Deciphering vesicle-assisted transport mechanisms in cytoplasm to cilium trafficking.

The cilium, a pivotal organelle crucial for cell signaling and proper cell function, relies on meticulous macromolecular transport from the cytoplasm for its formation and maintenance. While the intraflagellar transport (IFT) pathway has traditionally been the focus of extensive study concerning ciliogenesis and ciliary maintenance, recent research highlights a complementary and alternative mechanism-vesicle-assisted transport (VAT) in cytoplasm to cilium trafficking. Despite its potential significance, the VAT pathway remains largely uncharacterized. This review explores recent studies providing evidence for the dynamics of vesicle-related diffusion and transport within the live primary cilium, employing high-speed super-resolution light microscopy. Additionally, we analyze the spatial distribution of vesicles in the cilium, mainly relying on electron microscopy data. By scrutinizing the VAT pathways that facilitate cargo transport into the cilium, with a specific emphasis on recent advancements and imaging data, our objective is to synthesize a comprehensive model of ciliary transport through the integration of IFT-VAT mechanisms.

The Clinical and Mutational Spectrum of Bardet-Biedl Syndrome in Saudi Arabia.

The retinal features of Bardet-Biedl syndrome (BBS) are insufficiently characterized in Arab populations. This retrospective study investigated the retinal features and genotypes of BBS in Saudi patients managed at a single tertiary eye care center. Data analysis of the identified 46 individuals from 31 families included visual acuity (VA), systemic manifestations, multimodal retinal imaging, electroretinography (ERG), family pedigrees, and genotypes. Patients were classified to have cone-rod, rod-cone, or generalized photoreceptor dystrophy based on the pattern of macular involvement on the retinal imaging. Results showed that nyctalopia and subnormal VA were the most common symptoms with 76% having VA ≤ 20/200 at the last visit (age: 5-35). Systemic features included obesity 91%, polydactyly 56.5%, and severe cognitive impairment 33%. The predominant retinal phenotype was cone-rod dystrophy 75%, 10% had rod-cone dystrophy and 15% had generalized photoreceptor dystrophy. ERGs were undetectable in 95% of patients. Among the 31 probands, 61% had biallelic variants in BBSome complex genes, 32% in chaperonin complex genes, and 6% had biallelic variants in ARL6; including six previously unreported variants. Interfamilial and intrafamilial variabilities were noted, without a clear genotype-phenotype correlation. Most BBS patients had advanced retinopathy and were legally blind by early adulthood, indicating a narrow therapeutic window for rescue strategies.

RABL2 promotes the outward transition zone passage of signaling proteins in cilia via ARL3.

Certain transmembrane and membrane-tethered signaling proteins export from cilia as BBSome cargoes via the outward BBSome transition zone (TZ) diffusion pathway, indispensable for maintaining their ciliary dynamics to enable cells to sense and transduce extracellular stimuli inside the cell. Murine Rab-like 2 (Rabl2) GTPase resembles Chlamydomonas Arf-like 3 (ARL3) GTPase in promoting outward TZ passage of the signaling protein cargo-laden BBSome. During this process, ARL3 binds to and recruits the retrograde IFT train-dissociated BBSome as its effector to diffuse through the TZ for ciliary retrieval, while how RABL2 and ARL3 cross talk in this event remains uncertain. Here, we report that Chlamydomonas RABL2 in a GTP-bound form (RABL2GTP) cycles through cilia via IFT as an IFT-B1 cargo, dissociates from retrograde IFT trains at a ciliary region right above the TZ, and converts to RABL2GDP for activating ARL3GDP as an ARL3 guanine nucleotide exchange factor. This confers ARL3GTP to detach from the ciliary membrane and become available for binding and recruiting the phospholipase D (PLD)-laden BBSome, autonomous of retrograde IFT association, to diffuse through the TZ for ciliary retrieval. Afterward, RABL2GDP exits cilia by being bound to the ARL3GTP/BBSome entity as a BBSome cargo. Our data identify ciliary signaling proteins exported from cilia via the RABL2-ARL3 cascade-mediated outward BBSome TZ diffusion pathway. According to this model, hedgehog signaling defect-induced Bardet-Biedl syndrome caused by RABL2 mutations in humans could be well explained in a mutation-specific manner, providing us with a mechanistic understanding behind the outward BBSome TZ passage required for proper ciliary signaling.

