Molecular and structural perspectives on protein trafficking to the primary cilium membrane.

The primary cilium is a dynamic subcellular compartment templated from the mother centriole or basal body. Cilia are solitary and tiny, but remarkably consequential in cellular pathways regulating proliferation, differentiation, and maintenance. Multiple transmembrane proteins such as G-protein-coupled receptors, channels, enzymes, and membrane-associated lipidated proteins are enriched in the ciliary membrane. The precise regulation of ciliary membrane content is essential for effective signal transduction and maintenance of tissue homeostasis. Surprisingly, a few conserved molecular factors, intraflagellar transport complex A and the tubby family adapter protein TULP3, mediate the transport of most membrane cargoes into cilia. Recent advances in cryogenic electron microscopy provide fundamental insights into these molecular players. Here, we review the molecular players mediating cargo delivery into the ciliary membrane through the lens of structural biology. These mechanistic insights into ciliary transport provide a framework for understanding of disease variants in ciliopathies, enable precise manipulation of cilia-mediated pathways, and provide a platform for the development of targeted therapeutics.

Selective targeting of mu opioid receptors to primary cilia.

Opioid receptors are therapeutically important G protein-coupled receptors (GPCRs) with diverse neuromodulatory effects. The functional consequences of opioid receptor activation are known to depend on receptor location in the plasma membrane, but mechanisms mediating selective localization of receptors to any particular membrane domain remain elusive. Here, we demonstrate the targeting of the mu opioid receptor (MOR) to the primary cilium, a discrete microdomain of the somatic plasma membrane, both in vivo and in cultured cells. We further show that ciliary targeting is specific to MORs, requires a 17-residue sequence unique to the MOR cytoplasmic tail, and additionally requires the Tubby-like protein 3 (TULP3) ciliary adaptor protein. Our results reveal the potential for opioid receptors to undergo selective localization to the primary cilium. We propose that ciliary targeting is mediated through an elaboration of the recycling pathway, directed by a specific C-terminal recycling sequence in cis and requiring TULP3 in trans.

Distribution of ciliary adaptor proteins tubby and TULP3 in the organ of Corti.

Tubby-like proteins are membrane-associated adaptors that mediate directional trafficking into primary cilia. In inner ear sensory epithelia, cilia-including the hair cell's kinocilium-play important roles as organizers of polarity, tissue architecture and cellular function. However, auditory dysfunction in tubby mutant mice was recently found to be related to a non-ciliary function of tubby, the organization of a protein complex in sensory hair bundles of auditory outer hair cells (OHCs). Targeting of signaling components into cilia in the cochlea might therefore rather rely on closely related tubby-like proteins (TULPs). In this study, we compared cellular and subcellular localization of tubby and TULP3 in the mouse inner ear sensory organs. Immunofluorescence microscopy confirmed the previously reported highly selective localization of tubby in the stereocilia tips of OHCs and revealed a previously unnoticed transient localization to kinocilia during early postnatal development. TULP3 was detected in the organ of Corti and vestibular sensory epithelium, where it displayed a complex spatiotemporal pattern. TULP3 localized to kinocilia of cochlear and vestibular hair cells in early postnatal development but disappeared subsequently before the onset of hearing. This pattern suggested a role in targeting ciliary components into kinocilia, possibly related to the developmental processes that shape the sensory epithelia. Concurrent with loss from kinocilia, pronounced TULP3 immunolabeling progressively appeared at microtubule bundles in non-sensory Pillar (PCs) and Deiters cells (DC). This subcellular localization may indicate a novel function of TULP proteins associated with the formation or regulation of microtubule-based cellular structures.

IFT-A structure reveals carriages for membrane protein transport into cilia.

