Primary cilia as dynamic and diverse signalling hubs in development and disease.

Primary cilia, antenna-like sensory organelles protruding from the surface of most vertebrate cell types, are essential for regulating signalling pathways during development and adult homeostasis. Mutations in genes affecting cilia cause an overlapping spectrum of >30 human diseases and syndromes, the ciliopathies. Given the immense structural and functional diversity of the mammalian cilia repertoire, there is a growing disconnect between patient genotype and associated phenotypes, with variable severity and expressivity characteristic of the ciliopathies as a group. Recent technological developments are rapidly advancing our understanding of the complex mechanisms that control biogenesis and function of primary cilia across a range of cell types and are starting to tackle this diversity. Here, we examine the structural and functional diversity of primary cilia, their dynamic regulation in different cellular and developmental contexts and their disruption in disease.

DLG1 functions upstream of SDCCAG3 and IFT20 to control ciliary targeting of polycystin-2.

Polarized vesicular trafficking directs specific receptors and ion channels to cilia, but the underlying mechanisms are poorly understood. Here we describe a role for DLG1, a core component of the Scribble polarity complex, in regulating ciliary protein trafficking in kidney epithelial cells. Conditional knockout of Dlg1 in mouse kidney causes ciliary elongation and cystogenesis, and cell-based proximity labeling proteomics and fluorescence microscopy show alterations in the ciliary proteome upon loss of DLG1. Specifically, the retromer-associated protein SDCCAG3, IFT20, and polycystin-2 (PC2) are reduced in the cilia of DLG1-deficient cells compared to control cells. This phenotype is recapitulated in vivo and rescuable by re-expression of wild-type DLG1, but not a Congenital Anomalies of the Kidney and Urinary Tract (CAKUT)-associated DLG1 variant, p.T489R. Finally, biochemical approaches and Alpha Fold modelling suggest that SDCCAG3 and IFT20 form a complex that associates, at least indirectly, with DLG1. Our work identifies a key role for DLG1 in regulating ciliary protein composition and suggests that ciliary dysfunction of the p.T489R DLG1 variant may contribute to CAKUT.

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 cell-type-specific kinesin-3 motor, KLP-6, which moves within cilia independently of IFT and regulates ciliary content and function. Here, we provide evidence that a KLP-6 homolog, KIF13B, undergoes bursts of bidirectional movement within primary cilia of cultured immortalized human retinal pigment epithelial (hTERT-RPE1) cells. Anterograde and retrograde intraciliary velocities of KIF13B were similar to those of IFT (as assayed using IFT172-eGFP), but intraciliary movement of KIF13B required its own motor domain and appeared to be cell-type specific. Our work provides the first demonstration of motor-driven, intraciliary movement by a vertebrate kinesin other than kinesin-2 motors.

Endocytic Control of Cellular Signaling at the Primary Cilium.

Primary cilia are dynamic signaling organelles that project from the cell surface to sense diverse chemical, physical and morphogenetic cues. Ciliary defects therefore cause diseases (ciliopathies) that affect multiple organs in developing and adult organisms. Cilia-mediated signaling involves the orchestrated movement of signaling proteins in and out of the ciliary compartment, including movement of receptors such as the Sonic Hedgehog (Shh) receptor Patched 1 (PTCH1), Smoothened (SMO), and various other G protein-coupled receptors (GPCRs), as well as transforming growth factor ß (TGF-ß) receptors I and II (TGF-ß-RI/II). We provide here a current understanding of trafficking events associated with cilia-mediated signaling, with emphasis on the involvement of clathrin-dependent receptor-mediated endocytosis in regulating ciliary Shh and TGF-ß signaling.

Functional characterization of the first missense variant in CEP78, a founder allele associated with cone-rod dystrophy, hearing loss, and reduced male fertility.

