0000000000017583
AUTHOR
Nasrin Sorusch
PDZD7 connects the Usher protein complex to the intraflagellar transport machinery
Several Usher syndrome (USH)-associated proteins are known to localize to the connecting cilium of photoreceptor cells. The unconventional myosin MYO7A (USH1B) was long accepted as the transport molecule responsible for the ciliary localization of USH proteins. However, based on the typical location of several of the USH proteins along the ciliary axoneme, the involvement of the main ciliary trafficking machinery, intraflagellar transport (IFT), seems apparent. The USH-associated scaffold protein PDZD7 is known to interact with SANS, Usherin, GPR98 and Whirlin, all of which can be found in the connecting cilium. Here, we report that PDZD7 provides the physical link of the USH-protein networ…
KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome
Background Joubert syndrome (JBTS) and related disorders are defined by cerebellar malformation (molar tooth sign), together with neurological symptoms of variable expressivity. The ciliary basis of Joubert syndrome related disorders frequently extends the phenotype to tissues such as the eye, kidney, skeleton and craniofacial structures. Results Using autozygome and exome analyses, we identified a null mutation in KIAA0556 in a multiplex consanguineous family with hallmark features of mild Joubert syndrome. Patient-derived fibroblasts displayed reduced ciliogenesis potential and abnormally elongated cilia. Investigation of disease pathophysiology revealed that Kiaa0556-/- null mice possess…
A high-throughput genome-wide siRNA screen for ciliogenesis identifies new ciliary functional components and ciliopathy genes.
Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe the first whole genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource for investigation and interventions into the processes that are critical for the ciliary system. In total, we identified 83 candidate ciliogenesis and ciliopathy genes, including 15 components of the ubiquitin-proteasome system. The validated hits also include 12 encoding G-protein-coupled receptors, and three encoding pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. Com…
Disruption of the retinitis pigmentosa 28 gene Fam161a in mice affects photoreceptor ciliary structure and leads to progressive retinal degeneration.
Mutations in the FAM161A gene were previously identified as the cause for autosomal-recessive retinitis pigmentosa 28. To study the effects of Fam161a dysfunction in vivo, we generated gene-trapped Fam161a(GT/GT) mice with a disruption of its C-terminal domain essential for protein-protein interactions. We confirmed the absence of the full-length Fam161a protein in the retina of Fam161a(GT/GT) mice using western blots and showed weak expression of a truncated Fam161a protein by immunohistochemistry. Histological analyses demonstrated that photoreceptor segments were disorganized in young Fam161a(GT/GT) mice and that the outer retina was completely lost at 6 months of age. Reactive microglia…
Usher Syndrome Protein Network Functions in the Retina and their Relation to Other Retinal Ciliopathies
The human Usher syndrome (USH) is the most frequent cause of combined hereditary deaf-blindness. USH is genetically and clinically heterogeneous: 15 chromosomal loci assigned to 3 clinical types, USH1-3. All USH1 and 2 proteins are organized into protein networks by the scaffold proteins harmonin (USH1C), whirlin (USH2D) and SANS (USH1G). This has contributed essentially to our current understanding of the USH protein function in the eye and the ear and explains why defects in proteins of different families cause very similar phenotypes. Ongoing in depth analyses of USH protein networks in the eye indicated cytoskeletal functions as well as roles in molecular transport processes and ciliary…
Usherin defects lead to early-onset retinal dysfunction in zebrafish
Mutations in USH2A are the most frequent cause of Usher syndrome and autosomal recessive nonsyndromic retinitis pigmentosa. To unravel the pathogenic mechanisms underlying USH2A-associated retinal degeneration and to evaluate future therapeutic strategies that could potentially halt the progression of this devastating disorder, an animal model is needed. The available Ush2a knock-out mouse model does not mimic the human phenotype, because it presents with only a mild and late-onset retinal degeneration. Using CRISPR/Cas9-technology, we introduced protein-truncating germline lesions into the zebrafish ush2a gene (ush2a(rmc1): c.2337_2342delinsAC; p.Cys780GlnfsTer32 and ush2a(b1245): c.15520_…
An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes
Item does not contain fulltext Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin-proteasome system, 12 G-protein-coupled receptors, and 3 pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localize to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequen…
The giant spectrin βV couples the molecular motors to phototransduction and Usher syndrome type I proteins along their trafficking route.
