1
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Yap YT, Pan J, Xu J, Yuan S, Niu C, Zheng C, Li W, Zhou T, Li T, Zhang Y, Holtzman MJ, Pazour GJ, Hess RA, Kelly CV, Touré A, Brody SL, Zhang Z. Role of intraflagellar transport protein IFT140 in the formation and function of motile cilia in mammals. Cell Mol Life Sci 2025; 82:198. [PMID: 40348912 PMCID: PMC12065702 DOI: 10.1007/s00018-025-05710-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2024] [Revised: 04/05/2025] [Accepted: 04/10/2025] [Indexed: 05/14/2025]
Abstract
Cilia are microtubular structures extending from the surface of most mammalian cells. They can be categorized as motile cilia and primary sensory cilia. Both types possess intraflagellar transport (IFT) machinery, composed of unique protein complexes that travel along the microtubules to deliver proteins for ciliary and flagellar assembly, disassembly, and homeostasis. Although the role of IFT in primary cilia formation has been well studied, little is known about its role in mammalian motile cilia assembly. We generated conditional knockout mice by breeding floxed Ift140 mice with the FOXJ1-Cre transgenic mouse line to specifically delete Ift140 from cells that assemble motile cilia. Mice with Ift140 deficiency did not have laterality defects or gross; however most died prior to sexual maturity. Those mutants that survived to adulthood were completely infertile. Males demonstrated abnormal spermatogenesis associated with reduced sperm count and motility, together with short length flagella, and abnormal morphology. Cilia length was diminished in the epithelial cells of the efferent ductules and airways. Cilia from cultured tracheal epithelial cells were also short and had reduced beat frequency (CBF). Ultrastructural studies revealed the presence of inner and outer dynein arms, but an abnormal central apparatus, and the accumulation of particles within the cilia. Overall, the short length and abnormal localization of ciliary proteins in Ift140 conditional mutants resulted in inadequate cilia function despite proper localization of the dynein motor complexes. We propose a key role of Ift140 for motile cilia assembly in certain tissues and suggest that genetic alterations of IFT140 could be associated with motile ciliopathies.
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Affiliation(s)
- Yi Tian Yap
- Department of Physiology, Wayne State University, 275 E Hancock Street, Detroit, MI, 48201, USA
| | - Jiehong Pan
- Division of Pulmonary and Critical Care Medicine, Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA
| | - Jian Xu
- Division of Pulmonary and Critical Care Medicine, Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA
| | - Shuo Yuan
- Department of Physiology, Wayne State University, 275 E Hancock Street, Detroit, MI, 48201, USA
- Department of Occupational and Environmental Health, School of Public Health, Wuhan University of Science and Technology, Wuhan, Hubei, China
| | - Changmin Niu
- Department of Physiology, Wayne State University, 275 E Hancock Street, Detroit, MI, 48201, USA
- School of Nursing, School of Public Health, Yangzhou University, Yangzhou, Jiangsu, China
| | - Cheng Zheng
- Department of Physiology, Wayne State University, 275 E Hancock Street, Detroit, MI, 48201, USA
- Department of Occupational and Environmental Health, School of Public Health, Wuhan University of Science and Technology, Wuhan, Hubei, China
| | - Wei Li
- Department of Physiology, Wayne State University, 275 E Hancock Street, Detroit, MI, 48201, USA
| | - Ting Zhou
- Department of Physiology, Wayne State University, 275 E Hancock Street, Detroit, MI, 48201, USA
- Department of Occupational and Environmental Health, School of Public Health, Wuhan University of Science and Technology, Wuhan, Hubei, China
| | - Tao Li
- Department of Physiology, Wayne State University, 275 E Hancock Street, Detroit, MI, 48201, USA
- Institute of Reproductive Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China
| | - Yong Zhang
- Division of Pulmonary and Critical Care Medicine, Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA
| | - Michael J Holtzman
- Division of Pulmonary and Critical Care Medicine, Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA
| | - Gregory J Pazour
- Program in Molecular Medicine, University of Massachusetts Chan Medical School, Worcester, MA, USA
| | - Rex A Hess
- Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois, 2001S. Lincoln, Urbana, IL, USA
| | - Christopher V Kelly
- Department of Physics and Astronomy, Wayne State University, Detroit, MI, USA
| | - Aminata Touré
- Université Grenoble Alpes, INSERM U1209, CNRS UMR 5309, Institut pour l'Avancée des Biosciences (IAB), Team Physiology and Pathophysiology of Sperm cells, 38000, Grenoble, France
| | - Steven L Brody
- Division of Pulmonary and Critical Care Medicine, Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA
| | - Zhibing Zhang
- Department of Physiology, Wayne State University, 275 E Hancock Street, Detroit, MI, 48201, USA.
- Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI, USA.
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2
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Nitoiu A, Zhang Q, Tavares E, Li JM, Ahmed K, Green-Sanderson K, Rashid M, Morcos SM, Maynes JT, Campos EI, Sheffield VC, Vincent A, Héon E. Defective IFT57 underlies a novel cause of Bardet-Biedl syndrome. Hum Mol Genet 2025:ddaf058. [PMID: 40273360 DOI: 10.1093/hmg/ddaf058] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2025] [Accepted: 04/02/2025] [Indexed: 04/26/2025] Open
Abstract
A 29-year-old male presented with rod-cone degeneration leading to legal blindness, post-axial polydactyly, obesity, cognitive impairment, and fatty liver, features suggestive of a clinical diagnosis of Bardet-Biedl Syndrome (BBS). Following negative clinical genetic testing, genome analysis identified biallelic variants in IFT57: p.(Val397Glu) and p.(Lys225Asnfs*17). IFT57 is part of complex B of the intraflagellar transport (IFT) proteins, which is an adaptor to the anterograde transport of proteins, bringing cargo from the base of the primary cilia to the tip. Variants in IFT57 have not yet been associated with BBS or human retinal degeneration, but biallelic splicing variants were associated with a distinct ciliopathy: oral-facial-digital syndrome. Using patient-derived fibroblasts, IFT57-knockouts (KO) of RPE1, and mIMCD3 cells, we showed that p.(Lys225Asnfs*17) is subjected to non-sense mediated decay, and that p.(Val397Glu) is the predominant variant which leads to cilia defects. Exogenous expression of the p.(Val397Glu) variant partially restored structural and functional primary cilia defects, and of the anterograde transport in Ift57-KO mIMCD3 cells but it did not rescue primary cilia in retinal IFT57-KO-RPE1 cells. The cell autonomous effect, likely explains the retinal dystrophy in our proband with BBS.
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Affiliation(s)
- Alexandra Nitoiu
- Genetics and Genome Biology, Peter Gilgan Centre for Research and Learning, 686 Bay Street, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada
- Institute of Medical Science, Temerty Faculty of Medicine, 1 King's College Circle, University of Toronto, Toronto, Ontario M5S 1A8, Canada
| | - Qihong Zhang
- Department of Pediatrics, University of Iowa Carver College of Medicine, 200 Hawkins Drive, Iowa City, Iowa 52242, United States
| | - Erika Tavares
- Genetics and Genome Biology, Peter Gilgan Centre for Research and Learning, 686 Bay Street, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada
| | - Janice Min Li
- Genetics and Genome Biology, Peter Gilgan Centre for Research and Learning, 686 Bay Street, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada
| | - Kashif Ahmed
- Genetics and Genome Biology, Peter Gilgan Centre for Research and Learning, 686 Bay Street, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada
| | - Kit Green-Sanderson
- Genetics and Genome Biology, Peter Gilgan Centre for Research and Learning, 686 Bay Street, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada
| | - Mahnoor Rashid
- Genetics and Genome Biology, Peter Gilgan Centre for Research and Learning, 686 Bay Street, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada
| | - Shahir M Morcos
- Genetics and Genome Biology, Peter Gilgan Centre for Research and Learning, 686 Bay Street, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada
- Department of Molecular Genetics, Medical Sciences Building, 1 King's College, University of Toronto, Toronto, Ontario M5S 1A8, Canada
| | - Jayson T Maynes
- Department of Anesthesia and Pain Medicine, Peter Gilgan Centre for Research and Learning, 686 Bay Street, The Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada
- Program in Molecular Medicine, Peter Gilgan Centre for Research and Learning, 686 Bay Street, The Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada
| | - Eric I Campos
- Genetics and Genome Biology, Peter Gilgan Centre for Research and Learning, 686 Bay Street, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada
- Department of Molecular Genetics, Medical Sciences Building, 1 King's College, University of Toronto, Toronto, Ontario M5S 1A8, Canada
| | - Val C Sheffield
- Department of Pediatrics, University of Iowa Carver College of Medicine, 200 Hawkins Drive, Iowa City, Iowa 52242, United States
| | - Ajoy Vincent
- Genetics and Genome Biology, Peter Gilgan Centre for Research and Learning, 686 Bay Street, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada
- Institute of Medical Science, Temerty Faculty of Medicine, 1 King's College Circle, University of Toronto, Toronto, Ontario M5S 1A8, Canada
- Ophthalmology and Vision Sciences, 555 University Avenue, Hospital for Sick Children, University of Toronto, Toronto, Ontario M5G 1X8, Canada
| | - Elise Héon
- Genetics and Genome Biology, Peter Gilgan Centre for Research and Learning, 686 Bay Street, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada
- Institute of Medical Science, Temerty Faculty of Medicine, 1 King's College Circle, University of Toronto, Toronto, Ontario M5S 1A8, Canada
- Ophthalmology and Vision Sciences, 555 University Avenue, Hospital for Sick Children, University of Toronto, Toronto, Ontario M5G 1X8, Canada
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3
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Mitra A, Gioukakis E, Mul W, Peterman EJG. Delivery of intraflagellar transport proteins to the ciliary base and assembly into trains. SCIENCE ADVANCES 2025; 11:eadr1716. [PMID: 40184459 PMCID: PMC11970479 DOI: 10.1126/sciadv.adr1716] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/18/2024] [Accepted: 03/03/2025] [Indexed: 04/06/2025]
Abstract
Anterograde intraflagellar transport (IFT) trains, composed of IFT-B, IFT-A, and BBSome subcomplexes, are responsible for transporting ciliary proteins into the cilium. How IFT subcomplexes reach the ciliary base and assemble into IFT trains is poorly understood. Here, we perform quantitative single-molecule imaging in Caenorhabditis elegans chemosensory cilia to uncover how IFT subcomplexes arrive at the base, organize in IFT trains, and enter the cilium. We find that BBSomes reach the base via diffusion where they either associate with assembling IFT trains or with the membrane surrounding the base. In contrast, IFT-B and IFT-A reach the base via directed transport most likely on vesicles that stop at distinct locations near the base. Individual subcomplexes detach from the vesicles into a diffusive pool and associate to assembling trains. Our results show that IFT-B is first incorporated into IFT trains, followed by IFT-A, and finally BBSomes, indicating that the assembly of IFT trains is a highly regulated, step-wise process.
