Control of exocytotic processes: cytological and physiological studies of trichocyst mutants in Paramecium tetraurelia.

Abstract

The trichocysts of Paramecium tetraurelia consitute a favorable system for studying secretory processes because of the numerous available mutations that block, at various stages, the development of these secretory vesicles, their migration towards and interaction with the cell surface, and their exocytosis .Previous studies of several mutants provided information (a) on the assembly and function of the intramembranous particles arrays in the plasma membrane at trichocyst attachment sites, (b) on the autonomous motility of trichocysts, required for attachment to the cortex, and (c) on a diffusible cytoplasmic factor whose interaction with both trichocyst and plasma membrane is required for exocytosis to take place.We describe here the properties of four more mutants deficient in exocytosis ability, nd6, nd7, tam38, and tam6, which were analyzed by freeze-fracture, microinjection of trichocysts, and assay for repair of the mutational defect through cell-cell interaction during conjugation with wild-type cells. As well as providing confirmation of previous conclusions, our observations show that the mutations nd6 and tam6 (which display striking abnormalities in their plasma membrane particle arrays and are reparable through cell-cell contact but not by microinjection of cytoplasm) affect two distinct properties of the plasma membrane, whereas the other two mutations affect different properties of the trichocysts . Altogether, the mutants so far analyzed now provide a rather comprehensive view of the steps and functions involved in secretory processes in Paramecium and demonstrate that two steps of these processes, trichocyst attachment to the plasma membrane and exocytosis, depend upon specific properties of both the secretory vesicle and the plasma membrane .The trichocysts of Paramecium are secretory vesicles whose formation in the cytoplasm, migration to the cell cortex, interaction with the plasma membrane, and exocytosis are easily observable by light and electron microscopy and are also amenable to genetic dissection. A number of mutations are available that block trichocyst development and exocytosis at various stages . These mutations disclose different steps and functions that might not be suspected or identified in other systems. The trichocyst system offers two further advantages for studying secretory processes .First, in the region of contact between trichocyst and plasma membrane, organized arrays of intramembrane particles are visible on freeze-fracture replicas of both the plasma and the

Expression of the green fluorescent protein in Paramecium tetraurelia

“…Stocks and cell culturing P. tetraurelia, nd6/nd6 with 51S background (Lefort-Tran et al, 1981) and wild-type strain 7S (Sonneborn, 1975), was cultured at 22 8C in a growth medium of buffered wheat-grass extract inoculated with Enterobacter aerogenes (Sonneborn, 1970). Single cells were isolated and grown for more than twenty fissions before each clone was starved to induce autogamy (Berger, 1986).…”

Veratridine-Mediated Ca 2+ Dynamics and Exocytosis in Paramecium Cells

“…Wild-type cells (strain 7S), a trichocyst-free strain "trichless" (tl) [39], and a "pawn" mutant (d4-500r) devoid of ciliary voltage dependent Ca channels [20,44] but with normal secretory capacity were all grown at 25°C, the nondischarge mutant nd9 [3,4,30,40] To chelate [Ca 2+ ] o to ∼30 nM, i.e., slightly below resting levels (50-100 nM), we added 4.5 mM EGTA to the extracellular medium, as in ref. [26].…”

“…This normally occurs by influx of Ca o 2+ after formation of an exocytotic pore [37,38]. The nd9 mutant we used was grown at a nonpermissive temperature of 28°C, so it cannot secrete any of its trichocysts, although they are docked in great numbers at the cell membrane [3,4,30,40]. Their trichocyst contents can decondense in vitro, but since nd9-28°C cells cannot form an exocytotic fusion pore, Ca 2+ must artifically obtain access to trichocyst contents to provoke their ("internal") decondensation [40].…”

Ca2+ oscillations mediated by exogenous GTP in Paramecium cells: assessment of possible Ca2+ sources

“…Paramecium tetraurelia cells, mutant nd6 (without trichocyst discharge [38]), were cultured in dried lettuce medium, monoxenically inoculated with Enterobacter aerogenes as feeding bacteria, at [Ca 2+ ] o = 50 M. In addition we used the mutant pawnB [39] and ginA [40]. All cell lines used additionally contained the nd6 mutation.…”