scholarly journals Knockdown of ttc26 disrupts ciliogenesis of the photoreceptor cells and the pronephros in zebrafish

2012 ◽  
Vol 23 (16) ◽  
pp. 3069-3078 ◽  
Author(s):  
Qi Zhang ◽  
Qin Liu ◽  
Chrissy Austin ◽  
Iain Drummond ◽  
Eric A. Pierce
Keyword(s):  
Transition Zone ◽  
Primary Cilia ◽  
Collecting Duct ◽  
Genetic Disorders ◽  
Intraflagellar Transport ◽  
Photoreceptor Cells ◽  
Morpholino Knockdown ◽  
Sensory Cilia ◽  
Vertebrate Model

In our effort to understand genetic disorders of the photoreceptor cells of the retina, we have focused on intraflagellar transport in photoreceptor sensory cilia. From previous mouse proteomic data we identified a cilia protein Ttc26, orthologue of dyf-13 in Caenorhabditis elegans, as a target. We localized Ttc26 to the transition zone of photoreceptor and to the transition zone of cilia in cultured murine inner medullary collecting duct 3 (mIMCD3) renal cells. Knockdown of Ttc26 in mIMCD3 cells produced shortened and defective primary cilia, as revealed by immunofluorescence and scanning electron microscopy. To study Ttc26 function in sensory cilia in vivo, we utilized a zebrafish vertebrate model system. Morpholino knockdown of ttc26 in zebrafish embryos caused ciliary defects in the pronephric kidney at 27 h postfertilization and distension/dilation of pronephros at 5 d postfertilization (dpf). In the eyes, the outer segments of photoreceptor cells appeared shortened or absent, whereas cellular lamination appeared normal in retinas at 5 dpf. This suggests that loss of ttc26 function prevents normal ciliogenesis and differentiation in the photoreceptor cells, and that ttc26 is required for normal development and differentiation in retina and pronephros. Our studies support the importance of Ttc26 function in ciliogenesis and suggest that screening for TTC26 mutations in human ciliopathies is justified.

scholarly journals MAK and CCRK kinases regulate kinesin-2 motility in C. elegans neuronal cilia

2017 ◽  
Author(s):  
Peishan Yi ◽  
Chao Xie ◽  
Guangshuo Ou
Keyword(s):  
Cell Cycle ◽  
Sensory Neurons ◽  
Intraflagellar Transport ◽  
Time Lapse ◽  
Genetic Screen ◽  
Forward Genetic Screen ◽  
C Elegans ◽  
Neuronal Cilia ◽  
Sensory Cilia

AbstractKinesin-2 motors power the anterograde intraflagellar transport (IFT), a highly ordered process that assembles and maintains cilia. It remains elusive how kinesin-2 motors are regulated in vivo. Here we perform forward genetic screen to isolate suppressors that rescue the ciliary defects in the constitutive active mutation of OSM-3-kinesin (G444E) in C. elegans sensory neurons. We identify the C. elegans DYF-5 and DYF-18, which encode the homologs of mammalian male germ cell-associated kinase (MAK) and cell cycle-related kinase (CCRK). Using time-lapse fluorescence microscopy, we show that DYF-5 and DYF-18 are IFT cargo molecules and are enriched at the distal segments of sensory cilia. Mutations of dyf-5 and dyf-18 generate the elongated cilia and ectopic localization of kinesin-II at the ciliary distal segments. Genetic analyses reveal that dyf-5 and dyf-18 are also important for stabilizing the interaction between IFT particle and OSM-3-kinesin. Our data suggest that DYF-5 and DYF-18 act in the same pathway to promote handover between kinesin-II and OSM-3 in sensory cilia.

scholarly journals Prominins control ciliary length throughout the animal kingdom: New lessons from human prominin-1 and zebrafish prominin-3

2020 ◽  
Vol 295 (18) ◽  
pp. 6007-6022 ◽  
Author(s):  
József Jászai ◽  
Kristina Thamm ◽  
Jana Karbanová ◽  
Peggy Janich ◽  
Christine A. Fargeas ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Photoreceptor Cells ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Loss Of Function ◽  
Animal Kingdom ◽  
Left Right Asymmetry ◽  
And Function ◽  
The Impact

