Regulation of phospholipid translocation in the plasma membrane by tubulin

MUHLBERGER, TAMARA; BALACH M; MONESTEROLO, NOELIA E.; CASALE, CESAR H.; CAMPETELLI, ALEXIS N.

Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

The asymmetrical distribution of phospholipids in biological membranes plays major physiological roles at the cellular level. This asymmetry is generated and kept by transporters. Among them, the lipid flippases belongs to the family of P4-ATPases. Some P-ATPases, such as NKA and PMCA, are regulated by tubulin. In erythrocytes from hypertensive and diabetic subjects, tubulin is increased in the plasma membrane (PM) and as a consequence, the NKA and PMCA activity results partially inhibited. The objective of this work was to evaluate the putative inhibition of flippases by tubulin, both in vitro and in vivo. Here, we used Inside-Out vesicles (IOVs) from erythrocyte ghosts and the NBD labeled phosphathydilserine analog to test our hypothesis. When IOVs were incubated with purified tubulin, the NBD-PS translocation rate in these vesicles was decreased by 40% when compared with controls without tubulin, suggesting that tubulin inhibit the flippase activity in vitro. For the in vivo assay, erythrocytes were loaded with NBD-PS and the rate of NBD-PS translocation was assayed by flow cytometry. The results showed that the NBD-PS translocation rate is 20% slower in hypertensive and diabetic erythrocytes when compared with the control (erythrocytes from normal subjects). In addition, when tubulin levels are pharmacologically modified in the PM of the erythropoietic K562 cells, then percentage of NBD-PS translocated into the cell decreases in taxol-treated and in starved cells (conditions that promotes the association of tubulin to the PM) and the opposite effect was observed in cells treated with nocodazol (a condition that removes tubulin from the PM). These results are consistent with those obtained in in vitro experiments. Finally, we are currently performing pull down experiments in order to test the putative interaction between ATP11C, the main flippase in erythrocytes and tubulin. These experiments will bring some light to understand this inhibition mechanism.