Control of membrane fusion in exocytosis. Physiological studies on a Paramecium mutant blocked in the final step of the trichocyst extrusion process.

Abstract

Previous studies on exocytosis in Paramecium using mutants affecting trichocyst extrusion permitted us to analyze the assembly and function of three intramembrane particle arrays ("ring" and "rosette" in the plasma membrane, "annulus" in the trichocyst membrane) involved in the interaction between these two membranes .Using a conditional mutation, nd9, which blocks rosette assembly and prevents exocytosis at the nonpermissive temperature, we have analyzed the effect of temperature on the secretory capacity of nd9 cells . By combining several techniques (physiological studies, microinjections, inhibition of fatty acid synthesis, and freeze-fracture analysis) we demonstrate (a) that the product of the mutated allele nd9 is not thermolabile but that its activity is dependent upon temperature-induced changes in the membrane lipid composition and (b) that the product of the nd9 locus is a diffusible cytoplasmic component whose interaction with both plasma membrane and trichocyst membrane is required for rosette assembly and exocytosis .The data provide physiological evidence for the existence of a molecular complex(es) linking the two membranes and involved in the control of membrane fusion ; we discuss the possible nature and function of these links .For three main reasons, the trichocysts of Paramecium constitute a model system for analysis of the mechanisms of exocytosis and membrane fusion . Firstly, a number of mutations are available that block the trichocyst cycle at various stages (trichocyst development, attachment to the plasma membrane, or exocytosis) . Secondly, exocytosis can be triggered at will and observed under the J . CELL. BIOLOGY

Genetic approach to regulated exocytosis using functional complementation in Paramecium: identification of the ND7 gene required for membrane fusion.

“…The Paramecium system presents an additional unique advantage: the site of action of the products of the ND genes can be established by transferring cytoplasm containing a few trichocysts from one cell type to another in appropriate combinations of donor and recipient cells and checking the exocytotic capacity of the injected cells (Aufderheide, 1978;Lefort-Tran et al, 1981). Some ND gene products have thus been localized in the trichocyst compartment (e.g., ND7p), in the cytosol (e.g., ND9p) or in the plasma membrane (e.g., ND6p; Aufderheide, 1978;Beisson et al, 1980;LefortTran et al, 1981).…”

Serous Cells in the Parotid Glands of Two Species of Tamarins: Polarized Secretory Granules

“…In these protozoa, trichocysts, which are internally polarized elliptical secretory structures (Bannister, 1972), become joined to the plasmalemma only at their anterior, rounded end in a junction that involves a particulate annulus on their limiting membranes, and a little knob at the tip of the trichocyst (Beisson et al, 1980). In Tetrahymena, when an elliptical mucocyst approaches the plasma membrane, an annulus of particles forms at its leading end (Satir et al, 1973).…”

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].…”