the PMCA transiently expressed in human embryonic kidney (HEK293) cells, which allowed us to observe and quantify these relationships in a live cell, for the first time. We found that disrupting the actin cytoskeleton with cytochalasin D significantly increased PMCA-mediated Ca2 + extrusion (~ 50?100%) whereas pre-treatment with the F-actin stabilizing agent jasplakinolide caused its full inhibition. When the microtubule network was disrupted by pretreatment of the cells with colchicine, we observed a significant decrease in PMCA activity (~ 40?60% inhibition) in agreement with the previously reported role of acetylated tubulin on the calcium pump. In none of these cases was there a difference in the level of expression of the pump at the cell surface, thus suggesting that the specific activity of the pump was the regulated parameter. Our results indicate that PMCA activity is profoundly affected by the polymerization state of the cortical cytoskeleton in living cells.