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© 2015 Yee et al.Mutations in genes encoding cilia proteins cause human ciliopathies, diverse disorders affecting many tissues. Individual genes can be linked to ciliopathies with dramatically different phenotypes, suggesting that genetic modifiers may par

© 2015 Roberson et al.The Meckel syndrome (MKS) complex functions at the transition zone, located between the basal body and axoneme, to regulate the localization of ciliary membrane proteins. We investigated the role of Tmem231, a two-pass transmembrane p

Cilia are conserved, microtubule-based cell surface projections that emanate from basal bodies, membranedocked centrioles. The beating of motile cilia and flagella enables cells to swim and epithelia to displace fluids. In contrast, most primary cilia do n

We analyzed here how formin-like 1 β (FMNL1β), an actin cytoskeleton-regulatory protein, regulates microtubule-organizing center (MTOC) and multivesicular bodies (MVB) polarization and exosome secretion at an immune synapse (IS) model in a phosphorylation-dependent manner. IS formation was associated with transient recruitment of FMNL1β to the IS, which was independent of protein kinase C δ (PKCδ). Simultaneous RNA interference of all FMNL1 isoforms prevented MTOC/MVB polarization and exosome secretion, which were restored by FMNL1βWT expression. However, expression of the non-phosphorylatable mutant FMNL1βS1086A did not restore neither MTOC/MVB polarization nor exosome secretion to control levels, supporting the crucial role of S1086 phosphorylation in MTOC/MVB polarization and exosome secretion. In contrast, the phosphomimetic mutant, FMNL1βS1086D, restored MTOC/MVB polarization and exosome secretion. Conversely, FMNL1βS1086D mutant did not recover the deficient MTOC/MVB polarization occurring in PKCδ-interfered clones, indicating that S1086 FMNL1β phosphorylation alone is not sufficient for MTOC/MVB polarization and exosome secretion. FMNL1 interference inhibited the depletion of F-actin at the central region of the immune synapse (cIS), which is necessary for MTOC/MVB polarization. FMNL1βWT and FMNL1βS1086D, but not FMNL1βS1086A expression, restored F-actin depletion at the cIS. Thus, actin cytoskeleton reorganization at the IS underlies the effects of all these FMNL1β variants on polarized secretory traffic. FMNL1 was found in the IS made by primary T lymphocytes, both in T cell receptor (TCR) and chimeric antigen receptor (CAR)-evoked synapses. Taken together, these results point out a crucial role of S1086 phosphorylation in FMNL1β activation, leading to cortical actin reorganization and subsequent control of MTOC/MVB polarization and exosome secretion., Competing Interests: JR, SF, IS, PB, AN, AP, FG, VC, MI No competing interests declared, (© 2024, Ruiz-Navarro et al.)

Ellis van Creveld syndrome and Weyers acrofacial dysostosis are two rare genetic diseases affecting skeletal development. They are both ciliopathies, as they are due to malfunction of primary cilia, microtubule-based plasma membrane protrusions that function as cellular antennae and are required for Hedgehog signaling, a key pathway during skeletal morphogenesis. These ciliopathies are caused by mutations affecting the EVC-EVC2 complex, a transmembrane protein heterodimer that regulates Hedgehog signaling from inside primary cilia. Despite the importance of this complex, the mechanisms underlying its stability, targeting and function are poorly understood. To address this, we characterized the endogenous EVC protein interactome in control and Evc -null cells. This proteomic screen confirmed EVC's main known interactors (EVC2, IQCE, EFCAB7), while revealing new ones, including USP7, a deubiquitinating enzyme involved in Hedgehog signaling. We therefore looked at EVC-EVC2 complex ubiquitination. Such ubiquitination exists but is independent of USP7 (and of USP48, also involved in Hh signaling). We did find, however, that monoubiquitination of EVC-EVC2 cytosolic tails greatly reduces their protein levels. On the other hand, modification of EVC-EVC2 cytosolic tails with the small ubiquitin-related modifier SUMO3 has a different effect, enhancing complex accumulation at the EvC zone, immediately distal to the ciliary transition zone, possibly via increased binding to the EFCAB7-IQCE complex. Lastly, we find that EvC zone targeting of EVC-EVC2 depends on two separate EFCAB7-binding motifs within EVC2's Weyers-deleted peptide. Only one of these motifs had been characterized previously, so we have mapped the second herein. Altogether, our data shed light on EVC-EVC2 complex regulatory mechanisms, with implications for ciliopathies., Competing Interests: Author GM was employed by DBGen Ocular Genomics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Barbeito, Martin-Morales, Palencia-Campos, Cerrolaza, Rivas-Santos, Gallego-Colastra, Caparros-Martin, Martin-Bravo, Martin-Hurtado, Sánchez-Bellver, Marfany, Ruiz-Perez and Garcia-Gonzalo.)

