The fidelity of mitosis depends upon equal partitioning of the replicated genome between daughter cells. During mitosis, stable, bi-oriented attachment of sister chromatids to opposite spindle poles via kinetochore-microtubule attachments ensures that, upon chromatid separation in anaphase, one copy segregates to each daughter cell. Formation of kinetochore-microtubule attachments is an inherently stochastic process and thus varies in duration. Therefore to prevent chromosome segregation errors, improperly attached kinetochores catalyze formation of the mitotic checkpoint complex (MCC), which prevents anaphase onset by inhibiting the anaphase-promoting complex (APC). This surveillance mechanism is known as the spindle assembly checkpoint (SAC).
The duration of mitotic arrest under conditions that prevent satisfaction of the SAC is often used as an indication of SAC “strength”. Although poorly understood, variation in SAC strength is widely observed, suggesting that SAC activity may be context dependent and adaptable. As many chemo-therapeutics rely on the SAC to arrest mitosis in cancer cells, while weakened SAC function leads to aneuploidy and has been associated with tumour development in both model systems and human cancers, understanding the mechanism(s) underlying variability in SAC strength in vivo is both a fundamentally interesting biological question and relevant to cancer treatment.
We have shown that GSCs have a stronger SAC than somatic cells in the early embryo (somatic blastomeres), providing a tractable system in which the in vivo modulation of SAC activity can be explored. Two notable features differentiate GSCs from somatic blastomeres – their relatively small cell size and their germline identity. By using the C. elegans embryo we were able to ask whether either (or both) contributed to enhanced SAC strength in GSCs. GSCs are derived from a single founder cell, the P4 blastomere, which is specified during early embryogenesis by four successive asymmetric divisions. In addition, all cell divisions in the embryo occur without cell growth in interphase, such that cell size decreases as embryogenesis progresses.
To assess SAC strength in embryonic cells with different cell sizes and within different lineages, we developed an inducible monopolar spindle assay using a fast-acting temperature-sensitive allele of a gene required for centrosome duplication. Cells with monopolar spindles fail to satisfy the SAC due to the persistence of erroneous kinetochore attachments, and the duration of mitotic delay in monopolar cells can be used as a proxy for SAC strength. We found that while smaller cells tended to have a stronger SAC, germline blastomeres displayed a stronger SAC than comparably-sized somatic cells, suggesting that both cell size and cell identity contribute to SAC activity. We further showed that a stronger germline SAC depended on the asymmetric partitioning or control of checkpoint regulators downstream of the highly conserved PAR polarity proteins, which regulate the asymmetric divisions that specify the germline.
Ongoing research in the lab seeks to determine the mechanistic basis for elevated checkpoint activity in germline cells, using quantitative live imaging assays to compare the behavior of key checkpoint and kinetochore components in germline versus somatic cells. We are also taking advantage of the strength of C. elegans genetics to parse the novel interaction between PAR proteins and the SAC.