CrCDPK3 is present in the flagella of <i>C. reinhardtii</i>.

<p>(A) Schematic diagram of CrCDPK3 gene showing exons (grey) and introns (white). (B) Immunoblot analysis of <i>Chlamydomonas</i> cell lysates, bacterial expressed GST-CrCDPK3 and GST shows that anti-CrCDPK3 antibody is specific. Molecular weights are given in kilo-daltons. (C) CrCDPK3 is present in the cell body and flagella evidenced by immuoblotting with anti-CrCDPK3 antibody. 1 x indicates that approximately two flagella were loaded per cell body. 50 x indicates equal flagellar and cell body protein. (D) Isolated flagella (F), membrane/matrix (M and M) and axonemal (Ax) fractions were analyzed by immunoblotting with antibodies as indicated. (E) Immunostaining of cells expressing CrCDPK3-HA (a) or not (b) with antibodies against 3xHA tag and α-tubulin. Bars, 5 µm.</p

<i>C. reinhardtii</i> CDPKs.

<p>(A) Relatedness of CDPKs in <i>Chlamydomonas</i>. Protein sequences of the identified CDPKs annotated in <i>C. reinhardtii</i> genome v4 were aligned by using clastalx-2.1 and analyzed by the Phylip program (<a href="http://evolution.genetics.washington.edu/phylip.html" target="_blank">http://evolution.genetics.washington.edu/phylip.html</a>). The branch lengths are proportional to divergence with the scale of “0.1” representing 10% change. (B) Schematic diagram of protein domains of three CrCDPKs identified in the flagellar proteome.</p

Characterization of CrCDPK3 during flagellar shortening.

<p>(A) Cells were treated with 20 mM NaPPi to induce flagellar shortening followed by cell fixation and flagellar length measurement. No apparent difference in shortening was observed between two RNAi and wild type strains. (B) Flagellar increase of CrCDPK3 upon induction of flagellar shortening. Cells were treated with 20 mM NaPPi or 0.2 M sucrose followed by flagellar isolation and immunoblotting with antibodies indicated. (C) Formation of flagellar CrCDPK3 complex and its disruption upon induction of flagellar shortening. Flagella were isolated from steady state cells and cells treated with 20 mM NaPPi for 10 min followed by extraction of membrane/matrix fractions, which were analyzed by a 10-25% sucrose gradient and immunoblotting. Note that CrCDPK3 formed a complex around 20 S in steady state flagella and was disrupted upon inducing flagellar shortening.</p

Requirement of calcium and CrCDPK3 for flagellar regeneration.

<p>(A) After deflagellation by mechanical shearing, 2.3 mM EGTA (final concentration) was added to the cell samples or not. At 120 min after deflagellation, CaCl₂ was added to EGTA treated samples to reach 0.36 mM calcium present in normal medium. Samples were fixed at different times for flagellar length measurement. (B) Titration of [Ca²⁺]_e to determine calcium-dependent flagellar regeneration. After deflagellation, flagellar regeneration was allowed to proceed at different [Ca²⁺]_es. (C) Flagellar regeneration of CrCDPK3 RNAi strains at lower [Ca²⁺]_es. (D) Flagellar regeneration after transferring cells grown on agar plates into liquid medium. (E) Cell samples before (time 0) and at different times during flagellar regeneration after deflagellation were subjected to immunoblot analysis with anti-CrCDPK3 and anti-JPK1 antibodies. (F) Immunoblot analysis of CrCDPK3 in flagella from steady state cells and cells undergoing flagellar regeneration for 20 min. Equal flagelar proteins were loaded. IFT139 was used as positive control, which was shown to increase in regenerating flagella, and FMG1 used as loading control.</p

Phenotypic analysis of flagellar length, phototaxis and mating in CrCDPK3 RNAi strains.

<p>(A) Examination of CrCDPK3 protein level in RNAi strains. CrCDPK3 expression was analyzed by immunoblotting with anti-CrCDPK3 and anti-JPK1 antibodies. JPK1 was used as loading control. (B) Flagellar length measurements of wild type and two RNAi strains. Data are expressed as means ± SD in this and following figures. (C) Assay of phototaxis in 24-well microtiter plates. Note, cells accumulate on one side of the well after illumination. (D) Wild type 6145C (mt^-) strains were transformed with miRNA constructs to generate RNAi strains in mt^- background. Protein expression of CrCDPK3 was examined by immunoblotting with JPK1 as loading control. (E) Rate of zygote formation. Zygote formation was scored 30 min after mixing mt⁺ and mt^- gametes generated from either pairs of wild types or RNAi strains.</p

CrCDPK3 is defective in flagellar elongation induced by LiCl.

