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Joseph C Besharse - One of the best experts on this subject based on the ideXlab platform.
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transmembrane assemblage of the photoreceptor connecting Cilium and motile Cilium transition zone contain a common immunologic epitope
Cytoskeleton, 1990Co-Authors: Cynthia J Horst, Joseph C Besharse, Lincoln V JohnsonAbstract:The photoreceptor connecting Cilium bears a unique transmembrane assemblage which stably links cell surface glycoconjugates with the underlying axonemal cytoskeleton. Structural similarities between the photoreceptor connecting Cilium and the transition zone of motile cilia suggests that this assemblage may also be present in motile cilia. Using a subcellular fraction enriched in detergent-extracted photoreceptor axonemes, three high molecular mass glycoconjugates (425, 600, and 700 kD) were previously identified as potential components of the assemblage. Through oligosaccharide characterization and binding of a specific monoclonal antibody, we have verified the localization of the 425 kD glycoconjugate to the transmembrane assemblage. Binding of the lectin peanut agglutinin (PNA) to the 425 kD glycoconjugate on nitrocellulose blots, and to isolated detergentextracted axonemes, was assessed following treatment with the enzymes neuraminidase and O-glycanase. Changes in binding to the 425 kD glycoconjugate precisely paralleled changes in binding to intact axonemes, supporting the hypothesis that the 425 kD glycoconjugate is a component of the transmembrane assemblage. Furthermore, the results suggest that the 425 kD glycoconjugate contains sialated galactose-N-acetylgalactosamine oligosaccharides which are Olinked to the protein backbone. To directly assess the distribution of the 425 kD glycoconjugate, we produced a monoclonal antibody directed against this glycoconjugate. The antibody, K26, recognizes only the 425 kD on transblots of the axoneme fraction. K26 immunoreactivity of intact axonemes is identical to that seen by PNA staining. K26 staining of isolated photoreceptors and whole retina is uniquely localized to the region of the connecting Cilium. Thus, in the photoreceptor, the 425 kD is not only a component of the transmembrane assemblage but is also completely restricted to the connecting Cilium. Based on morphological similarities, the photoreceptor connecting Cilium is thought to be homologous to the transition zone of the motile Cilium. As such, we have stained oviduct epithelium with the K26 monoclonal antibody. Immunoreactivity is restricted to the region of the transition zone at the base of motile cilia. This demonstrates that the photoreceptor connecting Cilium and motile Cilium transition zone are immunologically related.
Uwe Wolfrum - One of the best experts on this subject based on the ideXlab platform.
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protein networks and complexes in photoreceptor cilia
Sub-cellular biochemistry, 2007Co-Authors: Ronald Roepman, Uwe WolfrumAbstract:Vertebrate photoreceptor cells are ciliated sensory cells specialized for single photon detection. The photoreceptor outer segment corresponds to the ciliary shaft of a prototypic Cilium. In the outer segment compartment, the ciliary membrane is highly modified into membranous disks which are enveloped by the plasma membrane in rod cells. At these outer segment disks, the visual transduction cascade--a prototypical G-protein coupled receptor transduction pathway is arranged. The light sensitive outer segments are linked by the socalled connecting Cilium with the inner segment, the photoreceptor compartment which contains all organelles necessary for cell metabolism. The connecting Cilium correlates with the transition zone, the short junction between the basal body and the axoneme of a prototypic Cilium. The connecting Cilium and the calycal processes, including the periciliary ridge complex, as well as the basal body complex are in close functional association with each other. In the latter ciliary compartments, the export and import from/into the outer segment of the photoreceptor cell are controlled and regulated. In all subciliary compartments, proteins are arranged in functional multiprotein complexes. In the outer segment, signaling components are arranged into complexes which provide specificity and speed for the signaling and serve in adaptation. Centrin-G-protein complexes may regulate the light driven translocation of the visual G-protein transducin through the connecting Cilium. Intraflagellar transport (IFT) complexes may serve in intersegmental exchange of molecules. The import/export of molecules is thought to be regulated by proteins arranged in networks at the basal body complex. Proteins of the interactome related to the human Usher syndrome are localized in the connecting Cilium and may participate in the ciliary transport, but are also arranged at interfaces between the inner segment and the connecting Cilium where they probably control the cargo handover between the transport systems of the inner segment and these of the Cilium. Furthermore, USH protein complexes may further provide mechanical stabilization to membrane specializations of the calycal processes and the connecting Cilium. The protein complex in which the retinitis pigmentosa GTPase regulator (RPGR) participates in the ciliary compartments also plays a key role in the function and maintenance of photoreceptor cells. It further associates through the presumed scaffolding protein RPGRIP1 with the nephrocystin protein network. Although many of these proteins have been also found in prototypic cilia or primary cilia, the arrangements of the proteins in complexes can be specific for vertebrate photoreceptor cells. Defects of proteins in these complexes lead to photoreceptor cell death and retinal degeneration, underlying syndromic and non-syndromic blindness.
