Cortical Cytoskeleton Dynamics Regulates PMCA2 activity

DALGHI MG., 1FERREIRA-GOMES M., 2MONTALBETTI N., 2SIMONIN A., 3STREHLER EE., 2HEDIGER MA., 1ROSSI JP.; ROSSI, JPFC.

San Miguel de Tucumán

Congreso; 242. III Latin American Federation of Biophysical Societies (LAFeBS) IX IberoAmerican Congress of Biophysics XLV Reunion Anual SAB 2016; 2016

Sociedad Argentina de Biofísica

 PosterMembrane Transporters and Channels  Cortical Cytoskeleton Dynamics Regulates PMCA2activity  1,2*DalghiMG., 1Ferreira-Gomes M., 2Montalbetti N., 2SimoninA., 3Strehler EE., 2Hediger MA., 1Rossi JP.1IQUIFIB? Instituto de Química y Fisicoquímica Biológicas, Conicet/UBA; 2Institute of Biochemistry and MolecularMedicine, and Swiss National Centre of Competence in Research, NCCR TransCure,University of Bern, 3Departmentof Biochemistry and Molecular Biology, Mayo Clinic College of Medicine,Rochester, USA.*Present address: Department of Medicine, University of Pittsburgh, USA, mgd29@pitt.edu We have previously shown that purified actin candirectly bind to hPMCA4b and exert a dual modulation on its Ca2+-ATPaseactivity: on one side F-actin inhibits PMCA [1] while short actin oligomers maybe responsible for PMCA activation [2]. These studies required to be performedusing purified proteins given the nature of the biophysical and biochemicalapproaches used.  In order to assesswhether this functional interaction may be of physiological relevance, wedecided to characterize this phenomenon in the context of a living cell bymonitoring in real-time the changes in the cytosolic calcium levels ([Ca2+]CYT).We tested the influence of drugs that change the actin and microtubulespolymerization state on the activity and membrane expression of the PMCAtransiently expressed in human embryonic kidney (HEK293) cells. We found that disruptingthe actin cytoskeleton with cytochalasin D significantly increasedPMCA-mediated Ca2+ extrusion (~50-100%) at the time thatpre-treatment with the F-actin stabilizing agent jasplakinolide caused its fullinhibition. These results are in full agreement with our previous in vitro observations. When themicrotubule network was disrupted by pretreatment of the cells with colchicine,we observed a significantly decrease in PMCA activity (~40-60% inhibition) inagreement with the previously reported role of acetylated tubulin on the calciumpump [3]. In none of these cases we observe a difference in the level ofexpression of the pump at the cell surface. Taken together, these datademonstrate that our and other?s previous observations on the in vitro effect of the actin and tubulincytoskeleton on PMCA activity is also evident in a living cell model.   References:[1] Vanagas et al., Cell BiochemBiophys 66:187-198, 2013.[2] Dalghi et al., J BiolChem 288:23380-23393, 2013.[3] Monesterolo etal., FEBS J. 275:3567-3579, 2008.