Functional Dissection of Mitosis Using Immortalized Fibroblasts from the Indian Muntjac, a Placental Mammal with Only...

During cell division in eukaryotes a microtubule-based network undergoes drastic changes and remodeling to assemble a mitotic spindle competent to segregate chromosomes. Several model systems have been widely used to dissect the molecular and structural mechanisms behind mitotic spindle assembly and function. These include budding and fission yeasts, which are ideal for genetic and molecular approaches, but show limitations in high-resolution live-cell imaging, while being evolutionarily distant from humans. On the other hand, systems that were historically used for their exceptional properties for live-cell imaging of mitosis (e.g., newt lung cells and Haemanthus endosperm cells) lack the necessary genomic tools for molecular studies. In a CRISPR-Cas9 era, human cultured cells have conquered the privilege to be positioned among the most powerful genetically manipulatable systems, but their high chromosome number remains a significant bottleneck for the molecular dissection of mitosis in mammals. We believe that we can significantly broaden this scenario by establishing a unique placental mammal model system that combines the powerful genetic tools and low chromosome number of fission yeast and Drosophila melanogaster, with the exceptional cytological features of a rat kangaroo cell. This system is based on hTERT-immortalized fibroblasts from a female Indian muntjac, a placental mammal with the lowest known chromosome number (n = 3). Here we describe a series of methodologies established in our laboratory for the study of mitosis in Indian muntjac. These include standard techniques such as immunofluorescence, western blotting, and FISH, but also several state-of-the-art methodologies, including live-cell imaging, cell confinement, RNAi, super-resolution STED microscopy, and laser microsurgery.