title: Flagellar regeneration requires cytoplasmic microtubule depolymerization and kinesin-13.
abstract: In ciliated cells, two types of microtubules can be categorized: cytoplasmic and axonemal. It has been shown that axonemal tubulins come from a 'cytoplasmic pool' during cilia regeneration. However, the identity and regulation of this 'pool' is not understood. Previously, we have shown that Chlamydomonas kinesin-13 (CrKin13) is phosphorylated during flagellar regeneration, and required for proper flagellar assembly. In the present study, we show that CrKin13 regulates depolymerization of cytoplasmic microtubules to control flagellar regeneration. After flagellar loss and before flagellar regeneration, cytoplasmic microtubules were quickly depolymerized, which was evidenced by the appearance of sparse and shorter microtubule arrays and increased free tubulins in the cell body. Knockdown of CrKin13 expression by RNA interference inhibited depolymerization of cytoplasmic microtubules and impaired flagellar regeneration. In vitro assay showed that CrKin13 possessed microtubule depolymerization activity. CrKin13 underwent phosphorylation during microtubule depolymerization, and phosphorylation induced targeting of CrKin13 to microtubules. The phosphorylation of CrKin13 occurred at residues S100, T469 and S522 as determined by mass spectrometry. Abrogation of CrKin13 phosphorylation at S100 but not at other residues by inducing point mutation prevented CrKin13 targeting to microtubules. We propose that CrKin13 depolymerizes cytoplasmic microtubules to provide tubulin precursors for flagellar regeneration.
authors: Protein Science Laboratory of the Ministry of Education, School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors: Liang
authors: L
authors: Wang
authors: Tian
authors: T
authors: Piao
authors: Muqing
authors: M
authors: Cao
authors: Tao
authors: T
authors: Qin
authors: Lei
authors: L
authors: Huang
authors: Haiteng
authors: H
authors: Deng
authors: Tonglin
authors: T
authors: Mao
authors: Junmin
authors: J
authors: Pan
mesh_terms: D002478:Cells, Cultured; D002696:Chlamydomonas / Q000502:physiology*; D002923:Cilia / Q000648:ultrastructure*; D005407:Flagella / Q000502:physiology* / Q000648:ultrastructure; D005818:Genetic Engineering; D016547:Kinesins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation / Q000235:genetics; D030821:Plants, Genetically Modified; D058105:Polymerization; D055503:Protein Multimerization / Q000235:genetics; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics; D012038:Regeneration; D014404:Tubulin / Q000378:metabolism
chemical_list: D034741:RNA, Small Interfering; D014404:Tubulin; D016547:Kinesins
keywords: Chlamydomonas; Cilia and flagella; Flagellar assembly; Kinesin-13; Protein phosphorylation