Chenevert Janet, Robert Morgane L V, Sallé Jérémy, Cacchia Sébastien, Lorca Thierry, Castro Anna, McDougall Alex, Minc Nicolas, Castagnetti Stefania, Dumont Julien, Lacroix Benjamin
Sorbonne Universités, CNRS, Laboratoire de Biologie du Développement de Villefranche-sur-mer (LBDV), Villefranche-sur-mer, France.
Université de Montpellier, Centre de Recherche en Biologie cellulaire de Montpellier (CRBM), CNRS UMR 5237, Montpellier Cedex 5, France.
Methods Mol Biol. 2024;2740:187-210. doi: 10.1007/978-1-0716-3557-5_12.
During eukaryotic cell division a microtubule-based structure, the mitotic spindle, aligns and segregates chromosomes between daughter cells. Understanding how this cellular structure is assembled and coordinated in space and in time requires measuring microtubule dynamics and visualizing spindle assembly with high temporal and spatial resolution. Visualization is often achieved by the introduction and the detection of molecular probes and fluorescence microscopy. Microtubules and mitotic spindles are highly conserved across eukaryotes; however, several technical limitations have restricted these investigations to only a few species. The ability to monitor microtubule and chromosome choreography in a wide range of species is fundamental to reveal conserved mechanisms or unravel unconventional strategies that certain forms of life have developed to ensure faithful partitioning of chromosomes during cell division. Here, we describe a technique based on injection of purified proteins that enables the visualization of microtubules and chromosomes with a high contrast in several divergent marine embryos. We also provide analysis methods and tools to extract microtubule dynamics and monitor spindle assembly. These techniques can be adapted to a wide variety of species in order to measure microtubule dynamics and spindle assembly kinetics when genetic tools are not available or in parallel to the development of such techniques in non-model organisms.
在真核细胞分裂过程中,一种基于微管的结构——有丝分裂纺锤体,会在子细胞之间排列并分离染色体。要了解这种细胞结构是如何在空间和时间上组装与协调的,就需要测量微管动力学,并以高时空分辨率观察纺锤体组装过程。可视化通常通过引入和检测分子探针以及荧光显微镜来实现。微管和有丝分裂纺锤体在真核生物中高度保守;然而,一些技术限制使得这些研究仅局限于少数物种。在广泛的物种中监测微管和染色体编排的能力,对于揭示保守机制或解开某些生命形式为确保细胞分裂期间染色体准确分配而发展出的非常规策略至关重要。在此,我们描述了一种基于注射纯化蛋白的技术,该技术能够在几种不同的海洋胚胎中以高对比度观察微管和染色体。我们还提供了分析方法和工具来提取微管动力学并监测纺锤体组装。当遗传工具不可用时,或者与非模式生物中此类技术的开发并行时,这些技术可以适用于多种物种,以测量微管动力学和纺锤体组装动力学。