Inactivating variants in the centrosomal CEP78 gene have been found in cone-rod dystrophy with hearing loss (CRDHL), a particular phenotype distinct from Usher syndrome. Here, we identified and functionally characterized the first CEP78 missense variant c.449T>C, p.(Leu150Ser) in three CRDHL families. The variant was found in a biallelic state in two Belgian families and in a compound heterozygous state-in trans with c.1462-1G>T-in a third German family. Haplotype reconstruction showed a founder effect. Homology modeling revealed a detrimental effect of p.(Leu150Ser) on protein stability, which was corroborated in patients' fibroblasts. Elongated primary cilia without clear ultrastructural abnormalities in sperm or nasal brushes suggest impaired cilia assembly. Two affected males from different families displayed sperm abnormalities causing infertility. One of these is a heterozygous carrier of a complex allele in SPAG17, a ciliary gene previously associated with autosomal recessive male infertility. Taken together, our data indicate that a missense founder allele in CEP78 underlies the same sensorineural CRDHL phenotype previously associated with inactivating variants. Interestingly, the CEP78 phenotype has been possibly expanded with male infertility. Finally, CEP78 loss-of-function variants may have an underestimated role in misdiagnosed Usher syndrome, with or without sperm abnormalities.

PDGFRß and oncogenic mutant PDGFRα D842V promote disassembly of primary cilia through a PLCγ- and AURKA-dependent mechanism.

Primary cilia are microtubule-based sensory organelles projecting from most quiescent mammalian cells, which disassemble in cells cultured in serum-deprived conditions upon re-addition of serum or growth factors. Platelet-derived growth factors (PDGF) are implicated in deciliation, but the specific receptor isoforms and mechanisms involved are unclear. We report that PDGFRß promotes deciliation in cultured cells and provide evidence implicating PLCγ and intracellular Ca(2+) release in this process. Activation of wild-type PDGFRα alone did not elicit deciliation. However, expression of constitutively active PDGFRα D842V mutant receptor, which potently activates PLCγ (also known as PLCG1), caused significant deciliation, and this phenotype was rescued by inhibiting PDGFRα D842V kinase activity or AURKA. We propose that PDGFRß and PDGFRα D842V promote deciliation through PLCγ-mediated Ca(2+) release from intracellular stores, causing activation of calmodulin and AURKA-triggered deciliation.

ATMIN is a transcriptional regulator of both lung morphogenesis and ciliogenesis.

Initially identified in DNA damage repair, ATM-interactor (ATMIN) further functions as a transcriptional regulator of lung morphogenesis. Here we analyse three mouse mutants, Atmin(gpg6/gpg6), Atmin(H210Q/H210Q) and Dynll1(GT/GT), revealing how ATMIN and its transcriptional target dynein light chain LC8-type 1 (DYNLL1) are required for normal lung morphogenesis and ciliogenesis. Expression screening of ciliogenic genes confirmed Dynll1 to be controlled by ATMIN and further revealed moderately altered expression of known intraflagellar transport (IFT) protein-encoding loci in Atmin mutant embryos. Significantly, Dynll1(GT/GT) embryonic cilia exhibited shortening and bulging, highly similar to the characterised retrograde IFT phenotype of Dync2h1. Depletion of ATMIN or DYNLL1 in cultured cells recapitulated the in vivo ciliogenesis phenotypes and expression of DYNLL1 or the related DYNLL2 rescued the effects of loss of ATMIN, demonstrating that ATMIN primarily promotes ciliogenesis by regulating Dynll1 expression. Furthermore, DYNLL1 as well as DYNLL2 localised to cilia in puncta, consistent with IFT particles, and physically interacted with WDR34, a mammalian homologue of the Chlamydomonas cytoplasmic dynein 2 intermediate chain that also localised to the cilium. This study extends the established Atmin-Dynll1 relationship into a developmental and a ciliary context, uncovering a novel series of interactions between DYNLL1, WDR34 and ATMIN. This identifies potential novel components of cytoplasmic dynein 2 and furthermore provides fresh insights into the molecular pathogenesis of human skeletal ciliopathies.

A divergent calponin homology (NN-CH) domain defines a novel family: implications for evolution of ciliary IFT complex B proteins.

