A microtubule depolymerizing kinesin functions during both flagellar disassembly and flagellar assembly in Chlamydomonas

Self Cite

102

SummaryIn 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.

Section: Introductionmentioning

confidence: 84%

Flagellar regeneration requires cytoplasmic microtubule depolymerization and kinesin-13

Wang

Piao

Cao

et al. 2013

Self Cite

102

“…EB3 interacts with the MT depolymerizing kinesin-13 MCAK (Lee et al, 2008). Kinesin-13 family proteins have been implicated in cilium disassembly in protists (Blaineau et al, 2007;Dawson et al, 2007;Piao et al, 2009), and EB3 might, therefore, stabilize the axoneme by counteracting such proteins.…”

Section: Discussionmentioning

confidence: 99%

EB1 and EB3 promote cilia biogenesis by several centrosome-related mechanisms

Schrøder

Larsen

Komarova

et al. 2011

101

SummaryThe microtubule (MT) plus-end-tracking protein EB1 is required for assembly of primary cilia in mouse fibroblasts, but the mechanisms involved and the roles of the related proteins EB2 and EB3 in ciliogenesis are unknown. Using protein depletion experiments and expression of dominant-negative constructs we show here that EB1 and EB3, but not EB2, are required for assembly of primary cilia in cultured cells. Electron microscopy and live imaging showed that cells lacking EB1 or EB3 are defective in MT minus-end anchoring at the centrosome and/or basal body, and possess abnormally short cilia stumps surrounded by vesicles. Further, GST pulldown assays, mass spectrometry and immunoprecipitation indicated that EB1 and EB3 interact with proteins implicated in MT minusend anchoring or vesicular trafficking to the cilia base, suggesting that EB1 and EB3 promote ciliogenesis by facilitating such trafficking. In addition, we show that EB3 is localized to the tip of motile cilia in bronchial epithelial cells and affects the formation of centriole-associated rootlet filaments. Collectively, our findings indicate that EBs affect biogenesis of cilia by several centrosomerelated mechanisms and support the idea that different EB1-EB3 dimer species have distinct functions within cells.

“…Mice with mutations of the Kif19a gene showed elongated cilia in brain, oviduct and tracheal epithelial cells, suggesting an important role for this protein in ciliary length regulation (Niwa et al, 2012). Although the Chlamydomonas homolog of kinesin-13 family proteins, CrKinesin-13 was not seen at the flagellar tip, RNAimediated depletion of this protein resulted in cells with shorter flagella that failed to regenerate after deflagellation (Piao et al, 2009), suggesting a role for CrKinesin-13 in flagellar assembly. Similarly, depletion of Kif24, a kinesin-13 family protein that specifically depolymerizes centriolar MTs, resulted in aberrant cilia (Kobayashi et al, 2011).…”

Section: Introductionmentioning

confidence: 98%

Centrosomal protein CEP104/ChlamydomonasFAP256 moves to the ciliary tip during cilia assembly

Tammana

Diener

et al. 2013

SummaryThe ciliary tip has been implicated in ciliary assembly and disassembly, and signaling, yet information on its protein composition is limited. Using comparative, quantitative proteomics based on the fact that tip proteins will be approximately twice as concentrated in half-length compared with full-length flagella, we have identified FAP256 as a tip protein in Chlamydomonas. FAP256 localizes to the tips of both central pair and outer doublet microtubules (MTs) and it remains at the tip during flagellar assembly and disassembly. Similarly, its vertebrate counterpart, CEP104, localizes on the distal ends of both centrioles of nondividing cells until the mother centriole forms a cilium and then localizes at the tip of the elongating cilium. A null mutant of FAP256 in Chlamydomonas and RNAi in vertebrate cells showed that FAP256/CEP104 is required for ciliogenesis in a high percentage of cells. In those cells that could form cilia, there were structural deformities at the ciliary tips.