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First published online 27 September 2005
doi: 10.1242/jcs.02585

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Calmodulin and PF6 are components of a complex that localizes to the C1 microtubule of the flagellar central apparatus

Dartmouth College, Department of Biological Sciences, 301 Gilman Hall, Hanover, NH 03755, USA

View larger version (62K): [in a new window]   Fig. 1. (A,B) Western blots of axonemes and silver-stained gel of immunoprecipitates. (A) Western blot of axonemes isolated from wild-type (wt) and pf6-2 Chlamydomonas reinhardtii cells probed with our anti-PF6 antibodies. A band with a molecular weight consistent with that of PF6 is present in wild-type axonemes and absent in pf6-2 axonemes. (B) Silver-stained gel of immunoprecipitates obtained from wild-type (wt) and pf6-2 axonemal extracts using our anti-PF6 antibodies. In wild-type axonemal extracts, five polypeptides in addition to PF6 are specifically precipitated using anti-PF6 antibodies. These polypeptides are not precipitated in pf6-2 axonemal extracts. The precipitating immunoglobulin heavy and light chains are labeled HC and LC, respectively.

View larger version (63K): [in a new window]   Fig. 2. (A,B) Western blots of sucrose gradient fractions and immunoprecipitates. (A) Western blots of axonemal extracts obtained from wild-type, pf18 and pf16 axonemes fractionated on 5-20% sucrose gradients. Blots were probed with our anti-calmodulin antibodies. Calmodulin sediments in three distinct peaks. The 12S peak of calmodulin is lacking or severely reduced in central pair-defective mutants. (B) Western blots of immunoprecipitates obtained from wild-type (wt) and pf6-2 axonemal extracts using our anti-PF6 antibodies. The blot was probed with anti-calmodulin antibodies. Calmodulin is precipitated by anti-PF6 antibodies from wild-type extracts.

View larger version (37K): [in a new window]   Fig. 3. Western blots of axonemes isolated from wild-type (wt) and pf6-2 cells probed with antibodies generated against PF6-IP1, -IP2, -IP3, -IP4 and calmodulin (CaM). (A) Blots of 4-16% gradient polyacrylamide gels. (B) Blots of 15% polyacrylamide gels. The anti-PF6-IP4 antibody recognizes a polypeptide of the correct size (asterisk). With the exception of calmodulin, each of the proteins is missing from pf6-2 axonemes.

View larger version (67K): [in a new window]   Fig. 4. (A,B) Silver-stained gel and corresponding western blots of immunoprecipitates. (A) Silver-stained gel of immunoprecipitation experiments. Axonemal extracts were prepared from wild-type (wt) and radial spoke-defective (pf14) strains. The antibody used for precipitation is noted at the top of each lane. Our anti-calmodulin antibodies precipitate all six members of the PF6-containing complex as well as several other polypeptides (dots and dashed lines). A subset of these polypeptides is precipitated from wild-type axonemal extracts, but not from axonemal extracts isolated from the radial spokeless mutant, pf14 (dots). Therefore, these polypeptides probably represent radial spoke components that are associated with calmodulin. Three additional polypeptides (dashed lines) that are present in radial spoke-defective mutants are also precipitated using our anti-calmodulin antibodies. As these polypeptides were not precipitated by any of our other antibodies, they are probably not components of the PF6-containing complex. The precipitating immunoglobulin heavy and light chains are labeled HC and LC, respectively. (B) Corresponding western blots for immunoprecipitations performed from wild-type extracts. The blots were probed with the antibodies against the proteins listed to the right of each panel.

View larger version (68K): [in a new window]   Fig. 5. Western blots of axonemal extracts obtained from wild-type axonemes fractionated on 5-20% sucrose gradients. Blots were probed with antibodies generated against the proteins noted to the right of each panel. PF6 and PF6-IP1, -IP2, -IP3 and -IP4 sediment in two peaks.

View larger version (87K): [in a new window]   Fig. 6. (A,B) Silver-stained gels and corresponding western blots of immunoprecipitates. (A) Silver-stained gels of immunoprecipitation experiments performed from pooled sucrose gradient fractions. The precipitating antibodies (Ab) and the fractions (F) used for each experiment are noted at the top of the lanes. The panel on the left is an 8% polyacrylamide gel whereas the panel on the right is a 12% polyacrylamide gel. The precipitating immunoglobulin heavy and light chains are labeled HC and LC, respectively. (B) Corresponding western blots using our anti-calmodulin and anti-PF6-IP4 antibodies. All six polypeptides in the PF6 complex precipitate from the 12S fraction. Only subsets of these polypeptides are precipitated from the 6S fraction.

View larger version (44K): [in a new window]   Fig. 7. (A,B) Silver-stained gels and corresponding western blots of immunoprecipitates. (A) Silver-stained gel of immunoprecipitation experiments using undialyzed, high-salt axonemal extracts obtained from wild-type flagella. The precipitating antibody is indicated at the top of each lane. The immunoglobulin heavy and light chains are labeled HC and LC, respectively. (B) Corresponding western blots from immunoprecipitation experiments. The blots were probed with the antibodies indicated to the right of each panel. In high-salt conditions, PF6-IP1 and calmodulin co-precipitate, and PF6-IP2, -IP3 and -IP4 co-precipitate. Only very minimal amounts of complex components precipitate with PF6.

View larger version (64K): [in a new window]   Fig. 8. Western blots of undialyzed high-salt axonemal extracts obtained from wild-type axonemes fractionated on 5-20% sucrose gradients. Blots were probed with antibodies generated against the proteins noted to the right of each panel. Under high-salt conditions, all six members of the PF6 complex sediment at 6S.

View larger version (67K): [in a new window]   Fig. 9. Western blot of gel overlay assay. Axonemes from wild-type (wt) and mutant axonemes were subjected to SDS-PAGE and transferred to nitrocellulose membrane. The blot was then incubated with bacterially expressed PF6-IP3. The bound PF6-IP3 was detected using S-protein overlay (see Materials and Methods). PF6-IP3 binds to a polypeptide of molecular weight consistent with that of PF6-IP2 (asterisk). This polypeptide is absent in axonemes isolated from pf6, pf16 and pf18, also consistent with PF6-IP2.

View larger version (38K): [in a new window]   Fig. 10. Models of C1a components and their interactions based on the immunoprecipitation and sucrose gradient data in both high-salt and low-salt conditions.