Kinesin-14 HSET may not oppose kinesin-5 Eg5 activity in RPE-1 cells
Résumé
Human hTERT-RPE-1 cells were immortalized, via telomerase expression, from normal somatic retinal pigment epithelium cells (Jiang et al., 1999). These cells are considered normal diploid cells, as they have the proper number of chromosomes and do not exhibit transformed phenotypes. As this cell line has gained prominence as a model system for research on spindle assembly and chromosome segregation, we sought to define its complete spindle assembly parameters throughout mitosis, as well as test the function of kinesin-5 and kinesin-14, two important motors involved in spindle assembly previously reported in cancer cells. Using CRISPR/Cas9 gene-editing, we created an RPE-1 cell line expressing at its genomic loci both alleles of auxin-inducible degron-degradable kinesin-14 eYFP-AID-HSET. We then performed confocal live-cell imaging to visualize HSET, as well as using the vital dye SiR-Tubulin and Hoechst to visualize the microtubules and DNA, respectively. Live-cell imaging enabled tracking mitosis from start to finish, with clear visualization of prophase, metaphase, anaphase and telophase spindles (Fig. 1A). The movies enabled the measurement of spindle length over time, where we observed three distinct spindle elongation phases, correlating with phase I/prophase, phase II/metaphase, and phase III/anaphase (Fig. 1E). The complete spindle elongation parameters for RPE-1 cells are reported (Fig. 1G).
Domaines
Sciences du Vivant [q-bio]
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