MOTIVATION: Microtubules are dynamic polymers of tubulin dimers that undergo continuous assembly and disassembly. A mounting number of microtubule-associated proteins (MAPs) regulate the dynamic behavior of microtubules and hence the assembly and disassembly of disparate microtubule structures within the cell. Despite recent advances in identification and functional characterization of MAPs, a substantial number of microtubule accessory factors have not been functionally annotated. Here, using profile-to-profile comparisons and structure modeling, we show that the yeast outer kinetochore components NDC80 and NUF2 share evolutionary ancestry with a novel protein family in mammals comprising, besides NDC80/HEC1 and NUF2, three Intraflagellar Transport (IFT) complex B subunits (IFT81, IFT57, CLUAP1) as well as six proteins with poorly defined function (FAM98A-C, CCDC22, CCDC93 and C14orf166). We show that these proteins consist of a divergent N-terminal calponin homology (CH)-like domain adjoined to an array of C-terminal heptad repeats predicted to form a coiled-coil arrangement. We have named the divergent CH-like domain NN-CH after the founding members NDC80 and NUF2.

The nucleotide-binding proteins Nubp1 and Nubp2 are negative regulators of ciliogenesis.

Nucleotide-binding proteins Nubp1 and Nubp2 are MRP/MinD-type P-loop NTPases with sequence similarity to bacterial division site-determining proteins and are conserved, essential proteins throughout the Eukaryotes. They have been implicated, together with their interacting minus-end directed motor protein KIFC5A, in the regulation of centriole duplication in mammalian cells. Here we show that Nubp1 and Nubp2 are integral components of centrioles throughout the cell cycle, recruited independently of KIFC5A. We further demonstrate their localization at the basal body of the primary cilium in quiescent vertebrate cells or invertebrate sensory cilia, as well as in the motile cilia of mouse cells and in the flagella of Chlamydomonas. RNAi-mediated silencing of nubp-1 in C. elegans causes the formation of morphologically aberrant and additional cilia in sensory neurons. Correspondingly, downregulation of Nubp1 or Nubp2 in mouse quiescent NIH 3T3 cells markedly increases the number of ciliated cells, while knockdown of KIFC5A dramatically reduces ciliogenesis. Simultaneous double silencing of Nubp1 + KIFC5A restores the percentage of ciliated cells to control levels. We document the normal ciliary recruitment, during these silencing regimes, of basal body proteins critical for ciliogenesis, namely CP110, CEP290, cenexin, Chibby, AurA, Rab8, and BBS7. Interestingly, we uncover novel interactions of Nubp1 with several members of the CCT/TRiC molecular chaperone complex, which we find enriched at the basal body and recruited independently of the Nubps or KIFC5A. Our combined results for Nubp1, Nubp2, and KIFC5A and their striking effects on cilium formation suggest a central regulatory role for these proteins, likely involving CCT/TRiC chaperone activity, in ciliogenesis.

DLG1 functions upstream of SDCCAG3 and IFT20 to control ciliary targeting of polycystin-2.

Polarized vesicular trafficking directs specific receptors and ion channels to cilia, but the underlying mechanisms are poorly understood. Here we describe a role for DLG1, a core component of the Scribble polarity complex, in regulating ciliary protein trafficking in kidney epithelial cells. Conditional knockout of Dlg1 in mouse kidney caused ciliary elongation and cystogenesis, and cell-based proximity labelling proteomics and fluorescence microscopy showed alterations in the ciliary proteome upon loss of DLG1. Specifically, the retromer-associated protein SDCCAG3, IFT20 and polycystin-2 (PC2) were reduced in cilia of DLG1 deficient cells compared to control cells. This phenotype was recapitulated in vivo and rescuable by re-expression of wildtype DLG1, but not a Congenital Anomalies of the Kidney and Urinary Tract (CAKUT)-associated DLG1 variant, p.T489R. Finally, biochemical approaches and Alpha Fold modelling suggested that SDCCAG3 and IFT20 form a complex that associates, at least indirectly, with DLG1. Our work identifies a key role for DLG1 in regulating ciliary protein composition and suggests that ciliary dysfunction of the p.T489R DLG1 variant may contribute to CAKUT.

EB1 and EB3 promote cilia biogenesis by several centrosome-related mechanisms.