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Affiliation(s)
| | - Evangelos Gioukakis
- Department of Physics and Astronomy and LaserLaB, Vrije Universiteit Amsterdam, Amsterdam, Netherlands
| | - Wouter Mul
- Department of Physics and Astronomy and LaserLaB, Vrije Universiteit Amsterdam, Amsterdam, Netherlands
| | - Erwin J. G. Peterman
- Department of Physics and Astronomy and LaserLaB, Vrije Universiteit Amsterdam, Amsterdam, Netherlands
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4
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Tasaki K, Satoda Y, Chiba S, Shin HW, Katoh Y, Nakayama K. Mutually independent and cilia-independent assembly of IFT-A and IFT-B complexes at mother centriole. Mol Biol Cell 2025; 36:ar48. [PMID: 40020180 PMCID: PMC12005097 DOI: 10.1091/mbc.e24-11-0509] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2024] [Revised: 02/06/2025] [Accepted: 02/10/2025] [Indexed: 03/05/2025] Open
Abstract
The intraflagellar transport (IFT) machinery, containing the IFT-A and IFT-B complexes and powered by dynein-2 and kinesin-2 motors, is crucial for bidirectional trafficking of ciliary proteins and their import/export across the transition zone (TZ). Stepwise assembly of anterograde IFT trains was proposed previously; that is, the IFT-B complex first forms a TZ-tethered scaffold with sequential incorporation of IFT-A, dynein-2, and finally kinesin-2. However, IFT-A and IFT-B complexes also demonstrate distinct localization to the basal body/mother centriole. We show that IFT-A, IFT-B, and dynein-2 complexes are recruited to the mother centriole independently of ciliogenesis. Furthermore, mother centriole recruitment of IFT-A and IFT-B can occur in the absence of IFT-B and IFT-A, respectively, and dynein-2 recruitment is independent of IFT-A and IFT-B. Expansion microscopy revealed that the IFT-A/IFT-B pool at the basal body is distinct from that at the TZ. We conclude that IFT-A and IFT-B are recruited to the mother centriole in a mutually independent and ciliogenesis-independent manner before IFT train assembly.
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Affiliation(s)
- Koshi Tasaki
- Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan
| | - Yuuki Satoda
- Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan
| | - Shuhei Chiba
- Laboratory of Molecular and Cellular Biology, Tohoku University, Aobayama, Sendai, Miyagi 980-8578, Japan
| | - Hye-Won Shin
- Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan
| | - Yohei Katoh
- Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan
| | - Kazuhisa Nakayama
- Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan
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5
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Yamamoto R, Sahashi Y, Shimo-Kon R, Sakato-Antoku M, Suzuki M, Luo L, Tanaka H, Ishikawa T, Yagi T, King SM, Kurisu G, Kon T. Chlamydomonas FBB18 is a ubiquitin-like protein essential for the cytoplasmic preassembly of various ciliary dyneins. Proc Natl Acad Sci U S A 2025; 122:e2423948122. [PMID: 40106351 PMCID: PMC11962417 DOI: 10.1073/pnas.2423948122] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2024] [Accepted: 02/14/2025] [Indexed: 03/22/2025] Open
Abstract
Motile cilia are organelles found on many eukaryotic cells that play critical roles in development and fertility. Human CFAP298 has been implicated in the transport/assembly of ciliary dyneins, and defects in this protein cause primary ciliary dyskinesia. However, neither the exact function nor the structure of CFAP298 have been elucidated. Here, we took advantage of Chlamydomonas, a ciliated alga, to study the structure and function of FBB18, an ortholog of CFAP298. Multiple ciliary dyneins were greatly reduced in cilia of Chlamydomonas fbb18 mutants. In addition, we found that both the stability of ciliary dynein heavy chains (HCs) and the association between HCs and intermediate/light chains (IC/LCs) are greatly reduced in fbb18 cytoplasm, strongly suggesting that FBB18 functions in the cytoplasmic assembly (the so-called "preassembly") of dynein complexes from HC/IC/LCs. Furthermore, X-ray crystallography revealed that FBB18 forms a bilobed structure with globular domains at both ends of the molecule, connected by an α-helical bundle. Unexpectedly, one globular domain shows high similarity to ubiquitin, a small protein critical for the modification of a variety of protein complexes, and this ubiquitin-like domain is indispensable for the molecular function of FBB18. Our results demonstrate that FBB18, a specialized member of the ubiquitin-like protein family, plays a critical role in dynein preassembly, most likely by mediating diverse interactions between dynein HCs, molecular chaperone(s), and other preassembly factor(s) using the ubiquitin-like domain as well as other regions, and by facilitating the proper folding of dynein HCs.
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Affiliation(s)
- Ryosuke Yamamoto
- Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka560-0043, Japan
| | - Yui Sahashi
- Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka560-0043, Japan
| | - Rieko Shimo-Kon
- Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka560-0043, Japan
| | - Miho Sakato-Antoku
- Department of Molecular Biology and Biophysics, University of Connecticut Health Center, Farmington, CT06030-3305
| | - Miyuka Suzuki
- Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka560-0043, Japan
| | - Leo Luo
- Department of Biology and Chemistry, Paul Scherrer Institute, Villigen5232, Switzerland
- Department of Biology, ETH Zurich, Zurich8093, Switzerland
| | - Hideaki Tanaka
- Institute for Protein Research, Osaka University, Osaka565-0871, Japan
| | - Takashi Ishikawa
- Department of Biology and Chemistry, Paul Scherrer Institute, Villigen5232, Switzerland
- Department of Biology, ETH Zurich, Zurich8093, Switzerland
| | - Toshiki Yagi
- Department of Life and Environmental Sciences, Faculty of Bioresource Sciences, Prefectural University of Hiroshima, Hiroshima727-0023, Japan
| | - Stephen M. King
- Department of Molecular Biology and Biophysics, University of Connecticut Health Center, Farmington, CT06030-3305
| | - Genji Kurisu
- Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka560-0043, Japan
- Institute for Protein Research, Osaka University, Osaka565-0871, Japan
| | - Takahide Kon
- Department of Biological Sciences, Graduate School of Science, Osaka University, Osaka560-0043, Japan
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6
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Abstract
Primary and motile cilia are eukaryotic organelles that perform crucial roles in cellular signalling and motility. Intraflagellar transport (IFT) contributes to the formation of the highly specialized ciliary proteome by active and selective transport of soluble and membrane proteins into and out of cilia. IFT is performed by the IFT-A and IFT-B protein complexes, which together link cargoes to the microtubule motors kinesin and dynein. In this Review, we discuss recent structural and mechanistic insights on how the IFT complexes are first recruited to the base of the cilium, how they polymerize into an anterograde IFT train, and how this complex imports cargoes from the cytoplasm. We will describe insights into how kinesin-driven anterograde trains are carried to the ciliary tip, where they are remodelled into dynein-driven retrograde trains for cargo export. We will also present how the interplay between IFT-A and IFT-B complexes, motor proteins and cargo adaptors is regulated for bidirectional ciliary transport.
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7
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Trevena RL, Veire BM, Chamberlain TJ, Moravec CE, Pelegri F. Embryonic Lethality, Juvenile Growth Variation, and Adult Sterility Correlate With Phylogenetic Distance of Danionin Hybrids. Evol Dev 2025; 27:e12495. [PMID: 39639649 PMCID: PMC11621593 DOI: 10.1111/ede.12495] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2024] [Revised: 10/01/2024] [Accepted: 11/08/2024] [Indexed: 12/07/2024]
Abstract
Hybrid incompatibility, which plays a pivotal role in speciation, is expected to correlate with greater phylogenetic distance. Here, we investigate the fitness of interspecies hybrids within the Danionin subfamily, which includes the model species, Danio rerio, and its relatives - Danio kyathit, Danio albolineatus, Danio margaritatus, and Devario aequipinnatus. We generated hybrids through in vitro fertilization, using Danio rerio as the maternal species, with normal fertilization rates showing no incompatibilities in sperm-egg interactions within these two genera. Generally, all hybrids exhibit normal patterns and timelines in early developmental transitions, from cleavage stages to the initiation of epiboly, although inter-genera Danio-Devario hybrids subsequently exhibit fully penetrant embryonic lethality. Intra-genus Danio hybrids, on the other hand, can survive through embryogenesis and into adulthood. However, rates of survival during these stages diminish according to phylogenetic distance, with increasing early lethality in hybrids from more distantly related species. Additionally, Danio hybrids exhibit increased growth rate variability during juvenile stages. All Danio hybrids have reduced testes sizes, sperm counts, and sperm viabilities, with sperm displaying defects in flagellum formation and integrity. Adult male intra-genus hybrids are invariably sterile, except in the case of Danio rerio hybrids with the closely related Danio kyathit, which produced a backcrossed F2 generation that did not survive juvenile stages. Our studies highlight a loss of hybrid compatibility at various life stages in the Danio and Devario genera, based on deleterious effects and reduced developmental robustness, emphasizing a correlation between the severity of incompatibility outcomes and the degree of phylogenetic relatedness.
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Affiliation(s)
- Ryan L. Trevena
- Laboratory of GeneticsUniversity of Wisconsin‐MadisonMadisonWisconsinUSA
| | - Benton M. Veire
- Laboratory of GeneticsUniversity of Wisconsin‐MadisonMadisonWisconsinUSA
| | | | - Cara E. Moravec
- Laboratory of GeneticsUniversity of Wisconsin‐MadisonMadisonWisconsinUSA
| | - Francisco Pelegri
- Laboratory of GeneticsUniversity of Wisconsin‐MadisonMadisonWisconsinUSA
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8
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Wang J, Kidmose RT, Boegholm N, Zacharia NK, Thomsen MB, Christensen A, Malik T, Lechtreck K, Lorentzen E. Integrative in silico and biochemical analyses demonstrate direct Arl3-mediated ODA16 release from the intraflagellar transport machinery. J Biol Chem 2025; 301:108237. [PMID: 39880089 PMCID: PMC11879689 DOI: 10.1016/j.jbc.2025.108237] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2024] [Revised: 12/27/2024] [Accepted: 01/17/2025] [Indexed: 01/31/2025] Open
Abstract
Outer dynein arms (ODAs) are essential for ciliary motility and are preassembled in the cytoplasm before trafficking into cilia by intraflagellar transport (IFT). ODA16 is a key adaptor protein that links ODAs to the IFT machinery via direct interaction with the IFT46 protein. However, the molecular mechanisms regulating the assembly, transport, and release of ODAs remain poorly understood. Here, we employ AlphaPulldown, an in silico screening method, to identify direct interactors of ODA16, including the dynein adaptor IDA3 and the small GTPase Arl3. We use structural modeling, biochemical, and biophysical assays on Chlamydomonas and human proteins to elucidate the interactions and regulatory mechanisms governing the IFT of ODAs. We identify a conserved N-terminal motif in Chlamydomonas IFT46 that mediates its binding to one side of the ODA16 structure. Biochemical dissection reveals that IDA3 and Arl3 bind to the same surface of ODA16 (the C-terminal β-propeller face), which is opposite to the IFT46 binding site, enabling them to dissociate ODA16 from IFT46, likely through an allosteric mechanism. Our findings provide mechanistic insights into the concerted actions of IFT and adaptor proteins in ODA transport and regulation.
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Affiliation(s)
- Jiaolong Wang
- Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark
| | - Rune T Kidmose
- Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark
| | - Niels Boegholm
- Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark
| | - Nevin K Zacharia
- Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark
| | - Mads B Thomsen
- Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark
| | - Anni Christensen
- Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark
| | - Tara Malik
- Department of Cellular Biology, University of Georgia, Athens, Georgia, USA
| | - Karl Lechtreck
- Department of Cellular Biology, University of Georgia, Athens, Georgia, USA
| | - Esben Lorentzen
- Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark.
