Danilchik Michael V, Bedrick Steven D, Brown Elizabeth E, Ray Kimberly
Department Biological Structure and Function SD, Oregon Health Sciences University, Portland, OR 97201-3097 USA.
J Cell Sci. 2003 Jan 15;116(Pt 2):273-83. doi: 10.1242/jcs.00217.
In dividing Xenopus eggs, furrowing is accompanied by expansion of a new domain of plasma membrane in the cleavage plane. The source of the new membrane is known to include a store of oogenetically produced exocytotic vesicles, but the site where their exocytosis occurs has not been described. Previous work revealed a V-shaped array of microtubule bundles at the base of advancing furrows. Cold shock or exposure to nocodazole halted expansion of the new membrane domain, which suggests that these microtubules are involved in the localized exocytosis. In the present report, scanning electron microscopy revealed collections of pits or craters, up to approximately 1.5 micro m in diameter. These pits are evidently fusion pores at sites of recent exocytosis, clustered in the immediate vicinity of the deepening furrow base and therefore near the furrow microtubules. Confocal microscopy near the furrow base of live embryos labeled with the membrane dye FM1-43 captured time-lapse sequences of individual exocytotic events in which irregular patches of approximately 20 micro m(2) of unlabeled membrane abruptly displaced pre-existing FM1-43-labeled surface. In some cases, stable fusion pores, approximately 2 micro m in diameter, were seen at the surface for up to several minutes before suddenly delivering patches of unlabeled membrane. To test whether the presence of furrow microtubule bundles near the surface plays a role in directing or concentrating this localized exocytosis, membrane expansion was examined in embryos exposed to D(2)O to induce formation of microtubule monasters randomly under the surface. D(2)O treatment resulted in a rapid, uniform expansion of the egg surface via random, ectopic exocytosis of vesicles. This D(2)O-induced membrane expansion was completely blocked with nocodazole, indicating that the ectopic exocytosis was microtubule-dependent. Results indicate that exocytotic vesicles are present throughout the egg subcortex, and that the presence of microtubules near the surface is sufficient to mobilize them for exocytosis at the end of the cell cycle.
在非洲爪蟾卵分裂过程中，卵裂沟的形成伴随着卵裂平面上新的质膜区域的扩展。已知新膜的来源包括一批卵原细胞产生的胞吐小泡，但它们发生胞吐作用的位点尚未得到描述。先前的研究揭示，在前进的卵裂沟底部有一个呈V形排列的微管束。冷休克或用诺考达唑处理会使新膜区域的扩展停止，这表明这些微管参与了局部胞吐作用。在本报告中，扫描电子显微镜显示有一些凹坑或火山口状结构，直径可达约1.5微米。这些凹坑显然是近期胞吐作用位点处的融合孔，聚集在加深的卵裂沟底部附近，因此靠近沟微管。用膜染料FM1-43标记的活胚胎在卵裂沟底部附近进行共聚焦显微镜观察，捕捉到了单个胞吐事件的延时序列，其中约20微米²的未标记膜的不规则斑块突然取代了先前FM1-43标记的表面。在某些情况下，可在表面看到直径约2微米的稳定融合孔，持续数分钟后才突然释放未标记膜的斑块。为了测试表面附近的沟微管束是否在引导或集中这种局部胞吐作用中发挥作用，对暴露于重水（D₂O）以诱导表面下随机形成微管单星体的胚胎中的膜扩展进行了研究。重水处理导致卵表面通过小泡的随机异位胞吐作用迅速均匀扩展。这种重水诱导的膜扩展被诺考达唑完全阻断，表明异位胞吐作用依赖于微管。结果表明，胞吐小泡存在于整个卵的亚皮层中，并且表面附近微管的存在足以促使它们在细胞周期结束时进行胞吐作用。
