Cells are highly sensitive to their environment, with cell cycle checkpoints at the G1/S and G2/M transitions to prevent cell division when circumstances are unfavourable. However, in the complex environment of a living organism, cells are likely to face a multitude of grey zones, with sub-optimal, but not absolutely inhibitory, conditions. Moreover, in tissues with a high rate of cellular turnover, blocking mitosis completely may be incompatible with sustaining tissue function. Thus mitosis in vivo likely occurs under a wide range of physiological conditions. Whether mitosis is adapted accordingly is unknown and constitutes a significant gap in our understanding of the cell biology of cell division.
We have developed the first method to live image mitosis in situ in C. elegans germline stem cells (GSCs), establishing GSCs as a model for the study of mitosis in vivo and affording the unique opportunity to assess how manipulation of organismal physiology impacts the cell biology of mitosis. By tracking centrosome pairs over time, we developed a robust and relatively high throughput method to monitor mitotic progression in GSCs. We use the distinctive changes in spindle length that accompany nuclear envelope breakdown and anaphase onset as landmarks to define a period of mitosis, encompassesing prometaphase through metaphase, which we call “congression”. The duration of congression, so defined, will be determined by how rapidly chromosomes become properly attached to the spindle, how well the spindle assembly checkpoint (SAC) responds to defects in kinetochore-microtubule attachment and how quickly anaphase is initiated upon SAC silencing.
We then asked how physiological and environmental changes known to effect cell cycle progression in GSCs impacted the duration of congression. Limited nutrient availability can block or reduce stem cell proliferation in a wide range of organisms, including C. elegans, where GSCs experience a cell cycle arrest or delay upon starvation or caloric restriction, respectively. We found that caloric restriction also delays anaphase onset in GSCs and may, in the absence of SAC surveillance, reduce mitotic fidelity, resulting in an increase in the incidence of chromosome segregation errors.
GSCs also exhibit distinct proliferative behaviors during germline development and in response to reproductive demand as the worm ages. GSCs undergo an expansion phase of growth during development to increase the size of the stem cell pool. In adulthood, they switch to a maintenance phase of growth, during which self-renewing divisions balance those producing differentiating progeny, and the size of the stem cell pool remains relatively constant. In older animals, at the end of self-fertile reproduction, GSC proliferation decreases as cells progressively enter quiescence. We have found that the duration of congression correlates with these changes in GSC proliferation, increasing slightly during maintenance and markedly at the transition to quiescence.
These results suggest that mitosis, like other parts of the cell cycle, is sensitive to physiological conditions and may be subject to regulation via signalling pathways which impact cell cycle progression, but are not known to regulate mitosis directly. Current projects in the lab are using a combination of genetics, quantitative live imaging and de novo tool development to investigate the molecular basis for altered GSC mitotic progression and/or mitotic fidelity under different physiological conditions and the signaling mechanism(s) by which these changes are regulated.
Building upon the wealth of mechanistic studies carried out in cell culture models, we aim to generate a comprehensive picture of mitosis, revealing where mitosis is vulnerable, where it is subject to active regulation and how it can be adapted to preserve fidelity under a range of physiological conditions. As most C. elegans genes perform evolutionarily conserved functions, these results may provide insight into the in vivo regulation of mitosis in other organisms, particularly those with analogous adult stem cell populations.