The microtubule (MT) plus-end-tracking protein EB1 is required for assembly of primary cilia in mouse fibroblasts, but the mechanisms involved and the roles of the related proteins EB2 and EB3 in ciliogenesis are unknown. Using protein depletion experiments and expression of dominant-negative constructs we show here that EB1 and EB3, but not EB2, are required for assembly of primary cilia in cultured cells. Electron microscopy and live imaging showed that cells lacking EB1 or EB3 are defective in MT minus-end anchoring at the centrosome and/or basal body, and possess abnormally short cilia stumps surrounded by vesicles. Further, GST pull-down assays, mass spectrometry and immunoprecipitation indicated that EB1 and EB3 interact with proteins implicated in MT minus-end anchoring or vesicular trafficking to the cilia base, suggesting that EB1 and EB3 promote ciliogenesis by facilitating such trafficking. In addition, we show that EB3 is localized to the tip of motile cilia in bronchial epithelial cells and affects the formation of centriole-associated rootlet filaments. Collectively, our findings indicate that EBs affect biogenesis of cilia by several centrosome-related mechanisms and support the idea that different EB1-EB3 dimer species have distinct functions within cells.

Primary cilia and coordination of receptor tyrosine kinase (RTK) signalling.

Primary cilia are microtubule-based sensory organelles that coordinate signalling pathways in cell-cycle control, migration, differentiation and other cellular processes critical during development and for tissue homeostasis. Accordingly, defects in assembly or function of primary cilia lead to a plethora of developmental disorders and pathological conditions now known as ciliopathies. In this review, we summarize the current status of the role of primary cilia in coordinating receptor tyrosine kinase (RTK) signalling pathways. Further, we present potential mechanisms of signalling crosstalk and networking in the primary cilium and discuss how defects in ciliary RTK signalling are linked to human diseases and disorders.

A targeted multi-proteomics approach generates a blueprint of the ciliary ubiquitinome.

Establishment and maintenance of the primary cilium as a signaling-competent organelle requires a high degree of fine tuning, which is at least in part achieved by a variety of post-translational modifications. One such modification is ubiquitination. The small and highly conserved ubiquitin protein possesses a unique versatility in regulating protein function via its ability to build mono and polyubiquitin chains onto target proteins. We aimed to take an unbiased approach to generate a comprehensive blueprint of the ciliary ubiquitinome by deploying a multi-proteomics approach using both ciliary-targeted ubiquitin affinity proteomics, as well as ubiquitin-binding domain-based proximity labelling in two different mammalian cell lines. This resulted in the identification of several key proteins involved in signaling, cytoskeletal remodeling and membrane and protein trafficking. Interestingly, using two different approaches in IMCD3 and RPE1 cells, respectively, we uncovered several novel mechanisms that regulate cilia function. In our IMCD3 proximity labeling cell line model, we found a highly enriched group of ESCRT-dependent clathrin-mediated endocytosis-related proteins, suggesting an important and novel role for this pathway in the regulation of ciliary homeostasis and function. In contrast, in RPE1 cells we found that several structural components of caveolae (CAV1, CAVIN1, and EHD2) were highly enriched in our cilia affinity proteomics screen. Consistently, the presence of caveolae at the ciliary pocket and ubiquitination of CAV1 specifically, were found likely to play a role in the regulation of ciliary length in these cells. Cilia length measurements demonstrated increased ciliary length in RPE1 cells stably expressing a ubiquitination impaired CAV1 mutant protein. Furthermore, live cell imaging in the same cells revealed decreased CAV1 protein turnover at the cilium as the possible cause for this phenotype. In conclusion, we have generated a comprehensive list of cilia-specific proteins that are subject to regulation via ubiquitination which can serve to further our understanding of cilia biology in health and disease.

3D-Structured Illumination Microscopy of Centrosomes in Human Cell Lines.

