Reorganization of filamentous actin and myosin-II in zebrafish eggs correlates temporally and spatially with cortical granule exocytosis

The zebrafish egg provides a useful experimental system to study events of fertilization, including exocytosis, We show by differential interference c ontrast videomicroscopy that cortical granules are: (1) released nonsynchro nously over the egg surface and (2) mobilized to the plasma membrane in two phases, depending upon vesicle size and location, Turbidometric assay meas urements of the timing and extent of exocytosis revealed a steady release o f small granules during the first 30 seconds of egg activation, This was fo llowed by an explosive discharge of large granules, beginning at 30 seconds and continuing for 1-2 minutes, Stages of single granule exocytosis and su bsequent remodeling of the egg surface were imaged by either realtime or ti me-lapse videomicroscopy as well as scanning electron microscopy. Cortical granule translocation and fusion with the plasma membrane were followed by the concurrent expansion of a fusion pore and release of granule contents, A dramatic rearrangement of the egg surface followed exocytosis. Cortical c rypts (sites of evacuated granules) displayed a purse-string-like acts as a physical barrier to secretion and is locally disassembled prior to granule release, Experimental results showed a reduction of rhodamine-phalloidin a nd antimyosin staining at putative sites of secretion, acceleration of the timing and extent of granule release in eggs pretreated with cytochalasin D , and dose-dependent inhibition of exocytosis in permeabilized eggs preincu bated with phalloidin, An increase in assembled actin was detected by fluor ometric assay during the period of exocytosis, Localization studies showed that F-actin and myosin-II codistributed with an inward-moving, membrane-de limited zone of cytoplasm that circumscribed cortical crypts during their t ransformation. Furthermore, cortical crypts displayed a distinct delay in t ransformation when incubated continuously with cytochalasin D following egg activation, We propose that closure of cortical crypts is driven by a cont ractile ring whose forces depend upon dynamic actin filaments and perhaps a ctomyosin interactions.