Keroles Mark B, Dusseault Sonya K, Liu Chuchu, Mohammed Masood R, Vadakkan Christy M, Amiel Jessica H, Abel Samantha N, Bensoussan Elena R, Russell Benjamin L, Baker James
Department of Biology, University of Miami.
J Vis Exp. 2014 Jan 23(83):e51239. doi: 10.3791/51239.
The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 µm of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.
长期以来，果蝇作为细胞与发育生物学的模型已产生了一系列工具。这些技术共同使得从各种方法学角度对细胞与发育生物学进行分析成为可能。实时成像作为一种新兴方法，可用于观察动态细胞过程，如细胞分裂或细胞运动。在分离出未鉴定的假定细胞周期蛋白中的突变后，利用实时成像原位观察有丝分裂变得至关重要。果蝇中的大多数实时成像研究都集中在胚胎阶段，由于其体积小且光学透明度高，便于操作和观察。然而，在这些阶段，细胞周期并不寻常，因为它缺少一个或两个间隙期。相比之下，果蝇蛹翅的细胞具有典型的细胞周期，并在约20小时的蛹发育过程中经历一段快速有丝分裂期。很容易识别和分离出合适阶段的蛹以原位捕捉有丝分裂。完整蛹的固定在成像过程中提供了可操作性和耐用性的最佳组合，使实验能够运行数小时，对细胞和动物活力的影响最小。该方法能够观察到小至或小于果蝇染色体的特征。通过调整显微镜设置和固定细节，可扩展样本制备以观察相邻细胞的膜动态以及荧光标记蛋白，如微管蛋白。该方法适用于所有测试的荧光蛋白，并且能够在各种时间尺度上捕捉亚微米级特征。虽然使用传统共聚焦显微镜时仅限于蛹外层20微米，但这种在体内观察蛹组织中蛋白质和细胞动态的方法可能在这些组织的细胞与发育生物学研究中普遍有用。
