The Role Of PRC1 In Cytokinesis

Regulation of the cell cycle involves steps crucial to the cell.Two key classes of regulatory molecules,cyclins and cyclin-dependent kinases (CDKs),determine a cell's progress through the cell cycle.Cyclins form the regulatory subunits and CDKs the catalytic subunits of an activated heterodimer;cyclins have no catalytic activity and CDKs are inactive in the absence of a partner cyclin.When activated by a bound cyclin,CDKs perform a common biochemical reaction called phosphorylation that activates or inactivates target proteins to orchestrate coordinated entry into the next phase of the cell cycle.During cell division,it is Cyclin B1-CDK1 complex that its activation causes initiation of mitosis,and subsequently,its deactivation causes the cell to exit mitosis.The central machine in cell division is the bipolar mitotic spindle, an apparatus that partitions the duplicated genome of a mother cell equally into two daughter cells.The spindle is composed largely of microtubules—relatively rigid but highly dynamic tubes formed by the polymerization of tubulin proteins.During anaphase a remarkable transition in spindle structure occurs.A conspicuous network of antiparallel nonkinetochore interdigitating microtubules(MTs) assembles between separating chromosomes.This unique structure is referred to as the spindle midzone.The midzone is believed to be required for the maintenance of overall spindle architecture,spindle elongation, and cleavage furrow positioning.PRC1(Protein Regulating Cytokinesis 1) originally was identified as a Cdk substrate in an invitrophosphorylation screen and was subsequently shown to be a midzone-associated protein required for cytokinesis.PRC1 forms oligomers in vivo and has MT-binding and -bundling activities.Cdk phosphorylation of PRC1 appears to be important for suppressing PRC1 MT-bundling activity in early mitosis.In addition,PRCI specifically interacts with the kinesin motor protein,Kif4.During the metaphase-to-anaphase transition,Kif4 translocates PRC1 along MTs to the plus ends of interdigitating mitotic spindles,and the timing of this translocation is controlled by Cdk phosphorylation of PRCI.PRC1 is thus a good candidate for bundling interdigitating MTs to form the midzone.In this study,the molecular structure of PRC1 was analyzed and its oligermerization domain was mapped by in vitro and in viva experiments and then this domain' s role on the formation of mitotic spindle midzone was investigated.Furthermore,the regulation role of phosphorylation of PRC1 by CDK1/Cyclin B1 kinase was also studied.The aim of this study is to elucidate the molecular mechanism of formation of mitotic spindle midzone by microtubules binding and bundling protein,PRC1.Objectives:To analyze the molecular structure and biological characterize of microtubule binding and bundling protein,PRC1 and then study its determinant role on formation of mitotic spindle midzone.Methods:1.Study PRC1 oligomerization in vitro using induction of bacteria;2.Study PRC1 oligomerization in vivo using cultured HeLa cells;3.Study oligomerization of endogenous PRC1 in HeLa cells using sucrose gradient sedimentation;4.Study subcellular localization of phosphorylated PRC1 using immunofluorescence staining in fixed HeLa cells;5.Study role of PRC1 in mitosis using small interference RNA technique;6.Study rescue of PRC1 RNAi using expression of exogenous proteins;7.Study phenotype of expression of Cyclin B1Δ90 in HeLa cells using living cell imaging;8.Study formation of mitotic spindle midzone using 3D immunoflurorescence reconstruction imaging. Results:1.PRC1 can self-oligomerize to tetramer both in vitro and in vivo;2.The oligomerizaiton domain of PRC1 localizes on its NH2-terminus 184 amino acids;3.Endogenous PRC1 can oligomerize except phosphorylated form;4.PRC1 is required for the formation of mitotic spindle midzone;5.Kif4 is not required for MT bundling activity of PRC1.Conclusions:PRC1 can self-oligomerize into tetramer in ceils however phoshorylation negatively inhibits this activity.During metaphase-to-anaphase transition,dephosphorylation of PRC1 promotes its oligomerization activity so as to help the formation of mitotic spindle midzone.