IN-SITU COMPOSITIONAL ANALYSIS OF ACIDOCALCISOMES IN TRYPANOSOMA-CRUZI

We measured the elemental content of different compartments in Trypano soma cruzi epimastigotes using quick freezing, ultracryomicrotomy, and electron probe microanalysis. Vacuoles identified by high electron de nsity contained (in units of mmol/kg dry weight +/- S.E.) large amount s of phosphorus (1390 +/- 13), magnesium (646 +/- 19), calcium (171 +/ - 5), sodium (161 +/- 18), and zinc (148 +/- 6). No other compartment had appreciable calcium or zinc content. Iron (128 +/- 16 mmol/kg) was detected only in vacuoles distinct from the electron-dense vacuoles a nd other organelles. Incubation of cells for 70 min in culture medium in the presence of ionomycin plus nigericin led to a very significant 3- or 2-fold increase in potassium in the electron-dense vacuoles and the iron-rich vacuoles, respectively, with no significant change in th e other elements investigated, This indicated the acidic nature of the vacuoles and demonstrated that the electron-dense vacuoles correspond to what were described previously as acidocalcisomes, i.e. acidic com partments rich in Ca2+. The acidocalcisomes were investigated by separ ation of epimastigote fractions on Percoll gradients in combination wi th Triton WR-1339 treatment. This detergent caused a rapid vacuolation ; these vacuoles were shown by electron microscopy to be largely trans parent, with a diffuse matrix. Percoll gradient fractionation demonstr ated decreases in the density of various organelle markers in detergen t-treated cells compared with controls. Large decreases in the density of the acidocalcisome and the mitochondrion were seen, as well as sma ller decreases in the density of the other markers. Conventional elect ron microscopy of epimastigotes loaded with gold-labeled transferrin i ndicated that the endosomal system was separate from vacuoles that pro bably corresponded to the calcium-containing organelles detected by el ectron probe microanalysis. The combined results provide evidence that acidocalcisomes are organelles different from lysosomes or other orga nelles previously described in these parasites.