Biochemical analyses of flagellar fractions of the BBS mutants revealed that several proteins associated with the flagellar membrane and matrix and likely to be involved in signal transduction are abnormally accumulated in the flagella; this apparently impairs the ability of the organelle to carry out phototactic steering because phototaxis is transiently restored tobbs4cells after the regeneration of new flagella

Biochemical analyses of flagellar fractions of the BBS mutants revealed that several proteins associated with the flagellar membrane and matrix and likely to be involved in signal transduction are abnormally accumulated in the flagella; this apparently impairs the ability of the organelle to carry out phototactic steering because phototaxis is transiently restored tobbs4cells after the regeneration of new flagella. hypogonadism, rodcone dystrophy, polydactyly, truncal obesity, and renal abnormalities (Katsanis et al., 2001;Tobin and Rabbit polyclonal to RABEPK Beales, 2007;Zaghloul and Katsanis, 2009). This combination of features partially overlaps with the pleiotropic phenotype of disorders caused by defective cilia, which is consistent with the hypothesis that cilia are involved in the etiology of BBS (Rosenbaum and Witman, 2002;Beales, 2005;Blacque and Leroux, 2006). Schaftoside In humans, 12 genes (BBS112) have been linked to BBS (Blacque and Leroux, 2006;Stoetzel et al., 2006;Stoetzel et al., 2007); the proteins (BBS1/2/4/5/7/8/9) encoded by 7 of these genes plus BBIP10 form a complex termed the BBSome (Nachury et al., 2007;Loktev et al., 2008). Experimental data from various species support a role for the BBSome in cilia (Tobin and Beales, 2007). Several BBSome proteins have been localized to Schaftoside basal bodies and cilia in mammalian cells (Ansley et al., 2003;Mykytyn and Sheffield, 2004) and have been shown to undergo intraflagellar transport (IFT) inCaenorhabditis elegans(Blacque et al., 2004). IFT is the bidirectional movement of protein particles consisting of IFT complex A and complex B driven by kinesin 2 (anterograde IFT) and cytoplasmic dynein 1b/2 (retrograde IFT) along the axoneme (Cole et al., 1998;Rosenbaum and Witman, 2002). IFT is required for flagellar assembly, maintenance, and signaling, and the loss of bona fide IFT components results in a failure to assemble cilia, which Schaftoside causes embryonic lethality in mice (Nonaka et al., 1998;Jonassen et al., 2008). In contrast, BBS1/2/4 knockout mice are viable, and cilia are at least initially assembled in the majority of tissues, with sperm flagella being an exception. Thus, the BBSome is dispensable for ciliary assembly in most cell types (Blacque et al., 2004;Kulaga et al., 2004;Mykytyn et al., 2004;Yen et al., 2006;Nachury et al., 2007;Loktev et al., 2008;Mukhopadhyay et al., 2008). The BBSome subunits are highly conserved and widely distributed among organisms with cilia, suggesting that the function of the BBSome has also been conserved. BBS proteins have been implicated in intracellular transport (Kim et al., 2004;Yen et al., 2006;Gerdes et al., 2007) and protein transport to the cilium and ciliary membrane (Nachury et al., 2007;Berbari et al., 2008;Shah et al., 2008). InC. elegans, BBS7 and -8 are thought to function in controlling IFT by maintaining an association of IFT complexes A and B (Blacque et al., 2004;Ou et al., 2005;Pan et al., 2006). Despite these advances, the functions of the BBSome in cilia remain unclear. This has been caused, in part, by the lack of a genetic model from which BBSome-defective cilia can be isolated and biochemically analyzed. In this study, we explore the function of the BBSome inChlamydomonas reinhardtii, which has advantages that allowed us to determine the structural, biochemical, and physiological consequences for cilia when BBSome function is impaired. We show thatC. reinhardtiiflagella contain a complex, including at least BBS1, -4, -5, -7, and -8, that is orthologous to the mammalian BBSome. Simultaneous tracking of BBS4-GFP and IFT20-mCherry in living cells by total internal reflection fluorescence (TIRF) microscopy (TIRFM) revealed that the BBSome is moved anterogradely and retrogradely in the Schaftoside flagellum in association with a subset of IFT particles. Consistent with this, we find that BBSome proteins are present in flagella at substoichiometric.