Biochemical Characterization And Functional Dissection Of Cytoskeleton Protein SEPT7 During Mitosis

Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubules and the kinetochore,a multiprotein complex assembled onto centromeric DNA of the chromosome.CENP-E is a microtubule-based kinesin motor protein located on the outer kinetochore and is responsible for a stable microtubule-kinetochore attachment.Earlier studies from our laboratory demonstrate that CENP-E forms a link between chromosome attachment and spindle checkpoint. To delineate the molecular mechanisms underlying CENP-E function and regulation during mitosis,we conducted a systemic identification of proteins interacting with CENP-E using yeast genetic screen,in which more than a dozen of candidates were identified.One of such candidates is SEPT7,a GTPase composing a novel filamentous structure essential for cytokinesis.The first part of my research work focused on the characterization of CENP-E-septin interaction and functional relevance in mitosis.Septin(SEPT)belongs to a family of conserved cytoskeletal proteins,which share common structural domains and form heteropolymeric filaments in several organisms.SEPT filaments localized to the metaphase spindle during mitosis. Previous studies indicated that SEPT2 depletion results in chromosome misalignment correlated with loss of CENP-E from the kinetochores of congressing chromosomes. However,it has remained elusive as to whether CENP-E physically interacts with SEPT and how this interaction orchestrates chromosome segregation in mitosis.Here we show SEPT7 is required for a stable kinetochore localization of CENP-E.SEPT7 stabilizes the kinetochore association of CENP-E by directly interacting with its C-terminal domain.The region of SEPT7 associating with CENP-E was mapped to its C-terminal domain by GST pull-down and yeast two-hybrid assays. Immunoflourescence study shows that SEPT7 filaments distribute along the mitotic spindle and terminate at the kinetochore marked by CENP-E.Remarkably, suppression of synthesis of SEPT7 by siRNA abrogated the localization of CENP-E to the kinetochore and caused aberrant chromosome congression.These mitotic defects and kinetochore localization of CENP-E can be successfully rescued by introducing exogenous GFP-SEPT7 into the SEPT7-depleted cells.These SEPT7-suppressed cells display reduced tension at kinetochores of bi-orientated chromosomes and activate mitotic spindle checkpoint marked by Mad2 and BubR1 labelings on these misaligned chromosomes.We propose that SEPT7 forms a link between kinetochore localization of CENP-E and the mitotic spindle checkpoint.These findings reveal a key role for the SEPT7-CENP-E interaction in distribution of CENP-E to the kinetochore and mainteinance the kinetochore-microtubule attachment.To delineate the structure-functional relationship of SEPT7 molecule related to SEPT2-SEPT6-SEPT7 complex assembly,I carried out biochemical characterization of human SEPT7.My study shows that human SEPT7 can assemble into filaments in vitro and in vivo.The N-terminal SEPT7,including GTPase domain and a short region at the amino terminus,is required for its filaments localization,self-association and interaction with SEPT2 and SEPT6.Although GTPase activity is indispensable for SEPT7 filaments assembly,it barely affects the formation of hetero-polymeric septin complex and filaments.Furthermore,SEPT7 can bundle SEPT2 and SEPT6 to form an integral filaments structure in vivo.Thus,these observations provide insight into a better understanding of structure-functional relationship of SEPT7.Currently,I am carrying our biophotonic study to illustrate the molecular dynamics of SEPT7 molecule and its relation to chromosome segregation in mitosis.Taken together,my finding of SEPT7-CENP-E interaction provides novel insight into a better understanding of CENP-E molecular dynamics in mitosis.In addition,the molecular delineation of SEPT7 structure-functional relationship established here provides framework by which we illustrate the function and regulation of septin filaments during mitosis.Given the fact that aberrant spindle plasticity and dynamics are involved in pathogenesis of chromosome instability phenotype,better understandings of the molecular mechanisms of CENP-E-SEPT7 interaction will help to correct aberrant chromosome segregation and perhaps provide potential therapeutic strategies.