Endogenous B-ring oxysterols inhibit the Hedgehog component Smoothened in a manner distinct from cyclopamine or side-chain oxysterols.

Cellular lipids are speculated to act as key intermediates in Hedgehog signal transduction, but their precise identity and function remain enigmatic. In an effort to identify such lipids, we pursued a Hedgehog pathway inhibitory activity that is particularly abundant in flagellar lipids of Chlamydomonas reinhardtii, resulting in the purification and identification of ergosterol endoperoxide, a B-ring oxysterol. A mammalian analog of ergosterol, 7-dehydrocholesterol (7-DHC), accumulates in Smith-Lemli-Opitz syndrome, a human genetic disease that phenocopies deficient Hedgehog signaling and is caused by genetic loss of 7-DHC reductase. We found that depleting endogenous 7-DHC with methyl-ß-cyclodextrin treatment enhances Hedgehog activation by a pathway agonist. Conversely, exogenous addition of 3ß,5α-dihydroxycholest-7-en-6-one, a naturally occurring B-ring oxysterol derived from 7-DHC that also accumulates in Smith-Lemli-Opitz syndrome, blocked Hedgehog signaling by inhibiting activation of the essential transduction component Smoothened, through a mechanism distinct from Smoothened modulation by other lipids.

Uni-directional ciliary membrane protein trafficking by a cytoplasmic retrograde IFT motor and ciliary ectosome shedding.

The role of the primary cilium in key signaling pathways depends on dynamic regulation of ciliary membrane protein composition, yet we know little about the motors or membrane events that regulate ciliary membrane protein trafficking in existing organelles. Recently, we showed that cilium-generated signaling in Chlamydomonas induced rapid, anterograde IFT-independent, cytoplasmic microtubule-dependent redistribution of the membrane polypeptide, SAG1-C65, from the plasma membrane to the periciliary region and the ciliary membrane. Here, we report that the retrograde IFT motor, cytoplasmic dynein 1b, is required in the cytoplasm for this rapid redistribution. Furthermore, signaling-induced trafficking of SAG1-C65 into cilia is unidirectional and the entire complement of cellular SAG1-C65 is shed during signaling and can be recovered in the form of ciliary ectosomes that retain signal-inducing activity. Thus, during signaling, cells regulate ciliary membrane protein composition through cytoplasmic action of the retrograde IFT motor and shedding of ciliary ectosomes.

Regulated membrane protein entry into flagella is facilitated by cytoplasmic microtubules and does not require IFT.

The membrane protein composition of the primary cilium, a key sensory organelle, is dynamically regulated during cilium-generated signaling [1, 2]. During ciliogenesis, ciliary membrane proteins, along with structural and signaling proteins, are carried through the multicomponent, intensely studied ciliary diffusion barrier at the base of the organelle [3-8] by intraflagellar transport (IFT) [9-18]. A favored model is that signaling-triggered accumulation of previously excluded membrane proteins in fully formed cilia [19-21] also requires IFT, but direct evidence is lacking. Here, in studies of regulated entry of a membrane protein into the flagellum of Chlamydomonas, we show that cells use an IFT-independent mechanism to breach the diffusion barrier at the flagellar base. In resting cells, a flagellar signaling component [22], the integral membrane polypeptide SAG1-C65, is uniformly distributed over the plasma membrane and excluded from the flagellar membrane. Flagellar adhesion-induced signaling triggers rapid, striking redistribution of the protein to the apical ends of the cells concomitantly with entry into the flagella. Protein polarization and flagellar enrichment are facilitated by cytoplasmic microtubules. Using a conditional anterograde IFT mutant, we demonstrate that the IFT machinery is not required for regulated SAG1-C65 entry into flagella. Thus, integral membrane proteins can negotiate passage through the ciliary diffusion barrier without the need for a motor.