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Identification of novel molecular components of the photoreceptor connecting Cilium by immunoscreens.
Experimental Eye Research, 2001Co-Authors: Angelika Schmitt, Uwe WolfrumAbstract:Abstract The connecting Cilium of photoreceptor cells is the only intracellular link between the morphologically, functionally and biochemically different compartments of the inner and outer segments. The non-motile modified Cilium plays an important role in the organization and the function of photoreceptor cells, namely in delivery and turnover of enzymes and substrates of the visual transduction cascade, and the photosensitive membranes of the outer segment. The protein components of the Cilium participate in the intracellular transport through the Cilium, in the outer segment disk morphogenesis and in the maintenance of discrete membrane domains. In order to identify yet unknown cytoskeletal components of the connecting Cilium, a combined biochemical and molecular biological strategy was applied. For this purpose, antibodies were raised against proteins of photoreceptor cell axonemes. Using this AX-4-antiserum, a rat retina cDNA expression library was immunoscreened and clones encoding partial sequences of (i) already known photoreceptor specific proteins; (ii) ubiquitously expressed proteins; (iii) clones with homologies to retinal ESTs; and (iv) clones coding for cytoskeletal proteins were isolated. Further analysis revealed that these candidate clones have homologies to Drosophila flightless I, mouse APC-binding protein EB2, human microtubule associated protein 4 (MAP4), human centrin 3, human cytoplasmic dynein intermediate chain 2C, and human dynamitin. The immunoscreening approach used here was successfully applied to isolate genes encoding yet unknown cytoskeletal proteins of photoreceptor cell axonemes. The obtained information will provide further insight into the role of the connecting Cilium in photoreceptor cell function.
Kathryn V. Anderson - One of the best experts on this subject based on the ideXlab platform.
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The kinesin-4 protein Kif7 regulates mammalian Hedgehog signalling by organizing the Cilium tip compartment
Nature Cell Biology, 2014Co-Authors: Mu He, Radhika Subramanian, Fiona Bangs, Tatiana Omelchenko, Karel F. Liem Jr, Tarun M. Kapoor, Kathryn V. AndersonAbstract:Mammalian Hedgehog (Hh) signal transduction requires a primary Cilium, a microtubule-based organelle, and the Gli–Sufu complexes that mediate Hh signalling, which are enriched at cilia tips. Kif7, a kinesin-4 family protein, is a conserved regulator of the Hh signalling pathway and a human ciliopathy protein. Here we show that Kif7 localizes to the Cilium tip, the site of microtubule plus ends, where it limits Cilium length and controls Cilium structure. Purified recombinant Kif7 binds the plus ends of growing microtubules in vitro , where it reduces the rate of microtubule growth and increases the frequency of microtubule catastrophe. Kif7 is not required for normal intraflagellar transport or for trafficking of Hh pathway proteins into cilia. Instead, a central function of Kif7 in the mammalian Hh pathway is to control Cilium architecture and to create a single Cilium tip compartment, where Gli–Sufu activity can be correctly regulated. Anderson and colleagues report that the kinesin-4 family member Kif7 binds to microtubule plus ends at Cilium tips to regulate their length and structure, and to ensure the fidelity of Hedgehog signalling.
