The Roles Of NudCL2 In Cell Migration And Cell Division

NudC(Nuclear distribution genc C)was first found in Aspergillus nidulas and is involved in nuclear movement regulation.NudC exhibits its biologic function through regulating the protein stability of LIS 1(Lissencephaly 1),whose mutation causes type I lissencenphaly.In mammals,NudC family consist 3 structure related members:NudC,NudCL and NudCL2.All members of NudC family share a conserved p23 domain,which possesses chaperone activity both in conjunction with and sovereign to Hsp90.Our laboratory and others have demonstrated that NudC family members are involved in the regulation of cell cycle,cell migration and ciliogenesis through regulating the stability of different proteins.Our lab first identified and cloned NudCL2 gene,and found it can regulate the LIS 1/dynein pathway by stabilizing LIS1 with Hsp90.However,little is known about other functions of NudCL2.In this study,we systematically analysed the interactome of NudCL2 by using immunoprecipition and protein mass spectrum.We found NudCL2 interacts with many cytoskeleton proteins(eg:NM IIA)and nuclei binding proteins(eg:Sccl),suggesting NudCL2 may play important roles in cell migration and cell division.Part I:The Role of NudCL2 in Cell MigrationCell migration is a fundamental biologic process that cells move towads particular direction under the migration signal.Cell migration plays crucial roles in embryonic development,immune response,tissue regeneration and tumorigenesis.Cell migration is tightly and precisely controlled by many regulators,such as non-muscle myosin Ⅱ(NM II),which is an actin binding protein.Our research showed depletion of NudCL2 causes the similar phenotypes with depletion of NM IIA,a major member of NM Ⅱ,including decreased stress fibers,more and larger lamellipodia in leading edge,tail retraction failure,decreased focal adhesion numbers and faster cell migration.Further studies revealed that NudCL2 incorporates with Hsp90 to regulate the NM IIA stability and cellular actin skeleton.We also found ectopic expression of Hsp90 can rescue the phenotypes caused by NudCL2 depletion but not vice versa.More importantly,we confirmed NudCL2 indeed is a cochaperone of Hsp90 through ATPase activity assay,thermal inactivation assay and refolding assay in vitro.These results indicate NudCL2 can regulate actin dynamics and cell migration through stabilizing NM IIA with Hsp90.Part Ⅱ:The Role of NudCL2 in Cell DivisionCell divison is the process by which a parent cell divides into two daughter cells:During cell divison,equally segregation of genetic material is most important.To this aim,eukarycyte generates sister chromatid cohesion mechanism,which is mediated by cohesin complex.Cohesin consists of four subunits:Rad21(also called Sccl),Smcl,Smc3 and STAG(including SA1 and SA2).We found NudCL2 interacts with Sccl and the regulators of cohesin from our mass spectrum data.We also confirmed NudCL2 interacts with cohesin on chromosomes by biochemical assays.Further investigation revealed that NudCL2 localizes at centromere and shows the similar dynamic pattern with cohesin during mitosis.Functional study showed depletion of NudCL2 causes premature sister chromatid separation,consequently leads mitosis defects and unstable kinetochore structure.Moreover,we blocked NudCL2 by anti-NudCL2 antibody in mouse oocytes and found premature sister chromatid separation before metaphase of meiosis Ⅱ,which implys NudCL2 possesses physiological significance through cohesin regulation.In summary,we systematically analysed the biological function of NudCL2.Our data suggest NudCL2 acts as a cochaperone of Hsp90 to regulate the stability of NM IIA and cell migration.NudCL2 also interacts with cohesin to mediate sister chromatid cohesion and to regulate cell division.Potentially,this study would provide a new therapy target for cell migration related and cell division related diseases.