Prominins (proms) are transmembrane glycoproteins conserved throughout the animal kingdom. They are associated with plasma membrane protrusions, such as primary cilia, as well as extracellular vesicles derived thereof. Primary cilia host numerous signaling pathways affected in diseases known as ciliopathies. Human PROM1 (CD133) is detected in both somatic and cancer stem cells and is also expressed in terminally differentiated epithelial and photoreceptor cells. Genetic mutations in the PROM1 gene result in retinal degeneration by impairing the proper formation of the outer segment of photoreceptors, a modified cilium. Here, we investigated the impact of proms on two distinct examples of ciliogenesis. First, we demonstrate that the overexpression of a dominant-negative mutant variant of human PROM1 (i.e. mutation Y819F/Y828F) significantly decreases ciliary length in Madin–Darby canine kidney cells. These results contrast strongly to the previously observed enhancing effect of WT PROM1 on ciliary length. Mechanistically, the mutation impeded the interaction of PROM1 with ADP-ribosylation factor–like protein 13B, a key regulator of ciliary length. Second, we observed that in vivo knockdown of prom3 in zebrafish alters the number and length of monocilia in the Kupffer's vesicle, resulting in molecular and anatomical defects in the left-right asymmetry. These distinct loss-of-function approaches in two biological systems reveal that prom proteins are critical for the integrity and function of cilia. Our data provide new insights into ciliogenesis and might be of particular interest for investigations of the etiologies of ciliopathies.

scholarly journals Fifteen years of research on oral–facial–digital syndromes: from 1 to 16 causal genes

2017 ◽  
Vol 54 (6) ◽  
pp. 371-380 ◽  
Author(s):  
Ange-Line Bruel ◽  
Brunella Franco ◽  
Yannis Duffourd ◽  
Julien Thevenon ◽  
Laurence Jego ◽  
...  
Keyword(s):  
Transition Zone ◽  
Genetic Disorders ◽  
Intraflagellar Transport ◽  
Heterozygous Mutation ◽  
Type I ◽  
Gene Encoding ◽  
Distinct Subgroup ◽  
Functional Studies ◽  
New Genes ◽  
Wide Range

Oral–facial–digital syndromes (OFDS) gather rare genetic disorders characterised by facial, oral and digital abnormalities associated with a wide range of additional features (polycystic kidney disease, cerebral malformations and several others) to delineate a growing list of OFDS subtypes. The most frequent, OFD type I, is caused by a heterozygous mutation in theOFD1gene encoding a centrosomal protein. The wide clinical heterogeneity of OFDS suggests the involvement of other ciliary genes. For 15 years, we have aimed to identify the molecular bases of OFDS. This effort has been greatly helped by the recent development of whole-exome sequencing (WES). Here, we present all our published and unpublished results for WES in 24 cases with OFDS. We identified causal variants in five new genes (C2CD3,TMEM107,INTU,KIAA0753andIFT57) and related the clinical spectrum of four genes in other ciliopathies (C5orf42,TMEM138,TMEM231andWDPCP) to OFDS. Mutations were also detected in two genes previously implicated in OFDS. Functional studies revealed the involvement of centriole elongation, transition zone and intraflagellar transport defects in OFDS, thus characterising three ciliary protein modules: the complex KIAA0753-FOPNL-OFD1, a regulator of centriole elongation; the Meckel-Gruber syndrome module, a major component of the transition zone; and the CPLANE complex necessary for IFT-A assembly. OFDS now appear to be a distinct subgroup of ciliopathies with wide heterogeneity, which makes the initial classification obsolete. A clinical classification restricted to the three frequent/well-delineated subtypes could be proposed, and for patients who do not fit one of these three main subtypes, a further classification could be based on the genotype.

scholarly journals Photoreceptor cilia, in contrast to primary cilia, grant entry to a partially assembled BBSome

2021 ◽  
Author(s):  
Ying Hsu ◽  
Seongjin Seo ◽  
Val C Sheffield
Keyword(s):  
Sucrose Gradient ◽  
Primary Cilia ◽  
Intraflagellar Transport ◽  
Photoreceptor Cells ◽  
Small Gtpase ◽  
Functional Differences ◽  
Retinal Photoreceptors ◽  
The Status ◽  
Specific Regulation ◽  
Gradient Fractionation

