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 .
Highlights
Chlamydomonas reinhardtii, a green alga, has been used as a model system for studies of various cellular processes [1]
calcium-dependent kinases (CDPKs) are unique among calcium sensors because they combine calcium sensing and decoding within one single molecule with a kinase domain at the N-terminus and several calcium-binding EF-hand domains at the C-terminus
We examined whether CrCDPK3 was involved in calcium-dependent mating that requires flagellar adhesion
Summary
Chlamydomonas reinhardtii, a green alga, has been used as a model system for studies of various cellular processes [1]. A universal second messenger, has been reported to be intimately involved in a variety of flagellar related activities including phototaxis as well as flagellar beating [8], [9], flagellar gliding [10], deflagellation [11] and mating which depends on flagellar adhesion [12], [13], flagellar outgrowth and shortening [14], [15] [16], One mechanism by which calcium exerts its divergent regulation of flagellar activities is through phosphorylation of flagellar proteins. Flagellar or ciliary localization of CDPKs has been reported in Paramecium [27], [28], and green algae C. eugametos [29] and C. reinhardtii [30] while their physiological functions remain unknown
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