Cortical Cytoskeleton Dynamics Regulates Plasma Membrane Calcium ATPase Isoform-2 (PMCA2) activity

DALGHI M, ROSSI JP.

BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2017 vol. 1864 p. 1413 - 1424

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 actinoligomers may contribute to PMCA activation. These studies had to be performed with purified proteins giventhe nature of the biophysical and biochemical approaches used. To assess whether a functional interactionbetween the PMCAs and the cortical cytoskeleton is of physiological relevance, we characterized thisphenomenon in the context of a living cell by monitoring in real-time the changes in the cytosolic calciumlevels ([Ca2+]CYT). In this study, we tested the influence of drugs that change the actin and microtubulepolymerization state on the activity and membrane expression of the PMCA transiently expressed in humanembryonic kidney (HEK293) cells, which allowed us to observe and quantify these relationships in a live cell, forthe first time. We found that disrupting the actin cytoskeleton with cytochalasin D significantly increased PMCAmediatedCa2+ extrusion (~50?100%) whereas pre-treatment with the F-actin stabilizing agent jasplakinolidecaused its full inhibition. When the microtubule network was disrupted by pretreatment of the cells withcolchicine, we observed a significant decrease in PMCA activity (~40?60% inhibition) in agreement with thepreviously reported role of acetylated tubulin on the calcium pump. In none of these cases was there a differencein the level of expression of the pump at the cell surface, thus suggesting that the specific activity of the pumpwas the regulated parameter. Our results indicate that PMCA activity is profoundly affected by thepolymerization state of the cortical cytoskeleton in living cells.