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the kinesin 4 protein kif7 regulates mammalian hedgehog signalling by organizing the Cilium tip compartment
Nature Cell Biology, 2014Co-Authors: Radhika Subramanian, Fiona Bangs, Tatiana Omelchenko, Tarun M. Kapoor, Karel F Liem, Kathryn V. AndersonAbstract:Mammalian Hedgehog (Hh) signal transduction requires a primary Cilium, a microtubule-based organelle, and the Gli-Sufu complexes that mediate Hh signalling, which are enriched at cilia tips. Kif7, a kinesin-4 family protein, is a conserved regulator of the Hh signalling pathway and a human ciliopathy protein. Here we show that Kif7 localizes to the Cilium tip, the site of microtubule plus ends, where it limits Cilium length and controls Cilium structure. Purified recombinant Kif7 binds the plus ends of growing microtubules in vitro, where it reduces the rate of microtubule growth and increases the frequency of microtubule catastrophe. Kif7 is not required for normal intraflagellar transport or for trafficking of Hh pathway proteins into cilia. Instead, a central function of Kif7 in the mammalian Hh pathway is to control Cilium architecture and to create a single Cilium tip compartment, where Gli-Sufu activity can be correctly regulated.
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The primary Cilium: a signalling centre during vertebrate development
Nature Reviews Genetics, 2010Co-Authors: Sarah C. Goetz, Kathryn V. AndersonAbstract:The primary Cilium has recently stepped into the spotlight, as a flood of data show that this organelle has crucial roles in vertebrate development and human genetic diseases. Cilia are required for the response to developmental signals, and evidence is accumulating that the primary Cilium is specialized for hedgehog signal transduction. The formation of cilia, in turn, is regulated by other signalling pathways, possibly including the planar cell polarity pathway. The Cilium therefore represents a nexus for signalling pathways during development. The connections between cilia and developmental signalling have begun to clarify the basis of human diseases associated with ciliary dysfunction.
Cynthia J Horst - One of the best experts on this subject based on the ideXlab platform.
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transmembrane assemblage of the photoreceptor connecting Cilium and motile Cilium transition zone contain a common immunologic epitope
Cytoskeleton, 1990Co-Authors: Cynthia J Horst, Joseph C Besharse, Lincoln V JohnsonAbstract:The photoreceptor connecting Cilium bears a unique transmembrane assemblage which stably links cell surface glycoconjugates with the underlying axonemal cytoskeleton. Structural similarities between the photoreceptor connecting Cilium and the transition zone of motile cilia suggests that this assemblage may also be present in motile cilia. Using a subcellular fraction enriched in detergent-extracted photoreceptor axonemes, three high molecular mass glycoconjugates (425, 600, and 700 kD) were previously identified as potential components of the assemblage. Through oligosaccharide characterization and binding of a specific monoclonal antibody, we have verified the localization of the 425 kD glycoconjugate to the transmembrane assemblage. Binding of the lectin peanut agglutinin (PNA) to the 425 kD glycoconjugate on nitrocellulose blots, and to isolated detergentextracted axonemes, was assessed following treatment with the enzymes neuraminidase and O-glycanase. Changes in binding to the 425 kD glycoconjugate precisely paralleled changes in binding to intact axonemes, supporting the hypothesis that the 425 kD glycoconjugate is a component of the transmembrane assemblage. Furthermore, the results suggest that the 425 kD glycoconjugate contains sialated galactose-N-acetylgalactosamine oligosaccharides which are Olinked to the protein backbone. To directly assess the distribution of the 425 kD glycoconjugate, we produced a monoclonal antibody directed against this glycoconjugate. The antibody, K26, recognizes only the 425 kD on transblots of the axoneme fraction. K26 immunoreactivity of intact axonemes is identical to that seen by PNA staining. K26 staining of isolated photoreceptors and whole retina is uniquely localized to the region of the connecting Cilium. Thus, in the photoreceptor, the 425 kD is not only a component of the transmembrane assemblage but is also completely restricted to the connecting Cilium. Based on morphological similarities, the photoreceptor connecting Cilium is thought to be homologous to the transition zone of the motile Cilium. As such, we have stained oviduct epithelium with the K26 monoclonal antibody. Immunoreactivity is restricted to the region of the transition zone at the base of motile cilia. This demonstrates that the photoreceptor connecting Cilium and motile Cilium transition zone are immunologically related.