Changes in expression of mesothelial BBS genes in 2D and 3D after lithium chloride and ammonium sulphate induction of primary cilium disturbance: a pilot study.

BACKGROUND: Malignant pleural mesothelioma (MPM), a rare and aggressive pleural tumor, has significant histological and molecular heterogeneity. Primary Cilium (PC), an organelle of emerging importance in malignancies, has been scarcely investigated in MPM. A critical molecular complex for the PC function is the BBSome and here we aimed at assessing its expression patterns in ordinary 2D and spheroid 3D cell cultures. METHODS: A human benign mesothelial cell line (MeT-5A), MPM cell lines (M14K, epithelioid MPM; MSTO, biphasic MPM), and primary MPM cells (pMPM) were used. Primers specific for the human BBS1, 2, 4, 5, 7, 9, 18 transcripts were designed, and quantitative real-time PCR (qRT-PCR) was done with ß-actin as the gene of reference. The relative gene expression across 2D and 3D cultures was analyzed by the expression factor (mean of 1/ΔCt values). With the 2-∆∆Ct method the gene expression fold changes were assessed from qRT-PCR data. Molecular changes using the PC-modulating drugs ammonium sulfate (AS) and lithium chloride (LC) were also determined. RESULTS: PC was present in all cells used in the study at approximately 15% of the observed area. BBSome transcripts were differentially expressed in different dimensions of cell culture (2D vs. 3D) in all cell lines and pMPM. Treatment with AS and LC affected the expression of the ciliary BBS2 and BBS18 genes in the benign as well as in the MPM cells. CONCLUSIONS: These data indicate distinct BBSome molecular profiles in human benign and MPM cells cultured in 2D and 3D dimensions and support the notion that PC genes should be investigated as potential MPM therapeutic targets.

Organization, functions, and mechanisms of the BBSome in development, ciliopathies, and beyond.

The BBSome is an octameric protein complex that regulates ciliary transport and signaling. Mutations in BBSome subunits are closely associated with ciliary defects and lead to ciliopathies, notably Bardet-Biedl syndrome. Over the past few years, there has been significant progress in elucidating the molecular organization and functions of the BBSome complex. An improved understanding of BBSome-mediated biological events and molecular mechanisms is expected to help advance the development of diagnostic and therapeutic approaches for BBSome-related diseases. Here, we review the current literature on the structural assembly, transport regulation, and molecular functions of the BBSome, emphasizing its roles in cilium-related processes. We also provide perspectives on the pathological role of the BBSome in ciliopathies as well as how these can be exploited for therapeutic benefit.

Seriously cilia: A tiny organelle illuminates evolution, disease, and intercellular communication.

The borders between cell and developmental biology, which have always been permeable, have largely dissolved. One manifestation is the blossoming of cilia biology, with cell and developmental approaches (increasingly complemented by human genetics, structural insights, and computational analysis) fruitfully advancing understanding of this fascinating, multifunctional organelle. The last eukaryotic common ancestor probably possessed a motile cilium, providing evolution with ample opportunity to adapt cilia to many jobs. Over the last decades, we have learned how non-motile, primary cilia play important roles in intercellular communication. Reflecting their diverse motility and signaling functions, compromised cilia cause a diverse range of diseases collectively called "ciliopathies." In this review, we highlight how cilia signal, focusing on how second messengers generated in cilia convey distinct information; how cilia are a potential source of signals to other cells; how evolution may have shaped ciliary function; and how cilia research may address thorny outstanding questions.

Neuronal primary cilia integrate peripheral signals with metabolic drives.

Neuronal primary cilia have recently emerged as important contributors to the central regulation of energy homeostasis. As non-motile, microtubule-based organelles, primary cilia serve as signaling antennae for metabolic status. The impairment of ciliary structure or function can produce ciliopathies for which obesity is a hallmark phenotype and global ablation of cilia induces non-syndromic adiposity in mouse models. This organelle is not only a hub for metabolic signaling, but also for catecholamine neuromodulation that shapes neuronal circuitry in response to sensory input. The objective of this review is to highlight current research investigating the mechanisms of primary cilium-regulated metabolic drives for maintaining energy homeostasis.

The ancestral ESCRT protein TOM1L2 selects ubiquitinated cargoes for retrieval from cilia.