Proteins related to the ubiquitin-proteasome system play an important role during the differentiation and ciliogenesis of photoreceptor cells. Mutations in several genes involved in ubiquitination and proteostasis have been identified as causative of inherited retinal dystrophies (IRDs) and ciliopathies. USP48 is a deubiquitinating enzyme whose role in the retina is still unexplored although previous studies indicate its relevance for neurosensory organs. In this work, we describe that a pool of endogenous USP48 localises to the basal body in retinal cells and provide data that supports the function of USP48 in the photoreceptor cilium. We also demonstrate that USP48 interacts with the IRD-associated proteins ARL3 and UNC119a, and stabilise their protein levels using different mechanisms. Our results suggest that USP48 may act in the regulation/stabilisation of key ciliary proteins for photoreceptor function, in the modulation of intracellular protein transport, and in ciliary trafficking to the photoreceptor outer segment.

Orofaciodigital syndrome (OFD) is a genetically heterogeneous ciliopathy characterized by anomalies of the oral cavity, face, and digits. We describe individuals with OFD from three unrelated families having bi-allelic loss-of-function variants in SCNM1 as the cause of their condition. SCNM1 encodes a protein recently shown to be a component of the human minor spliceosome. However, so far the effect of loss of SCNM1 function on human cells had not been assessed. Using a comparative transcriptome analysis between fibroblasts derived from an OFD-affected individual harboring SCNM1 mutations and control fibroblasts, we identified a set of genes with defective minor intron (U12) processing in the fibroblasts of the affected subject. These results were reproduced in SCNM1 knockout hTERT RPE-1 (RPE-1) cells engineered by CRISPR-Cas9-mediated editing and in SCNM1 siRNA-treated RPE-1 cultures. Notably, expression of TMEM107 and FAM92A encoding primary cilia and basal body proteins, respectively, and that of DERL2, ZC3H8, and C17orf75, were severely reduced in SCNM1-deficient cells. Primary fibroblasts containing SCNM1 mutations, as well as SCNM1 knockout and SCNM1 knockdown RPE-1 cells, were also found with abnormally elongated cilia. Conversely, cilia length and expression of SCNM1-regulated genes were restored in SCNM1-deficient fibroblasts following reintroduction of SCNM1 via retroviral delivery. Additionally, functional analysis in SCNM1-retrotransduced fibroblasts showed that SCNM1 is a positive mediator of Hedgehog (Hh) signaling. Our findings demonstrate that defective U12 intron splicing can lead to a typical ciliopathy such as OFD and reveal that primary cilia length and Hh signaling are regulated by the minor spliceosome through SCNM1 activity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Primary cilia are sensory membrane protrusions whose dysfunction causes ciliopathies. INPP5E is a ciliary phosphoinositide phosphatase mutated in ciliopathies like Joubert syndrome. INPP5E regulates numerous ciliary functions, but how it accumulates in cilia remains poorly understood. Herein, we show INPP5E ciliary targeting requires its folded catalytic domain and is controlled by four conserved ciliary localization signals (CLSs): LLxPIR motif (CLS1), W383 (CLS2), FDRxLYL motif (CLS3) and CaaX box (CLS4). We answer two long-standing questions in the field. First, partial CLS1-CLS4 redundancy explains why CLS4 is dispensable for ciliary targeting. Second, the essential need for CLS2 clarifies why CLS3-CLS4 are together insufficient for ciliary accumulation. Furthermore, we reveal that some Joubert syndrome mutations perturb INPP5E ciliary targeting, and clarify how each CLS works: (i) CLS4 recruits PDE6D, RPGR and ARL13B, (ii) CLS2-CLS3 regulate association to TULP3, ARL13B, and CEP164, and (iii) CLS1 and CLS4 cooperate in ATG16L1 binding. Altogether, we shed light on the mechanisms of INPP5E ciliary targeting, revealing a complexity without known parallels among ciliary cargoes., Competing Interests: DC, RM, PB, AD, AL, BS, GH, OP, MR, MI, TI, FG No competing interests declared, SG has consulted for Arvinas Inc, (© 2022, Cilleros-Rodriguez, Martin-Morales et al.)