Intraflagellar transport (IFT) trains are massive molecular machines that traffic proteins between cilia and the cell body. Each IFT train is a dynamic polymer of two large complexes (IFT-A and -B) and motor proteins, posing a formidable challenge to mechanistic understanding. Here, we reconstituted the complete human IFT-A complex and obtained its structure using cryo-EM. Combined with AlphaFold prediction and genome-editing studies, our results illuminate how IFT-A polymerizes, interacts with IFT-B, and uses an array of ß-propeller and TPR domains to create "carriages" of the IFT train that engage TULP adaptor proteins. We show that IFT-A⋅TULP carriages are essential for cilia localization of diverse membrane proteins, as well as ICK-the key kinase regulating IFT train turnaround. These data establish a structural link between IFT-A's distinct functions, provide a blueprint for IFT-A in the train, and shed light on how IFT evolved from a proto-coatomer ancestor.

TULP3 silencing suppresses cell proliferation, migration and invasion in gastric cancer via the PTEN/Akt/Snail pathway.

BACKGROUND: Tubby-like protein 3 (TULP3) is a member of the tubby family, has been related to the development of nervous system by gene knockout researches. Nevertheless, the role of TULP3 in the gastric cancer is not clear. METHODS: Western blotting and real-time polymerase chain reaction (PCR) were employed for the quantitative detection of TULP3 expression in the gastric cancer and consecutive non-cancerous tissues, and gastric cancer cells. The roles of TULP3 in invasion, migration as well as proliferation of the gastric cancer cell in vivo and in vitro through utilizing colony formation, MTT, wound-healing, transwell and mouse xenograft model. Western blotting assay was implemented in order to clarify the potential molecular mechanisms. Furthermore, electron microscopy and western blot were evaluated TULP3 expression in gastric cancer patient extracted serum exosomes. RESULTS: TULP3 expression levels were remarkably upregulated in the gastric cancer tissues and cells. Subsequent functional assays demonstrated that TULP3 downregulation suppressed invasion, migration as well as the proliferation of the gastric cancer cell. Mechanism assays depicted that the PTEN/Akt/Snail signaling pathway can inhibit invasion, migration as well as the proliferation of the gastric cancer cell via TULP3 silencing. Finally, we found that the expression of TULP3 could be determined in the extracted serum exosomes. The expression of TULP3 in gastric cancer group was higher in comparison with normal group. CONCLUSIONS: Our results reveal that TULP3 might serve as a potential prognostic biomarker and therapeutic target for the treatment of gastric cancer.

HTR6 and SSTR3 targeting to primary cilia.

Primary cilia are hair-like projections of the cell membrane supported by an inner microtubule scaffold, the axoneme, which polymerizes out of a membrane-docked centriole at the ciliary base. By working as specialized signaling compartments, primary cilia provide an optimal environment for many G protein-coupled receptors (GPCRs) and their effectors to efficiently transmit their signals to the rest of the cell. For this to occur, however, all necessary receptors and signal transducers must first accumulate at the ciliary membrane. Serotonin receptor 6 (HTR6) and Somatostatin receptor 3 (SSTR3) are two GPCRs whose signaling in brain neuronal cilia affects cognition and is implicated in psychiatric, neurodegenerative, and oncologic diseases. Over a decade ago, the third intracellular loops (IC3s) of HTR6 and SSTR3 were shown to contain ciliary localization sequences (CLSs) that, when grafted onto non-ciliary GPCRs, could drive their ciliary accumulation. Nevertheless, these CLSs were dispensable for ciliary targeting of HTR6 and SSTR3, suggesting the presence of additional CLSs, which we have recently identified in their C-terminal tails. Herein, we review the discovery and mapping of these CLSs, as well as the state of the art regarding how these CLSs may orchestrate ciliary accumulation of these GPCRs by controlling when and where they interact with the ciliary entry and exit machinery via adaptors such as TULP3, RABL2 and the BBSome.

A conserved acetylation switch enables pharmacological control of tubby-like protein stability.