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9
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Wang H, Li Y, Li X, Sun Z, Yu F, Pashang A, Kulasiri D, Li HW, Chen H, Hou H, Zhang Y. The Primary Cilia are Associated with the Axon Initial Segment in Neurons. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2025; 12:e2407405. [PMID: 39804991 PMCID: PMC11884599 DOI: 10.1002/advs.202407405] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/02/2024] [Revised: 12/16/2024] [Indexed: 01/16/2025]
Abstract
The primary cilia serve as pivotal mediators of environmental signals and play crucial roles in neuronal responses. Disruption of ciliary function has been implicated in neuronal circuit disorders and aberrant neuronal excitability. However, the precise mechanisms remain elusive. To study the link between the primary cilia and neuronal excitability, manipulation of somatostatin receptor 3 (SSTR3) is investigated, as an example of how alterations in ciliary signaling may affect neuronal activity. It is found that aberrant SSTR3 expression perturbed not only ciliary morphology but also disrupted ciliary signaling cascades. Genetic deletion of SSTR3 resulted in perturbed spatial memory and synaptic plasticity. The axon initial segment (AIS) is a specialized region in the axon where action potentials are initiated. Interestingly, loss of ciliary SSTR3 led to decrease of Akt-dependent cyclic AMP-response element binding protein (CREB)-mediated transcription at the AIS, specifically downregulating AIS master organizer adaptor protein ankyrin G (AnkG) expression. In addition, alterations of other ciliary proteins serotonin 6 receptor (5-HT6R)and intraflagellar transport protein 88 (IFT88) also induced length changes of the AIS. The findings elucidate a specific interaction between the primary cilia and AIS, providing insight into the impact of the primary cilia on neuronal excitability and circuit integrity.
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Affiliation(s)
- Han Wang
- State Key Laboratory of Membrane BiologySchool of Life SciencesPeking UniversityBeijing100871China
| | - Yu Li
- State Key Laboratory of Membrane BiologySchool of Life SciencesPeking UniversityBeijing100871China
| | - Xin Li
- Beijing Life Science AcademyBeijing102200China
| | - Zehui Sun
- State Key Laboratory of Membrane BiologySchool of Life SciencesPeking UniversityBeijing100871China
| | - Fengdan Yu
- State Key Laboratory of Membrane BiologySchool of Life SciencesPeking UniversityBeijing100871China
| | - Abolghasem Pashang
- Centre for Advanced Computational Solutions (C‐fACS)AGLS facultyLincoln UniversityCanterbury7647New Zealand
| | - Don Kulasiri
- Centre for Advanced Computational Solutions (C‐fACS)AGLS facultyLincoln UniversityCanterbury7647New Zealand
| | - Hung Wing Li
- Department of ChemistryThe Chinese University of Hong KongHong Kong999077China
| | - Huan Chen
- Beijing Life Science AcademyBeijing102200China
| | - Hongwei Hou
- Beijing Life Science AcademyBeijing102200China
| | - Yan Zhang
- State Key Laboratory of Membrane BiologySchool of Life SciencesPeking UniversityBeijing100871China
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10
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Li W, Niu C, Yap YT, Li T, Zheng C, Goswami M, Kandiraju S, Dhikhirullahi O, Xu J, Zhang J, Kelly CV, Zhang Z. Two-directional trafficking of the IFT25 protein in the developing mouse sperm flagella. Biol Reprod 2025; 112:309-318. [PMID: 39561113 PMCID: PMC12032603 DOI: 10.1093/biolre/ioae171] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2024] [Revised: 10/02/2024] [Accepted: 11/18/2024] [Indexed: 11/21/2024] Open
Abstract
Intraflagellar transport 25 is a component of the intraflagellar transport 25-B complex. In mice, even though this intraflagellar transport component is not required for cilia formation in somatic cells, it is essential for sperm formation. However, the intracellular localization of this protein in male germ cells is not known given no reliable antibodies are available for histologic studies, and the dynamic trafficking in the developing sperm flagella is not clear. To examine localization of the protein in male germ cells and further investigate the mechanism of intraflagellar transport in sperm formation, particularly to look into the dynamic trafficking of the protein, we generated a mouse intraflagellar transport 25-green fluorescent protein knock-in mouse model using the clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats associated protein 9 system, with the mouse intraflagellar transport 25 protein fused with a green fluorescent protein tag in the C-terminus. Three independent lines were analyzed. Western blotting using both anti-intraflagellar transport 25 and anti-green fluorescent protein antibodies showed that the intraflagellar transport 25-green fluorescent protein fusion protein was highly abundant only in the testis, which is consistent with the endogenous intraflagellar transport 25 protein. Examination of localization of the intraflagellar transport 25-green fluorescent protein in isolated germ cells revealed that the fusion protein was present in the cytoplasm of spermatocytes and round spermatids and a strong signal was present in the developing sperm flagellar. The homozygous knock-in mice had normal spermatogenesis, fertility and sperm parameters. Diffusion analysis of intraflagellar transport 25 within the developing flagellar revealed the presence of both mobile and immobile fractions as revealed by fluorescence recovery after photobleaching. Kymograph and fluorescence recovery after photobleaching analyses demonstrate the transport of intraflagellar transport 25-green fluorescent protein within the developing tail demonstrate no apparent preference for trafficking toward and away from the cell body. The speed of trafficking depends on the stage of sperm development, ranging from highly mobile unrestricted diffusion initially, mobile punctate structures in developing sperm, and immobile punctate structures in mature sperm. Our studies demonstrate that mouse intraflagellar transport 25 travels along the developing sperm flagella in two directions that might be essential for functional sperm formation.
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Affiliation(s)
- Wei Li
- Department of Physiology, Wayne State University, Detroit, MI, USA
| | - Changmin Niu
- Department of Physiology, Wayne State University, Detroit, MI, USA
- School of Nursing School of Public Health, Yangzhou University, Yangzhou, Jiangsu, China
| | - Yi Tian Yap
- Department of Physiology, Wayne State University, Detroit, MI, USA
| | - Tao Li
- Department of Physiology, Wayne State University, Detroit, MI, USA
- Institute of Reproductive Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, PR China
| | - Cheng Zheng
- Department of Physiology, Wayne State University, Detroit, MI, USA
- Department of Occupational and Environmental Health, School of Public Health, Wuhan University of Science and Technology, Wuhan, Hubei, PR China
| | | | | | | | - Jie Xu
- Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical School, Ann Arbor, MI, USA
| | - Jifeng Zhang
- Center for Advanced Models for Translational Sciences and Therapeutics, University of Michigan Medical School, Ann Arbor, MI, USA
| | - Christopher V Kelly
- Department of Physics and Astronomy, Wayne State University, Detroit, MI, USA
| | - Zhibing Zhang
- Department of Physiology, Wayne State University, Detroit, MI, USA
- Department of Obstetrics and Gynecology, Wayne State University, Detroit, MI, USA
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11
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Corrales RM, Vincent J, Crobu L, Neish R, Nepal B, Espeut J, Pasquier G, Gillard G, Cazevieille C, Mottram JC, Wetzel DM, Sterkers Y, Rogowski K, Lévêque MF. Tubulin detyrosination shapes Leishmania cytoskeletal architecture and virulence. Proc Natl Acad Sci U S A 2025; 122:e2415296122. [PMID: 39808657 PMCID: PMC11761321 DOI: 10.1073/pnas.2415296122] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2024] [Accepted: 11/22/2024] [Indexed: 01/16/2025] Open
Abstract
Tubulin detyrosination has been implicated in various human disorders and is important for regulating microtubule dynamics. While in most organisms this modification is restricted to α-tubulin, in trypanosomatid parasites, it occurs on both α- and β-tubulin. Here, we show that in Leishmania, a single vasohibin (LmVASH) enzyme is responsible for differential kinetics of α- and β-tubulin detyrosination. LmVASH knockout parasites, which are completely devoid of detyrosination, show decreased levels of glutamylation and exhibit a strongly diminished pathogenicity in mice, correlating with decreased proliferation in macrophages. Reduced virulence is associated with altered morphogenesis and flagellum remodeling in detyrosination-deficient amastigotes. Flagellum shortening in the absence of detyrosination is caused by hyperactivity of a microtubule-depolymerizing Kinesin-13 homolog, demonstrating its function as a key reader of the trypanosomatid-tubulin code. Taken together, our work establishes the importance of tubulin detyrosination in remodeling the microtubule-based cytoskeleton required for efficient proliferation in the mammalian host. This highlights tubulin detyrosination as a potential target for therapeutic action against leishmaniasis.
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Affiliation(s)
- Rosa Milagros Corrales
- Maladies infectieuses et Vecteurs: Ecologie, Génétique, Evolution et Contrôle, University of Montpellier, CNRS, Institut de Recherche pour le Développement, Montpellier34095, France
| | - Jeremy Vincent
- Maladies infectieuses et Vecteurs: Ecologie, Génétique, Evolution et Contrôle, University of Montpellier, CNRS, Institut de Recherche pour le Développement, Montpellier34095, France
| | - Lucien Crobu
- Maladies infectieuses et Vecteurs: Ecologie, Génétique, Evolution et Contrôle, University of Montpellier, CNRS, Institut de Recherche pour le Développement, Montpellier34095, France
| | - Rachel Neish
- York Biomedical Research Institute, Department of Biology, University of York, YorkYO10 5DD, United Kingdom
| | - Binita Nepal
- Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX75390
- Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX75390
| | - Julien Espeut
- Tubulin Code team, Institute of Human Genetics, CNRS, Université Montpellier, Montpellier34090, France
| | - Grégoire Pasquier
- Maladies infectieuses et Vecteurs: Ecologie, Génétique, Evolution et Contrôle, University of Montpellier, CNRS, Institut de Recherche pour le Développement, Montpellier34095, France
| | - Ghislain Gillard
- Tubulin Code team, Institute of Human Genetics, CNRS, Université Montpellier, Montpellier34090, France
| | - Chantal Cazevieille
- Université Montpellier, INSERM U1298, Institute for Neurosciences of Montpellier, Montpellier34090, France
| | - Jeremy C. Mottram
- York Biomedical Research Institute, Department of Biology, University of York, YorkYO10 5DD, United Kingdom
| | - Dawn M. Wetzel
- Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX75390
- Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX75390
| | - Yvon Sterkers
- Maladies infectieuses et Vecteurs: Ecologie, Génétique, Evolution et Contrôle, University of Montpellier, CNRS, Institut de Recherche pour le Développement, Montpellier34095, France
| | - Krzysztof Rogowski
- Tubulin Code team, Institute of Human Genetics, CNRS, Université Montpellier, Montpellier34090, France
| | - Maude F. Lévêque
- Maladies infectieuses et Vecteurs: Ecologie, Génétique, Evolution et Contrôle, University of Montpellier, CNRS, Institut de Recherche pour le Développement, Montpellier34095, France
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12
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Liu Y, Fang Y, Dhikhirullahi O, Zhang L, Zhang Z. Intraflagellar Transport (IFT) and Sperm Formation. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2025; 1469:395-409. [PMID: 40301266 DOI: 10.1007/978-3-031-82990-1_17] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/01/2025]
Abstract
Intraflagellar transport (IFT) is a conserved mechanism for cilia formation. Twenty-two IFT components form the IFT-A complex (six components) and IFT-B complex (sixteen components). Driven by kinesin and dynein motor proteins, these IFT complexes are involved in the trafficking of proteins needed for cilia assembly by anterograde transport and retrograde transport. IFT core components also associate with other proteins for cilia formation. Mutations in IFT core components result in ciliogenesis defects and human diseases, including male infertility. Sperm flagella are specialized motile cilia that not only have core axoneme structure but also possess accessory structures. IFT is required to assemble these structures to form functional sperm. This summary highlights the regulatory roles of specific IFT proteins in spermatogenesis. A deeper understanding of IFT-related mechanisms can shed light on the etiology and pathophysiology of certain male infertility cases, as well as provide insights for the development of novel male contraceptives.
