Abstract
Microtubule (MT) structures appear and disappear dependent on the phases of cell cycle. These structures are disassembled to tubulin dimers after fulfilling their function, and then tubulin dimers incorporate to other MT structure that required at the next phase.
In order to study the mechanism of cell-cycle dependent assembly and disassembly of MT structures, we searched the substrates of cyclin-dependent kinases (CDKs). We developed the method of Percoll density gradient centrifugation applicable to Arabidopsis MM2d cells and prepared MM2d miniprotoplasts (evacuolated protoplasts). We performed in vitro kinase assay using CDKA, CDKB1, CDKB2 and microtubule-associated proteins (MAPs) purified from crude extract of MM2s miniprotoplasts. As a result, we identified the multiple substrates that were phosphorylated by CDKA, CDKB1 and CDKB2. We discuss the roles of CDKs phosphorylation for the regulation of MT dynamics and the mechanism of assembly and disassembly of MT structures.
