Warecki Brandt, Bast Ian, Sullivan William
Department of Molecular, Cell, and Developmental Biology, University of California, Santa Cruz, Santa Cruz, CA, USA.
Methods Mol Biol. 2022;2415:37-46. doi: 10.1007/978-1-0716-1904-9_3.
The dramatic changes of subcellular structures during mitosis are best visualized by live imaging. In general, live imaging requires the expression of proteins of interest fused to fluorophores and a model system amenable to live microscopy. Drosophila melanogaster is an attractive model in which to perform live imaging because of the numerous transgenic stocks bearing fluorescently tagged transgenes as well as the ability to precisely manipulate gene expression. Traditionally, the early Drosophila embryo has been used for live fluorescent analysis of mitotic events such as spindle formation and chromosome segregation. More recent studies demonstrate that the Drosophila third instar neuroblasts have a number of properties that make them well suited for live analysis: (1) neuroblasts are distinct cells surrounded by plasma membranes; (2) neuroblasts undergo a complete cell cycle, consisting of G1, S, G2, and M phases; and (3) neuroblasts gene expression is not influenced by maternal load, and so the genetics are therefore relatively more simple. Finally, the Drosophila neuroblast is arguably the best system for live imaging asymmetric stem cell divisions. Here, we detail a method for live imaging Drosophila larval neuroblasts.
有丝分裂期间亚细胞结构的剧烈变化通过实时成像能得到最佳呈现。一般来说，实时成像需要将感兴趣的蛋白质与荧光团融合表达，以及一个适用于活体显微镜观察的模型系统。黑腹果蝇是进行实时成像的一个有吸引力的模型，因为有众多携带荧光标记转基因的转基因品系，以及精确操纵基因表达的能力。传统上，果蝇早期胚胎已被用于对有丝分裂事件如纺锤体形成和染色体分离进行实时荧光分析。最近的研究表明，果蝇三龄神经母细胞具有许多使其非常适合进行实时分析的特性：（1）神经母细胞是被质膜包围的独特细胞；（2）神经母细胞经历完整的细胞周期，包括G1、S、G2和M期；（3）神经母细胞的基因表达不受母体负荷影响，因此遗传学相对更简单。最后，果蝇神经母细胞可以说是实时成像不对称干细胞分裂的最佳系统。在这里，我们详细介绍一种对果蝇幼虫神经母细胞进行实时成像的方法。