International audience; Mutations in the myosin VIIa gene cause Usher syndrome type IB (USH1B), characterized by deaf-blindness. A delay of opsin trafficking has been observed in the retinal photoreceptor cells of myosin VIIa-deficient mice. We identified spectrin bV, the mammalian b-heavy spectrin, as a myosin VIIa-and rhodopsin-interacting partner in photoreceptor cells. Spectrin bV displays a polarized distribution from the Golgi apparatus to the base of the outer segment, which, unlike that of other b spectrins, matches the trafficking route of opsin and other phototransduction proteins. Formation of spectrin bV-rhodopsin complex could be detected in the differentiating photoreceptors a…
SANS (USH1G) Molecularly Links the Human Usher Syndrome Protein Network to the Intraflagellar Transport Module by Direct Binding to IFT-B Proteins.
The human Usher syndrome (USH) is a retinal ciliopathy, characterized by profound congenital deafness, variable vestibular dysfunction and pre-pubertal onset of retinitis pigmentosa. In the effected sensory cells, USH protein networks are assumed to function in ciliary transport processes. The USH1G protein SANS is a scaffold of the ciliary/periciliary USH protein network of photoreceptor cells. Moreover, SANS is associated with microtubules, the transport routes for protein delivery toward the cilium. To enlighten the role of SANS in ciliary transport processes, we aimed to identify transport related proteins associated with SANS. The intraflagellar transport (IFT) system is a conserved me…
The Usher syndrome 1G protein SANS participates in the transport of ciliary cargo in photoreceptor cells
Human Usher syndrome (USH) is the most common form of combined deaf-blindness, characterized by profound congenital deafness, constant vestibular dysfunction and pre-pubertal onset of retinitis pigmentosa. The USH1G protein SANS (scaffold protein containing ankyrin repeats and SAM domain) is associated with microtubules and mediates a transport related periciliary protein network in photoreceptor cells. Here we aim to enlighten the involvement of SANS in ciliary transport of photoreceptor cells by identifying proteins associated with SANS in transport complexes. In Y2H screen of retinal cDNA library we identified the direct binding of SANS to dynactin-1 (p150Glued), a subunit of the dynacti…
Additional file 1: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome
The phylogenetic distribution and sequence conservation of KIAA0556 orthologs in eukaryotes. Presence and sequence conservation of KIAA0556 are projected on the eukaryotic species tree to visualise the phylogenetic distribution of KIAA0556 orthologues as well as the distribution of the triple-repeat and quadruple-repeat configurations of the DUF4457 domains of unknown function. The black circles and white circles indicate which eukaryotic species contain or lack cilia/flagella. Recent KIAA0556 duplicates in Branchiostoma floridae and Paramecium tetraurelia are denoted by x2. *Dictyostelium discoideum protein sequence contains many â Nâ s (uncalled bases) in the N-terminal part of the sequen…
The retinitis pigmentosa 28 protein FAM161A is a novel ciliary protein involved in intermolecular protein interaction and microtubule association
Loss-of-function mutations in the gene encoding FAM161A were recently discovered as the cause for RP28, an autosomal recessive form of retinitis pigmentosa. To initiate the characterization of the cellular role of FAM161A in the retina, we focused on its subcellular localization and conducted in vitro studies to identify FAM161A-interacting proteins and associated cellular structures. Immunohistochemistry revealed the presence of mouse FAM161A in the photoreceptor inner segments, the synaptic regions of the outer and inner plexiform layers and the ganglion cells. In mouse and human retinal sections from unfixed eyes, FAM161A localized to the ciliary region linking photoreceptor outer and in…
Additional file 1: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome
The phylogenetic distribution and sequence conservation of KIAA0556 orthologs in eukaryotes. Presence and sequence conservation of KIAA0556 are projected on the eukaryotic species tree to visualise the phylogenetic distribution of KIAA0556 orthologues as well as the distribution of the triple-repeat and quadruple-repeat configurations of the DUF4457 domains of unknown function. The black circles and white circles indicate which eukaryotic species contain or lack cilia/flagella. Recent KIAA0556 duplicates in Branchiostoma floridae and Paramecium tetraurelia are denoted by x2. *Dictyostelium discoideum protein sequence contains many â Nâ s (uncalled bases) in the N-terminal part of the sequen…
Additional file 6: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome
