Regulation of flagellar assembly by glycogen synthase kinase 3 in Chlamydomonas reinhardtii

Abstract

Chlamydomonas reinhardtii controls flagellar assembly such that flagella are of an equal and predetermined length. Previous studies demonstrated that lithium, an inhibitor of glycogen synthase kinase 3 (GSK3), induced flagellar elongation, suggesting that a lithium-sensitive signal transduction pathway regulated flagellar length (S. Nakamura, H. Takino, and M. K. Kojima, Cell Struct. Funct. 12:369-374, 1987). Here, we demonstrate that lithium treatment depletes the pool of flagellar proteins from the cell body and that the heterotrimeric kinesin Fla10p accumulates in flagella. We identify GSK3 in Chlamydomonas and demonstrate that its kinase activity is inhibited by lithium in vitro. The tyrosine-phosphorylated, active form of GSK3 was enriched in flagella and GSK3 associated with the axoneme in a phosphorylation-dependent manner. The level of active GSK3 correlated with flagellar length; early during flagellar regeneration, active GSK3 increased over basal levels. This increase in active GSK3 was rapidly lost within 30 min of regeneration as the level of active GSK3 decreased relative to the predeflagellation level. Taken together, these results suggest a possible role for GSK3 in regulating the assembly and length of flagella.

FIG. 1.

Lithium chloride induces elongation of flagella. (A-E) Histograms (n = 104 to 107 cells for each measurement) show distributions of flagellar lengths following drug treatment. Wild-type cells were incubated for 60 min with M media (A), 25 mM NaCl (B), 25 mM KCl (C), 25 mM NH₄Cl (D), or 25 mM LiCl (E). Asterisks in this and subsequent histograms indicate the average lengths of flagella on control cells. Arrows in each histogram indicate the average lengths of flagella for each cell population. (F-G) DIC images of wild-type cells incubated for 60 min with M media (F) or 25 mM LiCl (G). Bars: 6 μm.

FIG. 2.

Prolonged incubation with lithium induces an aflagellate phenotype. (A, B) Histograms show the distribution of flagellar lengths following incubation with lithium. Cells were incubated without (A) (n = 109 cells) or with (B) 25 mM LiCl (n = 108 cells) for 48 h. Arrows indicate the average lengths of flagella for each cell population. (C) DIC images of control or lithium-treated cells illustrate effects on flagellar length. Arrows identify small bulbs at flagellar tips. Bars: 6 μm.

FIG. 3.

Protein synthesis is not required for lithium-induced elongation of flagella. (A-B) Histograms (n = 105 to 111 cells per measurement) show the distribution of flagellar lengths following treatment with 25 mM LiCl for 60 min. Wild-type cells were pretreated without (A) or with (B) 10 μg of cycloheximide/ml. After 10 min, LiCl was added to a final concentration of 25 mM. Arrows indicate the average lengths of flagella for each cell population. (C) Immunoblots reveal the lithium-induced depletion of flagellar precursors from cell bodies. Equal numbers of cell bodies from lithium (+) and control treated (−) cells were probed with antibodies against axonemal structural proteins (MBO2p, IC140), an axonemally associated protein (Fla10p), and the chloroplast protein OEE1.

FIG. 4.

Lithium inhibits regeneration of flagella. (A-D) Histograms (n = 105 to 111 cells per measurement) show distribution of flagellar lengths before and after regeneration. Wild-type cells were preincubated without (A, C) or with (B, D) 10 μg of cycloheximide/ml. After 10 min, 25 mM LiCl was added (C, D) and and the incubation was continued for an additional 60 min. Arrows indicate the average lengths of flagella for each cell population. (E) RNA blot analysis of flagellar transcripts during regeneration. Twenty micrograms of total RNA from either flagellated cells (lanes F) incubated without (Control) or with LiCl and cells regenerating flagella (lanes R) in the absence (Control) or presence of LiCl was loaded in each lane. The transcripts for RSP3 and PF20 were detected by a hybridization probe from the corresponding gene. Blots were reprobed with CRY1 (the gene encoding ribosomal protein S14) to demonstrate equal loading. A faint second message that is unaffected by LiCl was identified with the PF20 probe.

FIG. 5.

