Abstract
Microtubules segregate chromosomes by attaching to macromolecular kinetochores. Only microtubule-end attached kinetochores can be pulled apart; how these end-on attachments are selectively recognised and stabilised is not known. Using the kinetochore and microtubule-associated protein, Astrin, as a molecular probe, we show that end-on attachments are rapidly stabilised by spatially-restricted delivery of PP1 near the C-terminus of Ndc80, a core kinetochore-microtubule linker. PP1 is delivered by the evolutionarily conserved tail of Astrin and this promotes Astrin's own enrichment creating a highly-responsive positive feedback, independent of biorientation. Abrogating Astrin:PP1-delivery disrupts attachment stability, which is not rescued by inhibiting Aurora-B, an attachment destabiliser, but is reversed by artificially tethering PP1 near the C-terminus of Ndc80. Constitutive Astrin:PP1-delivery disrupts chromosome congression and segregation, revealing a dynamic mechanism for stabilising attachments. Thus, Astrin-PP1 mediates a dynamic 'lock' that selectively and rapidly stabilises end-on attachments, independent of biorientation, and ensures proper chromosome segregation.
Highlights
Chromosome segregation can not be initiated until all chromosomes are properly attached to microtubules from opposing spindle poles - a state called biorientation
We showed that Astrin is selectively enriched at end-on attached kinetochores and is required to stabilise end-on attachments, independent of biorientation (Shrestha and Draviam, 2013; Shrestha et al, 2017)
No rescue of the localisation defect was observed when Astrin-D70 or À4A was fused to an N-terminal YFP tag in mCherry-GBP-PP1g co-expressing cells (Figure 6—figure supplement 1A,B). These studies show that the kinetochore enrichment of Astrin is dependent on spatially-defined delivery of PP1 phosphatase at Astrin’s C-terminus (Figure 6C), which we show is proximal to the C-terminus of Ndc80 (Figure 1)
Summary
Chromosome segregation can not be initiated until all chromosomes are properly attached to microtubules from opposing spindle poles - a state called biorientation. End-on attachments can impart pulling forces to segregate chromosomes apart and in the absence of end-on attachments, checkpoint proteins are retained at the kinetochore which prevents premature chromosome segregation (reviewed in Musacchio and Desai, 2017; Cheeseman, 2014). A highly responsive set of kinases and phosphatases together monitor and signal kinetochore-microtubule (KT-MT) attachment status (reviewed in Saurin and Kops, 2016; Vallardi et al, 2017). Whether a similar highly responsive kinase-phosphatase feedback loop exists to selectively and rapidly stabilise end-on attachments is not known; this is important to establish as rapid stabilisation of end-on attachments is essential for withstanding microtubule-end mediated pulling forces that may otherwise detach kinetochore-microtubule attachments as soon as they form
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