ARL3 Mediates BBSome Ciliary Turnover by Promoting Its Outward Diffusion through the Transition Zone

Ciliary receptors and their certain downstream signaling components undergo intraflagellar transport (IFT) as BBSome cargoes to maintain their ciliary dynamics for sensing and transducing extracellular stimuli inside the cell. Cargo laden BBSomes shed from retrograde IFT at the proximal ciliary region above the transition zone (TZ) followed by diffusing through the TZ for ciliary retrieval, while how the BBSome barrier passage is controlled remains elusive. Here, we show that the BBSome is a major effector of the Arf-like 3 (ARL3) GTPase in Chlamydomonas. Under physiological condition, ARL3GDP binds the membrane for diffusing into and residing in cilia. Following a nucleotide conversion, ARL3GTP dissociates with the ciliary membrane and binds and recruits the IFT-detached and cargo (phospholipase D, PLD)-laden BBSome at the proximal ciliary region to diffuse through the TZ and out of cilia. ARL3 deficiency impairs ciliary signaling, e.g. phototaxis of Chlamydomonas cells, by disrupting BBSome ciliary retrieval, providing a mechanistic understanding behind BBSome ciliary turnover required for ciliary signaling.

Ciliary Generation of a Peptidergic Sexual Signal

Peptidergic intercellular communication occurs throughout the eukaryotes, and regulates a wide range of physiological and behavioral responses. Cilia are sensory and secretory organelles that both receive information from the environment and transmit signals. Cilia derived vesicles (ectosomes), formed by outward budding of the ciliary membrane, carry enzymes and other bioactive products; this process represents an ancient mode of regulated secretion. Our previous study revealed the presence of the peptide amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM), in cilia and its key role in ciliogenesis. Furthermore, PAM and its amidated products are released in ciliary ectosomes from the green alga Chlamydomonas reinhardtii. One amidated product (GATI-amide) serves as a chemotactic modulator for C. reinhardtii gametes, attracting minus gametes while repelling plus gametes. Here we dissect the complex processing pathway that leads to formation of this amidated peptidergic sexual signal specifically on the ectosomes of plus gametes. We also identify a potential prohormone convertase that undergoes domain rearrangement during ectosomal secretion as a substrate for PAM. Analysis of this pathway affords insight into how single-celled organisms lacking dense core vesicles engage in regulated secretion, and provides a paradigm for understanding how amidated peptides that transmit sexual and other signals through cilia are generated.

A WDR35-dependent coat protein complex transports ciliary membrane cargo vesicles to cilia

Intraflagellar transport (IFT) is a highly conserved mechanism for motor-driven transport of cargo within cilia, but how this cargo is selectively transported to cilia is unclear. WDR35/IFT121 is a component of the IFT-A complex best known for its role in ciliary retrograde transport. In the absence of WDR35, small mutant cilia form but fail to enrich in diverse classes of ciliary membrane proteins. In Wdr35 mouse mutants, the non-core IFT-A components are degraded and core components accumulate at the ciliary base. We reveal deep sequence homology of WDR35 and other IFT-A subunits to α and ß' COPI coatomer subunits, and demonstrate an accumulation of 'coat-less' vesicles which fail to fuse with Wdr35 mutant cilia. We determine that recombinant non-core IFT-As can bind directly to lipids and provide the first in-situ evidence of a novel coat function for WDR35, likely with other IFT-A proteins, in delivering ciliary membrane cargo necessary for cilia elongation.

CCRK/CDK20 regulates ciliary retrograde protein trafficking via interacting with BROMI/TBC1D32

CCRK/CDK20 was reported to interact with BROMI/TBC1D32 and regulate ciliary Hedgehog signaling. In various organisms, mutations in the orthologs of CCRK and those of the kinase ICK/CILK1, which is phosphorylated by CCRK, are known to result in cilia elongation. Furthermore, we recently showed that ICK regulates retrograde ciliary protein trafficking and/or the turnaround event at the ciliary tips, and that its mutations result in the elimination of intraflagellar transport (IFT) proteins that have overaccumulated at the bulged ciliary tips as extracellular vesicles, in addition to cilia elongation. However, how these proteins cooperate to regulate ciliary protein trafficking has remained unclear. We here show that the phenotypes of CCRK-knockout (KO) cells closely resemble those of ICK-KO cells; namely, the overaccumulation of IFT proteins at the bulged ciliary tips, which appear to be eliminated as extracellular vesicles, and the enrichment of GPR161 and Smoothened on the ciliary membrane. The abnormal phenotypes of CCRK-KO cells were rescued by the exogenous expression of wild-type CCRK but not its kinase-dead mutant or a mutant defective in BROMI binding. These results together indicate that CCRK regulates the turnaround process at the ciliary tips in concert with BROMI and probably via activating ICK.

Bardet-Biedl syndrome proteins modulate the release of bioactive extracellular vesicles

Primary cilia are microtubule based sensory organelles important for receiving and processing cellular signals. Recent studies have shown that cilia also release extracellular vesicles (EVs). Because EVs have been shown to exert various physiological functions, these findings have the potential to alter our understanding of how primary cilia regulate specific signalling pathways. So far the focus has been on lgEVs budding directly from the ciliary membrane. An association between cilia and MVB-derived smEVs has not yet been described. We show that ciliary mutant mammalian cells demonstrate increased secretion of small EVs (smEVs) and a change in EV composition. Characterisation of smEV cargo identified signalling molecules that are differentially loaded upon ciliary dysfunction. Furthermore, we show that these smEVs are biologically active and modulate the WNT response in recipient cells. These results provide us with insights into smEV-dependent ciliary signalling mechanisms which might underly ciliopathy disease pathogenesis.