Alignment of GSK3 from Chlamydomonas with various GSK3 amino acid sequences. Cr, C. reinhardtii GSK3 (accession no. AY621077); Pp, Physcomitrella patens shaggy-related protein kinase 2 (accession no. AAQ23107.1); Os, Oryza sativa, shaggy-related protein kinase γ (accession no. BAB40983.1); Ph, petunia hybrid shaggy kinase 4 (accession no. S5115); Ms, Medicago sativa MSK2 (accession no. P51138); At, Arabidopsis thaliana shaggy-related protein kinase γ (accession no. NP_187235.1); Hs, Homo sapiens GSK3β (accession no. P49841); Ce, C. elegans (accession no. AD45354); Dm, Drosophila melanogaster sgg protein kinase (accession no. CAA37419.1); Dd, Dictyostelium discoideum (accession no. P51136); and Ci, Ciona intestinalis (accession no. BAA92186). Roman numerals indicate the 11 kinase subdomains. The regulatory tyrosine that must be phosphorylated for GSK3 activity is identified with an asterisk.

FIG. 6.

GSK3 is a flagellar protein. (A) RNA blot analysis of the GSK3 transcript. Twenty micrograms of RNA from flagellated cells (F) or cells regenerating flagella (R) was loaded on each lane, and the GSK3 transcript was identified with a probe corresponding to the 3′ region of the gene. (B) Immunoblot analysis of the GSK3 protein. The same amount of protein from whole cells (WC), cell bodies (CB), or flagella (Flg) was loaded in each lane, and the protein was identified with either an antibody generated against recombinant Xenopus GSK3β (α-GSK3β) or an antibody generated from a tyrosine-phosphorylated peptide from the activation loop (α-PY-GSK3). (C) Identification of axonemal location of GSK3. Flagella (Flg) were fractionated into the membrane+matrix fraction (S) or the axonemes (P) in the absence (−Pase Inhib) or presence (+Pase Inhib) of phosphatase inhibitors. Equal amounts of proteins were loaded in each lane. Proteins were visualized after immunoblots were probed with the anti-Xenopus GSK3β antibody.

FIG. 7.

Lithium inhibits GSK3 kinase activity in vitro. The membrane+matrix fraction or GSK3 immunoprecipitated from the membrane+matrix fraction of flagella was assayed in the absence (Media) or presence (Lithium) of lithium (25 mM) in an in vitro kinase assay using a phosphopeptide from glycogen synthase as a substrate. Results shown are representative of at least three independent experiments.

FIG. 8.

Levels of active GSK3 change with lithium treatment or flagellar regeneration. (A) Immunoblots reveal accumulation of Fla10p and tyrosine-phosphorylated GSK3 in flagella of lithium-treated cells. The same amount of flagellar protein from control-treated cells (−) or cells treated with 25 mM LiCl for 60 min (+) was loaded into each well. Blots were probed with antibodies against GSK3 (GSK3 and PY-GSK3), IFT components (IFT172 and Fla10p), and structural axonemal components (MBO2p, IC140, and β-tubulin). (B, C) The active form of GSK3 decreases during flagellar regeneration. (B) Cells were incubated with 25 mM LiCl for 60 min and deflagellated by pH shock, and the flagella were removed by centrifugation. Cells were allowed to regenerate flagella for 5 min (lane 5), 10 min (lane 10), 15 min (lane 15), 30 min (lane 30), 60 min (lane 60), 90 min (lane 90), and 120 min (lane 120). Protein from equal numbers of cells from each time point were loaded into wells, and the levels of active GSK3 were determined by immunoblotting with the anti-phosphotyrosine GSK3 antibody. The blot was reprobed with the anti-Xenopus GSK3β antibody to show total GSK3 levels. (C) The histogram shows the ratio of active GSK3/total GSK3 from the blot shown in panel B. Statistical analysis was preformed to determine the significance in the change of active GSK3/total GSK3 compared to control length during flagellar regeneration. Asterisks indicate P values of 0.0014 to <0.0006.

FIG. 9.

GSK3 RNAi phenocopies the lithium treatment of cells. (A) A schematic representation of the plasmid pGSK3IR. Open boxes indicate the NIT1 promoter and genomic and cDNA fragments of GSK3, respectively. The arrows indicate the 5′ to 3′ direction of genomic and cDNA fragments of GSK3. (B) Immunoblot analysis of GSK3 RNAi transformants. Cells were cultured for 4 days in media containing ammonia (lane −, RNAi repressing) or nitrate (lane +, RNAi inducing). Equal numbers of cells were loaded in each lane, and the proteins were identified with an antibody against GSK3β. The blot was reprobed with an antibody against β-tubulin to demonstrate equal loading of protein. (C, D) Histograms show the distribution of flagellar lengths from RNAi transformants 1H11 and 1B1. Cells were incubated under inducing (NO₃, black bars) or repressing (NH₄, gray bars) conditions. Asterisks and arrows indicate the average lengths of flagella in each cell population under noninducing and inducing conditions, respectively. (E) DIC images of RNAi transformant 1B1 under RNAi inducing conditions (nitrate). (F) DIC images of RNAi transformant 1B1 under RNAi repressing conditions (ammonia). Bars: 6 μm.