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FAP206 is a microtubule-docking adapter for ciliary radial spoke 2 and dynein c.

Vasudevan KK, Song K, Alford LM, Sale WS, Dymek EE, Smith EF, Hennessey T, Joachimiak E, Urbanska P, Wloga D, Dentler W, Nicastro D, Gaertig J - Mol. Biol. Cell (2014)

Bottom Line: A knockout of the conserved ciliary protein FAP206 in the ciliate Tetrahymena resulted in slow cell motility.Occasionally, RS2 assembled but lacked both the front prong of its microtubule base and dynein c, whose tail is attached to the front prong.Overexpressed GFP-FAP206 decorated nonciliary microtubules in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Biology, University of Georgia, Athens, GA 30602.

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FAP206 interacts with CSC. (A) A Western blot of purified cilia isolated from Tetrahymena strains that are wild type, lack genes encoding the Tetrahymena homologues of CSC proteins (FAP61/CaM-IP3 or FAP251/CaM-IP4), or lack FAP206, probed with antibodies specific to FAP91/CaM-IP2 of C. reinhardtii (top; Dymek et al., 2011), 12G10 monoclonal α-tubulin (middle), or polyglycylated tubulin (bottom). Note that the levels of the anti-FAP91/CaM-IP2 band are strongly reduced in the strains lacking the CSC protein homologues, indicating that the antibodies are specific to the Tetrahymena FAP91 homologue. Furthermore, the levels of the FAP91 homologue are strongly reduced in the strain lacking FAP206, indicating that axonemal assembly of FAP91 and likely other CSC components depends on the presence of FAP206. Images of the entire blots are shown in Supplemental Figure S1. (B) Western blot of axonemes of wild type and FAP206-KO probed with anti-FAP91 and 12G10 anti–α-tubulin antibodies. (C) Silver stained SDS–PAGE gels showing an immunoprecipitate obtained from the radial spoke-enriched supernatants of C. reinhardtii axonemes. The positions of known CSC components and major radial spoke proteins are marked. The two bands (boxed area) that migrated with an apparent molecular weight of 80 kDa were found to contain the Chlamydomonas FAP206 homologue (CHLREDRAFT_171124). (D) Immunoprecipitates obtained with the anti-FAP91/CaM-IP2 antibodies from the radial spoke–enriched supernatant of Chlamydomonas axonemes that were either wild type or spokeless pf14 mutant. Note the absence of the FAP206 bands, indicating that the CSC-FAP206 interaction requires RSP3 as an intermediate.
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Figure 7: FAP206 interacts with CSC. (A) A Western blot of purified cilia isolated from Tetrahymena strains that are wild type, lack genes encoding the Tetrahymena homologues of CSC proteins (FAP61/CaM-IP3 or FAP251/CaM-IP4), or lack FAP206, probed with antibodies specific to FAP91/CaM-IP2 of C. reinhardtii (top; Dymek et al., 2011), 12G10 monoclonal α-tubulin (middle), or polyglycylated tubulin (bottom). Note that the levels of the anti-FAP91/CaM-IP2 band are strongly reduced in the strains lacking the CSC protein homologues, indicating that the antibodies are specific to the Tetrahymena FAP91 homologue. Furthermore, the levels of the FAP91 homologue are strongly reduced in the strain lacking FAP206, indicating that axonemal assembly of FAP91 and likely other CSC components depends on the presence of FAP206. Images of the entire blots are shown in Supplemental Figure S1. (B) Western blot of axonemes of wild type and FAP206-KO probed with anti-FAP91 and 12G10 anti–α-tubulin antibodies. (C) Silver stained SDS–PAGE gels showing an immunoprecipitate obtained from the radial spoke-enriched supernatants of C. reinhardtii axonemes. The positions of known CSC components and major radial spoke proteins are marked. The two bands (boxed area) that migrated with an apparent molecular weight of 80 kDa were found to contain the Chlamydomonas FAP206 homologue (CHLREDRAFT_171124). (D) Immunoprecipitates obtained with the anti-FAP91/CaM-IP2 antibodies from the radial spoke–enriched supernatant of Chlamydomonas axonemes that were either wild type or spokeless pf14 mutant. Note the absence of the FAP206 bands, indicating that the CSC-FAP206 interaction requires RSP3 as an intermediate.