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., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

G protein-coupled receptors (GPCRs) are the most common pharmacological target in human clinical practice. To perform their functions, many GPCRs must accumulate inside primary cilia, microtubule-based plasma membrane protrusions working as cellular antennae. Nevertheless, the molecular mechanisms underlying GPCR ciliary targeting remain poorly understood. Serotonin receptor 6 (HTR6) and somatostatin receptor 3 (SSTR3) are two brain-enriched ciliary GPCRs involved in cognition and pathologies such as Alzheimer's disease and cancer. Although the third intracellular loops (IC3) of HTR6 and SSTR3 suffice to target non-ciliary GPCRs to cilia, these IC3s are dispensable for ciliary targeting of HTR6 and SSTR3 themselves, suggesting these GPCRs contain additional ciliary targeting sequences (CTSs). Herein, we discover and characterize novel CTSs in HTR6 and SSTR3 C-terminal tails (CT). These CT-CTSs (CTS2) act redundantly with IC3-CTSs (CTS1), each being sufficient for ciliary targeting. In HTR6, RKQ and LPG motifs are critical for CTS1 and CTS2 function, respectively, whereas in SSTR3 these roles are mostly fulfilled by AP[AS]CQ motifs in IC3 and juxtamembrane residues in CT. Furthermore, we shed light on how these CTSs promote ciliary targeting by modulating binding to ciliary trafficking adapters TULP3 and RABL2., (© 2020 Barbeito et al.)

HERC1 is a ubiquitin ligase protein, which, when mutated, induces several malformations and intellectual disability in humans. The animal model of HERC1 mutation is the mouse tambaleante characterized by: (1) overproduction of the protein; (2) cerebellar Purkinje cells death by autophagy; (3) dysregulation of autophagy in spinal cord motor neurons, and CA3 and neocortical pyramidal neurons; (4) impairment of associative learning, linked to altered spinogenesis and absence of LTP in the lateral amygdala; and, (5) motor impairment due to delayed action potential transmission, decrease synaptic transmission efficiency and altered myelination in the peripheral nervous system. To investigate the putative role of HERC1 in the presynaptic dynamics we have performed a series of experiments in cultured tambaleante hippocampal neurons by using transmission electron microscopy, FM1-43 destaining and immunocytochemistry. Our results show: (1) a decrease in the number of synaptic vesicles; (2) reduced active zones; (3) less clathrin immunoreactivity and less presynaptic endings over the hippocampal main dendritic trees; which contrast with (4) a greater number of endosomes and autophagosomes in the presynaptic endings of the tambaleante neurons relative to control ones. Altogether these results show an important role of HERC1 in the regulation of presynaptic membrane dynamics.

