Regulatory Mechanisms Underlying Spindle Plasticity In Mitosis

The most important event during mitosis is the movements and separation of chromosomes, it is orchestrated by the interaction between spindle microtubules and kinetochore. CENP-E (Centromere-associated protein E) is a kinesin-related motor protein, which is required for stable microtubule-kinetochore attachment and maintenance of the active spindle checkpoint signaling. We are interested in finding the proteins interacting with CENP-E and building a complete CENP-E-interacting network. Using the combination of yeast-two-hybrid screening and tandem affinity purification technology, we identified SKAP and SEPT7as new CENP-E binding proteins. My previous work focused on the interactions between CENP-E and these two proteins, here I mainly dissect their biochemical characterization and molecular functions.The first part of my work focused on SKAP (Small kinetochore associated protein), it is a spindle and microtubule associated protein. Recently we proved that SKAP could form a complex with CENP-E. It collaborates with CENP-E to contribute to kinetochore-microtubule attachment and orchestrates chromosome dynamics in mitosis. But it has still remained elusive which protein(s) specifies its kinetochore localization and regulates its functions underlying spindle plasticity. Through MBP pull-down and yeast-two-hybrid assay, we indicated that SKAP directly interacts with Misl3via its C terminal and this interaction stabilizes kinetochore localization of SKAP. Knocking-down of Mis13by small interfering RNA abrogates the localization of SKAP to the kinetochore. And we also found that SKAP is essential for kinetochore oscillation and dynamics of microtubule plus ends. Moreover, SKAP promotes the kinetochore localization of Clasp1by physically interacting with it. Overall, our studies indicated that SKAP represents an important function in regulating spindle plasticity.The second part of my work was concerned with SEPT7. Septin is the fourth class of cytoskeleton. Our previous work demonstrated that SEPT7is a novel interacting parterner of CENP-E. SEPT7connects septin filaments and kinetochores, regulates the interaction between microtubules and kinetochores. Using mass spectrometric identification coupled with biochemical assay, we identified acetylation of SEPT7. It is a previously uncharacterized post-translational modification of human septins. Human SEPT7is a substrate of acetyl transferase Tip60and the acetylation sites were mapped to K175and K201. As determined by GST pull-down assy and FRET (fluorescence resonance energy transfer) assay, we revealed that acetylation regulates the binding activity of SEPT7and affects the formation of septin filaments. Expressing non-acetylatable SEPT7in HeLa and MDCK cells alter the distribution of septin filaments and cause aberrant chromosome alignment and separation. Taken together, my work demonstrated novel functions of SEPT7acetylation and may be helpful to decipher the molecular mechanisms underlying septin assembly and spindle plasticity in mitosis.