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Affiliation(s)
- Yunhao Liu
- School of Public Health, Wuhan University of Science and Technology, Wuhan, Hubei, China
| | - Yu Fang
- School of Public Health, Wuhan University of Science and Technology, Wuhan, Hubei, China
| | | | - Ling Zhang
- School of Public Health, Wuhan University of Science and Technology, Wuhan, Hubei, China
| | - Zhibing Zhang
- Department of Physiology, Wayne State University, Detroit, MI, USA.
- Department of Obstetrics & Gynecology, Wayne State University, Detroit, MI, USA.
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13
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Niehrs C, Da Silva F, Seidl C. Cilia as Wnt signaling organelles. Trends Cell Biol 2025; 35:24-32. [PMID: 38697898 DOI: 10.1016/j.tcb.2024.04.001] [Citation(s) in RCA: 6] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2024] [Revised: 04/03/2024] [Accepted: 04/08/2024] [Indexed: 05/05/2024]
Abstract
Cilia and Wnt signaling have a complex relationship, wherein Wnt regulates cilia and, conversely, cilia may affect Wnt signaling. Recently, it was shown that Wnt receptors are present in flagella, primary cilia, and multicilia, where they transmit an intraciliary signal that is independent of β-catenin. Intraciliary Wnt signaling promotes ciliogenesis, affecting male fertility, adipogenesis, and mucociliary clearance. Wnt also stimulates the beating of motile cilia, highlighting that these nanomotors, too, are chemosensory. Intraciliary Wnt signaling employs a Wnt-protein phosphatase 1 (PP1) signaling axis, involving the canonical Wnt pathway's inhibition of glycogen synthase kinase 3 (GSK3) to repress PP1 activity. Collectively, these findings support that cilia are Wnt signaling organelles, with implications for ciliopathies and cancer.
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Affiliation(s)
- Christof Niehrs
- Division of Molecular Embryology, DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany; Institute of Molecular Biology (IMB), 55128 Mainz, Germany.
| | - Fabio Da Silva
- Division of Molecular Embryology, DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany
| | - Carina Seidl
- Division of Molecular Embryology, DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany
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14
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Palicharla VR, Badgandi HB, Hwang SH, Legué E, Liem KF, Mukhopadhyay S. A defined tubby domain β-barrel surface region of TULP3 mediates ciliary trafficking of diverse cargoes. Mol Biol Cell 2025; 36:ar1. [PMID: 39565681 PMCID: PMC11742108 DOI: 10.1091/mbc.e24-09-0426] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2024] [Revised: 11/05/2024] [Accepted: 11/12/2024] [Indexed: 11/22/2024] Open
Abstract
The primary cilium is a paradigmatic subcellular compartment at the nexus of numerous cellular and morphogenetic pathways. The tubby family protein TULP3 acts as an adapter of the intraflagellar transport complex A in transporting integral membrane and membrane-associated lipidated proteins into cilia. However, the mechanisms by which TULP3 coordinates ciliary transport of diverse cargoes is not well understood. Here, we provide molecular insights into TULP3-mediated ciliary cargo recognition. We screened for critical TULP3 residues by proximity biotinylation-mass spectrometry, structural analysis, and testing TULP3 variants in human patients with hepatorenal fibrocystic disease and spina bifida. The TULP3 residues we identified 1) were located on one side of the β-barrel of the tubby domain away from the phosphoinositide binding site, 2) mediated ciliary trafficking of lipidated and transmembrane cargoes, and 3) determined proximity with these cargoes in vivo without affecting ciliary localization, phosphoinositide binding or hydrodynamic properties of TULP3. Overall, these findings implicate a specific region of one of the surfaces of the TULP3 β-barrel in ciliary trafficking of diverse cargoes. This region overlooks the β-strands 8-12 of the β-barrel and is away from the membrane anchoring phosphoinositide binding site. Targeting the TULP3-cargo interactions could provide therapeutics in ciliary trafficking diseases.
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Affiliation(s)
- Vivek Reddy Palicharla
- Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390
| | - Hemant B. Badgandi
- Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390
| | - Sun-Hee Hwang
- Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390
| | - Emilie Legué
- Vertebrate Developmental Biology Program, Department of Pediatrics, Yale University School of Medicine, New Haven, CT 06520
| | - Karel F. Liem
- Vertebrate Developmental Biology Program, Department of Pediatrics, Yale University School of Medicine, New Haven, CT 06520
| | - Saikat Mukhopadhyay
- Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390
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15
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Sun J, Yuan H, Yu Y, Li A, Zhao Z, Tang Y, Zheng F. Immunomodulatory potential of primary cilia in the skin. Front Immunol 2024; 15:1456875. [PMID: 39676858 PMCID: PMC11638010 DOI: 10.3389/fimmu.2024.1456875] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2024] [Accepted: 11/15/2024] [Indexed: 12/17/2024] Open
Abstract
Primary cilia (PC) are essential signaling hubs for proper epithelial formation and the maintenance of skin homeostasis. Found on most cells in the human body, including skin cells, PC facilitate signal transduction that allows ciliated cells to interact with the immune system via multiple pathways, helping to maintain immune system homeostasis. PC can be altered by various microenvironmental stimuli to develop corresponding regulatory functions. Both PC and ciliary signaling pathways have been shown to be involved in the immune processes of various skin lesions. However, the mechanisms by which PC regulate cellular functions and maintain immune homeostasis in tissues are highly complex, and our understanding of them in the skin remains limited. In this paper, we discuss key ciliary signaling pathways and ciliated cells in the skin, with a focus on their immunomodulatory functions. We have compiled evidence from various cells, tissues and disease models to help explore the potential immunomodulatory effects of PC in the skin and their molecular mechanisms.
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Affiliation(s)
- Jingwei Sun
- School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing, China
| | - Huimin Yuan
- Department of Dermatology, Beijing Tsinghua Changgung Hospital, School of Clinical Medicine, Tsinghua University, Beijing, China
| | - Yanru Yu
- School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing, China
| | - Aorou Li
- School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing, China
| | - Zihe Zhao
- School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing, China
| | - Yang Tang
- School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing, China
| | - Fengjie Zheng
- School of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing, China
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16
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Patel MB, Griffin PJ, Olson SF, Dai J, Hou Y, Malik T, Das P, Zhang G, Zhao W, Witman GB, Lechtreck KF. Distribution and bulk flow analyses of the intraflagellar transport (IFT) motor kinesin-2 support an "on-demand" model for Chlamydomonas ciliary length control. Cytoskeleton (Hoboken) 2024; 81:586-604. [PMID: 38456596 PMCID: PMC11380706 DOI: 10.1002/cm.21851] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2023] [Revised: 02/22/2024] [Accepted: 02/23/2024] [Indexed: 03/09/2024]
Abstract
Most cells tightly control the length of their cilia. The regulation likely involves intraflagellar transport (IFT), a bidirectional motility of multi-subunit particles organized into trains that deliver building blocks into the organelle. In Chlamydomonas, the anterograde IFT motor kinesin-2 consists of the motor subunits FLA8 and FLA10 and the nonmotor subunit KAP. KAP dissociates from IFT at the ciliary tip and diffuses back to the cell body. This observation led to the diffusion-as-a-ruler model of ciliary length control, which postulates that KAP is progressively sequestered into elongating cilia because its return to the cell body will require increasingly more time, limiting motor availability at the ciliary base, train assembly, building block supply, and ciliary growth. Here, we show that Chlamydomonas FLA8 also returns to the cell body by diffusion. However, more than 95% of KAP and FLA8 are present in the cell body and, at a given time, just ~1% of the motor participates in IFT. After repeated photobleaching of both cilia, IFT of fluorescent kinesin subunits continued indicating that kinesin-2 cycles from the large cell-body pool through the cilia and back. Furthermore, growing and full-length cilia contained similar amounts of kinesin-2 subunits and the size of the motor pool at the base changed only slightly with ciliary length. These observations are incompatible with the diffusion-as-a-ruler model, but rather support an "on-demand model," in which the cargo load of the trains is regulated to assemble cilia of the desired length.
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Affiliation(s)
- Mansi B Patel
- Department of Cellular Biology, University of Georgia, Athens, Georgia, USA
| | - Paul J Griffin
- Department of Cellular Biology, University of Georgia, Athens, Georgia, USA
| | - Spencer F Olson
- Department of Cellular Biology, University of Georgia, Athens, Georgia, USA
| | - Jin Dai
- Department of Cellular Biology, University of Georgia, Athens, Georgia, USA
| | - Yuqing Hou
- Department of Radiology, UMass Chan Medical School, Worcester, Massachusetts, USA
| | - Tara Malik
- Department of Cellular Biology, University of Georgia, Athens, Georgia, USA
| | - Poulomi Das
- Department of Cellular Biology, University of Georgia, Athens, Georgia, USA
| | - Gui Zhang
- Department of Cellular Biology, University of Georgia, Athens, Georgia, USA
| | - Winston Zhao
- Department of Radiology, UMass Chan Medical School, Worcester, Massachusetts, USA
| | - George B Witman
- Department of Radiology, UMass Chan Medical School, Worcester, Massachusetts, USA
| | - Karl F Lechtreck
- Department of Cellular Biology, University of Georgia, Athens, Georgia, USA
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17
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Ewerling A, May-Simera HL. Evolutionary trajectory for nuclear functions of ciliary transport complex proteins. Microbiol Mol Biol Rev 2024; 88:e0000624. [PMID: 38995044 PMCID: PMC11426024 DOI: 10.1128/mmbr.00006-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/13/2024] Open
Abstract
SUMMARYCilia and the nucleus were two defining features of the last eukaryotic common ancestor. In early eukaryotic evolution, these structures evolved through the diversification of a common membrane-coating ancestor, the protocoatomer. While in cilia, the descendants of this protein complex evolved into parts of the intraflagellar transport complexes and BBSome, the nucleus gained its selectivity by recruiting protocoatomer-like proteins to the nuclear envelope to form the selective nuclear pore complexes. Recent studies show a growing number of proteins shared between the proteomes of the respective organelles, and it is currently unknown how ciliary transport proteins could acquire nuclear functions and vice versa. The nuclear functions of ciliary proteins are still observable today and remain relevant for the understanding of the disease mechanisms behind ciliopathies. In this work, we review the evolutionary history of cilia and nucleus and their respective defining proteins and integrate current knowledge into theories for early eukaryotic evolution. We postulate a scenario where both compartments co-evolved and that fits current models of eukaryotic evolution, explaining how ciliary proteins and nucleoporins acquired their dual functions.
