NUCLEAR CALCIUM FLUX IN TRYPANOSOMA-BRUCEI CAN BE QUANTIFIED WITH TARGETED AEQUORIN

The following study was undertaken to determine if calcium ions move f rom the plasma membrane to the nucleus of Trypanosoma brucei. Nuclear and cytosolic calcium flux was measured with the calcium sensitive pho toprotein, aequorin which was targeted to various locations in stably transformed procyclic cells. Immunoblots revealed that the recombinant proteins, CYT-AEQ and NUC-AEQ were translated in transformants, and t hat CYT-AEQ was contained in a soluble fraction. Immunolocalization de monstrated that NUC-AEQ was contained within the trypanosome nucleus. To evaluate calcium movement from the plasma membrane to the nucleus i n live trypanosomes, aequorin was reconstituted in vivo with coelenter azine and luminescence was recorded. The resting levels of [Ca2+](cyt) and [Ca2+](nuc) were similar (314 +/- 43 and 287 +/- 28 nM, respectiv ely). When calcium influx across the plasma membrane was initiated wit h 2 mu M ionomycin, [Ca2+](cyt) and [Ca2+](nuc) each became elevated i n parallel to a new steady state which was approximately 2-fold above the resting level. Compound 48/80 initiated a calcium flux across the plasma membrane by a different mechanism from ionomycin, and in a mann er that was inhibited by the calcium channel antagonist, La3+. Compoun d 48/80 (8 mu g/ml) transiently elevated [Ca2+](cyt) to 1.73 +/- 0.3 m u M over the course of 20 s, and also generated a transient rise in [C a2+](nuc) which peaked at 1.32 + 0.29 mu M over the same time course. Overall, these data demonstrate that calcium moves into and out of the trypanosome nucleus in a manner which closely parallels changes in [C a2+](cyt). A small calcium ion gradient between nucleus and cytoplasm was also observed. Copyright (C) 1996 Elsevier Science B.V.