Final Report Summary - PRECISE (Spatiotemporal regulation of chromosome segregation fidelity)
At any given moment, 250 million cells are dividing in the human body through an essential process known as mitosis. Inaccuracy of mitosis leads directly to aneuploidy (gain or loss of chromosomes), a hallmark of several cancers and birth defects. The kinetochore (KT), a minute structure on each replicated sister-chromatid, promotes the rapid turnover of MTs to correct potential attachment errors during early mitotic stages. Upon anaphase onset, the KT then switches to bind MTs with higher affinity, so that the energy derived from their depolymerizing plus ends helps driving chromosome motion to the poles. While the molecular basis of the KT-MT interface is only now starting to be elucidated, how the multiple KT activities are regulated throughout mitosis remains unknown. Here we proposed to dissect from a molecular perspective how the interaction between spindle MTs and KTs controls chromosome segregation fidelity in space and time. Specifically, we aimed to dissect the molecular basis of mitotic spindle assembly pathways in animal somatic cells. Our findings established the constitutive nature of a centrosome-independent spindle assembly program and how this program is adjusted to the presence/absence of centrosomes in animal somatic cells (Moutinho-Pereira et al., PNAS, 2013). We also investigated the roles played by key microtubule modulators at the kinetochore interface in error-correction mechanisms and found that the MT- and KT-associated protein CLASP2 is progressively and distinctively phosphorylated by Cdk1 and Plk1 kinases, concomitant with the establishment of kinetochore-microtubule attachments (Maia et al. J. Cell Biol. 2012). Additionally, we demonstrated that mammalian CLASPs contribute to mitotic fidelity not only by regulating KT-MT attachments, but also by preventing irreversible spindle multipolarity through distinct molecular partnerships at kinetochores and centrosomes in response to the action of chromosome motors (Logarinho et al., Nat. Cell Biol. 2012). In this regard, we showed that Dynein and CENP-E at kinetochores drive congression of peripheral polar chromosomes by preventing arm-ejection forces by chromokinesins to work in the wrong direction (Barisic et al., Nat. Cell Biol, 2014). Moreover, we found that congression of pole-proximal chromosomes depended on specific post-translational detyrosination of spindle microtubules that point to the equator. We proposed that microtubule detyrosination, as part of the “tubulin code”, works as a navigation system for kinetochore-based chromosome motility during mitosis (Barisic et al., Science, 2015). In a parallel line, we have identified a conserved feedback control mechanism that delays chromosome decondensation and NER in response to incomplete chromosome separation during anaphase (Afonso, Matos et al., Science, 2014). Finally, we aimed to investigate the in vivo role of CLASPs in mammals. Our findings so far strongly support a critical role for this protein in fetal growth regulation, proper pulmonary maturation and nervous system function, being an essential protein for mammalian life after birth (Pereira et al., manuscript in preparation). Overall the results from this project help to elucidate fundamental mechanisms behind mitotic fidelity and will likely have implications for the etiology and treatment of human cancers.