Becker K A, Hart N H
Rutgers University, Department of Cell Biology and Neuroscience, Busch Campus, Nelson Biology Laboratories, Piscataway, NJ 08854-8082, USA.
J Cell Sci. 1999 Jan;112 ( Pt 1):97-110. doi: 10.1242/jcs.112.1.97.
The zebrafish egg provides a useful experimental system to study events of fertilization, including exocytosis. We show by differential interference contrast videomicroscopy that cortical granules are: (1) released nonsynchronously over the egg surface and (2) mobilized to the plasma membrane in two phases, depending upon vesicle size and location. Turbidometric assay measurements of the timing and extent of exocytosis revealed a steady release of small granules during the first 30 seconds of egg activation. This was followed by an explosive discharge of large granules, beginning at 30 seconds and continuing for 1-2 minutes. Stages of single granule exocytosis and subsequent remodeling of the egg surface were imaged by either real-time or time-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 crypts (sites of evacuated granules) displayed a purse-string-like contraction, resulting in their gradual flattening and disappearance from the egg surface. We tested the hypothesis that subplasmalemmal filamentous (F-) actin acts as a physical barrier to secretion and is locally disassembled prior to granule release. Experimental results showed a reduction of rhodamine-phalloidin and 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 preincubated with phalloidin. An increase in assembled actin was detected by fluorometric assay during the period of exocytosis. Localization studies showed that F-actin and myosin-II codistributed with an inward-moving, membrane-delimited zone of cytoplasm that circumscribed cortical crypts during their transformation. Furthermore, cortical crypts displayed a distinct delay in transformation when incubated continuously with cytochalasin D following egg activation. We propose that closure of cortical crypts is driven by a contractile ring whose forces depend upon dynamic actin filaments and perhaps actomyosin interactions.
斑马鱼卵为研究受精过程中的事件，包括胞吐作用，提供了一个有用的实验系统。我们通过微分干涉相差视频显微镜观察到，皮质颗粒：(1) 在卵表面非同步释放；(2) 根据囊泡大小和位置，分两个阶段向质膜移动。通过比浊法测定胞吐作用的时间和程度，发现在卵激活的前30秒内，小颗粒持续稳定释放。随后，大颗粒在30秒时开始爆发性释放，并持续1 - 2分钟。通过实时或延时视频显微镜以及扫描电子显微镜对单个颗粒胞吐作用的阶段和随后卵表面的重塑进行了成像。皮质颗粒向质膜的转运和融合伴随着融合孔的同时扩张和颗粒内容物的释放。胞吐作用后，卵表面发生了显著的重排。皮质隐窝（排空颗粒的部位）呈现出类似束带的收缩，导致它们逐渐变平并从卵表面消失。我们测试了以下假设：质膜下丝状（F-）肌动蛋白作为分泌的物理屏障，并在颗粒释放前局部解聚。实验结果表明，在假定的分泌部位，罗丹明-鬼笔环肽和抗肌球蛋白染色减少；用细胞松弛素D预处理的卵中，颗粒释放的时间和程度加快；用鬼笔环肽预孵育的通透卵中，胞吐作用受到剂量依赖性抑制。在胞吐作用期间，通过荧光测定法检测到组装的肌动蛋白增加。定位研究表明，F-肌动蛋白和肌球蛋白-II与一个向内移动的、由膜界定的细胞质区域共分布，该区域在皮质隐窝转化过程中围绕着它们。此外，卵激活后连续用细胞松弛素D孵育时，皮质隐窝的转化明显延迟。我们提出，皮质隐窝的闭合是由一个收缩环驱动的，其力量取决于动态肌动蛋白丝，可能还取决于肌动球蛋白相互作用。
