Abstract
Accurate chromosome segregation during cell division is crucial for propagating life and protects from cellular transformation. The SKAP:Astrin heterodimer localizes to spindle microtubules and to mature microtubule–kinetochore attachments during mitosis. Depletion of either subunit disrupts spindle structure and destabilizes kinetochore–microtubule attachments. Here, we identify molecular requirements for the inter-subunit interaction of SKAP and Astrin, and discuss requirements for their kinetochore recruitment. We also identify and characterize a microtubule-binding domain in SKAP, distinct from the SXIP motif that mediates end binding (EB) protein binding and plus end tracking, and show that it stimulates the growth-rate of microtubules, possibly through a direct interaction with tubulin. Mutations targeting this microtubule-binding domain impair microtubule plus-end tracking but not kinetochore targeting, and recapitulate many effects observed during depletion of SKAP. Collectively, our studies represent the first thorough mechanistic analysis of SKAP and Astrin, and significantly advance our functional understanding of these important mitotic proteins.
Highlights
Accurate chromosome segregation during cell division is crucial for propagating life and protects from cellular transformation
To identify interaction domains within the human SKAP: Astrin complex, we purified recombinant fragments of SKAP and Astrin that were systematically expressed in isolation of by co-expression in bacteria or insect cells
A C-terminal segment of SKAP (SKAP159–316), corresponding to the first and second predicted coiled-coil regions, was found to interact with the first coiled-coil region of Astrin (Astrin[482–850]). This result is consistent with a previous analysis in which segments with similar boundaries were expressed in mammalian cells and subjected to immunoprecipitation analysis[12]
Summary
Accurate chromosome segregation during cell division is crucial for propagating life and protects from cellular transformation. We identify and characterize a microtubule-binding domain in SKAP, distinct from the SXIP motif that mediates end binding (EB) protein binding and plus end tracking, and show that it stimulates the growth-rate of microtubules, possibly through a direct interaction with tubulin. SKAP and Astrin display identical localization patterns They associate with the spindle and the spindle poles in prophase and prometaphase, and become recruited to the outer kinetochores but only at the late stages of metaphase and telophase[6,7,11,12,13,14], when end-on attachments prevail. Subunits of the Knl1-complex, Mis[12] complex, Ndc80-complex (KMN) protein network in the outer kinetochore[22] were shown to be required for kinetochore localization of SKAP:Astrin in human cells, possibly through a direct interaction[11,18]. The precise mechanism of SKAP:Astrin localization to spindle poles, on the other hand, is unknown, but the complex has been implicated in the recruitment of Aurora A kinase to spindle poles[23]
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