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Affiliation(s)
- Alexander Ewerling
- Faculty of Biology, Institute of Molecular Physiology, Johannes Gutenberg-University, Mainz, Germany
| | - Helen Louise May-Simera
- Faculty of Biology, Institute of Molecular Physiology, Johannes Gutenberg-University, Mainz, Germany
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18
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Ye JW, Abbas T, Zhou JT, Chen J, Yang ML, Huang XH, Zhang H, Ma H, Ma A, Xu B, Murtaza G, Shi QH, Shi BL. Homozygous CCDC146 mutation causes oligoasthenoteratozoospermia in humans and mice. Zool Res 2024; 45:1073-1087. [PMID: 39245651 PMCID: PMC11491774 DOI: 10.24272/j.issn.2095-8137.2024.019] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2024] [Accepted: 06/11/2024] [Indexed: 09/10/2024] Open
Abstract
Infertility represents a significant health concern, with sperm quantity and quality being crucial determinants of male fertility. Oligoasthenoteratozoospermia (OAT) is characterized by reduced sperm motility, lower sperm concentration, and morphological abnormalities in sperm heads and flagella. Although variants in several genes have been implicated in OAT, its genetic etiologies and pathogenetic mechanisms remain inadequately understood. In this study, we identified a homozygous nonsense mutation (c.916C>T, p.Arg306*) in the coiled-coil domain containing 146 ( CCDC146) gene in an infertile male patient with OAT. This mutation resulted in the production of a truncated CCDC146 protein (amino acids 1-305), retaining only two out of five coiled-coil domains. To validate the pathogenicity of the CCDC146 mutation, we generated a mouse model ( Ccdc146 mut/mut ) with a similar mutation to that of the patient. Consistently, the Ccdc146 mut/mut mice exhibited infertility, characterized by significantly reduced sperm counts, diminished motility, and multiple defects in sperm heads and flagella. Furthermore, the levels of axonemal proteins, including DNAH17, DNAH1, and SPAG6, were significantly reduced in the sperm of Ccdc146 mut/mut mice. Additionally, both human and mouse CCDC146 interacted with intraflagellar transport protein 20 (IFT20), but this interaction was lost in the mutated versions, leading to the degradation of IFT20. This study identified a novel deleterious homozygous nonsense mutation in CCDC146 that causes male infertility, potentially by disrupting axonemal protein transportation. These findings offer valuable insights for genetic counseling and understanding the mechanisms underlying CCDC146 mutant-associated infertility in human males.
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Affiliation(s)
- Jing-Wei Ye
- Division of Reproduction and Genetics, First Affiliated Hospital of University of Science and Technology of China, Hefei National Laboratory for Physical Sciences at Microscale, School of Basic Medical Sciences, Division of Life Sciences and Medicine, Biomedical Sciences and Health Laboratory of Anhui Province, Institute of Health and Medicine, Hefei Comprehensive National Science Center, University of Science and Technology of China, Hefei, Anhui 230026, China
| | - Tanveer Abbas
- Division of Reproduction and Genetics, First Affiliated Hospital of University of Science and Technology of China, Hefei National Laboratory for Physical Sciences at Microscale, School of Basic Medical Sciences, Division of Life Sciences and Medicine, Biomedical Sciences and Health Laboratory of Anhui Province, Institute of Health and Medicine, Hefei Comprehensive National Science Center, University of Science and Technology of China, Hefei, Anhui 230026, China
| | - Jian-Teng Zhou
- Division of Reproduction and Genetics, First Affiliated Hospital of University of Science and Technology of China, Hefei National Laboratory for Physical Sciences at Microscale, School of Basic Medical Sciences, Division of Life Sciences and Medicine, Biomedical Sciences and Health Laboratory of Anhui Province, Institute of Health and Medicine, Hefei Comprehensive National Science Center, University of Science and Technology of China, Hefei, Anhui 230026, China
| | - Jing Chen
- Division of Reproduction and Genetics, First Affiliated Hospital of University of Science and Technology of China, Hefei National Laboratory for Physical Sciences at Microscale, School of Basic Medical Sciences, Division of Life Sciences and Medicine, Biomedical Sciences and Health Laboratory of Anhui Province, Institute of Health and Medicine, Hefei Comprehensive National Science Center, University of Science and Technology of China, Hefei, Anhui 230026, China
| | - Meng-Lei Yang
- Division of Reproduction and Genetics, First Affiliated Hospital of University of Science and Technology of China, Hefei National Laboratory for Physical Sciences at Microscale, School of Basic Medical Sciences, Division of Life Sciences and Medicine, Biomedical Sciences and Health Laboratory of Anhui Province, Institute of Health and Medicine, Hefei Comprehensive National Science Center, University of Science and Technology of China, Hefei, Anhui 230026, China
| | - Xiong-Heng Huang
- Division of Reproduction and Genetics, First Affiliated Hospital of University of Science and Technology of China, Hefei National Laboratory for Physical Sciences at Microscale, School of Basic Medical Sciences, Division of Life Sciences and Medicine, Biomedical Sciences and Health Laboratory of Anhui Province, Institute of Health and Medicine, Hefei Comprehensive National Science Center, University of Science and Technology of China, Hefei, Anhui 230026, China
| | - Huan Zhang
- Division of Reproduction and Genetics, First Affiliated Hospital of University of Science and Technology of China, Hefei National Laboratory for Physical Sciences at Microscale, School of Basic Medical Sciences, Division of Life Sciences and Medicine, Biomedical Sciences and Health Laboratory of Anhui Province, Institute of Health and Medicine, Hefei Comprehensive National Science Center, University of Science and Technology of China, Hefei, Anhui 230026, China
| | - Hui Ma
- Division of Reproduction and Genetics, First Affiliated Hospital of University of Science and Technology of China, Hefei National Laboratory for Physical Sciences at Microscale, School of Basic Medical Sciences, Division of Life Sciences and Medicine, Biomedical Sciences and Health Laboratory of Anhui Province, Institute of Health and Medicine, Hefei Comprehensive National Science Center, University of Science and Technology of China, Hefei, Anhui 230026, China
| | - Ao Ma
- Division of Reproduction and Genetics, First Affiliated Hospital of University of Science and Technology of China, Hefei National Laboratory for Physical Sciences at Microscale, School of Basic Medical Sciences, Division of Life Sciences and Medicine, Biomedical Sciences and Health Laboratory of Anhui Province, Institute of Health and Medicine, Hefei Comprehensive National Science Center, University of Science and Technology of China, Hefei, Anhui 230026, China
- Institute of Andrology, Nanjing Drum Tower Hospital, Affiliated Hospital of Nanjing University Medical School, Nanjing, Jiangsu 210008, China
| | - Bo Xu
- Division of Reproduction and Genetics, First Affiliated Hospital of University of Science and Technology of China, Hefei National Laboratory for Physical Sciences at Microscale, School of Basic Medical Sciences, Division of Life Sciences and Medicine, Biomedical Sciences and Health Laboratory of Anhui Province, Institute of Health and Medicine, Hefei Comprehensive National Science Center, University of Science and Technology of China, Hefei, Anhui 230026, China
| | - Ghulam Murtaza
- Division of Reproduction and Genetics, First Affiliated Hospital of University of Science and Technology of China, Hefei National Laboratory for Physical Sciences at Microscale, School of Basic Medical Sciences, Division of Life Sciences and Medicine, Biomedical Sciences and Health Laboratory of Anhui Province, Institute of Health and Medicine, Hefei Comprehensive National Science Center, University of Science and Technology of China, Hefei, Anhui 230026, China
| | - Qing-Hua Shi
- Division of Reproduction and Genetics, First Affiliated Hospital of University of Science and Technology of China, Hefei National Laboratory for Physical Sciences at Microscale, School of Basic Medical Sciences, Division of Life Sciences and Medicine, Biomedical Sciences and Health Laboratory of Anhui Province, Institute of Health and Medicine, Hefei Comprehensive National Science Center, University of Science and Technology of China, Hefei, Anhui 230026, China. E-mail:
| | - Bao-Lu Shi
- Division of Reproduction and Genetics, First Affiliated Hospital of University of Science and Technology of China, Hefei National Laboratory for Physical Sciences at Microscale, School of Basic Medical Sciences, Division of Life Sciences and Medicine, Biomedical Sciences and Health Laboratory of Anhui Province, Institute of Health and Medicine, Hefei Comprehensive National Science Center, University of Science and Technology of China, Hefei, Anhui 230026, China. E-mail:
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Boldizar H, Friedman A, Stanley T, Padilla M, Galdieri J, Sclar A, Stawicki TM. The role of cilia in the development, survival, and regeneration of hair cells. Biol Open 2024; 13:bio061690. [PMID: 39263863 PMCID: PMC11413933 DOI: 10.1242/bio.061690] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2024] [Accepted: 08/15/2024] [Indexed: 09/13/2024] Open
Abstract
Mutations impacting cilia genes lead to a class of human diseases known as ciliopathies. This is due to the role of cilia in the development, survival, and regeneration of many cell types. We investigated the extent to which disrupting cilia impacted these processes in lateral line hair cells of zebrafish. We found that mutations in two intraflagellar transport (IFT) genes, ift88 and dync2h1, which lead to the loss of kinocilia, caused increased hair cell apoptosis. IFT gene mutants also have a decreased mitochondrial membrane potential, and blocking the mitochondrial uniporter causes a loss of hair cells in wild-type zebrafish but not mutants, suggesting mitochondria dysfunction may contribute to the apoptosis seen in these mutants. These mutants also showed decreased proliferation during hair cell regeneration but did not show consistent changes in support cell number or proliferation during hair cell development. These results show that the loss of hair cells seen following disruption of cilia through either mutations in anterograde or retrograde IFT genes appears to be due to impacts on hair cell survival but not necessarily development in the zebrafish lateral line.
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Affiliation(s)
- Hope Boldizar
- Neuroscience Program, Lafayette College, Easton, PA 18042, USA
| | - Amanda Friedman
- Neuroscience Program, Lafayette College, Easton, PA 18042, USA
| | - Tess Stanley
- Neuroscience Program, Lafayette College, Easton, PA 18042, USA
| | - María Padilla
- Biology Department, Lafayette College, Easton, PA 18042, USA
| | | | - Arielle Sclar
- Neuroscience Program, Lafayette College, Easton, PA 18042, USA
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20
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Boegholm N, Petriman NA, Tanvir NM, Lorentzen E. Architecture of RabL2-associated complexes at the ciliary base: A structural modeling perspective: Deciphering the structural organization of ciliary RabL2 complexes. Bioessays 2024; 46:e2300222. [PMID: 38991980 DOI: 10.1002/bies.202300222] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/24/2023] [Revised: 06/28/2024] [Accepted: 07/01/2024] [Indexed: 07/13/2024]
Abstract
Cilia are slender, micrometer-long organelles present on the surface of eukaryotic cells. They function in signaling and locomotion and are constructed by intraflagellar transport (IFT). The assembly of IFT complexes into so-called IFT trains to initiate ciliary entry at the base of the cilium remains a matter of debate. Here, we use structural modeling to provide an architectural framework for how RabL2 is anchored at the ciliary base via CEP19 before being handed over to IFT trains for ciliary entry. Our models suggest that the N-terminal domain of CEP43 forms a homo-dimer to anchor at the subdistal appendages of cilia through a direct interaction with CEP350. A long linker region separates the N-terminal domain of CEP43 from the C-terminal domain, which captures CEP19 above the subdistal appendages and close to the distal appendages. Furthermore, we present a structural model for how RabL2-CEP19 associates with the IFT-B complex, providing insight into how RabL2 is handed over from CEP19 to the IFT complex. Interestingly, RabL2 association with the IFT-B complex appears to induce a significant conformational change in the IFT complex via a kink in the coiled-coils of the IFT81/74 proteins, which may prime the IFT machinery for entry into cilia.
