Increased ciliary trafficking of FLS2 upon induction of ciliary disassembly.

(A) FLS2 is a cell body protein and transported to cilia during ciliary disassembly as examined by immunostaining. fls2 cells expressing FLS2-HA were treated with or without NaPPi for 10 min followed by immunostaining with HA and α-tubulin antibodies. WT cells were used as control. Bar, 5 μm. (B) Analysis of ciliary transport of FLS2 by immunoblotting. fls2 cells expressing FLS2-HA were separated into cell bodies (CB) and cilia (C) fractions after treatment with or without NaPPi for 10 min followed by immunoblotting. WC, whole cells. (C) Ciliary FLS2 is associated with the axonemes. Cilia isolated from cells treated with or without NaPPi for 10 min were fractionated into membrane matrix (M+M) and axonemal fractions followed by immunoblotting with the indicated antibodies. FMG1, a ciliary membrane protein was used as a control for M+M fractions. (D) FLS2 is transported to cilia during ciliary disassembly in zygote development. Immunostaining analysis of FLS2 in mt+ and mt- gametes (G+ and G-), 0.5 hr (Z0.5h) and 2.5 hr (Z2.5h) zygotes. Bar, 5 μm. (E) FLS2 in cilia undergoes dephosphorylation during ciliary disassembly but its levels are unchanged. Cilia were isolated from cells treated with NaPPi for 10 or 120 min. The isolated cilia were treated with or without phosphatase (Ptase) followed by phos-tag immunoblotting. Please note that FLS2 in the 10 min sample without phosphatase treatment exhibited slower gel motility shift relative to other samples. (F) The kinase activity of FLS2 is not required for its ciliary transport. Whole cells (WC) or isolated cilia from kinase-dead K33R mutant cells treated with or without NaPPi for 10 min were subjected to immunoblot analysis. (G) FLS1 does not affect ciliary transport of FLS2. Cilia isolated from fls1 cells expressing FLS2-HA treated with or without NaPPi were analyzed by phos-tag immunoblotting. fls2 cells expressing FLS2-HA were used as control. Ciliary transport as well as gel mobility of FLS2 expressed in fls1 were similar to the control.</p

Identification of a CDK-like kinase, FLS2 that functions in ciliary disassembly.

(A) fls2 mutant is defective in cilia resorption but not steady state ciliary length. Wild type (WT) or fls2 cells were treated with or without 20 mM NaPPi for three hours followed by ciliary length measurement. Ciliary length data shown here and below are presented as mean±SD with n = 50 cilia. N.S., not significant (p>0.05). (B) fls2 mutant exhibits slower kinetics of ciliary disassembly. WT, fls2 and the rescued cells were induced for ciliary disassembly by addition of 20 mM NaPPi. Ciliary length was measured at the indicated times. (C) Diagrams of the gene structure of FLS2 with the indicated foreign DNA insertion site and the domain structure of the protein kinase encoded by FLS2. The AphVIII DNA fragment is inserted in the last exon of FLS2 between 3353 and 3354 nt and results in deletion of the 3352 nt. The left and right arrows show the positions of the primers used for RT-PCR. (D) Alignment of the protein kinase domain III and VIII of FLS2 with those of human CDK1, CDK-like kinases (CDKLs) and two Chlamydomonas CDKLs (LF5 and FLS1) that are implicated in ciliary functions. (E) Phylogenetic analysis places FLS2 in the group of CDKLs. A neighbor-joining phylogenetic tree was constructed by using an algorithm (www.phylogeny.fr) following the instruction. FLS2 was analyzed with the human CDKLs and two Chlamdydomonas CDKLs: FLS1 and LF5. The outgroup members include LF2 and LF4, two MAPK-related kinases in Chlamdyomonas; CDPK1, a Chlamydomonas calcium dependent kinase and HsCDK1, a cyclin-dependent kinase. The numbers above the line indicate the bootstrap values. The sequences of the kinase domains were used for the analysis. (F) FLS2 is not expressed in fls2 cells shown by RT-PCR. Gene expression of CBLP was used as a control. (G) An immunoblot showing expression of FLS2-HA in fls2 cells. WT and fls2 cells were used as controls.</p

FLS2 does not regulate CALK and IFT but suppresses CrKinesin13 phosphorylation independently of FLS1.