When not dividing, many cell types target their centrosome to the plasma membrane, where it nucleates assembly of a primary cilium, an antenna-like signaling structure consisting of nine concentric microtubule pairs surrounded by membrane. Primary cilia play important pathophysiological roles in many tissues, their dysfunction being associated with cancer and ciliopathies, a diverse group of congenital human diseases. Several recent studies have unveiled functional connections between primary cilia and NRF2 (nuclear factor erythroid 2-related factor 2), the master transcription factor orchestrating cytoprotective responses to oxidative and other cellular stresses. These NRF2-cilia relationships are reciprocal: primary cilia, by promoting autophagy, downregulate NRF2 activity. In turn, NRF2 transcriptionally regulates genes involved in ciliogenesis and Hedgehog (Hh) signaling, a cilia-dependent pathway with major roles in embryogenesis, stem cell function and tumorigenesis. Nevertheless, while we found that NRF2 stimulates ciliogenesis and Hh signaling, a more recent study reported that NRF2 negatively affects these processes. Herein, we review the available evidence linking NRF2 to primary cilia, suggest possible explanations to reconcile seemingly contradictory data, and discuss what the emerging interplay between primary cilia and NRF2 may mean for human health and disease.

The transcription factor NRF2 is a master regulator of cellular antioxidant and detoxification responses, but it also regulates other processes such as autophagy and pluripotency. In human embryonic stem cells (hESCs), NRF2 antagonizes neuroectoderm differentiation, which only occurs after NRF2 is repressed via a Primary Cilia-Autophagy-NRF2 (PAN) axis. However, the functional connections between NRF2 and primary cilia, microtubule-based plasma membrane protrusions that function as cellular antennae, remain poorly understood. For instance, nothing is known about whether NRF2 affects cilia, or whether cilia regulation of NRF2 extends beyond hESCs. Here, we show that NRF2 and primary cilia reciprocally regulate each other. First, we demonstrate that fibroblasts lacking primary cilia have higher NRF2 activity, which is rescued by autophagy-activating mTOR inhibitors, indicating that the PAN axis also operates in differentiated cells. Furthermore, NRF2 controls cilia formation and function. NRF2-null cells grow fewer and shorter cilia and display impaired Hedgehog signaling, a cilia-dependent pathway. These defects are not due to increased oxidative stress or ciliophagy, but rather to NRF2 promoting expression of multiple ciliogenic and Hedgehog pathway genes. Among these, we focused on GLI2 and GLI3, the transcription factors controlling Hh pathway output. Both their mRNA and protein levels are reduced in NRF2-null cells, consistent with their gene promoters containing consensus ARE sequences predicted to bind NRF2. Moreover, GLI2 and GLI3 fail to accumulate at the ciliary tip of NRF2-null cells upon Hh pathway activation. Given the importance of NRF2 and ciliary signaling in human disease, our data may have important biomedical implications.

Primary cilia are required for Smoothened to transduce vertebrate Hedgehog signals, but how Smoothened accumulates in cilia and is activated is incompletely understood. Here, we identify cilia-associated oxysterols that promote Smoothened accumulation in cilia and activate the Hedgehog pathway. Our data reveal that cilia-associated oxysterols bind to two distinct Smoothened domains to modulate Smoothened accumulation in cilia and tune the intensity of Hedgehog pathway activation. We find that the oxysterol synthase HSD11β2 participates in the production of Smoothened-activating oxysterols and promotes Hedgehog pathway activity. Inhibiting oxysterol biosynthesis impedes oncogenic Hedgehog pathway activation and attenuates the growth of Hedgehog pathway-associated medulloblastoma, suggesting that targeted inhibition of Smoothened-activating oxysterol production may be therapeutically useful for patients with Hedgehog-associated cancers., (Published by Elsevier Inc.)

Cilia are plasma membrane protrusions that act as cellular propellers or antennae. To perform these functions, cilia must maintain a composition distinct from those of the contiguous cytosol and plasma membrane. The specialized composition of the cilium depends on the ciliary gate, the region at the ciliary base separating the cilium from the rest of the cell. The ciliary gate's main structural features are electron dense struts connecting microtubules to the adjacent membrane. These structures include the transition fibers, which connect the distal basal body to the base of the ciliary membrane, and the Y-links, which connect the proximal axoneme and ciliary membrane within the transition zone. Both transition fibers and Y-links form early during ciliogenesis and play key roles in ciliary assembly and trafficking. Accordingly, many human ciliopathies are caused by mutations that perturb ciliary gate function., (Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.)

