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

Abstract

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 hTERTimmortalized fibroblasts from a female Indian muntjac, a placental mammal with the lowest known chromosome number (n ¼ 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.

Augmin-dependent microtubule self-organization drives kinetochore fiber maturation in mammals

“…For siRNA experiments, Indian muntjac fibroblasts were plated at 60%–70% confluence in 6-well plates or 22 × 22 mm no. 1.5 glass coverslips (Corning), previously coated with fibronectin (Sigma-Aldrich) as described in ( Almeida et al., 2020 ) for 24 h in normal medium. Cells were then starved with MEM supplemented with 5% FBS for 30 min siRNA transfection was performed using 5 μL of Lipofectamine RNAi Max (Invitrogen) and 50–100 nM of the respective siRNA, each diluted in 250 μL of serum free-medium (Opti-MEM, Gibco).…”

Kinetochore-mediated microtubule assembly and Augmin-dependent amplification drive k-fiber maturation in mammals

“…For siRNA experiments, Indian muntjac fibroblasts were plated at 60%-70% confluence in 6-well plates or 22x22 mm no. 1.5 glass coverslips (Corning), previously coated with fibronectin (Sigma-Aldrich) as described in (Almeida et al, 2020) for 24 hours in normal medium. Cells were then starved with MEM supplemented with 5% FBS for 30 minutes.…”

“…Indian muntjac fibroblasts were seeded on fibronectin coverslips 24h before the experiment, as shown in (Almeida et al, 2020). Cell were fixed with ice-cold methanol (Sigma) for 4 minutes at -20ºC; or 4% paraformaldehyde (Electron Microscopy Sciences) or, for STED microscopy, PFA 4% supplemented with 0.1%-0.2% glutaraldehyde (Electron Microscopy Sciences) for 10 minutes at room temperature (RT).…”

“…For siRNA experiments, Indian muntjac fibroblasts were plated at 60%-70% confluence in 6-well plates or 22x22 mm no. 1.5 glass coverslips (Corning), previously coated with fibronectin (Sigma-Aldrich) as described in [85] or using a wet-transfer system (if protein molecular weight >120kDa). Membranes were then blocked in 5% milk diluted in PBS 0.05% Tween and the primary antibodies were incubated overnight at 4ºC at the dilutions shown in Table S1.…”

“…Detection was performed with Clarity Western ECL Substrate (Bio-Rad). Quantification of blots was performed with a Bio-Rad ChemiDoc XRS system using the IMAGE LAB software and immunosignals were normalized to GAPDH, α -tubulin or vinculin expression depending on protein molecular weight.ImmunofluorescenceIndian muntjac fibroblasts were seeded on fibronectin coverslips 24h before the experiment, as shown in(Almeida et al, 2020). Cell were fixed with ice-cold methanol (Sigma) for 4 minutes at -20ºC; or 4% paraformaldehyde (Electron Microscopy Sciences) or, for STED microscopy, PFA 4% supplemented with 0.1%-0.2% glutaraldehyde (Electron Microscopy Sciences) for 10 minutes at room temperature (RT).…”