Regulation of Flagellar Biogenesis by a Calcium Dependent Protein Kinase in Chlamydomonas reinhardtii

Abstract

Chlamydomonas reinhardtii, a bi-flagellated green alga, is a model organism for studies of flagella or cilia related activities including cilia-based signaling, flagellar motility and flagellar biogenesis. Calcium has been shown to be a key regulator of these cellular processes whereas the signaling pathways linking calcium to these cellular functions are less understood. Calcium-dependent protein kinases (CDPKs), which are present in plants but not in animals, are also present in ciliated microorganisms which led us to examine their possible functions and mechanisms in flagellar related activities. By in silico analysis of Chlamydomonas genome we have identified 14 CDPKs and studied one of the flagellar localized CDPKs – CrCDPK3. CrCDPK3 was a protein of 485 amino acids and predicted to have a protein kinase domain at the N-terminus and four EF-hand motifs at the C-terminus. In flagella, CrCDPK3 was exclusively localized in the membrane matrix fraction and formed an unknown 20 S protein complex. Knockdown of CrCDPK3 expression by using artificial microRNA did not affect flagellar motility as well as flagellar adhesion and mating. Though flagellar shortening induced by treatment with sucrose or sodium pyrophosphate was not affected in RNAi strains, CrCDPK3 increased in the flagella, and pre-formed protein complex was disrupted. During flagellar regeneration, CrCDPK3 also increased in the flagella. When extracellular calcium was lowered to certain range by the addition of EGTA after deflagellation, flagellar regeneration was severely affected in RNAi cells compared with wild type cells. In addition, during flagellar elongation induced by LiCl, RNAi cells exhibited early onset of bulbed flagella. This work expands new functions of CDPKs in flagellar activities by showing involvement of CrCDPK3 in flagellar biogenesis in Chlamydomonas .

Two Calcium-Dependent Protein Kinases fromChlamydomonas reinhardtiiare transcriptionally regulated by nutrient starvation

“…A recent study of CDPK3 being distributed in both the cell body and flagellar membrane-matrix fraction of C. reinhardtii cells has been shown. 19 Moreover, whether the protein re-localizes upon an exposure to stress needs to be addressed. As far as the sub-cellular localization of CDPKs is concerned, they have been shown to occupy several parts of the plant cell, such as cytoplasm, peroxisomes, nucleus, endoplasmic reticulum, mitochondrial outer membrane, and plasma membrane.…”

“…[63][64][65][66][67] CDPK3 is localized in the C. reinhardtii membrane-matrix fraction of the flagella. 19 Another 65 kDa CDPK has been shown to be localized in the C. moewusii flagella. 41 In conclusion, the 2 CDPKs from C. reinhardtii cells are regulated by nutrient starvation (P and N); however, it was only CDPK3 that was upregulated upon acetate starvation.…”

The conserved ciliary protein Bug22 controls planar beating ofChlamydomonasflagella

“…Then tagged gene was cloned into a modified vector pHyg3 harboring a Hygromycine B resistance gene (Berthold et al, 2002). The final construct was linearized with ScaI and transformed into the bug22 mutant by using electroporation (Liang and Pan, 2013).…”

“…The pelleted axonemal fraction was either collected directly or further extracted with 0.6 M NaCl followed by 0.2 M KI, following a published protocol (Mitchell and Sale, 1999). Flagellar length measurement and capture of DIC images were essentially as described previously (Liang and Pan, 2013).…”

“…Analysis for SDS-PAGE and immunoblotting was essentially as described previously (Liang and Pan, 2013). Cells were collected by centrifugation and lysed with HMDEK buffer containing an EDTA-free protease inhibitor cocktail (Roche) prior to boiling in SDS-PAGE sample buffer.…”