Regulation of flagellar biogenesis by a calcium dependent protein kinase in Chlamydomonas reinhardtii

PLoS One. 2013 Jul 25;8(7):e69902. doi: 10.1371/journal.pone.0069902. Print 2013.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algal Proteins / genetics
  • Algal Proteins / metabolism*
  • Calcium / metabolism
  • Cell Membrane / drug effects
  • Cell Membrane / enzymology
  • Cell Membrane / ultrastructure
  • Chlamydomonas reinhardtii / drug effects
  • Chlamydomonas reinhardtii / enzymology
  • Chlamydomonas reinhardtii / genetics*
  • Chlamydomonas reinhardtii / ultrastructure
  • Diphosphates / pharmacology
  • Egtazic Acid / pharmacology
  • Flagella / drug effects
  • Flagella / enzymology*
  • Flagella / ultrastructure
  • Gene Expression Regulation, Plant*
  • Genome*
  • Isoenzymes / antagonists & inhibitors
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Lithium Chloride / pharmacology
  • Plant Development / genetics
  • Protein Kinases / genetics
  • Protein Kinases / metabolism*
  • Protein Structure, Tertiary
  • RNA, Small Interfering / genetics
  • RNA, Small Interfering / metabolism
  • Signal Transduction
  • Sucrose / pharmacology

Substances

  • Algal Proteins
  • Diphosphates
  • Isoenzymes
  • RNA, Small Interfering
  • Egtazic Acid
  • Sucrose
  • Protein Kinases
  • calcium-dependent protein kinase
  • Lithium Chloride
  • sodium pyrophosphate
  • Calcium

Grant support

This work was supported by National Basic Research Program of China (973 program) (2012CB945003, 2013CB910700) and National Natural Science Foundation of China (Grants 30830057, 30988004) (to JP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.