DIRECT VISUALIZATION OF A VAST CORTICAL CALCIUM COMPARTMENT IN PARAMECIUM BY SECONDARY-ION MASS-SPECTROMETRY (SIMS) MICROSCOPY - POSSIBLE INVOLVEMENT IN EXOCYTOSIS

The plasma membrane of ciliates is underlaid by a vast continuous arra y of membrane vesicles known as cortical alveoli. Previous work had sh own that a purified fraction of these vesicles actively pumps calcium, suggesting that alveoli may constitute a calcium-storage compartment. Here we provide direct confirmation of this hypothesis using in situ visualization of total cell calcium on sections of cryofixed and cryos ubstituted cells analyzed by SIMS (secondary ion mass spectrometry) mi croscopy a method never previously applied to protists. A narrow, cont inuous, Ca-emitting zone located all along the cell periphery was obse rved on sections including the cortex. In contrast, Na and K were even ly distributed throughout the cell. Various controls confirmed that em ission was from the alveoli, in particular, the emitting zone was stil l seen in mutants totally lacking trichocysts, the large exocytotic or ganelles docked at the cell surface, indicating that they make no majo r direct contribution to the emission. Calcium concentration within al veoli was quantified for the first time in SIMS microscopy using an ex ternal reference and was found to be in the range of 3 to 5 mM, a valu e similar to that for sarcoplasmic reticulum. After massive induction of trichocyst discharge, this concentration was found to decrease by a bout 50%, suggesting that the alveoli are the main source of the calci um involved in exocytosis.