Real-Time Confocal Imaging of Calcium Changes Upon Exocytosis in Paramecium

Abstract

Exocytosis in the unicellular eukaryote Paramecium occurs rapidly upon stimulation with the biological secretagogue lysozyme (100 μM) in a Ca2+-dependent manner. Prior to stimulation each cell contains several thousand dense core secretory vesicles (trichocysts) docked beneath the cell membrane which can be induced to fuse with the plasma membrane either globally or locally. Fusion of the vesicle is followed by secretion and expansion of the trichocyst content from a 4 μm protein crystalline array to a 40 μm spear-like protein structure that are visible in the light microscope. The exocytosis microdomain of the cell membrane has been well characterized in earlier freeze fracture studies. The specific site of exocytosis was shown to consist of a rosette (fusion rosette) of 9 to 11 intramembrane particles surrounded by two outer rings of smaller sized particles. It is below these fusion rosettes that each of the secretory vesicles is docked and primed to release upon stimulation. In the absence of assembled fusion rosette normal release of trichocysts is blocked, as demonstrated with rosette defective mutants1. The fusion rosette has been postulated to function as calcium gates involved in exocytosis3. Paramecium has three known distinct Ca2+ channels: 1) the ciliary voltage-gated channel, 2) the hyper-polarization-activated channel, and 3) the anterior mechanosensory channel. Recently, we have described experiments indicating the presence of a fourth type of Ca2+ channel, a specific receptor operated Ca2+ conductance associated with lysozyme stimulated exocytosis. In this study we have visualized local (temporal and spatial) calcium fluxes associated with discrete exocytic events, using real time confocal microscopy (Nikon, RCM 8000) and calcium imaging with Indo-1 dual emission dye.