(A) CALK phosphorylation is not affected in fls2. WT and fls2 cells were treated with NaPPi for ciliary disassembly. Cell lysates at the indicated time were analyzed by immunoblotting with CALK antibody. CALK underwent phosphorylation with slower gel migration, which were not affected in fls2. (B) FLS2 does not affect increasing transport of IFT proteins in cilia. WT or fls2 Cells were treated with or without 20 mM NaPPi for 10 min followed by cilia isolation and immunoblotting. IFT121, a subunit of IFT-A and IFT46, a subunit of IFT-B were analyzed. α-tubulin was used as a loading control. (C) Loss of FLS2 does not affect ciliary transport of CrKinesin13 but induces earlier onset of its phosphorylation. WT or fls2 cells were treated with NaPPi for the indicated times followed by cilia isolation and immunoblotting. The slower migration form of CrKinesin13 is due to phosphorylation as demonstrated previously. (D) The kinase activity of FLS2 is required for suppression of earlier onset of CrKinesin13 phosphorylation. Isolated cilia from cell samples that were treated with or without NaPPi for 10 min were subjected to immunoblot analysis with the indicated antibodies. (E) FLS1 phosphorylation is not altered in fls2. Cell samples as indicated were analyzed by immunoblotting with FLS1 antibody. (F) FLS2 phosphorylation is not altered in fls1. fls1 or fls2 cells expressing FLS2-HA were treated for the indicated times followed by Phos-tag immunoblotting. FLS2 phosphorylation as evidenced by patterns of gel migration was similar between fls1 and fls2 cells. (G) Generation of an fls1-kd/fls2 strain by RNAi depletion of FLS1 in fls2 cells. Cell samples as indicated were subjected to immunoblotting with anti-FLS1 antibody. (H) CrKinesin13 phosphorylation in fls1-kd/fls2 cells. Cilia were isolated from cell samples that were treated with NaPPi for 10 min and were then subjected to immunoblotting. (I) fls1-kd/fls2 cells show more severe defect in ciliary disassembly. Cells as indicated were induced for ciliary disassembly followed by ciliary length measurement at the indicated times. Bars indicate SD.</p

FLS2 is a cargo of IFT70 for ciliary transport.

(A) Ciliary transport of FLS2 depends on IFT. Temperature sensitive mutant fla10-1 cells expressing FLS2-HA were incubated at the indicated temperatures for 1h followed by treatment with NaPPi or not for 10 min. The cilia were then isolated for immunoblotting with the indicated antibodies. (B) FLS2 interacts with IFT70 shown in yeast two-hybrid assays. Subunits of IFT-B or IFT-A were transformed respectively into yeast cells with FLS2, followed by growth on selection medium lacking Leu and Trp (-2) or Leu, Trp, His and Ade (-4). (C-D) The C-terminal non-kinase region of FLS2 is required for its interaction with IFT70. Full-length FLS2 and its deletion variants were assayed for their interaction with IFT70 in yeast two-hybrid assays (C). A GST pull-down assay of bacterial expressed FLS2-CT and IFT70 (D). (E) Co-immunoprecipitation of FLS2 and IFT70. Cilia were isolated from control cells (WT) or fls2 cells expressing FLS2-HA during ciliary disassembly followed by immunoprecipitation with anti-HA antibody and immunoblotting. (F-G) TPR1-3 of IFT70 is essential for its interaction with FLS2. Full-length of IFT70 and its various segments (E) or various TPR deletion mutants (F) were subjected to yeast two-hybrid assays with FLS2.</p

<i>FLS2</i> regulates ciliary disassembly under physiological conditions and cell cycle progression.