Tubby-like proteins (TULPs) are characterized by a conserved C-terminal domain that binds phosphoinositides. Collectively, mammalian TULP1-4 proteins play essential roles in intracellular transport, cell differentiation, signaling, and motility. Yet, little is known about how the function of these proteins is regulated in cells. Here, we present the protein-protein interaction network of TULP3, a protein that is responsible for the trafficking of G-protein-coupled receptors to cilia and whose aberrant expression is associated with severe developmental disorders and polycystic kidney disease. We identify several protein interaction nodes linked to TULP3 that include enzymes involved in acetylation and ubiquitination. We show that acetylation of two key lysine residues on TULP3 by p300 increases TULP3 protein abundance and that deacetylation of these sites by HDAC1 decreases protein levels. Furthermore, we show that one of these sites is ubiquitinated in the absence of acetylation and that acetylation inversely correlates with ubiquitination of TULP3. This mechanism is evidently conserved across species and is active in zebrafish during development. Finally, we identify this same regulatory module in TULP1, TULP2, and TULP4 and demonstrate that the stability of these proteins is similarly modulated by an acetylation switch. This study unveils a signaling pathway that links nuclear enzymes to ciliary membrane receptors via TULP3, describes a dynamic mechanism for the regulation of all tubby-like proteins, and explores how to exploit it pharmacologically using drugs.

Mutations in Ciliary Trafficking Genes affect Sonic Hedgehog-dependent Neural Tube Patterning Differentially along the Anterior-Posterior Axis.

Cell specification in the ventral spinal cord is a well-studied model system to understand how tissue pattern develops in response to a morphogen gradient. Ventral cell types including motor neurons (MNs) are induced in the neural tube in response to graded Sonic Hedgehog (Shh) signaling. We performed a forward genetic screen in the mouse that incorporated a GFP-expressing transgene to visualize MNs to identify genes regulating ventral patterning. Here we contrast the neural patterning phenotypes of two mouse lines carrying induced mutations in ciliary trafficking genes. We show that a hypomorphic mutation in the gene Tubby-like protein 3 (Tulp3) resulted in a dorsal expansion of MNs consistent with an up-regulation of Shh signaling. Interestingly, patterning defects in Tulp3 mutants were restricted to posterior regions of the spinal cord as patterning was similar to WT in the anterior spinal cord. In contrast, a mutation in the ciliary trafficking gene cytoplasmic dynein 2 heavy chain 1 (Dync2h1), led to a complete loss of MNs in anterior regions of the spinal cord, indicating a strong down-regulation of Shh signaling. However, this severe phenotype was restricted to the cervical region as MNs developed posteriorly. Mutations in cilia trafficking genes affect Shh-dependent signaling in the neural tube differentially along the anterior-posterior (A-P) axis in a process that is not understood.

STAT3-induced up-regulation of lncRNA NEAT1 as a ceRNA facilitates abdominal aortic aneurysm formation by elevating TULP3.

Long noncoding RNAs (lncRNAs) were viewed as crucial participants in the pathogenesis of abdominal aortic aneurysm (AAA). LncRNA NEAT1 was recognized as an oncogenic gene in various diseases. However, its function and mechanism in AAA were not precisely documented. Here, we explored the functional role and molecular mechanism of NEAT1 in AAA. Functionally, the effect of NEAT1 on the proliferation was assessed by CCK-8 and EdU assay, while its impact on the apoptosis was evaluated through caspase-3/9 activity and TUNEL assays. As a result, we found that NEAT1 knockdown enhanced the proliferation and impaired the apoptosis of vascular smooth muscle cells (VSMCs). Reversely, overexpressed NEAT1 exerted anti-proliferation and pro-apoptosis effects in VSMCs. Mechanically, we found that STAT3 acted as a transcription factor and contributed to NEAT1 transcription by ChIP and luciferase reporter assays. In addition, NEAT1 was confirmed as a sponge of miR-4688 and thereby increase the expression of TULP3 in VSMCs via RIP assay and RNA pull-down assay. Rescue experiments indicted that TULP3 overexpressing countervailed the impact of NEAT1 depletion on AAA biological processes. Conclusively, lncRNA NEAT1 induced by STAT3 was identified as a ceRNA and facilitated AAA formation by targeting miR-4688/TULP3 axis.