Mentions: A detailed comparison of the subtomogram averages of all repeats of the FAP206-KO and wild-type axonemes revealed that in FAP206-KO, the electron density of RS1 is unaffected, RS2 is greatly reduced, but a faint signal is still detectable, and RS3 is mildly reduced (Figures 3, A and B, and 4, A and B). A reduced electron density in specific areas of a subtomogram average is an indication that the individual repeats are not identical. Heterogeneity among the individual 96-nm repeats could be either caused by flexibility (i.e., the position of a structure varies between the individual repeats) or because a structure is absent in a subset of repeats. We used automatic image classification (Heumann et al., 2011) to sort the axonemal repeats into homogeneous subgroups before generating class averages. For wild type, we could not identify classes that differed in the structures of radial spokes (Figures 3A and 4A). When we classified for differences in the RS2 structure among the FAP206-KO subtomograms, the majority of axonemal repeats (83%) lacked RS2 entirely (except for the side prong; Figure 3, D and F), whereas the remaining 17% (FAP206-KO [+RS2] class) had a nearly completely assembled RS2 but lacked parts of the microtubule base (Figure 3, C and E). A comparison of the wild-type RS2 structure with the FAP206-KO (+RS2) class revealed a fairly normal stem and head of RS2, whereas the entire front prong and the closely associated dynein c were missing, and the back prong was reduced (compare Figures 2G and 3C, and 2C and 3E; also see Supplemental Movie S7). We estimated the molecular weight of the front prong density that is missing in the FAP206-KO (+RS2) class average as ∼80 kDa (red region in Figure 2, C, D, and K), which is consistent with the predicted (73 kDa) and observed molecular weight (80 kDa; see later discussion of Figure 7D) of FAP206. The simplest explanation of these observations is that FAP206 forms the front prong of RS2.


FAP206 is a microtubule-docking adapter for ciliary radial spoke 2 and dynein c.

Vasudevan KK, Song K, Alford LM, Sale WS, Dymek EE, Smith EF, Hennessey T, Joachimiak E, Urbanska P, Wloga D, Dentler W, Nicastro D, Gaertig J - Mol. Biol. Cell (2014)