Cilia have a unique diffusion barrier ("gate") within their proximal region, termed transition zone (TZ), that compartmentalises signalling proteins within the organelle. The TZ is known to harbour two functional modules/complexes (Meckel syndrome [MKS] and Nephronophthisis [NPHP]) defined by genetic interaction, interdependent protein localisation (hierarchy), and proteomic studies. However, the composition and molecular organisation of these modules and their links to human ciliary disease are not completely understood. Here, we reveal Caenorhabditis elegans CEP-290 (mammalian Cep290/Mks4/Nphp6 orthologue) as a central assembly factor that is specific for established MKS module components and depends on the coiled coil region of MKS-5 (Rpgrip1L/Rpgrip1) for TZ localisation. Consistent with a critical role in ciliary gate function, CEP-290 prevents inappropriate entry of membrane-associated proteins into cilia and keeps ARL-13 (Arl13b) from leaking out of cilia via the TZ. We identify a novel MKS module component, TMEM-218 (Tmem218), that requires CEP-290 and other MKS module components for TZ localisation and functions together with the NPHP module to facilitate ciliogenesis. We show that TZ localisation of TMEM-138 (Tmem138) and CDKL-1 (Cdkl1/Cdkl2/Cdkl3/Cdlk4 related), not previously linked to a specific TZ module, similarly depends on CEP-290; surprisingly, neither TMEM-138 or CDKL-1 exhibit interdependent localisation or genetic interactions with core MKS or NPHP module components, suggesting they are part of a distinct, CEP-290-associated module. Lastly, we show that families presenting with Oral-Facial-Digital syndrome type 6 (OFD6) have likely pathogenic mutations in CEP-290-dependent TZ proteins, namely Tmem17, Tmem138, and Tmem231. Notably, patient fibroblasts harbouring mutated Tmem17, a protein not yet ciliopathy-associated, display ciliogenesis defects. Together, our findings expand the repertoire of MKS module-associated proteins--including the previously uncharacterised mammalian Tmem80--and suggest an MKS-5 and CEP-290-dependent assembly pathway for building a functional TZ.

Mutations in genes encoding cilia proteins cause human ciliopathies, diverse disorders affecting many tissues. Individual genes can be linked to ciliopathies with dramatically different phenotypes, suggesting that genetic modifiers may participate in their pathogenesis. The ciliary transition zone contains two protein complexes affected in the ciliopathies Meckel syndrome (MKS) and nephronophthisis (NPHP). The BBSome is a third protein complex, affected in the ciliopathy Bardet-Biedl syndrome (BBS). We tested whether mutations in MKS, NPHP and BBS complex genes modify the phenotypic consequences of one another in both C. elegans and mice. To this end, we identified TCTN-1, the C. elegans ortholog of vertebrate MKS complex components called Tectonics, as an evolutionarily conserved transition zone protein. Neither disruption of TCTN-1 alone or together with MKS complex components abrogated ciliary structure in C. elegans. In contrast, disruption of TCTN-1 together with either of two NPHP complex components, NPHP-1 or NPHP-4, compromised ciliary structure. Similarly, disruption of an NPHP complex component and the BBS complex component BBS-5 individually did not compromise ciliary structure, but together did. As in nematodes, disrupting two components of the mouse MKS complex did not cause additive phenotypes compared to single mutants. However, disrupting both Tctn1 and either Nphp1 or Nphp4 exacerbated defects in ciliogenesis and cilia-associated developmental signaling, as did disrupting both Tctn1 and the BBSome component Bbs1. Thus, we demonstrate that ciliary complexes act in parallel to support ciliary function and suggest that human ciliopathy phenotypes are altered by genetic interactions between different ciliary biochemical complexes.

