Ribas Juan Carlos, Cortés Juan Carlos G
Instituto de Biología Funcional y Genómica, Consejo Superior de Investigaciones Científicas (CSIC) / Universidad de Salamanca, C/ Zacarias Gonzalez 1, 37007, Salamanca, Spain.
Methods Mol Biol. 2016;1369:73-85. doi: 10.1007/978-1-4939-3145-3_6.
Fungal cleavage furrow formation during cytokinesis relays in the coordinated contraction of an actomyosin-based ring and the centripetal synthesis of both new plasma membrane and a special wall structure named division septum. Through transmission electron microscopy, the septum exhibits a three-layered structure with a central primary septum, flanked at both sides by the secondary septum. In contrast to the chitinous primary septum present in most of fungi, the fission yeast Schizosaccharomyces pombe does not contain chitin, instead it divides through the formation of a linear β(1,3)glucan-rich primary septum, which has been shown to be specifically stained by the fluorochrome Calcofluor white. Recent findings in S. pombe have revealed the importance of septum synthesis for the steady contraction of the ring during cytokinesis. Therefore, to study the molecular mechanisms that connect the extracellular septum wall with the other components of the cytokinetic machinery located in the plasma membrane and cytoplasm, new experimental approaches are needed. Here we describe the methods developed to image the septum structure by fluorescence microscopy, with a special focus in the analysis of septum progression by the use of time-lapse microscopy.
真菌在胞质分裂过程中形成分裂沟，这依赖于基于肌动球蛋白的环的协同收缩以及新质膜和一种名为隔膜的特殊壁结构的向心合成。通过透射电子显微镜观察，隔膜呈现出三层结构，中间是初级隔膜，两侧是次级隔膜。与大多数真菌中存在的几丁质初级隔膜不同，裂殖酵母粟酒裂殖酵母不含几丁质，而是通过形成富含线性β(1,3) - 葡聚糖的初级隔膜进行分裂，已证明这种隔膜能被荧光染料荧光增白剂特异性染色。粟酒裂殖酵母的最新研究结果揭示了隔膜合成对于胞质分裂过程中环的稳定收缩的重要性。因此，为了研究将细胞外隔膜壁与位于质膜和细胞质中的胞质分裂机制的其他成分联系起来的分子机制，需要新的实验方法。在这里，我们描述了通过荧光显微镜对隔膜结构进行成像所开发的方法，特别关注使用延时显微镜对隔膜进展的分析。