FAP206 interacts with CSC. (A) A Western blot of purified cilia isolated from Tetrahymena strains that are wild type, lack genes encoding the Tetrahymena homologues of CSC proteins (FAP61/CaM-IP3 or FAP251/CaM-IP4), or lack FAP206, probed with antibodies specific to FAP91/CaM-IP2 of C. reinhardtii (top; Dymek et al., 2011), 12G10 monoclonal α-tubulin (middle), or polyglycylated tubulin (bottom). Note that the levels of the anti-FAP91/CaM-IP2 band are strongly reduced in the strains lacking the CSC protein homologues, indicating that the antibodies are specific to the Tetrahymena FAP91 homologue. Furthermore, the levels of the FAP91 homologue are strongly reduced in the strain lacking FAP206, indicating that axonemal assembly of FAP91 and likely other CSC components depends on the presence of FAP206. Images of the entire blots are shown in Supplemental Figure S1. (B) Western blot of axonemes of wild type and FAP206-KO probed with anti-FAP91 and 12G10 anti–α-tubulin antibodies. (C) Silver stained SDS–PAGE gels showing an immunoprecipitate obtained from the radial spoke-enriched supernatants of C. reinhardtii axonemes. The positions of known CSC components and major radial spoke proteins are marked. The two bands (boxed area) that migrated with an apparent molecular weight of 80 kDa were found to contain the Chlamydomonas FAP206 homologue (CHLREDRAFT_171124). (D) Immunoprecipitates obtained with the anti-FAP91/CaM-IP2 antibodies from the radial spoke–enriched supernatant of Chlamydomonas axonemes that were either wild type or spokeless pf14 mutant. Note the absence of the FAP206 bands, indicating that the CSC-FAP206 interaction requires RSP3 as an intermediate.
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Figure 7: FAP206 interacts with CSC. (A) A Western blot of purified cilia isolated from Tetrahymena strains that are wild type, lack genes encoding the Tetrahymena homologues of CSC proteins (FAP61/CaM-IP3 or FAP251/CaM-IP4), or lack FAP206, probed with antibodies specific to FAP91/CaM-IP2 of C. reinhardtii (top; Dymek et al., 2011), 12G10 monoclonal α-tubulin (middle), or polyglycylated tubulin (bottom). Note that the levels of the anti-FAP91/CaM-IP2 band are strongly reduced in the strains lacking the CSC protein homologues, indicating that the antibodies are specific to the Tetrahymena FAP91 homologue. Furthermore, the levels of the FAP91 homologue are strongly reduced in the strain lacking FAP206, indicating that axonemal assembly of FAP91 and likely other CSC components depends on the presence of FAP206. Images of the entire blots are shown in Supplemental Figure S1. (B) Western blot of axonemes of wild type and FAP206-KO probed with anti-FAP91 and 12G10 anti–α-tubulin antibodies. (C) Silver stained SDS–PAGE gels showing an immunoprecipitate obtained from the radial spoke-enriched supernatants of C. reinhardtii axonemes. The positions of known CSC components and major radial spoke proteins are marked. The two bands (boxed area) that migrated with an apparent molecular weight of 80 kDa were found to contain the Chlamydomonas FAP206 homologue (CHLREDRAFT_171124). (D) Immunoprecipitates obtained with the anti-FAP91/CaM-IP2 antibodies from the radial spoke–enriched supernatant of Chlamydomonas axonemes that were either wild type or spokeless pf14 mutant. Note the absence of the FAP206 bands, indicating that the CSC-FAP206 interaction requires RSP3 as an intermediate.
Mentions: A detailed comparison of the subtomogram averages of all repeats of the FAP206-KO and wild-type axonemes revealed that in FAP206-KO, the electron density of RS1 is unaffected, RS2 is greatly reduced, but a faint signal is still detectable, and RS3 is mildly reduced (Figures 3, A and B, and 4, A and B). A reduced electron density in specific areas of a subtomogram average is an indication that the individual repeats are not identical. Heterogeneity among the individual 96-nm repeats could be either caused by flexibility (i.e., the position of a structure varies between the individual repeats) or because a structure is absent in a subset of repeats. We used automatic image classification (Heumann et al., 2011) to sort the axonemal repeats into homogeneous subgroups before generating class averages. For wild type, we could not identify classes that differed in the structures of radial spokes (Figures 3A and 4A). When we classified for differences in the RS2 structure among the FAP206-KO subtomograms, the majority of axonemal repeats (83%) lacked RS2 entirely (except for the side prong; Figure 3, D and F), whereas the remaining 17% (FAP206-KO [+RS2] class) had a nearly completely assembled RS2 but lacked parts of the microtubule base (Figure 3, C and E). A comparison of the wild-type RS2 structure with the FAP206-KO (+RS2) class revealed a fairly normal stem and head of RS2, whereas the entire front prong and the closely associated dynein c were missing, and the back prong was reduced (compare Figures 2G and 3C, and 2C and 3E; also see Supplemental Movie S7). We estimated the molecular weight of the front prong density that is missing in the FAP206-KO (+RS2) class average as ∼80 kDa (red region in Figure 2, C, D, and K), which is consistent with the predicted (73 kDa) and observed molecular weight (80 kDa; see later discussion of Figure 7D) of FAP206. The simplest explanation of these observations is that FAP206 forms the front prong of RS2.

Bottom Line: A knockout of the conserved ciliary protein FAP206 in the ciliate Tetrahymena resulted in slow cell motility.Occasionally, RS2 assembled but lacked both the front prong of its microtubule base and dynein c, whose tail is attached to the front prong.Overexpressed GFP-FAP206 decorated nonciliary microtubules in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Biology, University of Georgia, Athens, GA 30602.

Show MeSH
Related in: MedlinePlus