Primary cilia interpret vertebrate Hedgehog (Hh) signals. Why cilia are essential for signaling is unclear. One possibility is that some forms of signaling require a distinct membrane lipid composition, found at cilia. We found that the ciliary membrane contains a particular phosphoinositide, PI(4)P, whereas a different phosphoinositide, PI(4,5)P2, is restricted to the membrane of the ciliary base. This distribution is created by Inpp5e, a ciliary phosphoinositide 5-phosphatase. Without Inpp5e, ciliary PI(4,5)P2 levels are elevated and Hh signaling is disrupted. Inpp5e limits the ciliary levels of inhibitors of Hh signaling, including Gpr161 and the PI(4,5)P2-binding protein Tulp3. Increasing ciliary PI(4,5)P2 levels or conferring the ability to bind PI(4)P on Tulp3 increases the ciliary localization of Tulp3. Lowering Tulp3 in cells lacking Inpp5e reduces ciliary Gpr161 levels and restores Hh signaling. Therefore, Inpp5e regulates ciliary membrane phosphoinositide composition, and Tulp3 reads out ciliary phosphoinositides to control ciliary protein localization, enabling Hh signaling., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The Meckel syndrome (MKS) complex functions at the transition zone, located between the basal body and axoneme, to regulate the localization of ciliary membrane proteins. We investigated the role of Tmem231, a two-pass transmembrane protein, in MKS complex formation and function. Consistent with a role in transition zone function, mutation of mouse Tmem231 disrupts the localization of proteins including Arl13b and Inpp5e to cilia, resulting in phenotypes characteristic of MKS such as polydactyly and kidney cysts. Tmem231 and B9d1 are essential for each other and other complex components such as Mks1 to localize to the transition zone. As in mouse, the Caenorhabditis elegans orthologue of Tmem231 localizes to and controls transition zone formation and function, suggesting an evolutionarily conserved role for Tmem231. We identified TMEM231 mutations in orofaciodigital syndrome type 3 (OFD3) and MKS patients that compromise transition zone function. Thus, Tmem231 is critical for organizing the MKS complex and controlling ciliary composition, defects in which cause OFD3 and MKS., (© 2015 Roberson et al.)

Cilia are conserved, microtubule-based cell surface projections that emanate from basal bodies, membrane-docked centrioles. The beating of motile cilia and flagella enables cells to swim and epithelia to displace fluids. In contrast, most primary cilia do not beat but instead detect environmental or intercellular stimuli. Inborn defects in both kinds of cilia cause human ciliopathies, diseases with diverse manifestations such as heterotaxia and kidney cysts. These diseases are caused by defects in ciliogenesis or ciliary function. The signaling functions of cilia require regulation of ciliary composition, which depends on the control of protein traffic into and out of cilia.

Mutations affecting ciliary components cause ciliopathies. As described here, we investigated Tectonic1 (Tctn1), a regulator of mouse Hedgehog signaling, and found that it is essential for ciliogenesis in some, but not all, tissues. Cell types that do not require Tctn1 for ciliogenesis require it to localize select membrane-associated proteins to the cilium, including Arl13b, AC3, Smoothened and Pkd2. Tctn1 forms a complex with multiple ciliopathy proteins associated with Meckel and Joubert syndromes, including Mks1, Tmem216, Tmem67, Cep290, B9d1, Tctn2 and Cc2d2a. Components of this complex co-localize at the transition zone, a region between the basal body and ciliary axoneme. Like Tctn1, loss of Tctn2, Tmem67 or Cc2d2a causes tissue-specific defects in ciliogenesis and ciliary membrane composition. Consistent with a shared function for complex components, we identified a mutation in TCTN1 that causes Joubert syndrome. Thus, a transition zone complex of Meckel and Joubert syndrome proteins regulates ciliary assembly and trafficking, suggesting that transition zone dysfunction is the cause of these ciliopathies.

