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Cellular processes rely on proteins with temperature-dependent stability and activity. While thermosensitivity in biological networks is well-explored, the effect of temperature on complex mechanochemical assemblies, like the spindle, is rarely studied. We examined fission yeast spindle dynamics and chromosome segregation from 15⁰C to 40⁰C. Our findings reveal that these parameters follow U-shaped temperature-dependent curves but reach their minima at different temperatures. Specifically, spindle dynamics peak around 35⁰C, whereas chromosome segregation defects are minimized at 25⁰C. This suggests a scenario in which mitotic errors are tolerated to expedite rapid cell cycle progression., Competing Interests: The authors declare that there are no conflicts of interest present., (Copyright: © 2024 by the authors.)

Mitosis is usually shorter than other phases of the cell cycle and maintains a consistent duration despite variations in cell size and spindle size. This suggests the existence of a compensatory mechanism that ensures a short duration, possibly as a protective measure against irreversible damage, such as DNA damage. To explore the link between prolonged mitosis and DNA damage, we develop a microscopy-based assay utilizing Rad52-GFP as a marker for mitotic DNA damage. Through this assay, we provide evidence that mutants with prolonged mitosis exhibit increased Rad52 puncta, indicating an elevation in endogenous DNA damage., Competing Interests: The authors declare that there are no conflicts of interest present., (Copyright: © 2023 by the authors.)

We previously showed that the silkworm holocentric spindles are square-shaped, compared to the canonical oval shape of human monocentric spindles (Vanpoperinghe et al. 2021). Further, while kinesin-5 depletion resulted in monopolar spindles in both cells, kinesin-14 depletion affected only the silkworm cells, resulting in mal-shaped spindles (Vanpoperinghe et al. 2021). We now extend our study to quantify the effect of kinesin-5 and kinesin-14 on spindle assembly dynamics and chromosome segregation in holocentric silkworm BmN4 cells. We find that mal-shaped spindle and prolonged mitosis duration are highly correlated with chromosome segregation error, leading to aneuploidy and cell death in BmN4 cells. Further, double RNAi-mediated depletion of kinesin-5 and kinesin-14 partially rescue the monopolar spindle and mal-shaped spindle phenotypes in kinesin-5 and kinesin 14-depleted cells, respectively., (Copyright: © 2022 by the authors.)

Human retinal pigment epithelium RPE-1 cells are immortalized diploid wild-type cells. RPE-1 is increasingly used for studies of spindle assembly dynamics and chromosome segregation. Here, we imaged living RPE-1 cells using the spinning disk confocal microscope and report their complete spindle assembly dynamic parameters. Live-cell experiments enabled ascribing precise timing of function of the kinesin-5 Eg5 and kinesin-14 HSET throughout different phases of mitosis. Eg5 functions at prophase and metaphase, to assemble and maintain spindle bipolarity, respectively. Eg5 inhibition results in spindle collapse during prophase and metaphase, resulting in monoastral/monopolar spindles. HSET functions throughout mitosis to maintain spindle length. HSET degradation results in shorter spindles through all phases of mitosis. Double-inhibition of Eg5 and HSET produces only monoastral/monopolar spindles, indicating that Eg5 and HSET may not be antagonistic in wild-type RPE-1 cells, contrary to previous studies using cancer cells. In the context of spindle assembly, our results highlight potential important differences between RPE-1 and other cancer-derived cell lines., (Copyright: © 2022 by the authors.)

Proper chromosome segregation during mitosis requires both the assembly of a microtubule (MT)-based spindle and the assembly of DNA-centromere-based kinetochore structure. Kinetochore-to-MT attachment enables chromosome separation. Monocentric cells, such as found in human, have one unique kinetochore per chromosome. Holocentric cells, such as found in the silkworm, in contrast, have multiple kinetochore structures per chromosome. Interestingly, some human cancer chromosomes contain more than one kinetochore, a condition called di- and tricentric. Thus, comparing how wild-type mono- and holocentric cells perform mitosis may provide novel insights into cancer di- and tricentric cell mitosis. We present here live-cell imaging of human RPE1 and silkworm BmN4 cells, revealing striking differences in spindle architecture and dynamics, and highlighting differential kinesin function between mono- and holocentric cells., (Copyright: © 2021 by the authors.)