Many G protein-coupled receptors (GPCRs) reside within cilia of mammalian cells and must undergo regulated exit from cilia for the appropriate transduction of signals such as hedgehog morphogens. Lysine 63-linked ubiquitin (UbK63) chains mark GPCRs for regulated removal from cilia, but the molecular basis of UbK63 recognition inside cilia remains elusive. Here, we show that the BBSome-the trafficking complex in charge of retrieving GPCRs from cilia-engages the ancestral endosomal sorting factor target of Myb1-like 2 (TOM1L2) to recognize UbK63 chains within cilia of human and mouse cells. TOM1L2 directly binds to UbK63 chains and the BBSome, and targeted disruption of the TOM1L2/BBSome interaction results in the accumulation of TOM1L2, ubiquitin, and the GPCRs SSTR3, Smoothened, and GPR161 inside cilia. Furthermore, the single-cell alga Chlamydomonas also requires its TOM1L2 ortholog in order to clear ubiquitinated proteins from cilia. We conclude that TOM1L2 broadly enables the retrieval of UbK63-tagged proteins by the ciliary trafficking machinery.

Unraveling the intricate cargo-BBSome coupling mechanism at the ciliary tip.

Certain ciliary transmembrane and membrane-tethered signaling proteins migrate from the ciliary tip to base via retrograde intraflagellar transport (IFT), essential for maintaining their ciliary dynamics to enable cells to sense and transduce extracellular stimuli inside the cell. During this process, the BBSome functions as an adaptor between retrograde IFT trains and these signaling protein cargoes. The Arf-like 13 (ARL13) small GTPase resembles ARL6/BBS3 in facilitating these signaling cargoes to couple with the BBSome at the ciliary tip prior to loading onto retrograde IFT trains for transporting towards the ciliary base, while the molecular basis for how this intricate coupling event happens remains elusive. Here, we report that Chlamydomonas ARL13 only in a GTP-bound form (ARL13GTP) anchors to the membrane for diffusing into cilia. Upon entering cilia, ARL13 undergoes GTPase cycle for shuttling between the ciliary membrane (ARL13GTP) and matrix (ARL13GDP). To achieve this goal, the ciliary membrane-anchored BBS3GTP binds the ciliary matrix-residing ARL13GDP to activate the latter as an ARL13 guanine nucleotide exchange factor. At the ciliary tip, ARL13GTP recruits the ciliary matrix-residing and post-remodeled BBSome as an ARL13 effector to anchor to the ciliary membrane. This makes the BBSome spatiotemporally become available for the ciliary membrane-tethered phospholipase D (PLD) to couple with. Afterward, ARL13GTP hydrolyzes GTP for releasing the PLD-laden BBSome to load onto retrograde IFT trains. According to this model, hedgehog signaling defects associated with ARL13b and BBS3 mutations in humans could be satisfactorily explained, providing us a mechanistic understanding behind BBSome-cargo coupling required for proper ciliary signaling.

Ubiquitylation of BBSome is required for ciliary assembly and signaling.

Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by retinal degeneration, obesity, renal abnormalities, postaxial polydactyly, and developmental defects. Genes mutated in BBS encode for components and regulators of the BBSome, an octameric complex that controls the trafficking of cargos and receptors within the primary cilium. Although both structure and function of the BBSome have been extensively studied, the impact of ubiquitin signaling on BBSome is largely unknown. We identify the E3 ubiquitin ligase PJA2 as a novel resident of the ciliary compartment and regulator of the BBSome. Upon GPCR-cAMP stimulation, PJA2 ubiquitylates BBSome subunits. We demonstrate that ubiquitylation of BBS1 at lysine 143 increases the stability of the BBSome and promotes its binding to BBS3, an Arf-like GTPase protein controlling the targeting of the BBSome to the ciliary membrane. Downregulation of PJA2 or expression of a ubiquitylation-defective BBS1 mutant (BBS1K143R ) affects the trafficking of G-protein-coupled receptors (GPCRs) and Shh-dependent gene transcription. Expression of BBS1K143R in vivo impairs cilium formation, embryonic development, and photoreceptors' morphogenesis, thus recapitulating the BBS phenotype in the medaka fish model.

RABL4/IFT27 in a nucleotide-independent manner promotes phospholipase D ciliary retrieval via facilitating BBSome reassembly at the ciliary tip.