Kristen J Verhey - One of the best experts on this subject based on the ideXlab platform.
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NPHP proteins are binding partners of nucleoporins at the base of the primary Cilium.
PLOS ONE, 2019Co-Authors: T. Lynne Blasius, Daisuke Takao, Kristen J VerheyAbstract:Cilia are microtubule-based organelles that protrude from the surface of eukaryotic cells to generate motility and to sense and respond to environmental cues. In order to carry out these functions, the complement of proteins in the Cilium must be specific for the organelle. Regulation of protein entry into primary cilia has been shown to utilize mechanisms and components of nuclear gating, including nucleoporins of the nuclear pore complex (NPC). We show that nucleoporins also localize to the base of motile cilia on the surface of trachea epithelial cells. How nucleoporins are anchored at the Cilium base has been unclear as transmembrane nucleoporins, which anchor nucleoporins at the nuclear envelope, have not been found to localize at the Cilium. Here we use the directed yeast two-hybrid assay to identify direct interactions between nucleoporins and nephronophthisis proteins (NPHPs) which localize to the Cilium base and contribute to Cilium assembly and identity. We validate NPHP-nucleoporin interactions in mammalian cells using the knocksideways assay and demonstrate that the interactions occur at the base of the primary Cilium using bimolecular fluorescence complementation. We propose that NPHP proteins anchor nucleoporins at the base of primary cilia to regulate protein entry into the organelle.
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the ciliary lumen accommodates passive diffusion and vesicle trafficking in cytoplasmic ciliary transport
bioRxiv, 2019Co-Authors: Andrew Ruba, Kristen J Verhey, Daisuke Takao, Jingjie Yu, Athanasios Evangelou, Rachel Higgins, Saovleak Khim, Weidong YangAbstract:Abstract Transport of membrane and cytosolic proteins into the primary Cilium is essential for its role in cellular signaling. Using single molecule microscopy, we mapped the movement of membrane and soluble proteins at the base of the primary Cilium. In addition to the well-known intraflagellar transport (IFT) route, we identified two new pathways within the lumen of the primary Cilium - passive diffusional and vesicle transport routes - that are adopted by proteins for cytoplasmic-Cilium transport in live cells. Independent of the IFT path, approximately half of IFT motors (KIF3A) and cargo (α-tubulin) take the passive diffusion route and more than half of membrane-embedded G protein coupled receptors (SSTR3 and HTR6) use RAB8A-regulated vesicles to transport into and inside cilia. Furthermore, ciliary lumen transport is the preferred route for membrane proteins in the early stages of ciliogenesis and inhibition of SSTR3 vesicle transport completely blocks ciliogenesis. Furthermore, clathrin-mediated, signal-dependent internalization of SSTR3 also occurs through the ciliary lumen. These transport routes were also observed in Chlamydomonas reinhardtii flagella, suggesting their conserved roles in trafficking of ciliary proteins.
