Abstract
Partitioning duplicated chromosomes equally between daughter cells is a microtubule-mediated process essential to eukaryotic life. A multi-protein machine, the kinetochore, drives chromosome segregation by coupling the chromosomes to dynamic microtubule tips, even as the tips grow and shrink through the gain and loss of subunits. The kinetochore must harness, transmit, and sense mitotic forces, as a lack of tension signals incorrect chromosome-microtubule attachment and precipitates error correction mechanisms. But though the field has arrived at a 'parts list' of dozens of kinetochore proteins organized into subcomplexes, the path of force transmission through these components has remained unclear. Here we report reconstitution of functional Saccharomyces cerevisiae kinetochore assemblies from recombinantly expressed proteins. The reconstituted kinetochores are capable of self-assembling in vitro, coupling centromeric nucleosomes to dynamic microtubules, and withstanding mitotically relevant forces. They reveal two distinct pathways of force transmission and Ndc80c recruitment.
Highlights
Chromosome segregation is a complex imperative faced by all eukaryotes, as failure to accurately distribute genetic material to daughter cells in mitosis and meiosis causes potentially lethal aneuploidy (Cimini et al, 2001; Cimini, 2008; Santaguida and Amon, 2015)
Eukaryotic cells rely on a network of protein complexes (Figure 1A and Table 1) to tether centromeres to the dynamic spindle microtubules that pull them to opposite poles of the dividing cell (Musacchio and Desai, 2017)
We showed that the microtubule-binding Ndc80c and Dam1c complexes, when introduced free in solution, will associate with bead-bound MIND to form a load-bearing attachment to a dynamic microtubule tip (Kudalkar et al, 2015)
Summary
Chromosome segregation is a complex imperative faced by all eukaryotes, as failure to accurately distribute genetic material to daughter cells in mitosis and meiosis causes potentially lethal aneuploidy (Cimini et al, 2001; Cimini, 2008; Santaguida and Amon, 2015). Eukaryotic cells rely on a network of protein complexes (Figure 1A and Table 1) to tether centromeres to the dynamic spindle microtubules that pull them to opposite poles of the dividing cell (Musacchio and Desai, 2017) This network, the kinetochore, mediates the vital process of chromosome segregation by coupling microtubule dynamics to chromosome movement. We demonstrate that there are at least two possible paths of force transmission through the inner kinetochore: the Mif and OA protein complexes Each of these components can independently form load-bearing interactions with both MIND and the centromeric nucleosome
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