Abstract The BBSome is a protein complex consisting of BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, BBS9 and BBS18 that associates with intraflagellar transport complexes and specializes in ciliary trafficking. In primary cilia, ciliary entry requires the fully assembled BBSome as well as the small GTPase, ARL6 (BBS3). Retinal photoreceptors possess specialized cilia. In light of key structural and functional differences between primary and specialized cilia, we examined the principles of BBSome recruitment to photoreceptor cilia. We performed sucrose gradient fractionation using retinal lysates of Bbs2−/−, Bbs7−/−, Bbs8−/− and Bbs3−/− mice to determine the status of BBSome assembly, then determined localization of BBSome components using immunohistochemistry. Surprisingly, we found that a subcomplex of the BBSome containing at least BBS1, BBS5, BBS8 and BBS9 is recruited to cilia in the absence of BBS2 or BBS7. In contrast, a BBSome subcomplex consisting of BBS1, BBS2, BBS5, BBS7 and BBS9 is found in Bbs8−/− retinas and is denied ciliary entry in photoreceptor cells. In addition, the BBSome remains fully assembled in Bbs3−/− retinas and can be recruited to photoreceptor cilia in the absence of BBS3. We compared phenotypic severity of their retinal degeneration phenotypes. These findings demonstrate that unlike primary cilia, photoreceptor cilia admit a partially assembled BBSome meeting specific requirements. In addition, the recruitment of the BBSome to photoreceptor cilia does not require BBS3. These findings indicate that the ciliary entry of the BBSome is subjected to cell-specific regulation, particularly in cells with highly adapted forms of cilia such as photoreceptors.

scholarly journals Dynein-2 intermediate chains play crucial but distinct roles in primary cilia formation and function

2018 ◽  
Author(s):  
Laura Vuolo ◽  
Nicola L. Stevenson ◽  
Kate J. Heesom ◽  
David J. Stephens
Keyword(s):  
Transition Zone ◽  
Cell Lines ◽  
Primary Cilia ◽  
Point Mutations ◽  
Intraflagellar Transport ◽  
Jeune Syndrome ◽  
Cilia Formation ◽  
Microtubule Motor ◽  
And Function ◽  
Intermediate Chains

AbstractThe dynein-2 microtubule motor is the retrograde motor for intraflagellar transport. Mutations in dynein-2 components cause skeletal ciliopathies, notably Jeune syndrome. Dynein-2 comprises a heterodimer of two non-identical intermediate chains, WDR34 and WDR60. Here, we use knockout cell lines to demonstrate that each intermediate chain has a distinct role in cilia function. Both proteins are required to maintain a functional transition zone and for efficient bidirectional intraflagellar transport, only WDR34 is essential for axoneme extension. In contrast, only WDR60 is essential for co-assembly of the other subunits. Furthermore, WDR60 cannot compensate for loss of WDR34 or vice versa. This work defines a functional asymmetry to match the subunit asymmetry within the dynein-2 motor. Analysis of causative point mutations in WDR34 and WDR60 can partially restore function to knockout cells. Our data show that Jeune syndrome is caused by defects in transition zone architecture as well as intraflagellar transport.SUMMARYHere, Vuolo and colleagues use engineered knockout human cell lines to define roles for dynein-2 intermediate chains. WDR34 is required for axoneme extension, while WDR60 is not. Both subunits are required for cilia transition zone organization and bidirectional intraflagellar transport.

scholarly journals Autoinhibition regulates the motility of the C. elegans intraflagellar transport motor OSM-3

2006 ◽  
Vol 174 (7) ◽  
pp. 931-937 ◽  
Author(s):  
Miki Imanishi ◽  
Nicholas F. Endres ◽  
Arne Gennerich ◽  
Ronald D. Vale
Keyword(s):  
Atpase Activity ◽  
Single Molecule ◽  
Intramolecular Interaction ◽  
Optical Trap ◽  
Coiled Coil ◽  
Intraflagellar Transport ◽  
Wild Type ◽  
C Elegans ◽  
Sensory Cilia

