Supplementary Materials Supplemental Textiles (PDF) JCB_201607096_sm

Supplementary Materials Supplemental Textiles (PDF) JCB_201607096_sm. al., 2017). The pressure required for this process is definitely generated by kinetochores, large protein machines that assemble within the centromeric DNA of each ML 161 sister chromatid and form attachment sites for spindle microtubules (Westhorpe and Right, 2013; Cheeseman, 2014). Two unique congression mechanisms have been identified, which collectively make sure efficient chromosome positioning. Kinetochores located in the periphery of the spindle after nuclear envelope breakdown (NEB) engage the side wall of spindle microtubules, forming lateral attachments (Kapoor et al., 2006; Barisic et al., 2014; Auckland and McAinsh, 2015). Such kinetochores are enriched in the Kinesin-7 CENP-E, which methods toward the microtubule plus-end, pulling chromosomes to the equator. However, loss of CENP-E activity still allows the vast majority of chromosomes to congress (McEwen et al., 2001; Barisic et al., 2014; Bancroft et al., 2015) and only one-quarter of PtK1 cells contain laterally attached kinetochores (Kapoor et al., 2006). This is in part explained from the observation that sister kinetochore pairs can biorient in the 1st moments after NEB (Magidson et al., 2011). Indeed, biorientation is an absolute requirement for the eventual accurate segregation of sister chromatids and is advertised by (a) the conversion of lateral to end-on attachments (Magidson et al., 2011, 2015; Shrestha and Draviam, 2013; Drpic et al., 2015) and (b) stabilization of the bioriented state via the dephosphorylation of outer-kinetochore Aurora B substrates (Lampson et al., 2004; Liu et al., 2009; Welburn P57 et al., 2010; Lampson and Cheeseman, 2011). Sister pairs that instantaneously biorient do not necessarily require congression, as they are preferentially located in the spindle equator (Magidson et al., 2011). However, those that biorient inside a pole-proximal position must generate a directional push to align. This push is definitely thought to be produced by microtubule plus-end depolymerization in the kinetochore, which allows the pulling of chromosomes to the equator via the maintenance of attachment to the shortening dietary fiber (Cassimeris and Salmon, 1991; Skibbens et al., 1993, 1995; Khodjakov and Rieder, 1996; McEwen et al., 1997; Kapoor et al., 2006). Originally termed Pac-man (Gorbsky et al., 1987), this force-generating mechanism can be described as depolymerization-coupled pulling (DCP; Auckland and McAinsh, 2015). DCP demands the leading (poleward-moving [P]) kinetochore can maintain its attachment to depolymerizing microtubules, whereas the trailing (away-from-the-poleCmoving [AP]) kinetochore is definitely attached to polymerizing microtubules. In vitro reconstitution experiments have suggested the heterotrimeric spindle and kinetochore connected ML 161 (Ska) complex (Ska1-Ska2-Ska3/RAMA1) could mediate P kinetochore coupling to depolymerizing microtubules, because it can autonomously track depolymerizing plus-ends, bind curved protofilaments, and transduce the push generated by depolymerization to a polystyrene bead (Welburn et al., 2009; Schmidt et al., 2012). Moreover, siRNA-mediated depletion of the Ska complex in human being cells has been shown to cause congression defects, consistent with its involvement in DCP (Hanisch et al., 2006; Daum et al., 2009; Gaitanos et al., 2009; Theis et al., 2009; Welburn et al., 2009; Jeyaprakash et al., 2012; Schmidt et al., 2012; Abad ML 161 et al., 2014). Here, we use live-cell imaging of solitary kinetochores during congression to reveal how the Ska complex is required for a specific substep of DCP. We further show how bioriented kinetochores are maturing through progressive recruitment of the Ska complex and that this may reflect a mechanical self-check that is coupled to signaling from ML 161 your spindle assembly checkpoint (SAC). These findings contribute to explaining how kinetochores ensure that anaphase initiates only when all sister-pairs have formed adult bioriented attachments and congressed to the spindle equator. Results The Ska complex is required for the maintenance of biorientation during congression To assay the behavior of congressing chromosomes, we imaged mid-to-late prometaphase HeLa ML 161 cells expressing eGFP-CENP-A at 7.5-s intervals for 5 min. Because kinetochores can congress by both lateral sliding and DCP (Fig. 1 a), it was important to determine the latter events in our video clips. First, we focused on kinetochores located within the spindle (between pole and equator), because lateral sliding is largely restricted to the peripheral chromosomes (McEwen et al., 2001; Barisic et al., 2014). These kinetochores appeared bioriented based on glutaraldehyde fixation and -tubulin staining (Fig. 1.