<p>(A) Flagellar length increase after treatment with 25 mM LiCl for wild type and RNAi strains. (B) Differential interference contrast images of cells before treatment (a) and 180 min after treatment (b–f). b, elongated normal flagella; c, flagellum with one small bulb proximal to flagellar tip (arrow); d, one flagellum curled at the flagellar base; e, two curled flagella with one remaining flagellar tip (arrow); f, loss of curled flagella. (C) Statistical presentation of cells with different forms of flagella after LiCl treatment for wild type cells, and (D) for RNAi strain C60, and (E) for RNAi strain C70. (F) Increase of IFT proteins during flagellar elongation induced by LiCl. Flagella isolated from wild type or RNAi strains after 30 min treatment were analyzed by immunoblotting with antibodies against IFT139, IFT motor protein Fla10, and α-tubulin, which was used as loading control.</p

Identification of Regulators for Ciliary Disassembly by a Chemical Screen

Cilia are organelles for cellular signaling and motility. They are assembled in G0/G1 and disassembled prior to mitosis. Compared to what is known about ciliary assembly, less is understood about ciliary disassembly. To uncover new mechanisms of ciliary disassembly, we performed an unbiased chemical screen. Chlamydomonas reinhardtii cells were experimentally induced for ciliary disassembly by treatment with sodium pyrophosphate. An FDA approved drug library (HY-L022P-1, MedChemExpress) was used for the screening. Primary screening with further experiments has identified microtubule stabilizer taxanes, CDK4/6 inhibitor abemaciclib and Raf inhibitor dabrafenib being effective in inhibiting ciliary disassembly induced experimentally but also under physiological conditions. In addition, their effects on ciliary disassembly in mammalian cells has also been confirmed. Thus, our studies have not only revealed new mechanisms in ciliary disassembly but also provided new tools for studying ciliary disassembly. These discovered drugs may be used for therapeutic interventions of disorders involving ciliary degeneration such as retinopathies

Identification of Regulators for Ciliary Disassembly by a Chemical Screen

Cilia are organelles for cellular signaling and motility. They are assembled in G0/G1 and disassembled prior to mitosis. Compared to what is known about ciliary assembly, less is understood about ciliary disassembly. To uncover new mechanisms of ciliary disassembly, we performed an unbiased chemical screen. Chlamydomonas reinhardtii cells were experimentally induced for ciliary disassembly by treatment with sodium pyrophosphate. An FDA approved drug library (HY-L022P-1, MedChemExpress) was used for the screening. Primary screening with further experiments has identified microtubule stabilizer taxanes, CDK4/6 inhibitor abemaciclib and Raf inhibitor dabrafenib being effective in inhibiting ciliary disassembly induced experimentally but also under physiological conditions. In addition, their effects on ciliary disassembly in mammalian cells has also been confirmed. Thus, our studies have not only revealed new mechanisms in ciliary disassembly but also provided new tools for studying ciliary disassembly. These discovered drugs may be used for therapeutic interventions of disorders involving ciliary degeneration such as retinopathies

Identification of Regulators for Ciliary Disassembly by a Chemical Screen

Cilia are organelles for cellular signaling and motility. They are assembled in G0/G1 and disassembled prior to mitosis. Compared to what is known about ciliary assembly, less is understood about ciliary disassembly. To uncover new mechanisms of ciliary disassembly, we performed an unbiased chemical screen. Chlamydomonas reinhardtii cells were experimentally induced for ciliary disassembly by treatment with sodium pyrophosphate. An FDA approved drug library (HY-L022P-1, MedChemExpress) was used for the screening. Primary screening with further experiments has identified microtubule stabilizer taxanes, CDK4/6 inhibitor abemaciclib and Raf inhibitor dabrafenib being effective in inhibiting ciliary disassembly induced experimentally but also under physiological conditions. In addition, their effects on ciliary disassembly in mammalian cells has also been confirmed. Thus, our studies have not only revealed new mechanisms in ciliary disassembly but also provided new tools for studying ciliary disassembly. These discovered drugs may be used for therapeutic interventions of disorders involving ciliary degeneration such as retinopathies

Identification of Regulators for Ciliary Disassembly by a Chemical Screen

Cilia are organelles for cellular signaling and motility. They are assembled in G0/G1 and disassembled prior to mitosis. Compared to what is known about ciliary assembly, less is understood about ciliary disassembly. To uncover new mechanisms of ciliary disassembly, we performed an unbiased chemical screen. Chlamydomonas reinhardtii cells were experimentally induced for ciliary disassembly by treatment with sodium pyrophosphate. An FDA approved drug library (HY-L022P-1, MedChemExpress) was used for the screening. Primary screening with further experiments has identified microtubule stabilizer taxanes, CDK4/6 inhibitor abemaciclib and Raf inhibitor dabrafenib being effective in inhibiting ciliary disassembly induced experimentally but also under physiological conditions. In addition, their effects on ciliary disassembly in mammalian cells has also been confirmed. Thus, our studies have not only revealed new mechanisms in ciliary disassembly but also provided new tools for studying ciliary disassembly. These discovered drugs may be used for therapeutic interventions of disorders involving ciliary degeneration such as retinopathies