Nephronophthisis (NPHP), Joubert (JBTS), and Meckel-Gruber (MKS) syndromes are autosomal-recessive ciliopathies presenting with cystic kidneys, retinal degeneration, and cerebellar/neural tube malformation. Whether defects in kidney, retinal, or neural disease primarily involve ciliary, Hedgehog, or cell polarity pathways remains unclear. Using high-confidence proteomics, we identified 850 interactors copurifying with nine NPHP/JBTS/MKS proteins and discovered three connected modules: "NPHP1-4-8" functioning at the apical surface, "NPHP5-6" at centrosomes, and "MKS" linked to Hedgehog signaling. Assays for ciliogenesis and epithelial morphogenesis in 3D renal cultures link renal cystic disease to apical organization defects, whereas ciliary and Hedgehog pathway defects lead to retinal or neural deficits. Using 38 interactors as candidates, linkage and sequencing analysis of 250 patients identified ATXN10 and TCTN2 as new NPHP-JBTS genes, and our Tctn2 mouse knockout shows neural tube and Hedgehog signaling defects. Our study further illustrates the power of linking proteomic networks and human genetics to uncover critical disease pathways., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The Regulator of Chromosome Condensation 1 (RCC1) was identified over 20 years ago as a critical cell cycle regulator. By analyzing its amino acid sequence, RCC1 was found to consist of seven homologous repeats of 51-68 amino acid residues, which were later shown to adopt a seven-bladed beta-propeller fold. Since the initial identification of RCC1, a number of proteins have been discovered that contain one or more RCC1-like domains (RLDs). As we show here, these RCC1 superfamily proteins can be subdivided in five subgroups based on structural criteria. In recent years, a number of studies have been published regarding the functions of RCC1 superfamily proteins. From these studies, the emerging picture is that the RLD is a versatile domain which may perform many different functions, including guanine nucleotide exchange on small GTP-binding proteins, enzyme inhibition or interaction with proteins and lipids. Here, we review the available structural and functional data on RCC1 superfamily members, paying special attention to the human proteins and their involvement in disease.

Background: Although human embryonic stem cells (hESCs) hold great promise as a source of differentiated cells to treat several human diseases, many obstacles still need to be surmounted before this can become a reality. First among these, a robust chemically-defined system to expand hESCs in culture is still unavailable despite recent advances in the understanding of factors controlling hESC self-renewal., Methodology/principal Findings: In this study, we attempted to find new molecules that stimulate long term hESC self-renewal. In order to do this, we started from the observation that a commercially available serum replacement product has a strong positive effect on the expansion of undifferentiated hESCs when added to a previously reported chemically-defined medium. Subsequent experiments demonstrated that the active ingredient within the serum replacement is lipid-rich albumin. Furthermore, we show that this activity is trypsin-resistant, strongly suggesting that lipids and not albumin are responsible for the effect. Consistent with this, lipid-poor albumin shows no detectable activity. Finally, we identified the major lipids bound to the lipid-rich albumin and tested several lipid candidates for the effect., Conclusions/significance: Our discovery of the role played by albumin-associated lipids in stimulating hESC self-renewal constitutes a significant advance in the knowledge of how hESC pluripotency is maintained by extracellular factors and has important applications in the development of increasingly chemically defined hESC culture systems.

To be able to separate and analyze giant proteins and small proteins in the same electrophoretic gel, we have used a continuous SDS-PAGE gel formed by the combination of a low-percentage acrylamide gel and a gradient SDS-PAGE gel that we have named LAG gel. To get a good resolution for proteins of more than 200 kDa, we used an acrylamide/bisacrylamide ratio of 80:1 in the low-percentage acrylamide gel. To successfully resolve proteins in the 5-200 kDa range, we used a conventional 6-15% SDS-PAGE gradient gel with the standard acrylamide/bisacrylamide ratio of 40:1. We show that the LAG system can be successfully used in general applications of SDS-PAGE electrophoresis such as proteomics and immunobloting techniques. Thus, using this continuous LAG gel, it is possible to simultaneously analyze giant proteins, such as HERC1 and dynein, big proteins like clathrin heavy chain and small proteins like ARF. The LAG system has a good resolution, low cost, and high reproducibility. Moreover, to simultaneously analyze all proteins saves time. All these characteristics, together with the use of a standard apparatus found in any biochemistry laboratory, make the LAG system an easy tool to use.

