X-ray microanalysis of calcium binding sites in Paramecium with special reference to exocytosis

Abstract

In Paramecium cells Ca++-stimulated triggering of the exocytosis of secretory vesicles ("trichocysts") was achieved by ionophores X-537 A or A 23187. Under triggering conditions electron dense deposits were present in some "resting" trichocysts and regularly in discharging trichocysts; upon subsequent fixation deposits occurred on the trichocyst membrane (on the inner side or within the membrane) and on the "inner lamellar sheath" from where deposits seemed to "radiate" into the secretory materials. Similar results were obtained with glutardialdehyde fixation alone which also triggers exocytosis but only at low concentrations. Element analysis by energy dispersive x-ray microanalysis ascertained the presence of Ca and P in deposits occurring in trichocysts. Those "resting" trichocysts which were devoid of deposits did not contain Ca or P enriched. Hence, an abrupt Ca++-influx into individual trichocysts just before exocytosis seems to be involved in the triggering mechanism, possible in combination with the sudden activation of an ATPase system localized at those sites of the trichocysts which primarily contain the deposits. When paramecia were treated only with Ca++ and then fixed with OsO4 plus oxalate or merely with glutardialdehyde, electron scattering deposits were formed also on the inner side of the cell membrane and within the ciliary shaft (but rarely in trichocysts). Deposits obtained on cilia (including "ciliary granule plaques") also contained Ca, P and S. Cells contain osmiophilic "calcium-storing vacuoles" which were selectively rich in Ca and S but devoid of P.