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Affiliation(s)
- Niels Boegholm
- Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark
| | - Narcis A Petriman
- Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark
| | - Niaj M Tanvir
- Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark
| | - Esben Lorentzen
- Department of Molecular Biology and Genetics, Aarhus University, Aarhus C, Denmark
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21
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Lacey SE, Graziadei A, Pigino G. Extensive structural rearrangement of intraflagellar transport trains underpins bidirectional cargo transport. Cell 2024; 187:4621-4636.e18. [PMID: 39067443 PMCID: PMC11349379 DOI: 10.1016/j.cell.2024.06.041] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2024] [Revised: 05/06/2024] [Accepted: 06/28/2024] [Indexed: 07/30/2024]
Abstract
Bidirectional transport in cilia is carried out by polymers of the IFTA and IFTB protein complexes, called anterograde and retrograde intraflagellar transport (IFT) trains. Anterograde trains deliver cargoes from the cell to the cilium tip, then convert into retrograde trains for cargo export. We set out to understand how the IFT complexes can perform these two directly opposing roles before and after conversion. We use cryoelectron tomography and in situ cross-linking mass spectrometry to determine the structure of retrograde IFT trains and compare it with the known structure of anterograde trains. The retrograde train is a 2-fold symmetric polymer organized around a central thread of IFTA complexes. We conclude that anterograde-to-retrograde remodeling involves global rearrangements of the IFTA/B complexes and requires complete disassembly of the anterograde train. Finally, we describe how conformational changes to cargo-binding sites facilitate unidirectional cargo transport in a bidirectional system.
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22
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Fujii T, Liang L, Nakayama K, Katoh Y. Defects in diffusion barrier function of ciliary transition zone caused by ciliopathy variations of TMEM218. Hum Mol Genet 2024; 33:1442-1453. [PMID: 38751342 DOI: 10.1093/hmg/ddae083] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2024] [Revised: 05/01/2024] [Accepted: 05/07/2024] [Indexed: 08/09/2024] Open
Abstract
Primary cilia are antenna-like structures protruding from the surface of various eukaryotic cells, and have distinct protein compositions in their membranes. This distinct protein composition is maintained by the presence of the transition zone (TZ) at the ciliary base, which acts as a diffusion barrier between the ciliary and plasma membranes. Defects in cilia and the TZ are known to cause a group of disorders collectively called the ciliopathies, which demonstrate a broad spectrum of clinical features, such as perinatally lethal Meckel syndrome (MKS), relatively mild Joubert syndrome (JBTS), and nonsyndromic nephronophthisis (NPHP). Proteins constituting the TZ can be grouped into the MKS and NPHP modules. The MKS module is composed of several transmembrane proteins and three soluble proteins. TMEM218 was recently reported to be mutated in individuals diagnosed as MKS and JBTS. However, little is known about how TMEM218 mutations found in MKS and JBTS affect the functions of cilia. In this study, we found that ciliary membrane proteins were not localized to cilia in TMEM218-knockout cells, indicating impaired barrier function of the TZ. Furthermore, the exogenous expression of JBTS-associated TMEM218 variants but not MKS-associated variants in TMEM218-knockout cells restored the localization of ciliary membrane proteins. In particular, when expressed in TMEM218-knockout cells, the TMEM218(R115H) variant found in JBTS was able to restore the barrier function of cells, whereas the MKS variant TMEM218(R115C) could not. Thus, the severity of symptoms of MKS and JBTS individuals appears to correlate with the degree of their ciliary defects at the cellular level.
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Affiliation(s)
- Taiju Fujii
- Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
| | - Luxiaoxue Liang
- Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
| | - Kazuhisa Nakayama
- Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
| | - Yohei Katoh
- Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
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23
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Shao S, Chen Y, Deng H, Pan J. Quantitative proteomics reveals insights into the assembly of IFT trains and ciliary assembly. J Cell Sci 2024; 137:jcs262152. [PMID: 38853670 DOI: 10.1242/jcs.262152] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2024] [Accepted: 05/28/2024] [Indexed: 06/11/2024] Open
Abstract
Intraflagellar transport (IFT) is required for ciliary assembly. The IFT machinery comprises the IFT motors kinesin-2 and IFT dynein plus IFT-A and IFT-B complexes, which assemble into IFT trains in cilia. To gain mechanistic understanding of IFT and ciliary assembly, here, we performed an absolute quantification of IFT machinery in Chlamydomonas reinhardtii cilium. There are ∼756, ∼532, ∼276 and ∼350 molecules of IFT-B, IFT-A, IFT dynein and kinesin-2, respectively, per cilium. The amount of IFT-B is sufficient to sustain rapid ciliary growth in terms of tubulin delivery. The stoichiometric ratio of IFT-B:IFT-A:dynein is ∼3:2:1 whereas the IFT-B:IFT-A ratio in an IFT dynein mutant is 2:1, suggesting that there is a plastic interaction between IFT-A and IFT-B that can be influenced by IFT dynein. Considering diffusion of kinesin-2 during retrograde IFT, it is estimated that one kinesin-2 molecule drives eight molecules of IFT-B during anterograde IFT. These data provide new insights into the assembly of IFT trains and ciliary assembly.
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Affiliation(s)
- Shangjin Shao
- MOE Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China
- Laboratory for Marine Biology and Biotechnology, Qingdao Marine Science and Technology Center, Qingdao, Shandong Province 266000, China
| | - Yuling Chen
- MOE Key Laboratory of Bioinformatics, School of Life Sciences, Core Facility Center for Biomedical Analysis, Tsinghua University, Beijing 100084, China
| | - Haiteng Deng
- MOE Key Laboratory of Bioinformatics, School of Life Sciences, Core Facility Center for Biomedical Analysis, Tsinghua University, Beijing 100084, China
| | - Junmin Pan
- MOE Key Laboratory of Protein Sciences, School of Life Sciences, Tsinghua University, Beijing 100084, China
- Laboratory for Marine Biology and Biotechnology, Qingdao Marine Science and Technology Center, Qingdao, Shandong Province 266000, China
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24
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Reddy Palicharla V, Mukhopadhyay S. Molecular and structural perspectives on protein trafficking to the primary cilium membrane. Biochem Soc Trans 2024; 52:1473-1487. [PMID: 38864436 PMCID: PMC11346432 DOI: 10.1042/bst20231403] [Citation(s) in RCA: 6] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2024] [Revised: 05/23/2024] [Accepted: 05/24/2024] [Indexed: 06/13/2024]
Abstract
The primary cilium is a dynamic subcellular compartment templated from the mother centriole or basal body. Cilia are solitary and tiny, but remarkably consequential in cellular pathways regulating proliferation, differentiation, and maintenance. Multiple transmembrane proteins such as G-protein-coupled receptors, channels, enzymes, and membrane-associated lipidated proteins are enriched in the ciliary membrane. The precise regulation of ciliary membrane content is essential for effective signal transduction and maintenance of tissue homeostasis. Surprisingly, a few conserved molecular factors, intraflagellar transport complex A and the tubby family adapter protein TULP3, mediate the transport of most membrane cargoes into cilia. Recent advances in cryogenic electron microscopy provide fundamental insights into these molecular players. Here, we review the molecular players mediating cargo delivery into the ciliary membrane through the lens of structural biology. These mechanistic insights into ciliary transport provide a framework for understanding of disease variants in ciliopathies, enable precise manipulation of cilia-mediated pathways, and provide a platform for the development of targeted therapeutics.
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Affiliation(s)
- Vivek Reddy Palicharla
- Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, U.S.A
| | - Saikat Mukhopadhyay
- Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, U.S.A
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25
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Kuwasako K, Dang W, He F, Takahashi M, Tsuda K, Nagata T, Tanaka A, Kobayashi N, Kigawa T, Güntert P, Shirouzu M, Yokoyama S, Muto Y. 1H, 13C, and 15N resonance assignments and solution structure of the N-terminal divergent calponin homology (NN-CH) domain of human intraflagellar transport protein 54. BIOMOLECULAR NMR ASSIGNMENTS 2024; 18:71-78. [PMID: 38551798 DOI: 10.1007/s12104-024-10170-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 11/28/2023] [Accepted: 03/13/2024] [Indexed: 05/12/2024]
Abstract
The intraflagellar transport (IFT) machinery plays a crucial role in the bidirectional trafficking of components necessary for ciliary signaling, such as the Hedgehog, Wnt/PCR, and cAMP/PKA systems. Defects in some components of the IFT machinery cause dysfunction, leading to a wide range of human diseases and developmental disorders termed ciliopathies, such as nephronophthisis. The IFT machinery comprises three sub-complexes: BBsome, IFT-A, and IFT-B. The IFT protein 54 (IFT54) is an important component of the IFT-B sub-complex. In anterograde movement, IFT54 binds to active kinesin-II, walking along the cilia microtubule axoneme and carrying the dynein-2 complex in an inactive state, which works for retrograde movement. Several mutations in IFT54 are known to cause Senior-Loken syndrome, a ciliopathy. IFT54 possesses a divergent Calponin Homology (CH) domain termed as NN-CH domain at its N-terminus. However, several aspects of the function of the NN-CH domain of IFT54 are still obscure. Here, we report the 1H, 15N, and 13C resonance assignments of the NN-CH domain of human IFT54 and its solution structure. The NN-CH domain of human IFT54 adopts essentially the α1-α2-α3-α4-α5 topology as that of mouse IFT54, whose structure was determined by X-ray crystallographic study. The structural information and assignments obtained in this study shed light on the molecular function of the NN-CH domain in IFT54.
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Affiliation(s)
- Kanako Kuwasako
- RIKEN, Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045, Japan
- RIKEN Center for Biosystems Dynamics Research, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230- 0045, Japan
- Faculty of Pharmacy and Research Institute of Pharmaceutical Sciences, Musashino University, Tokyo, 202-8585, Japan
| | - Weirong Dang
- RIKEN, Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045, Japan
| | - Fahu He
- RIKEN, Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045, Japan
| | - Mari Takahashi
- RIKEN, Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045, Japan
- RIKEN Center for Biosystems Dynamics Research, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230- 0045, Japan
| | - Kengo Tsuda
- RIKEN, Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045, Japan
| | - Takashi Nagata
- RIKEN, Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045, Japan
- Institute of Advanced Energy, Graduate School of Energy Science, Kyoto University, Gokasho, Kyoto, Uji, 611-0011, Japan
| | - Akiko Tanaka
- RIKEN, Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045, Japan
| | - Naohiro Kobayashi
- RIKEN, Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045, Japan
- RIKEN Yokohama NMR Facility, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Japan
| | - Takanori Kigawa
- RIKEN, Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045, Japan
- RIKEN Center for Biosystems Dynamics Research, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230- 0045, Japan
| | - Peter Güntert
- Tatsuo Miyazawa Memorial Program, RIKEN Genomic Sciences Center, Yokohama, 230-0045, Japan
- Institute of Biophysical Chemistry, Goethe-University Frankfurt am Main, Max-von-Laue-Str. 9, Frankfurt am Main, 60438, Germany
- Institute of Molecular Physical Science, ETH Zurich, Vladimir-Prelog-Weg 2, Zurich, 8093, Switzerland
- Department of Chemistry, Tokyo Metropolitan University, 1-1 Minami-Ohsawa, Hachioji, Tokyo, 192- 0397, Japan
| | - Mikako Shirouzu
- RIKEN, Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045, Japan
- RIKEN Center for Biosystems Dynamics Research, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230- 0045, Japan
| | - Shigeyuki Yokoyama
- RIKEN, Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045, Japan.