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acute inhibition of heterotrimeric kinesin 2 function reveals mechanisms of intraflagellar transport in mammalian cilia
Current Biology, 2019Co-Authors: Martin F Engelke, Bridget Waas, Sarah Kearns, Ayana Suber, Allison Boss, Benjamin L Allen, Kristen J VerheyAbstract:Summary The trafficking of components within cilia, called intraflagellar transport (IFT), is powered by kinesin-2 and dynein-2 motors. Loss of function in any subunit of the heterotrimeric KIF3A/KIF3B/KAP kinesin-2 motor prevents ciliogenesis in mammalian cells and has hindered an understanding of how kinesin-2 motors function in Cilium assembly and IFT. We used a chemical-genetic approach to generate an inhibitable KIF3A/KIF3B/KAP kinesin-2 motor (i3A/i3B) that is capable of rescuing wild-type (WT) motor function for Cilium assembly and Hedgehog signaling in Kif3a/Kif3b double-knockout cells. We demonstrate that KIF3A/KIF3B function is required not just for Cilium assembly but also for Cilium maintenance, as inhibition of i3A/i3B blocks IFT within 2 min and leads to a complete loss of primary cilia within 8 h. In contrast, inhibition of dynein-2 has no effect on Cilium maintenance within the same time frame. The kinetics of cilia loss indicate that two processes contribute to ciliary disassembly in response to cessation of anterograde IFT: a slow shortening that is steady over time and a rapid deciliation that occurs with stochastic onset. We also demonstrate that the kinesin-2 family members KIF3A/KIF3C and KIF17 cannot rescue ciliogenesis in Kif3a/Kif3b double-knockout cells or delay the loss of assembled cilia upon i3A/i3B inhibition. These results demonstrate that KIF3A/KIF3B/KAP is the sole and essential motor for Cilium assembly and maintenance in mammalian cells. These findings highlight differences in how kinesin-2 motors were adapted for Cilium assembly and IFT function across species.
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regulation of Cilium length and intraflagellar transport by the rck kinases ick and mok in renal epithelial cells
PLOS ONE, 2014Co-Authors: Joost R Broekhuis, Kristen J Verhey, Gert JansenAbstract:Primary cilia are important sensory organelles. They exist in a wide variety of lengths, which could reflect different cell-specific functions. How Cilium length is regulated is unclear, but it probably involves intraflagellar transport (IFT), which transports protein complexes along the ciliary axoneme. Studies in various organisms have identified the small, conserved family of ros-cross hybridizing kinases (RCK) as regulators of Cilium length. Here we show that Intestinal Cell Kinase (ICK) and MAPK/MAK/MRK overlapping kinase (MOK), two members of this family, localize to cilia of mouse renal epithelial (IMCD-3) cells and negatively regulate Cilium length. To analyze the effects of ICK and MOK on the IFT machinery, we set up live imaging of five fluorescently tagged IFT proteins: KIF3B, a subunit of kinesin-II, the main anterograde IFT motor, complex A protein IFT43, complex B protein IFT20, BBSome protein BBS8 and homodimeric kinesin KIF17, whose function in mammalian cilia is unclear. Interestingly, all five proteins moved at ∼0.45 µm/s in anterograde and retrograde direction, suggesting they are all transported by the same machinery. Moreover, GFP tagged ICK and MOK moved at similar velocities as the IFT proteins, suggesting they are part of, or transported by the IFT machinery. Indeed, loss- or gain-of-function of ICK affected IFT speeds: knockdown increased anterograde velocities, whereas overexpression reduced retrograde speed. In contrast, MOK knockdown or overexpression did not affect IFT speeds. Finally, we found that the effects of ICK or MOK knockdown on Cilium length and IFT are suppressed by rapamycin treatment, suggesting that these effects require the mTORC1 pathway. Our results confirm the importance of RCK kinases as regulators of Cilium length and IFT. However, whereas some of our results suggest a direct correlation between Cilium length and IFT speed, other results indicate that Cilium length can be modulated independent of IFT speed.