OSM-3 is a Kinesin-2 family member from Caenorhabditis elegans that is involved in intraflagellar transport (IFT), a process essential for the construction and maintenance of sensory cilia. In this study, using a single-molecule fluorescence assay, we show that bacterially expressed OSM-3 in solution does not move processively (multiple steps along a microtubule without dissociation) and displays low microtubule-stimulated adenosine triphosphatase (ATPase) activity. However, a point mutation (G444E) in a predicted hinge region of OSM-3's coiled-coil stalk as well as a deletion of that hinge activate ATPase activity and induce robust processive movement. These hinge mutations also cause a conformational change in OSM-3, causing it to adopt a more extended conformation. The motility of wild-type OSM-3 also can be activated by attaching the motor to beads in an optical trap, a situation that may mimic attachment to IFT cargo. Our results suggest that OSM-3 motility is repressed by an intramolecular interaction that involves folding about a central hinge and that IFT cargo binding relieves this autoinhibition in vivo. Interestingly, the G444E allele in C. elegans produces similar ciliary defects to an osm-3–null mutation, suggesting that autoinhibition is important for OSM-3's biological function.

scholarly journals Inhibition of Pkhd1 Impairs Tubulomorphogenesis of Cultured IMCD Cells

2005 ◽  
Vol 16 (9) ◽  
pp. 4398-4409 ◽  
Author(s):  
Weiyi Mai ◽  
Dong Chen ◽  
Tianbing Ding ◽  
Ingyu Kim ◽  
Sujun Park ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Cell Function ◽  
Collecting Duct ◽  
Three Dimensional ◽  
Duct Cell ◽  
Cell Contact ◽  
Cytoskeleton Organization ◽  
Actin Cytoskeleton Organization

Fibrocystin/polyductin (FPC), the gene product of PKHD1, is responsible for autosomal recessive polycystic kidney disease (ARPKD). This disease is characterized by symmetrically large kidneys with ectasia of collecting ducts. In the kidney, FPC predominantly localizes to the apical domain of tubule cells, where it associates with the basal bodies/primary cilia; however, the functional role of this protein is still unknown. In this study, we established stable IMCD (mouse inner medullary collecting duct) cell lines, in which FPC was silenced by short hairpin RNA inhibition (shRNA). We showed that inhibition of FPC disrupted tubulomorphogenesis of IMCD cells grown in three-dimensional cultures. Pkhd1-silenced cells developed abnormalities in cell-cell contact, actin cytoskeleton organization, cell-ECM interactions, cell proliferation, and apoptosis, which may be mediated by dysregulation of extracellular-regulated kinase (ERK) and focal adhesion kinase (FAK) signaling. These alterations in cell function in vitro may explain the characteristics of ARPKD phenotypes in vivo.

scholarly journals Dynein-2 intermediate chains play crucial but distinct roles in primary cilia formation and function

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Laura Vuolo ◽  
Nicola L Stevenson ◽  
Kate J Heesom ◽  
David J Stephens
Keyword(s):  
Transition Zone ◽  
Primary Cilia ◽  
Complex Function ◽  
Intraflagellar Transport ◽  
Loss Of Function ◽  
Jeune Syndrome ◽  
Cilia Formation ◽  
Microtubule Motor ◽  
And Function ◽  
Intermediate Chains

The dynein-2 microtubule motor is the retrograde motor for intraflagellar transport. Mutations in dynein-2 components cause skeletal ciliopathies, notably Jeune syndrome. Dynein-2 contains a heterodimer of two non-identical intermediate chains, WDR34 and WDR60. Here, we use knockout cell lines to demonstrate that each intermediate chain has a distinct role in cilium function. Using quantitative proteomics, we show that WDR34 KO cells can assemble a dynein-2 motor complex that binds IFT proteins yet fails to extend an axoneme, indicating complex function is stalled. In contrast, WDR60 KO cells do extend axonemes but show reduced assembly of dynein-2 and binding to IFT proteins. Both proteins are required to maintain a functional transition zone and for efficient bidirectional intraflagellar transport. Our results indicate that the subunit asymmetry within the dynein-2 complex is matched with a functional asymmetry between the dynein-2 intermediate chains. Furthermore, this work reveals that loss of function of dynein-2 leads to defects in transition zone architecture, as well as intraflagellar transport.

scholarly journals Spinocerebellar ataxia type 11-associated alleles of Ttbk2 dominantly interfere with ciliogenesis and cilium stability