Tuberous sclerosis complex (TSC) is an autosomal dominant disease characterized by hamartoma formation in various organs. Two genes responsible for the disease, TSC1 and TSC2, have been identified. The TSC1 and TSC2 proteins, also called hamartin and tuberin, respectively, have been shown to regulate cell growth through inhibition of the mammalian target of rapamycin pathway. TSC1 is known to stabilize TSC2 by forming a complex with TSC2, which is a GTPase-activating protein for the Rheb small GTPase. We have identified HERC1 as a TSC2-interacting protein. HERC1 is a 532-kDa protein with an E3 ubiquitin ligase homology to E6AP carboxyl terminus (HECT) domain. We observed that the interaction of TSC1 with TSC2 appears to exclude TSC2 from interacting with HERC1. Disease mutations in TSC2, which result in its destabilization, allow binding to HERC1 in the presence of TSC1. Our study reveals a potential molecular mechanism of how TSC1 stabilizes TSC2 by excluding the HERC1 ubiquitin ligase from the TSC2 complex. Furthermore, these data reveal a possible biochemical basis of how certain disease mutations inactivate TSC2.

HERC proteins are defined as containing both HECT and RCC1-like domains in their amino acid sequences. Six HERC genes have turned up in the human genome which encode two different sorts of polypeptides: while the small HERC proteins possess little more than the two aforementioned domains, the large ones are giant proteins with a plethora of potentially important regions. It is now almost 10 years since the discovery of the first family member and information is starting to accumulate pointing to a general role for these proteins as ubiquitin ligases involved in membrane-trafficking events. In this review, the available data on these six members are discussed, together with an account of their evolution.

HERC1 is a giant multidomain protein involved in membrane trafficking through its interaction with vesicle coat proteins such as clathrin and ARF. Previously, it has been shown that the RCC1-like domain 1 (RLD1) of HERC1 stimulates guanine nucleotide dissociation on ARF1 and Rab proteins. In this study, we have analyzed whether HERC1 may also regulate ARF6 activity. We show that HERC1, through its RLD1, stimulates GDP release from ARF6 but, unexpectedly, it inhibits GDP/GTP exchange on ARF6 under conditions where ARNO stimulates it. Furthermore, we demonstrate that the activity of HERC1 as a guanine nucleotide release factor requires the presence of PI(4,5)P(2) bound to HERC1's RLD1. In agreement with this, we find that purified HERC1 contains PI(4,5)P(2) bound to the RLD1.

HERC1 is a very large protein involved in membrane traffic through both its ability to bind clathrin and its guanine nucleotide exchange factor (GEF) activity over ARF and Rab family GTPases. Herein, we show that HERC1 is recruited onto actin-rich surface protrusions in ARF6-transfected HeLa cells upon aluminum fluoride (AlF(4)(-)) treatment. Moreover, the fact that HERC1 overexpression does not stimulate protrusion formation in the absence of AlF(4)(-), in conditions where ARNO does, indicates that HERC1 is not acting as an ARF6-GEF in this system, but that instead its recruitment takes place downstream of ARF6 activation. Finally, we suggest a phosphoinositide-binding mechanism whereby HERC1 may translocate to these protrusions.

HERC proteins are characterized by having one or more RCC1-like domains as well as a C-terminal HECT domain in their amino acid sequences. This has led researchers to suggest that they may act as both guanine nucleotide exchange factors and E3 ubiquitin ligases. Here we describe a physical interaction between the HECT domain of HERC1, a giant protein involved in intracellular membrane traffic, and the M2 isoform of glycolytic enzyme pyruvate kinase (M2-PK). Partial colocalization of endogenous proteins was observed by immunofluorescence studies. This interaction neither induced M2-PK ubiquitination nor affected its enzymatic activity. The putative significance of the association is discussed.