Figard Lauren, Sokac Anna Marie
Program in Cell & Molecular Biology, Baylor College of Medicine (BCM), USA.
J Vis Exp. 2011 Mar 30(49):2503. doi: 10.3791/2503.
The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)(1-5). On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells. The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility(6), but is instead fueled largely by exocytosis of membrane from internal stores(7). Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle. Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)(8-19). In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques(9-10). Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.
发育中的黑腹果蝇胚胎会经历许多细胞形状变化，这些变化非常适合进行实时共聚焦成像。果蝇中的细胞形状变化与高等生物中的类似，它们驱动组织形态发生。因此，在许多情况下，对它们的研究对于理解人类疾病具有直接意义（表1）（1 - 5）。在亚细胞尺度上，这些细胞形状变化是从基因表达、信号转导、细胞极性、细胞骨架重塑到膜运输等一系列活动的产物。因此，果蝇胚胎不仅提供了评估与组织形态发生相关的细胞形状变化的背景，还提供了一个完全生理的环境来研究塑造细胞的亚细胞活动。这里描述的方案旨在对一种称为细胞化的特定细胞形状变化进行成像。细胞化是一个质膜剧烈生长的过程，它最终将合胞体胚胎转化为细胞胚盘。也就是说，在有丝分裂周期14的间期，质膜同时围绕约6000个皮质锚定的细胞核中的每一个内陷，以产生一层初级上皮细胞。与之前的观点相反，细胞化不是由肌球蛋白-2的收缩力驱动的（6），而是主要由来自内部储存的膜的胞吐作用推动的（7）。因此，细胞化是一个研究在需要质膜内陷或扩张的细胞形状变化过程中的膜运输的优秀系统，例如肌肉中的胞质分裂或横管（T管）形态发生。请注意，该方案很容易应用于果蝇胚胎中其他细胞形状变化的成像，只需要进行一些小的调整，例如改变胚胎收集的阶段，或者使用“胚胎胶水”将胚胎以特定方向固定（表1）（8 - 19）。在所有情况下，工作流程基本相同（图1）。使用标准的克隆和果蝇转基因方法来制备稳定的果蝇品系，这些品系表达与绿色荧光蛋白（GFP）或其变体融合的感兴趣的蛋白质，这些果蝇提供了可再生的胚胎来源。或者，通过直接的显微注射技术（9 - 10）将荧光蛋白/探针直接引入果蝇胚胎。然后，根据要成像的发育事件和细胞形状变化，在解剖显微镜下通过形态学收集和分期胚胎，最后在共聚焦显微镜上定位并固定以进行延时成像。
