Function And Mechanism Of CrKin13Underlying Flagellar Assembly And Disassembly In Chlamydomonas

Cilia (or flagella) in eukaryotes are highly conserved organelles and "9+2" microtubule based structures, protruding from the cell body. Cilia play animportant role in mammalian development, cell motility, cell cycle regulationand signal transduction. In mammalian cells, defects in cilia structure orfunction lead to varieties of human diseases, so recently many researchers focuson this area. Assembly and disassembly of cilla play a key role in cilia structureand function, but little is known about the mechanism of cilla assembly anddisassembly. Microtubule based cilia come from the cytoplasmic “precursorpool”, but little is understood about the regulation of cytoplasmic pool.Here, we use unicellular algae chlamydomonas as model organism forresearch on the molecular mechanism of flagellar assembly and disassembly.Studies have shown that a protein is phosphorylated during flagellarregeneration, named CrKinesin13/CrKin13, which belongs to microtubuledepolymerizing kinesin family. It is crucial for flagellar regeneration, becausethat it depolymerizes cytoplasmic microtubules to provide free tubulins forflagellar regeneration.In this paper, we further explore the mechanism of CrKin13on flagellarregeneration and shortening. Based on the previous studies, we further verifythat cytoplasmic microtubules are depolymerized at the early stage of flagellarregeneration. Using mutants and drug treatments, it is also found thatcytoplasmic microtubule depolymerization occurs before the flagellar loss, andcytoplasmic microtubules are depolymerized as long as CrKin13isphosphorylated, which is not related with flagella loss per se. Moreover, in vitroassay shows that both of phosphorylated and non-phosphorylated forms ofCrKin13possess ATP-dependent microtubule depolymerizing activity, but theactivity of phosphorylated form of CrKin13is lower. The phosphorylation ofCrKin13occurs at residues S100as primary site and S522, T469as secondarysites by mass spectrometry. Mutation of CrKin13phosphorylation site at S100but not at other residues prevents CrKin13targeting to cytoplasmicmicrotubules, which explains the phosphorylation of CrKin13at the early stage of flagellar regeneration. While the enrichment of CrKin13in the regeneratingflagella might be involved in the flagellar microtubule dynamics.Additionally, the kinase responsible for CrKin13’s phosphorylation and itspartner are also examined. TOG1screened as candidate by yeast two hybridmethod need to be further analyzed. To reveal the mechanism of targeting toshortening flagella, our results find that N-domain is necessary for flagellartargeting and M-, C-domain may be co-regulate the CrKin13targeting signals.Thus, we propose that CrKin13is regulated by its phosphorylation todepolymerize cytoplasmic microtubules to provide tubulin precursors duringflagellar regeneration, so it plays a key role in flagellar regeneration.