ARL3 and ARL13B GTPases participate in distinct steps of INPP5E targeting to the ciliary membrane

ABSTRACT INPP5E, a phosphoinositide 5-phosphatase, localizes on the ciliary membrane via its C-terminal prenyl moiety, and maintains the distinct ciliary phosphoinositide composition. The ARL3 GTPase contributes to the ciliary membrane localization of INPP5E by stimulating the release of PDE6D bound to prenylated INPP5E. Another GTPase, ARL13B, which is localized on the ciliary membrane, contributes to the ciliary membrane retention of INPP5E by directly binding to its ciliary targeting sequence. However, as ARL13B was shown to act as a guanine nucleotide exchange factor (GEF) for ARL3, it is also possible that ARL13B indirectly mediates the ciliary INPP5E localization via activating ARL3. We here show that INPP5E is delocalized from cilia in both ARL3-knockout (KO) and ARL13B-KO cells. However, some of the abnormal phenotypes were different between these KO cells, while others were found to be common, indicating the parallel roles of ARL3 and ARL13B, at least concerning some cellular functions. For several variants of ARL13B, their ability to interact with INPP5E, rather than their ability as an ARL3-GEF, was associated with whether they could rescue the ciliary localization of INPP5E in ARL13B-KO cells. These observations together indicate that ARL13B determines the ciliary localization of INPP5E, mainly by its direct binding to INPP5E.

Transient accumulation and bidirectional movement of KIF13B in primary cilia

Primary cilia are microtubule-based sensory organelles whose assembly and function rely on the conserved bidirectional intraflagellar transport (IFT) system, which is powered by anterograde kinesin-2 and retrograde cytoplasmic dynein 2 motors. Nematodes additionally employ a male-specific kinesin-3 motor, KLP-6, which regulates ciliary content and function by promoting release of bioactive extracellular vesicles (EVs) from cilia. Here we show by live cell imaging that a KLP-6 homolog, KIF13B, undergoes bursts of bidirectional movement within primary cilia of cultured mammalian cells at 0.64 +/- 0.07 μm/s in the anterograde direction and at 0.39 +/- 0.06 μm/s in the retrograde direction, reminiscent of conventional IFT. In addition, we found that KIF13B undergoes EV-like release from the ciliary tip whereas a ciliary membrane marker, SMO-tRFP, remains stably associated with cilia during such EV release. Our results suggest that KIF13B, similar to KLP-6, regulates ciliary membrane content by promoting ciliary EV release, possibly in coordination with conventional IFT.

Multi-dimensional and spatiotemporal correlative imaging at the plasma membrane of live cells to determine the continuum nano-to-micro scale lipid adaptation and collective motion

The primary cilium is a specialized plasma membrane protrusion with important receptors for signalling pathways. In polarized epithelial cells, the primary cilium assembles after the midbody remnant (MBR) encounters the centrosome at the apical surface. The membrane surrounding the MBR, namely remnant associated membrane patch (RAMP) once situated next to the centrosome, releases some of its lipid components to form a centrosome-associated membrane patch (CAMP) from which the ciliary membrane stems. The RAMP undergoes a spatiotemporal membrane refinement during the formation of the CAMP, which becomes highly enriched in condensed membranes with low lateral mobility. To better understand this process, we have developed a correlative imaging approach that yields quantitative information about the lipid lateral packing, its mobility and collective assembly at the plasma membrane at different spatial scales over time. Our work paves the way towards a quantitative understanding of lipid collective assembly at the plasma membrane spatiotemporally as a functional determinant in cell biology and its direct correlation with the membrane physicochemical state. These findings allowed us to gain a deeper insight into the mechanisms behind the biogenesis of the ciliary membrane of polarized epithelial cells.

Ca2+ elevations disrupt interactions between intraflagellar transport and the flagella membrane in Chlamydomonas

ABSTRACTThe movement of ciliary membrane proteins is directed by transient interactions with intraflagellar transport (IFT) trains. The green alga Chlamydomonas has adapted this process for gliding motility, using retrograde IFT motors to move adhesive glycoproteins in the flagella membrane. Ca2+ signalling contributes directly to the gliding process, although uncertainty remains over the mechanism through which it acts. Here, we show that flagella Ca2+ elevations initiate the movement of paused retrograde IFT trains, which accumulate at the distal end of adherent flagella, but do not influence other IFT processes. On highly adherent surfaces, flagella exhibit high-frequency Ca2+ elevations that prevent the accumulation of paused retrograde IFT trains. Flagella Ca2+ elevations disrupt the IFT-dependent movement of microspheres along the flagella membrane, suggesting that Ca2+ acts by directly disrupting an interaction between retrograde IFT trains and flagella membrane glycoproteins. By regulating the extent to which glycoproteins on the flagella surface interact with IFT motor proteins on the axoneme, this signalling mechanism allows precise control of traction force and gliding motility in adherent flagella.