Certain ciliary transmembrane and membrane-associated signaling proteins export from cilia as intraflagellar transport (IFT) cargoes in a BBSome-dependent manner. Upon reaching the ciliary tip via anterograde IFT, the BBSome disassembles before being reassembled to form an intact entity for cargo phospholipase D (PLD) coupling. During this BBSome remodeling process, Chlamydomonas Rab-like 4 GTPase IFT27, by binding its partner IFT25 to form the heterodimeric IFT25/27, is indispensable for BBSome reassembly. Here, we show that IFT27 binds IFT25 in an IFT27 nucleotide-independent manner. IFT25/27 and the IFT subcomplexes IFT-A and -B are irrelevant for maintaining the stability of one another. GTP-loading onto IFT27 enhances the IFT25/27 affinity for binding to the IFT-B subcomplex core IFT-B1 entity in cytoplasm, while GDP-bound IFT27 does not prevent IFT25/27 from entering and cycling through cilia by integrating into IFT-B1. Upon at the ciliary tip, IFT25/27 cycles on and off IFT-B1 and this process is irrelevant with the nucleotide state of IFT27. During BBSome remodeling at the ciliary tip, IFT25/27 promotes BBSome reassembly independent of IFT27 nucleotide state, making postremodeled BBSomes available for PLD to interact with. Thus, IFT25/27 facilitates BBSome-dependent PLD export from cilia via controlling availability of intact BBSomes at the ciliary tip, while IFT27 nucleotide state does not participate in this regulatory event.

T cell-specific deficiency in BBSome component BBS1 interferes with selective immune responses.

Bsardet Biedl syndrome (BBS) is a genetic condition associated with various clinical features including cutaneous disorders and certain autoimmune and inflammatory diseases pointing to a potential role of BBS proteins in the regulation of immune function. BBS1 protein, which is a key component of the BBSome, a protein complex involved in the regulation of cilia function and other cellular processes, has been implicated in the immune synapse assembly by promoting the centrosome polarization to the antigen-presenting cells. Here, we assessed the effect of disrupting the BBSome, through Bbs1 gene deletion, in T cells. Interestingly, mice lacking the Bbs1 gene specifically in T cells (T-BBS1-/-) displayed normal body weight, adiposity, and glucose handling, but have smaller spleens. However, T-BBS1-/- mice had no change in the proportion and absolute number of B cells and T cells in the spleen and lymph nodes. There was also no alteration in the CD4/CD8 lineage commitment or survival in the thymus of T-BBS1-/- mice. On the other hand, T-BBS1-/- mice treated with Imiquimod dermally exhibited a significantly higher percentage of CD3-positive splenocytes that was due to CD4 but not CD8 T cell predominance. Notably, we found that T-BBS1-/- mice had significantly decreased wound closure, an effect that was more pronounced in males indicating that the BBSome plays an important role in T cell-mediated skin repair. Together, these findings implicate the BBSome in the regulation of selective functions of T cells.

Cargo adapters expand the transport range of intraflagellar transport.

The assembly and maintenance of most cilia and eukaryotic flagella depends on intraflagellar transport (IFT), the bidirectional movement of multi-megadalton IFT trains along the axonemal microtubules. These IFT trains function as carriers, moving ciliary proteins between the cell body and the organelle. Whereas tubulin, the principal protein of cilia, binds directly to IFT particle proteins, the transport of other ciliary proteins and complexes requires adapters that link them to the trains. Large axonemal substructures, such as radial spokes, outer dynein arms and inner dynein arms, assemble in the cell body before attaching to IFT trains, using the adapters ARMC2, ODA16 and IDA3, respectively. Ciliary import of several membrane proteins involves the putative adapter tubby-like protein 3 (TULP3), whereas membrane protein export involves the BBSome, an octameric complex that co-migrates with IFT particles. Thus, cells employ a variety of adapters, each of which is substoichiometric to the core IFT machinery, to expand the cargo range of the IFT trains. This Review summarizes the individual and shared features of the known cargo adapters and discusses their possible role in regulating the transport capacity of the IFT pathway.

A cilia-independent function of BBSome mediated by DLK-MAPK signaling in C. elegans photosensation.

