Results: 4

1.
Fig. 3.

Fig. 3. From: A microtubule depolymerizing kinesin functions during both flagellar disassembly and flagellar assembly in Chlamydomonas.

Effect of CrKinesin-13 depletion on flagellar shortening. (A) Immunoblots of CrKinesin-13 RNAi transformants. (B) Flagellar regeneration in 21gr wild-type cells (predeflagellation length, 12.6 μm), RNAi transformant 92 (predeflagellation length, 6.5 μm), and RNAi transformant 93 (predeflagellation length, 8.0 μm). Curves for 21gr and RNAi-92 represent results from three independent experiments and for RNAi-93 from one experiment. Error bars indicate SEM. (C) Flagellar shortening induced by NaPPi in 21gr cells and RNAi transformants 92 and 93. The length of one flagellum on at least 50 cells was measured for each time point. Error bars show SEM.

Tian Piao, et al. Proc Natl Acad Sci U S A. 2009 March 24;106(12):4713-4718.
2.
Fig. 2.

Fig. 2. From: A microtubule depolymerizing kinesin functions during both flagellar disassembly and flagellar assembly in Chlamydomonas.

Modification of CrKinesin-13. (A) CrKinesin-13 is modified during flagellar regeneration. 21gr cells were triggered to grow new flagella by pH shock (error bars, SD); (B) by transfer from agar plates into liquid medium (error bars, SEM); and (C) by washing into fresh medium cells whose flagella had shortened approximately to half length during incubation in 20 mM NaPPi (error bars, SEM). A flagellum on at least 50 cells was measured for each time point. In (A), pdf is a sample taken just before deflagellation; the T = 0 sample was taken immediately after deflagellation. (B, inset) Cells scraped from agar directly into sample buffer, and a sample of the same cells 5 minutes after washing into liquid medium; all of the CrKinesin-13 was in the unphosphorylated form in the agar-grown cells.

Tian Piao, et al. Proc Natl Acad Sci U S A. 2009 March 24;106(12):4713-4718.
3.
Fig. 1.

Fig. 1. From: A microtubule depolymerizing kinesin functions during both flagellar disassembly and flagellar assembly in Chlamydomonas.

Posttranslational modification of CrKinesin-13 (A) MPM2 antibody immunoprecipitates a 70-kDa protein from pH-shocked cells. Cell lysates from control (con) cells and pH-shocked cells (DF) were incubated with MPM2 antibody, and the immunoprecipitates were analyzed by immunoblotting (Fig. S1) and silver staining. (B) CrKinesin-13 is phosphorylated during pH shock, and the phosphorylated CrKinesin-13 is the antigen recognized by the MPM2 antibody. Cell lysates from control and deflagellated cells were treated with or without phosphatase followed by immunoblot analysis with anti-CrKinesin-13 and mAb-MPM2. (C) CrKinesin-13 properties in mutants defective in flagellar assembly. The indicated mutant and wild-type (21gr) cells were analyzed by immunoblotting using the CrKinesin-13 antibody. (D) CrKinesin-13 is modified during the pathways activated by pH shock. The flagellar mutants were subjected to pH shock along with wild-type cells, frozen within 1 minute, and subsequently analyzed by SDS/PAGE and immunobloting. (E) The fa1 and fa2 mutants, which are defective in flagellar detachment, undergo pH shock-induced modification of CrKinesin-13. Immunoblot analysis of 21gr, fa1, and fa2 mutant cells was carried out before and after pH shock.

Tian Piao, et al. Proc Natl Acad Sci U S A. 2009 March 24;106(12):4713-4718.
4.
Fig. 4.

Fig. 4. From: A microtubule depolymerizing kinesin functions during both flagellar disassembly and flagellar assembly in Chlamydomonas.

Transport of CrKinesin-13 into flagella is triggered when the flagellar shortening pathway is activated and transport requires IFT. (A) Immunofluorescence of control cells under standard culture conditions. Staining with anti-CrKinesin-13 was detected in the basal body region, and little if any was detected in the flagella. (B) Immunoblot of whole cells (WC), cell bodies (CB), and flagella (F) with anti-CrKinesin-13. CrKinesin-13 was predominantly localized to the cell body (note that the pH shock used for deflagellation caused CrKinesin-13 phosphorylation). (1xF: One cell equivalent of flagella; 50xF: 50 cell equivalents of flagella.) Staining with alpha tubulin antibody documented equal loading. (C) Immunofluorescence of cells undergoing flagellar disassembly. CrKinesin-13 staining was detected along the length of the flagella and at the tip. (D) CrKinesin-13 in the flagella increased within 5 minutes after cells were placed in NaPPi. (E) Immunoblots of flagella from gametes and the zygotes that formed at 1 hour and 3 hours after gametes were mixed. CrKinesin-13 in the flagella increased during zygotic flagellar disassembly. G+, mt+ gametes; G−, mt− gametes; Z, zygotes. Alpha tubulin was used as loading control. (F) Co-immunoprecipitation of CrKinesin-13 and IFT particles. The flagellar membrane/matrix fractions of flagella isolated 5 minutes after transfer of cells to NaPPi were used for immunoprecipitation with anti-CrKinesin-13 or preimmune IgG. The immunoprecipitates were analyzed with anti-CrKinesin-13 and anti-IFT139 antibodies. (G) Failure of CrKinesin-13 to be transported into flagella in the fla10–1 mutant at the non-permissive temperature after induction of flagellar disassembly by NaPPi. fla10–1 Cells were incubated at 22 °C or 32 °C for 1 hour followed by incubation in 20 mM NaPPi for 5 minutes at the same temperatures, and the flagella were isolated and analyzed by immunoblotting. Alpha tubulin was used as a loading control and an anti-IFT139 antibody was used to detect IFT particles.

Tian Piao, et al. Proc Natl Acad Sci U S A. 2009 March 24;106(12):4713-4718.

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