- RIKEN Structural Biology Laboratory, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Japan.
- RIKEN Cluster for Science, Technology and Innovation Hub, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, Yokohama, 230-0045, Japan.
| | - Yutaka Muto
- RIKEN, Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, 230-0045, Japan.
- RIKEN Center for Biosystems Dynamics Research, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230- 0045, Japan.
- Faculty of Pharmacy and Research Institute of Pharmaceutical Sciences, Musashino University, Tokyo, 202-8585, Japan.
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26
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Mukhopadhyay AG, Toropova K, Daly L, Wells JN, Vuolo L, Mladenov M, Seda M, Jenkins D, Stephens DJ, Roberts AJ. Structure and tethering mechanism of dynein-2 intermediate chains in intraflagellar transport. EMBO J 2024; 43:1257-1272. [PMID: 38454149 PMCID: PMC10987677 DOI: 10.1038/s44318-024-00060-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2023] [Revised: 02/06/2024] [Accepted: 02/09/2024] [Indexed: 03/09/2024] Open
Abstract
Dynein-2 is a large multiprotein complex that powers retrograde intraflagellar transport (IFT) of cargoes within cilia/flagella, but the molecular mechanism underlying this function is still emerging. Distinctively, dynein-2 contains two identical force-generating heavy chains that interact with two different intermediate chains (WDR34 and WDR60). Here, we dissect regulation of dynein-2 function by WDR34 and WDR60 using an integrative approach including cryo-electron microscopy and CRISPR/Cas9-enabled cell biology. A 3.9 Å resolution structure shows how WDR34 and WDR60 use surprisingly different interactions to engage equivalent sites of the two heavy chains. We show that cilia can assemble in the absence of either WDR34 or WDR60 individually, but not both subunits. Dynein-2-dependent distribution of cargoes depends more strongly on WDR60, because the unique N-terminal extension of WDR60 facilitates dynein-2 targeting to cilia. Strikingly, this N-terminal extension can be transplanted onto WDR34 and retain function, suggesting it acts as a flexible tether to the IFT "trains" that assemble at the ciliary base. We discuss how use of unstructured tethers represents an emerging theme in IFT train interactions.
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Affiliation(s)
- Aakash G Mukhopadhyay
- Sir William Dunn School of Pathology, University of Oxford, Oxford, UK
- Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck, University of London, London, UK
| | - Katerina Toropova
- Sir William Dunn School of Pathology, University of Oxford, Oxford, UK
- Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck, University of London, London, UK
| | - Lydia Daly
- Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck, University of London, London, UK
- Randall Centre of Cell & Molecular Biophysics, King's College London, London, UK
| | - Jennifer N Wells
- Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck, University of London, London, UK
- MRC London Institute of Medical Sciences (LMS), London, UK
| | - Laura Vuolo
- Cell Biology Laboratories, School of Biochemistry, University of Bristol, Bristol, UK
| | - Miroslav Mladenov
- Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck, University of London, London, UK
- Cellular Signalling and Cytoskeletal Function Laboratory, The Francis Crick Institute, London, UK
| | - Marian Seda
- UCL Great Ormond Street Institute of Child Health, University College London, London, UK
| | - Dagan Jenkins
- UCL Great Ormond Street Institute of Child Health, University College London, London, UK
| | - David J Stephens
- Cell Biology Laboratories, School of Biochemistry, University of Bristol, Bristol, UK
| | - Anthony J Roberts
- Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.
- Institute of Structural and Molecular Biology, Department of Biological Sciences, Birkbeck, University of London, London, UK.
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27
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Brinzer RA, Winter AD, Page AP. The relationship between intraflagellar transport and upstream protein trafficking pathways and macrocyclic lactone resistance in Caenorhabditis elegans. G3 (BETHESDA, MD.) 2024; 14:jkae009. [PMID: 38227795 PMCID: PMC10917524 DOI: 10.1093/g3journal/jkae009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/23/2023] [Revised: 01/08/2024] [Accepted: 01/08/2024] [Indexed: 01/18/2024]
Abstract
Parasitic nematodes are globally important and place a heavy disease burden on infected humans, crops, and livestock, while commonly administered anthelmintics used for treatment are being rendered ineffective by increasing levels of resistance. It has recently been shown in the model nematode Caenorhabditis elegans that the sensory cilia of the amphid neurons play an important role in resistance toward macrocyclic lactones such as ivermectin (an avermectin) and moxidectin (a milbemycin) either through reduced uptake or intertissue signaling pathways. This study interrogated the extent to which ciliary defects relate to macrocyclic lactone resistance and dye-filling defects using a combination of forward genetics and targeted resistance screening approaches and confirmed the importance of intraflagellar transport in this process. This approach also identified the protein trafficking pathways used by the downstream effectors and the components of the ciliary basal body that are required for effector entry into these nonmotile structures. In total, 24 novel C. elegans anthelmintic survival-associated genes were identified in this study. When combined with previously known resistance genes, there are now 46 resistance-associated genes that are directly involved in amphid, cilia, and intraflagellar transport function.
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Affiliation(s)
- Robert A Brinzer
- School of Biodiversity, One Health and Veterinary Medicine, University of Glasgow, Scotland G61 1QH, UK
| | - Alan D Winter
- School of Biodiversity, One Health and Veterinary Medicine, University of Glasgow, Scotland G61 1QH, UK
| | - Antony P Page
- School of Biodiversity, One Health and Veterinary Medicine, University of Glasgow, Scotland G61 1QH, UK
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28
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Seo S, Sonn SK, Kweon HY, Jin J, Kume T, Ko JY, Park JH, Oh GT. Primary Cilium in Neural Crest Cells Crucial for Anterior Segment Development and Corneal Avascularity. Invest Ophthalmol Vis Sci 2024; 65:30. [PMID: 38517430 PMCID: PMC10981158 DOI: 10.1167/iovs.65.3.30] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2023] [Accepted: 02/26/2024] [Indexed: 03/23/2024] Open
Abstract
Purpose Intraflagellar transport 46 (IFT46) is an integral subunit of the IFT-B complex, playing a key role in the assembly and maintenance of primary cilia responsible for transducing signaling pathways. Despite its predominant expression in the basal body of cilia, the precise role of Ift46 in ocular development remains undetermined. This study aimed to elucidate the impact of neural crest (NC)-specific deletion of Ift46 on ocular development. Methods NC-specific conditional knockout mice for Ift46 (NC-Ift46F/F) were generated by crossing Ift46F mice with Wnt1-Cre2 mice, enabling the specific deletion of Ift46 in NC-derived cells (NCCs). Sonic Hedgehog (Shh) and Notch signaling activities in NC-Ift46F/F mice were evaluated using Gli1lacZ and CBF:H2B-Venus reporter mice, respectively. Cell fate mapping was conducted using ROSAmTmG reporter mice. Results The deletion of Ift46 in NCCs resulted in a spectrum of ocular abnormalities, including thickened corneal stroma, hypoplasia of the anterior chamber, irregular iris morphology, and corneal neovascularization. Notably, this deletion led to reduced Shh signal activity in the periocular mesenchyme, sustained expression of key transcription factors Foxc1, Foxc2 and Pitx2, along with persistent cell proliferation. Additionally, it induced increased Notch signaling activity and the development of ectopic neovascularization within the corneal stroma. Conclusions The absence of primary cilia due to Ift46 deficiency in NCCs is associated with anterior segment dysgenesis (ASD) and corneal neovascularization, suggesting a potential link to Axenfeld-Rieger syndrome, a disorder characterized by ASD. This underscores the pivotal role of primary cilia in ensuring proper anterior segment development and maintaining an avascular cornea.
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Affiliation(s)
- Seungwoon Seo
- Heart-Immune-Brain Network Research Center, Department of Life Science, Ewha Womans University, Seoul, Republic of Korea
- Imvastech Inc., Seoul, Republic of Korea
| | - Seong Keun Sonn
- Heart-Immune-Brain Network Research Center, Department of Life Science, Ewha Womans University, Seoul, Republic of Korea
| | - Hyae Yon Kweon
- Heart-Immune-Brain Network Research Center, Department of Life Science, Ewha Womans University, Seoul, Republic of Korea
| | - Jing Jin
- Heart-Immune-Brain Network Research Center, Department of Life Science, Ewha Womans University, Seoul, Republic of Korea
| | - Tsutomu Kume
- Feinberg Cardiovascular and Renal Research Institute, Northwestern University School of Medicine, Chicago, Illinois, United States
| | - Je Yeong Ko
- Department of Biological Science, Sookmyung Women's University, Seoul, Republic of Korea
| | - Jong Hoon Park
- Department of Biological Science, Sookmyung Women's University, Seoul, Republic of Korea
| | - Goo Taeg Oh
- Heart-Immune-Brain Network Research Center, Department of Life Science, Ewha Womans University, Seoul, Republic of Korea
- Imvastech Inc., Seoul, Republic of Korea
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29
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Penny GM, Dutcher SK. Gene dosage of independent dynein arm motor preassembly factors influences cilia assembly in Chlamydomonas reinhardtii. PLoS Genet 2024; 20:e1011038. [PMID: 38498551 PMCID: PMC11020789 DOI: 10.1371/journal.pgen.1011038] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2023] [Revised: 04/16/2024] [Accepted: 02/26/2024] [Indexed: 03/20/2024] Open
Abstract
Motile cilia assembly utilizes over 800 structural and cytoplasmic proteins. Variants in approximately 58 genes cause primary ciliary dyskinesia (PCD) in humans, including the dynein arm (pre)assembly factor (DNAAF) gene DNAAF4. In humans, outer dynein arms (ODAs) and inner dynein arms (IDAs) fail to assemble motile cilia when DNAAF4 function is disrupted. In Chlamydomonas reinhardtii, a ciliated unicellular alga, the DNAAF4 ortholog is called PF23. The pf23-1 mutant assembles short cilia and lacks IDAs, but partially retains ODAs. The cilia of a new null allele (pf23-4) completely lack ODAs and IDAs and are even shorter than cilia from pf23-1. In addition, PF23 plays a role in the cytoplasmic modification of IC138, a protein of the two-headed IDA (I1/f). As most PCD variants in humans are recessive, we sought to test if heterozygosity at two genes affects ciliary function using a second-site non-complementation (SSNC) screening approach. We asked if phenotypes were observed in diploids with pairwise heterozygous combinations of 21 well-characterized ciliary mutant Chlamydomonas strains. Vegetative cultures of single and double heterozygous diploid cells did not show SSNC for motility phenotypes. When protein synthesis is inhibited, wild-type Chlamydomonas cells utilize the pool of cytoplasmic proteins to assemble half-length cilia. In this sensitized assay, 8 double heterozygous diploids with pf23 and other DNAAF mutations show SSNC; they assemble shorter cilia than wild-type. In contrast, double heterozygosity of the other 203 strains showed no effect on ciliary assembly. Immunoblots of diploids heterozygous for pf23 and wdr92 or oda8 show that PF23 is reduced by half in these strains, and that PF23 dosage affects phenotype severity. Reductions in PF23 and another DNAAF in diploids affect the ability to assemble ODAs and IDAs and impedes ciliary assembly. Thus, dosage of multiple DNAAFs is an important factor in cilia assembly and regeneration.
