EFFECT OF CALCIUM ON THE DISTRIBUTION OF TUBULIN AND SPECTRIN IN HUMAN ERYTHROCYTES

ELISIO, EY; CAMPETELLI AN; MONESTEROLO N E; MUHLBERGER, T; CASALE C H; BALACH M; SANTANDER VS

Mar del Plata

Congreso; S A I C . S A F E . S A B . SAP 2 0 1 9; 2019

S A I C . S A F E . S A B . SAP 2 0 1 9

In previous work we demonstrated that membrane tubulin in erythrocytes of hypertensive patients is increased, PMCA is inhibited and associated with tubulin, which correlates with a decrease in erythrocyte deformability. On the other hand, tubulin associated with the membrane inhibits PMCA, which would cause an increase in intracellular calcium. Our goal is to determine the factors that cause the increase in tubulin in the erythrocyte membrane. At this time in our work we wonder what event occurs first in erythrocytes: 1-PMCA is inhibited by the increase in membrane tubulin, or 2-PMCA is inhibited by another factor and this causes the intracellular increase in calcium that causes the translocation of tubulin to the membrane. Given these hypotheses we decided to determine the effect of calcium (exogenous and endogenous) on the distribution of tubulin in erythrocytes. Our immunofluorescence and western blot experiments reveal that tubulin translocates from the sedimentable structure to the membrane due to the increase in exogenous calcium or by inhibition of PMCA with caloxin. This effect was similar to that found by the addition of taxol (a microtubule stabilizer) in the absence of exogenous calcium, however, we now find that the simultaneous addition of exogenous calcium and taxol prevents translocation of the tubulin to the membrane observed with exogenous calcium only. This was expected because taxol activates PMCA which does not allow accumulation of intracellular calcium. On the other hand, in our laboratory we demonstrate, in vitro, that tubulin forms a complex with spectrin (major component of the erythrocyte cytoskeleton). If calcium affects the distribution of tubulin in erythrocytes, it may also affect the location of the spectrin. In this sense we determine the distribution of spectrin in erythrocytes treated with exogenous calcium. We found that exogenous calcium causes a less homogeneous redistribution of spectrin in 50% of erythrocytes. This phenomenon does not change if taxol is added in addition to calcium. These data suggest that tubulin and spectrin, both components of the erythrocyte cytoskeleton, may change their cellular location by increasing intracellular calcium. The increase in calcium causes the translocation of tubulin to the membrane, while spectrin follows an independent redistribution of tubulin even though both proteins form an in vitro complex.