2018 ◽  
Author(s):  
Emily Bowie ◽  
Ryan Norris ◽  
Kathryn V. Anderson ◽  
Sarah C. Goetz
Keyword(s):  
Spinocerebellar Ataxia ◽  
Primary Cilia ◽  
Genetic Disorders ◽  
Dominant Negative ◽  
Full Length ◽  
Spinocerebellar Ataxia Type ◽  
Allelic Series ◽  
Shh Pathway ◽  
Ciliary Signaling

AbstractSpinocerebellar ataxia type 11 (SCA11) is a rare, dominantly inherited human ataxia characterized by atrophy of Purkinje neurons in the cerebellum. SCA11 is caused by mutations in the gene encoding the Serine/Threonine kinase Tau tubulin kinase 2 (TTBK2) that result in premature truncations of the protein. We previously showed that TTBK2 is a key regulator of the assembly of primary cilia in vivo. However, the mechanisms by which the SCA11-associated mutations disrupt TTBK2 function, and whether they interfere with ciliogenesis were unknown. In this work, we present evidence that SCA11-associated mutations are dominant negative alleles and that the resulting truncated protein (TTBK2SCA11) interferes with the function of full length TTBK2 in mediating ciliogenesis. A Ttbk2 allelic series revealed that upon partial reduction of full length TTBK2 function, TTBK2SCA11 can interfere with the activity of the residual wild-type protein to decrease cilia number and interrupt cilia-dependent Sonic hedgehog (SHH) signaling. Our studies have also revealed new functions for TTBK2 after cilia initiation in the control of cilia length, trafficking of a subset of SHH pathway components, including Smoothened (SMO), and cilia stability. These studies provide a molecular foundation to understand the cellular and molecular pathogenesis of human SCA11, and help account for the link between ciliary dysfunction and neurodegenerative diseases.Author SummaryDefects in primary cilia structure and function are linked to a number of recessive genetic disorders, now collectively referred to as ciliopathies. Most of the characteristics of these disorders arise from disruptions to embryonic development, with the requirements for primary cilia in adult tissues being less well-defined. We previously showed that a kinase associated with an adult-onset neurodegenerative condition is required for cilium assembly and ciliary signaling during development. Here, we show that the human disease-associated mutations act as mild dominant negatives, interfering with the function of the full length protein in cilia formation and ciliary signaling.

scholarly journals Intraflagellar transport-A deficiency attenuates ADPKD in a renal tubular- and maturation-dependent manner

2020 ◽  
Author(s):  
Wei Wang ◽  
Luciane M. Silva ◽  
Henry H. Wang ◽  
Matthew A. Kavanaugh ◽  
Tana S. Pottorf ◽  
...  
Keyword(s):  
Mouse Models ◽  
Primary Cilia ◽  
Protein Complexes ◽  
Collecting Duct ◽  
Intraflagellar Transport ◽  
Renal Tubules ◽  
Dependent Manner ◽  
Cortical Collecting Duct ◽  
Knock Out

AbstractPrimary cilia are sensory organelles that are built and maintained by intraflagellar transport (IFT) multi-protein complexes. Deletion of certain ciliary genes in Autosomal Dominant Polycystic Kidney Disease (ADPKD) mouse models markedly attenuates PKD severity, indicating that a component of cilia dysfunction may have potential therapeutic value. To broaden the role of ciliary dysfunction, here we investigate the role of global deletion of Ift-A gene, Thm1, in juvenile and adult ADPKD mouse models. In cyst-lining cells of both juvenile and adult ADPKD models, cortical collecting duct cilia lengths and cytoplasmic and nuclear levels of the nutrient sensor, O-linked β-Nacetylglucosamine (O-GlcNAc) were increased. Relative to juvenile Pkd2 conditional knock-out mice, deletion of Thm1 together with Pkd2 both increased and reduced cystogenesis in a tubule-specific manner without altering kidney function, inflammation, cilia lengths, and ERK, STAT3 and OGlcNAc signaling. In contrast, Thm1 deletion in adult ADPKD mouse models markedly attenuated almost all features of PKD, including renal cystogenesis, inflammation, cilia lengths, and ERK, STAT3 and O-GlcNAc signaling. These data suggest that differential factors in the microenvironments between renal tubules and between developing and mature kidneys influence cilia and ADPKD pathobiology. Further, since O-GlcNAcylation directly regulates ciliary homeostasis and the balance between glycolysis and oxidative phosphorylation, we propose that increased O-GlcNAcylation may promote certain key ADPKD pathological processes.

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