Bardet-Biedl syndrome (BBS) is a genetic disorder that affects primary cilia. BBSome, a protein complex composed of eight BBS proteins, regulates the structure and function of cilia, and its malfunction causes BBS in humans. Here, we report a cilia-independent function of BBSome. To identify genes that regulate the C. elegans photoreceptor protein LITE-1 in ciliated ASH photosensory neurons, we performed a genetic screen and isolated bbs mutants. Functional analysis revealed that BBSome regulates LITE-1 protein stability independently of cilia. Through another round of genetic screening, we found that this cilia-independent function of BBSome is mediated by DLK-MAPK signaling, which acts downstream of BBSome to control LITE-1 stability via Rab5-mediated endocytosis. BBSome exerts its function by regulating the expression of DLK. BBSome also regulates the expression of LZK, a mammalian DLK in human cells. These studies identify a cilia-independent function of BBSome and uncover DLK as an evolutionarily conserved BBSome effector.

Rab-like small GTPases in the regulation of ciliary Bardet-Biedl syndrome (BBS) complex transport.

Primary cilia, microtubule-based hair-like structures protruding from most cells, contain membranes enriched in signaling molecules and function as sensory and regulatory organelles critical for development and tissue homeostasis. Intraflagellar transport (IFT), cilia-specific bidirectional transport, is required for the assembly, maintenance, and function of cilia. BBSome, the coat complex, acts as the adaptor between the IFT complex and membrane proteins and is therefore essential for establishing the specific compartmentalization of signaling molecules in the cilia. Recent findings have revealed that three ciliary Rab-like small GTPases, IFT27, IFT22, and Rabl2, play critical regulatory roles in ciliary BBSome transport. In this review, we provide an overview of these three Rab-like small GTPases and their relationship with BBSome.

Loss of the ciliary gene Bbs4 results in defective thermogenesis due to metabolic inefficiency and impaired lipid metabolism.

Adipose tissue is central to the regulation of energy balance. While white adipose tissue (WAT) is responsible for triglyceride storage, brown adipose tissue specializes in energy expenditure. Deterioration of brown adipocyte function contributes to the development of metabolic complications like obesity and diabetes. These disorders are also leading symptoms of the Bardet-Biedl syndrome (BBS), a hereditary disorder in humans which is caused by dysfunctions of the primary cilium and which therefore belongs to the group of ciliopathies. The cilium is a hair-like organelle involved in cellular signal transduction. The BBSome, a supercomplex of several Bbs gene products, localizes to the basal body of cilia and is thought to be involved in protein sorting to and from the ciliary membrane. The effects of a functional BBSome on energy metabolism and lipid mobilization in brown and white adipocytes were tested in whole-body Bbs4 knockout mice that were subjected to metabolic challenges. Chronic cold exposure reveals cold-intolerance of knockout mice but also ameliorates the markers of metabolic pathology detected in knockouts prior to cold. Hepatic triglyceride content is markedly reduced in knockout mice while circulating lipids are elevated, altogether suggesting that defective lipid metabolism in adipose tissue creates increased demand for systemic lipid mobilization to meet energetic demands of reduced body temperatures. These findings taken together suggest that Bbs4 is essential for the regulation of adipose tissue lipid metabolism, representing a potential target to treat metabolic disorders.

In vivo imaging shows continued association of several IFT-A, IFT-B and dynein complexes while IFT trains U-turn at the tip.

Flagellar assembly depends on intraflagellar transport (IFT), a bidirectional motility of protein carriers, the IFT trains. The trains are periodic assemblies of IFT-A and IFT-B subcomplexes and the motors kinesin-2 and IFT dynein. At the tip, anterograde trains are remodeled for retrograde IFT, a process that in Chlamydomonas involves kinesin-2 release and train fragmentation. However, the degree of train disassembly at the tip remains unknown. Here, we performed two-color imaging of fluorescent protein-tagged IFT components, which indicates that IFT-A and IFT-B proteins from a given anterograde train usually return in the same set of retrograde trains. Similarly, concurrent turnaround was typical for IFT-B proteins and the IFT dynein subunit D1bLIC-GFP but severance was observed as well. Our data support a simple model of IFT turnaround, in which IFT-A, IFT-B and IFT dynein typically remain associated at the tip and segments of the anterograde trains convert directly into retrograde trains. Continuous association of IFT-A, IFT-B and IFT dynein during tip remodeling could balance protein entry and exit, preventing the build-up of IFT material in flagella.

Chlamydomonas LZTFL1 mediates phototaxis via controlling BBSome recruitment to the basal body and its reassembly at the ciliary tip.