Tulp3 Regulates Renal Cystogenesis by Trafficking of Cystoproteins to Cilia.

Polycystic kidney disease proteins, polycystin-1 and polycystin-2, localize to primary cilia. Polycystin knockouts have severe cystogenesis compared to ciliary disruption, whereas simultaneous ciliary loss suppresses excessive cyst growth. These data suggest the presence of a cystogenic activator that is inhibited by polycystins and an independent but relatively minor cystogenic inhibitor, either of which are cilia dependent. However, current genetic models targeting cilia completely ablate the compartment, making it difficult to uncouple cystoprotein function from ciliary localization. Thus, the role of cilium-generated signaling in cystogenesis is unclear. We recently demonstrated that the tubby family protein Tulp3 determines ciliary trafficking of polycystins in kidney collecting duct cells without affecting protein levels or cilia. Here, we demonstrate that embryonic-stage, nephron-specific Tulp3 knockout mice developed cystic kidneys, while retaining intact cilia. Cystic kidneys showed increased mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), mTOR, and persistently high cyclic AMP (cAMP) signaling, suggesting contribution of multiple factors to cystogenesis. Based on kidney-to-body-weight ratio, cystic index, and epithelial proliferation in developing tubules or cysts, the severity of cystogenesis upon Tulp3 deletion was intermediate between that caused by loss of polycystin-1 or cilia. However, concomitant Tulp3 loss did not inhibit cystogenesis in polycystin-1 knockouts, unlike ciliary disruption. Interestingly, ciliary trafficking of the small guanosine triphosphatase (GTPase) Arl13b, loss of which causes cystogenic severity similar to ciliary loss, was reduced prior to cyst initiation. Thus, we propose that cystogenesis in Tulp3 mutants results from a reduction of ciliary levels of polycystins, Arl13b, and Arl13b-dependent lipidated cargoes. Arl13b might be the ciliary factor that represses cystogenesis distinct from polycystins.

TULP3 is required for localization of membrane-associated proteins ARL13B and INPP5E to primary cilia.

The primary cilia are known as biosensors that transduce signals through the ciliary membrane proteins in vertebrate cells. The ciliary membrane contains transmembrane proteins and membrane-associated proteins. Tubby-like protein 3 (TULP3), a member of the tubby family, has been shown to interact with the intraflagellar transport-A complex (IFT-A) and to be involved in the ciliary localization of transmembrane proteins, although its role in the ciliary entry of membrane-associated proteins has remained unclear. Here, to determine whether TULP3 is required for the localization of ciliary membrane-associated proteins, we generated and analyzed TULP3-knockout (KO) hTERT RPE-1 (RPE1) cells. Immunofluorescence analysis demonstrated that ciliary formation was downregulated in TULP3-KO cells and that membrane-associated proteins, ADP-ribosylation factor-like 13B (ARL13B) and inositol polyphosphate-5-phosphatase E (INPP5E), failed to localize to primary cilia in TULP3-KO cells. These defects in the localization of ARL13B and INPP5E in TULP3-KO cells were rescued by the exogenous expression of wild-type TULP3, but not that of mutant TULP3 lacking the ability to bind IFT-A. In addition, the expression of TUB protein, another member of the tubby family whose endogenous expression is absent in RPE1 cells, also rescued the defective ciliary localization of ARL13B and INPP5E in TULP3-KO cells, suggesting that there is functional redundancy between TULP3 and TUB. Our findings indicate that TULP3 participates in ciliogenesis, and targets membrane-associated proteins to primary cilia via binding to IFT-A.