(A) fls2 is defective in ciliary disassembly during zygote development. Gametes of opposite mating types of fls2 were mixed to allow mating and zygote development followed by ciliary length measurement. The mating of wild type gametes was used as a control. Error bars indicate SD. (B) fls2 is defective in ciliary disassembly during cell cycle progression. WT, fls2 and rescued cells were synchronized by a light/dark (14h/10h) cycle. Ciliary length was scored beginning in the dark period when cells prepared to enter mitosis. (C) Cell cycle progression in fls2 mutant is retarded. Dividing cells were scored microscopically in the dark period of the light/dark cycle. Compared to WT and rescued cells, the peak of cell division in fls2 mutant was delayed around 3 hrs.</p

The C-terminal non-kinase region of FLS2 is required for its ciliary transport and proper ciliary disassembly.

(A) Deletion of TPR1, TPR2 or TPR3 of IFT70 abrogates its interaction with IFT52-IFT88 dimer. Cell lysates from bacterial cells expressing His-tagged full-length IFT70 and its various deletion mutants (IFT70*-His) were mixed, respectively, with cell lysates from cells expressing GST-IFT52 and IFT88-His followed by GST pull-down assay. His and GST antibodies were used for immunoblotting. (B) Expression of C-terminal deletion mutant of FLS2 in fls2 cells. fls2 cells expressing HA-tagged full-length (FL) FLS2 or its C-terminal deletion mutants (ΔCT) (three strains 11, 109, 191) were analyzed by immunoblotting. fls2 cells were used as a negative control. (C) The C-terminal region of FLS2 is required for its ciliary transport. Cilia were isolated from fls2 cells expressing full-length FLS2 or ΔCT mutant that were treated with or without NaPPi for 10 min followed by immunoblot analysis. (D) The C-terminal region of FLS2 does not affect its kinase activity. FLS2 was immunoprecipitated with anti-HA from cell samples as indicated and subjected to immunoblot analysis and in vitro kinase assay. In vitro kinase assay was performed as shown in Fig 3C. (E-F) Failed ciliary transport of FLS2 by C-terminal deletion induces CrKinesin13 phosphorylation and impairs ciliary disassembly. Cilia isolated from cell samples as indicated were analyzed by immunoblotting (E). fls2 cells expressing full-length (FL) FLS2 or ΔCT mutant were induced for ciliary disassembly by NaPPi treatment followed by ciliary length measurement at the indicated times. fls2 cells were used as control. Bars indicate SD.</p

FLS2 kinase activity is required for ciliary disassembly during which it undergoes dephosphorylation and inactivation.

(A) FLS2 expression is not altered during ciliary disassembly. fls2 rescued cells expressing FLS2-HA was induced for ciliary disassembly by addition of NaPPi followed by immunoblotting with the indicated antibodies. (B) FLS2 undergoes gradual dephosphorylation during ciliary disassembly. A phos-tag immunoblotting was performed using antibodies as indicated. Steady state cells were treated with or without phosphatase (Ptase) to demonstrate that the gel mobility shift was caused by changes in protein phosphorylation. (C) Dephosphorylation of FLS2 results in loss of its kinase activity. fls2 cells expressing wild type FLS2-HA or kinase dead mutant K33R-HA were treated with 20 mM NaPPi for the indicated times. Wild type (WT) cells were used as a negative control. Cell lysates were incubated with anti-HA antibodies for immunoprecipitation followed by in vitro kinase assay. ATPγS was used as ATP donor and myelin basic protein as substrate. Anti-thiophosphate ester antibody was used to detect substrate phosphorylation. (D) The kinase activity of FLS2 is required for ciliary disassembly. Cells from WT, fls2 and kinase-dead mutant K33R were induced for ciliary disassembly by NaPPi treatment. Ciliary length was measured at the indicated times. Bars indicate SD.</p