The Role And Mechanism Of WDR62 In Regulating Spindle Dynamics

The mitotic spindle is composed of bipolar arrays of microtubules(MTs)with the minus ends embedded at the spindle pole and the plus ends projecting toward the chromosome.MTs are highly dynamic structures,the dynamic changes in the spindle MTs,which are orchestrated by a variety of microtubule-associated proteins(MAPs),play an essential role in ensuring faithful chromosome segregation during mitosis.However,despite a wealth of knowledge on the regulation of the plus-end dynamics of spindle MTs,little is known about how the minus-end dynamics are regulated by the spindle pole-associated MAPs.WDR62 is a microcephaly(MCPH)-related,microtubule(MT)-associated protein(MAP)that localizes to the spindle pole and regulates spindle organization,but the MTbinding nature of WDR62,the more detailed functions of WDR62 in spindle dynamics and the underlying mechanisms remain elusive.Here,we show that WDR62 and its new binding partner,katanin,form a complex and regulate spindle dynamics at the spindle pole.Knocking out of WDR62 or katanin decreases the depolymerization rate of MT minus-end on spindle poles,increases the stability of spindle MTs,decreases the length and number of astral MTs,causes spindle orientation defects,and impairs the connection between spindle pole and centrosome causing an embedded centrosome.Pull-down assays show that WDR62 interacts with the WD domain of katanin p80 through a conserved middle region where an MCPHassociated mutation is positioned.Both cellular and in vitro assays indicate that WDR62 can recruit katanin to MTs and sever MTs.By combining cellular and in vitro assays,we demonstrate that WDR62 shows a preference for curved segments of dynamic GDP-MTs,as well as GMPCPP-and paclitaxel-stabilized MTs,suggesting that it recognizes extended MT lattice.In addition,the WDR62 N-terminal WD domain is necessary and sufficient for extended MT lattice binding.However,the MT-binding affinity of the WDR62 N-terminus is autoinhibited by its C-terminus through JNK phosphorylation-induced intramolecular interaction.Because of WDR62’s MT binding property,it seems that WDR62 alone is inefficient in recruiting katanin to sever dynamic GDP-MTs,while WDR62 complexed with TPX2/Aurora A can potently promote katanin-mediated severing of GDP-MTs in vitro.In cells,depletion of endogenous TPX2 or Aurora A using a auxin-inducible degron(AID)results in the disorganization of spindle and the impaired accumulation of WDR62 and katanin at spindle poles.Pull-down assays show that the N-terminus of WDR62 can also interact with Aurora A kinase domain directly,which MCPHassociated mutations in WDR62 N-terminus disrupt this interaction.Both cellular and in vitro assays demonstrate that these four proteins interact and assemble at the spindle pole in a hierarchy,with TPX2 at the apex and Aurora A,WDR62,and katanin at sequentially lower positions.We propose that WDR62 is an atypical MAP and functions as an adaptor protein between its recruiting factor TPX2/Aurora A and the effector katanin to orchestrate the regulation of spindle dynamics.