Many G protein-coupled receptors and other signaling proteins localize to the ciliary membrane for regulating diverse cellular processes. The BBSome composed of multiple Bardet-Biedl syndrome (BBS) proteins is an intraflagellar transport (IFT) cargo adaptor essential for sorting signaling proteins in and/or out of cilia via IFT. Leucine zipper transcription factor-like 1 (LZTFL1) protein mediates ciliary signaling by controlling BBSome ciliary content, reflecting how LZTFL1 mutations could cause BBS. However, the mechanistic mechanism underlying this process remains elusive thus far. Here, we show that LZTFL1 maintains BBSome ciliary dynamics by finely controlling BBSome recruitment to the basal body and its reassembly at the ciliary tip simultaneously in Chlamydomonas reinhardtii LZTFL1 directs BBSome recruitment to the basal body via promoting basal body targeting of Arf-like 6 GTPase BBS3, thus deciding the BBSome amount available for loading onto anterograde IFT trains for entering cilia. Meanwhile, LZTFL1 stabilizes the IFT25/27 component of the IFT-B1 subcomplex in the cell body so as to control its presence and amount at the basal body for entering cilia. Since IFT25/27 promotes BBSome reassembly at the ciliary tip for loading onto retrograde IFT trains, LZTFL1 thus also directs BBSome removal out of cilia. Therefore, LZTFL1 dysfunction deprives the BBSome of ciliary presence and generates Chlamydomonas cells defective in phototaxis. In summary, our data propose that LZTFL1 maintains BBSome dynamics in cilia by such a dual-mode system, providing insights into how LZTFL1 mediates ciliary signaling through maintaining BBSome ciliary dynamics and the pathogenetic mechanism of the BBS disorder as well.

Endothelial BBSome is essential for vascular, metabolic, and retinal functions.

OBJECTIVES: Endothelial cells that line the entire vascular system play a pivotal role in the control of various physiological processes, including metabolism. Additionally, endothelial dysfunction is associated with many pathological conditions, including obesity. Here, we assessed the role of the BBSome, a protein complex composed of eight Bardet-Biedl syndrome (BBS) proteins in endothelial cells. METHODS: We studied the effects of BBSome disruption in endothelial cells on vascular function, body weight, glucose homeostasis, and the liver and retina. For this, we generated mice with selective BBSome disruption in endothelial cells through Bbs1 gene deletion. RESULTS: We found that endothelial cell-specific BBSome disruption causes endothelial dysfunction, as indicated by the impaired acetylcholine-induced vasorelaxation in both the aorta and mesenteric artery. This was associated with an increase in the contractile response to thromboxane A2 receptor agonist (U46619) in the mesenteric artery. Mechanistically, we demonstrated that mice lacking the Bbs1 gene in endothelial cells show elevated vascular angiotensinogen gene expression, implicating renin-angiotensin system activation in the vascular changes evoked by endothelial BBSome deficiency. Strikingly, our data indicate that endothelial BBSome deficiency increases body weight and fat mass and causes hepatosteatosis along with alterations in hepatic expression of lipid metabolism-related genes and metabolomics profile. In addition, electroretinogram and optical coherence tomography analyses revealed functional and structural abnormalities in the retina, evoked by absence of the endothelial BBSome. CONCLUSIONS: Our findings demonstrate that the BBSome in endothelial cells is required for the regulation of vascular function, adiposity, hepatic lipid metabolism, and retinal function.

Bardet-Biedl syndrome 3 protein promotes ciliary exit of the signaling protein phospholipase D via the BBSome.

Certain ciliary signaling proteins couple with the BBSome, a conserved complex of Bardet-Biedl syndrome (BBS) proteins, to load onto retrograde intraflagellar transport (IFT) trains for their removal out of cilia in Chlamydomonas reinhardtii. Here, we show that loss of the Arf-like 6 (ARL6) GTPase BBS3 causes the signaling protein phospholipase D (PLD) to accumulate in cilia. Upon targeting to the basal body, BBSomes enter and cycle through cilia via IFT, while BBS3 in a GTP-bound state separates from BBSomes, associates with the membrane, and translocates from the basal body to cilia by diffusion. Upon arriving at the ciliary tip, GTP-bound BBS3 binds and recruits BBSomes to the ciliary membrane for interacting with PLD, thus making the PLD-laden BBSomes available to load onto retrograde IFT trains for ciliary exit. Therefore, BBS3 promotes PLD exit from cilia via the BBSome, providing a regulatory mechanism for ciliary signaling protein removal out of cilia.