The centrosome is the main microtubule-organizing center of animal cells, and is composed of two barrel-shaped microtubule-based centrioles embedded in protein dense pericentriolar material. Compositional and architectural re-organization of the centrosome drives its duplication, and enables its microtubule-organizing activity and capability to form the primary cilium, which extends from the mature (mother) centriole, as the cell exits the cell cycle. Centrosomes and primary cilia are essential to human health, signified by the causal role of centrosome- and cilia-aberrations in numerous congenic disorders, as well as in the etiology and progression of cancer. The list of disease-associated centrosomal proteins and their proximitomes is steadily expanding, emphasizing the need for high resolution mapping of such proteins to specific substructures of the organelle. Here, we provide a detailed 3D-structured illumination microscopy (3D-SIM) protocol for comparative localization analysis of fluorescently labeled proteins at the centrosome in fixed human cell lines, at approximately 120 nm lateral and 300 nm axial resolution. The procedure was optimized to work with primary antibodies previously known to depend on more disruptive fixation reagents, yet largely preserves centriole and centrosome architecture, as shown by transposing acquired images of landmark proteins on previously published transmission electron microscopy (TEM) images of centrosomes. Even more advantageously, it is compatible with fluorescent protein tags. Finally, we introduce an internal reference to ensure correct 3D channel alignment. This protocol hence enables flexible, swift, and information-rich localization and interdependence analyses of centrosomal proteins, as well as their disorder-associated mutations.

EB1 is required for primary cilia assembly in fibroblasts.

EB1 is a small microtubule (MT)-binding protein that associates preferentially with MT plus ends and plays a role in regulating MT dynamics. EB1 also targets other MT-associated proteins to the plus end and thereby regulates interactions of MTs with the cell cortex, mitotic kinetochores, and different cellular organelles [1, 2]. EB1 also localizes to centrosomes and is required for centrosomal MT anchoring and organization of the MT network [3, 4]. We previously showed that EB1 localizes to the flagellar tip and proximal region of the basal body in Chlamydomonas[5], but the function of EB1 in the cilium/flagellum is unknown. We depleted EB1 from NIH3T3 fibroblasts by using siRNA and found that EB1 depletion causes a approximately 50% reduction in the efficiency of primary cilia assembly in serum-starved cells. Expression of dominant-negative EB1 also inhibited cilia formation, and expression of mutant dominant-negative EB1 constructs suggested that binding of EB1 to p150(Glued) is important for cilia assembly. Finally, expression of a C-terminal fragment of the centrosomal protein CAP350, which removes EB1 from the centrosome but not MT plus ends [6], also inhibited ciliogenesis. We conclude that localization of EB1 at the centriole/basal body is required for primary cilia assembly in fibroblasts.

Analysis of Caveolin in Primary Cilia.

Recent evidence has indicated that caveolins are localized at the base of primary cilia, which are microtubule-based sensory organelles present on the cell surface, and that Caveolin-1 (CAV1) plays important roles in regulating ciliary membrane composition and function. Here we describe methods to analyze the localization and function of CAV1 in primary cilia of cultured mammalian cells. These include methods for culturing and transfecting mammalian cells with a CAV1-encoding plasmid or small interfering RNA (siRNA), analysis of mammalian cells by immunofluorescence microscopy (IFM) with antibodies against ciliary markers and CAV1, as well as methods for analyzing ciliary CAV1 function in siRNA-treated cells by IFM and cell-based signaling assays.

TGFß Signaling Increases Net Acid Extrusion, Proliferation and Invasion in Panc-1 Pancreatic Cancer Cells: SMAD4 Dependence and Link to Merlin/NF2 Signaling.

Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer-related death, with a 5-year survival of <10% and severely limited treatment options. PDAC hallmarks include profound metabolic acid production and aggressive local proliferation and invasiveness. This phenotype is supported by upregulated net acid extrusion and epithelial-to-mesenchymal transition (EMT), the latter typically induced by aberrant transforming growth factor-ß (TGFß) signaling. It is, however, unknown whether TGFß-induced EMT and upregulation of acid extrusion are causally related. Here, we show that mRNA and protein expression of the net acid extruding transporters Na+/H+ exchanger 1 (NHE1, SLC9A1) and Na+, HCO 3 - cotransporter 1 (NBCn1, SLC4A7) are increased in a panel of human PDAC cell lines compared to immortalized human pancreatic ductal epithelial (HPDE) cells. Treatment of Panc-1 cells (which express SMAD4, required for canonical TGFß signaling) with TGFß-1 for 48 h elicited classical EMT with down- and upregulation of epithelial and mesenchymal markers, respectively, in a manner inhibited by SMAD4 knockdown. Accordingly, less pronounced EMT was induced in BxPC-3 cells, which do not express SMAD4. TGFß-1 treatment elicited a SMAD4-dependent increase in NHE1 expression, and a smaller, SMAD4-independent increase in NBCn1 in Panc-1 cells. Consistent with this, TGFß-1 treatment led to elevated intracellular pH and increased net acid extrusion capacity in Panc-1 cells, but not in BxPC-3 cells, in an NHE1-dependent manner. Proliferation was increased in Panc-1 cells and decreased in BxPC-3 cells, upon TGFß-1 treatment, and this, as well as EMT per se, was unaffected by NHE1- or NBCn1 inhibition. TGFß-1-induced EMT was associated with a 4-fold increase in Panc-1 cell invasiveness, which further increased ~10-fold upon knockdown of the tumor suppressor Merlin (Neurofibromatosis type 2). Knockdown of NHE1 or NBCn1 abolished Merlin-induced invasiveness, but not that induced by TGFß-1 alone. In conclusion, NHE1 and NBCn1 expression and NHE-dependent acid extrusion are upregulated during TGFß-1-induced EMT of Panc-1 cells. NHE1 upregulation is SMAD4-dependent, and SMAD4-deficient BxPC-3 cells show no change in pHi regulation. NHE1 and NBCn1 are not required for EMT per se or EMT-associated proliferation changes, but are essential for the potentiation of invasiveness induced by Merlin knockdown.

Concerted action of kinesins KIF5B and KIF13B promotes efficient secretory vesicle transport to microtubule plus ends.

Intracellular transport relies on multiple kinesins, but it is poorly understood which kinesins are present on particular cargos, what their contributions are and whether they act simultaneously on the same cargo. Here, we show that Rab6-positive secretory vesicles are transported from the Golgi apparatus to the cell periphery by kinesin-1 KIF5B and kinesin-3 KIF13B, which determine the location of secretion events. KIF5B plays a dominant role, whereas KIF13B helps Rab6 vesicles to reach freshly polymerized microtubule ends, to which KIF5B binds poorly, likely because its cofactors, MAP7-family proteins, are slow in populating these ends. Sub-pixel localization demonstrated that during microtubule plus-end directed transport, both kinesins localize to the vesicle front and can be engaged on the same vesicle. When vesicles reverse direction, KIF13B relocates to the middle of the vesicle, while KIF5B shifts to the back, suggesting that KIF5B but not KIF13B undergoes a tug-of-war with a minus-end directed motor.

Dissecting the molecular mechanisms of intraflagellar transport in chlamydomonas.

BACKGROUND: The assembly and maintenance of eukaryotic cilia and flagella are mediated by intraflagellar transport (IFT), a bidirectional microtubule (MT)-based transport system. The IFT system consists of anterograde (kinesin-2) and retrograde (cDynein1b) motor complexes and IFT particles comprising two complexes, A and B. In the current model for IFT, kinesin-2 carries cDynein1b, IFT particles, and axonemal precursors from the flagellar base to the tip, and cDynein1b transports kinesin-2, IFT particles, and axonemal turnover products from the tip back to the base. Most of the components of the IFT system have been identified and characterized, but the mechanisms by which these different components are coordinated and regulated at the flagellar base and tip are unclear. RESULTS: Using a variety of Chlamydomonas mutants, we confirm that cDynein1b requires kinesin-2 for transport toward the tip and show that during retrograde IFT, kinesin-2 can exit the flagella independent of the cDynein1b light intermediate chain (LIC) and IFT particles. Furthermore, using biochemical approaches, we find that IFT complex B can associate with cDynein1b independent of complex A and cDynein1b LIC. Finally, using electron microscopy, we show that the IFT tip turnaround point most likely is localized distal to the plus end of the outer-doublet B MTs. CONCLUSION: Our results support a model for IFT in which tip turnaround involves (1) dissociation of IFT complexes A and B and release of inactive cDynein1b from complex B, (2) binding of complex A to active cDynein1b, and (3) reassociation of complex B with A prior to retrograde IFT.