Results of the SF-TAP analysis with over-expressed N-terminally SF-TAP-tagged KIAA0556 in HEK293T cells. Shown is the number of unique identified peptides as well as the sequence coverage for each protein detected by mass spectrometry. Proteins identified in >1 out 17 SF-TAP control experiments (empty vector) were removed. (XLSX 28 kb)
Additional file 4: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome
IFT analysis in C. elegans K04F10.2( tm1830 ) mutants. a Intraflagellar transport rates in wild-type and K04F10.2(tm1830) mutant worms. Shown are the anterograde and retrograde velocities (μm.s-1/standard deviation (SD)) of GFP-tagged IFT proteins along amphid and phasmid channel cilia (combined; top rows), or phasmid cilia only (bottom rows). t-test pairwise comparison with wild-type controls, n number of particles, N measured number of amphids and phasmids. OSM-3 is the worm orthologue of KIF17; CHE-11 is the worm orthologue of IFT140; OSM-6 is the worm orthologue of IFT52. b Representative fluorescence images of phasmid cilia showing normal IFT protein localisations and distributions in …
Additional file 8: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome
Post-embryonic tissue expression of C. elegans katanin genes mei-1 , mei-2 and F47G4.4. Shown are fluorescence images of worms expressing a transcriptional GFP reporter under the control of the indicated geneâ s promoter, which stains the entire cell in which it is expressed. DiI (red) co-stain identifies six pairs of ciliated amphid neurons and both pairs of ciliated phasmid neurons. Arrowheads denote cells with both red and green signals. Other ciliated head cells are identifiable by long dendritic processes (arrows) extending to the anterior end of the worm. Scale bars, 20Â Îźm (all images similarly scaled). (JPG 611 kb)
Additional file 2: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome
Alignment of IFT25 with permutated KIAA0556 repeat sequences. When aligned using HHpred, a significant part of the Chlamydomonas IFT25 N-terminus was unmatched with human KIAA0556 and significant sequence remained at the C-terminus of the repeats, suggesting a circular permutation relationship between the repeats and IFT25. Shown is a HHpred alignment of IFT25 orthologues with permutated repeat sequences (r1–4) from KIAA0556 orthologues, which results in improved sequence matches. In each permutated repeat sequence, 30–40 amino acids from the beginning of each repeat have been added to the end of the same repeat (denoted by red box) using manual editing. The precise number of amino acids tr…
Additional file 3: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome
Ciliary phenotypes that are unaffected in C. elegans K04F10.2( tm1830 ) mutants. a K04F10.2 mutants possess normal fluorescent dye (DiI) filling in amphid (head) and phasmid (tail) neurons. Scale bars, 15 μm. b The lengths and morphologies of various sensory neuronal cilia are normal in K04F10.2 mutants. Shown are fluorescence images of cilia from worms expressing str-1p::GFP (AWB neuron), gcy-5p::GFP (ASER neuron) and OSM-6::GFP (PHA/B neurons) transgenes. Numbers (± standard error of the mean) refer to cilium lengths. Scale bars, 2 μm. c–e K04F10.2 mutants possess normal sensory benzaldehyde chemoattraction (n = 10), osmotic avoidance (n = 10), and foraging/roaming (n = 34) behaviours. ch…
Additional file 7: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome
Supplementary information to the data in Fig. 8 . a Schematic representation of all the different KIAA0556 fragments used to screen our selection of 200+ ciliary proteins. The predicted protein repeat domains, shown in Additional files 1 and 2, are depicted as d1 to d4. Constructs were generated containing isolated domains as well as a combination of domains. b Single transfections of PalMyr-KIAA0556 and mRFP-KATNBL1, showing that membrane localisation of the mRFP tagged protein is indeed dependent on the interaction with the PalMyr-tagged protein. (JPG 491 kb)
Additional file 5: of KIAA0556 is a novel ciliary basal body component mutated in Joubert syndrome
Data supplementary to the nocodazole destabilization assay shown in Fig. 7 . a, b Replicate images of DMSO or nocodazole-treated hTERT-RPE1 cells. Cells were transfected with SF-TAP-tagged KIAA0556 (detected with anti-FLAG immunostaining; green) or GFP-KIAA0556 and counterstained with anti-acetylated tubulin (red) and DAPI (blue). Cells with high KIAA0556 expression are characterised by a filamentous staining pattern and spots of accumulated KIAA0556 signal. In non-transfected cells, 10 minute nocodazole treatment resulted in the loss of a stabilised MT network (see especially the high exposure images), as judged by loss of (almost) all cytoplasmic acetylated tubulin staining and/or the abs…