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Affiliation(s)
- Gervette M. Penny
- Department of Genetics, Washington University in Saint Louis, Saint Louis,Missouri, United States of America
| | - Susan K. Dutcher
- Department of Genetics, Washington University in Saint Louis, Saint Louis,Missouri, United States of America
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Fitzsimons LA, Tasouri E, Willaredt MA, Stetson D, Gojak C, Kirsch J, Gardner HAR, Gorgas K, Tucker KL. Primary cilia are critical for tracheoesophageal septation. Dev Dyn 2024; 253:312-332. [PMID: 37776236 PMCID: PMC10922539 DOI: 10.1002/dvdy.660] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2023] [Revised: 08/14/2023] [Accepted: 09/09/2023] [Indexed: 10/02/2023] Open
Abstract
INTRODUCTION Primary cilia play pivotal roles in the patterning and morphogenesis of a wide variety of organs during mammalian development. Here we examined murine foregut septation in the cobblestone mutant, a hypomorphic allele of the gene encoding the intraflagellar transport protein IFT88, a protein essential for normal cilia function. RESULTS We reveal a crucial role for primary cilia in foregut division, since their dramatic decrease in cilia in both the foregut endoderm and mesenchyme of mutant embryos resulted in a proximal tracheoesophageal septation defects and in the formation of distal tracheo(broncho)esophageal fistulae similar to the most common congenital tracheoesophageal malformations in humans. Interestingly, the dorsoventral patterning determining the dorsal digestive and the ventral respiratory endoderm remained intact, whereas Hedgehog signaling was aberrantly activated. CONCLUSIONS Our results demonstrate the cobblestone mutant to represent one of the very few mouse models that display both correct endodermal dorsoventral specification but defective compartmentalization of the proximal foregut. It stands exemplary for a tracheoesophageal ciliopathy, offering the possibility to elucidate the molecular mechanisms how primary cilia orchestrate the septation process. The plethora of malformations observed in the cobblestone embryo allow for a deeper insight into a putative link between primary cilia and human VATER/VACTERL syndromes.
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Affiliation(s)
- Lindsey Avery Fitzsimons
- Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, Maine, U.S.A
- Dept. of Biomedical Sciences, Center for Excellence in the Neurosciences, College of Osteopathic Medicine, University of New England, Biddeford, Maine 04005, U.S.A
| | - Evangelia Tasouri
- Interdisciplinary Center for Neurosciences, University of Heidelberg, 69120 Heidelberg, Germany
- Institute of Anatomy and Cell Biology, University of Heidelberg, 69120 Heidelberg, Germany
| | - Marc August Willaredt
- Interdisciplinary Center for Neurosciences, University of Heidelberg, 69120 Heidelberg, Germany
- Institute of Anatomy and Cell Biology, University of Heidelberg, 69120 Heidelberg, Germany
| | - Daniel Stetson
- AstraZeneca Pharmaceuticals LP, 35 Gatehouse Drive, Waltham, Massachusetts 02451, U.S.A
| | - Christian Gojak
- Interdisciplinary Center for Neurosciences, University of Heidelberg, 69120 Heidelberg, Germany
- Institute of Anatomy and Cell Biology, University of Heidelberg, 69120 Heidelberg, Germany
| | - Joachim Kirsch
- Institute of Anatomy and Cell Biology, University of Heidelberg, 69120 Heidelberg, Germany
| | | | - Karin Gorgas
- Institute of Anatomy and Cell Biology, University of Heidelberg, 69120 Heidelberg, Germany
| | - Kerry L. Tucker
- Graduate School of Biomedical Sciences and Engineering, University of Maine, Orono, Maine, U.S.A
- Dept. of Biomedical Sciences, Center for Excellence in the Neurosciences, College of Osteopathic Medicine, University of New England, Biddeford, Maine 04005, U.S.A
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31
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Leggere JC, Hibbard JV, Papoulas O, Lee C, Pearson CG, Marcotte EM, Wallingford JB. Label-free proteomic comparison reveals ciliary and nonciliary phenotypes of IFT-A mutants. Mol Biol Cell 2024; 35:ar39. [PMID: 38170584 PMCID: PMC10916875 DOI: 10.1091/mbc.e23-03-0084] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2023] [Revised: 12/11/2023] [Accepted: 12/18/2023] [Indexed: 01/05/2024] Open
Abstract
DIFFRAC is a powerful method for systematically comparing proteome content and organization between samples in a high-throughput manner. By subjecting control and experimental protein extracts to native chromatography and quantifying the contents of each fraction using mass spectrometry, it enables the quantitative detection of alterations to protein complexes and abundances. Here, we applied DIFFRAC to investigate the consequences of genetic loss of Ift122, a subunit of the intraflagellar transport-A (IFT-A) protein complex that plays a vital role in the formation and function of cilia and flagella, on the proteome of Tetrahymena thermophila. A single DIFFRAC experiment was sufficient to detect changes in protein behavior that mirrored known effects of IFT-A loss and revealed new biology. We uncovered several novel IFT-A-regulated proteins, which we validated through live imaging in Xenopus multiciliated cells, shedding new light on both the ciliary and non-ciliary functions of IFT-A. Our findings underscore the robustness of DIFFRAC for revealing proteomic changes in response to genetic or biochemical perturbation.
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Affiliation(s)
- Janelle C. Leggere
- Department of Molecular Biosciences, University of Texas at Austin, TX 78712
| | - Jaime V.K. Hibbard
- Department of Molecular Biosciences, University of Texas at Austin, TX 78712
| | - Ophelia Papoulas
- Department of Molecular Biosciences, University of Texas at Austin, TX 78712
| | - Chanjae Lee
- Department of Molecular Biosciences, University of Texas at Austin, TX 78712
| | - Chad G. Pearson
- Anschutz Medical Campus, Department of Cell and Developmental Biology, University of Colorado, Aurora, CO 80045
| | - Edward M. Marcotte
- Department of Molecular Biosciences, University of Texas at Austin, TX 78712
| | - John B. Wallingford
- Department of Molecular Biosciences, University of Texas at Austin, TX 78712
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Moran AL, Louzao-Martinez L, Norris DP, Peters DJM, Blacque OE. Transport and barrier mechanisms that regulate ciliary compartmentalization and ciliopathies. Nat Rev Nephrol 2024; 20:83-100. [PMID: 37872350 DOI: 10.1038/s41581-023-00773-2] [Citation(s) in RCA: 4] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/19/2023] [Indexed: 10/25/2023]
Abstract
Primary cilia act as cell surface antennae, coordinating cellular responses to sensory inputs and signalling molecules that regulate developmental and homeostatic pathways. Cilia are therefore critical to physiological processes, and defects in ciliary components are associated with a large group of inherited pleiotropic disorders - known collectively as ciliopathies - that have a broad spectrum of phenotypes and affect many or most tissues, including the kidney. A central feature of the cilium is its compartmentalized structure, which imparts its unique molecular composition and signalling environment despite its membrane and cytosol being contiguous with those of the cell. Such compartmentalization is achieved via active transport pathways that bring protein cargoes to and from the cilium, as well as gating pathways at the ciliary base that establish diffusion barriers to protein exchange into and out of the organelle. Many ciliopathy-linked proteins, including those involved in kidney development and homeostasis, are components of the compartmentalizing machinery. New insights into the major compartmentalizing pathways at the cilium, namely, ciliary gating, intraflagellar transport, lipidated protein flagellar transport and ciliary extracellular vesicle release pathways, have improved our understanding of the mechanisms that underpin ciliary disease and associated renal disorders.
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Affiliation(s)
- Ailis L Moran
- School of Biomolecular and Biomedical Science, University College Dublin, Dublin, Ireland
| | - Laura Louzao-Martinez
- Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands
| | | | - Dorien J M Peters
- Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands.
| | - Oliver E Blacque
- School of Biomolecular and Biomedical Science, University College Dublin, Dublin, Ireland.
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Beyer T, Martins T, Srikaran JJ, Seda M, Peskett E, Klose F, Junger K, Beales PL, Ueffing M, Boldt K, Jenkins D. Affinity Purification of Intraflagellar Transport (IFT) Proteins in Mice Using Endogenous Streptavidin/FLAG Tags. Methods Mol Biol 2024; 2725:199-212. [PMID: 37856026 DOI: 10.1007/978-1-0716-3507-0_12] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2023]
Abstract
Biological complexity is achieved through elaborate interactions between relatively few individual components. Affinity purification (AP) has allowed these networks of protein-protein interactions that regulate key biological processes to be interrogated systematically. In order to perform these studies at the required scale, easily transfectable immortalized cell lines have typically been used. Gene-editing now affords the systematic creation of isogenic mouse models carrying endogenous tags for affinity proteomics. This may allow protein-protein interactions to be characterized in the appropriate tissue for a particular biological process or disease phenotype under physiological conditions, and for interaction landscapes to be compared across tissues. Here we demonstrate application to intraflagellar transport (IFT) proteins, which are WD40-domain-containing proteins that are essential for the formation and function of all types of cilia. We describe a method to generate mice with an endogenous C-terminal streptavidin/FLAG tag, using Ift80 as an example, and demonstrate the successful implementation of AP in this model. This method can easily be adapted for N- and C-terminal tagging of many other proteins in vivo.
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Affiliation(s)
- Tina Beyer
- Institute for Ophthalmic Research, Center for Ophthalmology, University of Tübingen, Tübingen, Germany
| | - Tiago Martins
- UCL Great Ormond Street Institute of Child Health, University College London, London, UK
| | | | - Marian Seda
- UCL Great Ormond Street Institute of Child Health, University College London, London, UK
| | - Emma Peskett
- UCL Great Ormond Street Institute of Child Health, University College London, London, UK
| | - Franziska Klose
- Institute for Ophthalmic Research, Center for Ophthalmology, University of Tübingen, Tübingen, Germany
| | - Katrin Junger
- Institute for Ophthalmic Research, Center for Ophthalmology, University of Tübingen, Tübingen, Germany
| | - Philip L Beales
- UCL Great Ormond Street Institute of Child Health, University College London, London, UK
| | - Marius Ueffing
- Institute for Ophthalmic Research, Center for Ophthalmology, University of Tübingen, Tübingen, Germany
| | - Karsten Boldt
- Institute for Ophthalmic Research, Center for Ophthalmology, University of Tübingen, Tübingen, Germany
| | - Dagan Jenkins
- UCL Great Ormond Street Institute of Child Health, University College London, London, UK.
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Shaikh Qureshi WM, Hentges KE. Functions of cilia in cardiac development and disease. Ann Hum Genet 2024; 88:4-26. [PMID: 37872827 PMCID: PMC10952336 DOI: 10.1111/ahg.12534] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2023] [Revised: 09/08/2023] [Accepted: 10/02/2023] [Indexed: 10/25/2023]
Abstract
Errors in embryonic cardiac development are a leading cause of congenital heart defects (CHDs), including morphological abnormalities of the heart that are often detected after birth. In the past few decades, an emerging role for cilia in the pathogenesis of CHD has been identified, but this topic still largely remains an unexplored area. Mouse forward genetic screens and whole exome sequencing analysis of CHD patients have identified enrichment for de novo mutations in ciliary genes or non-ciliary genes, which regulate cilia-related pathways, linking cilia function to aberrant cardiac development. Key events in cardiac morphogenesis, including left-right asymmetric development of the heart, are dependent upon cilia function. Cilia dysfunction during left-right axis formation contributes to CHD as evidenced by the substantial proportion of heterotaxy patients displaying complex CHD. Cilia-transduced signaling also regulates later events during heart development such as cardiac valve formation, outflow tract septation, ventricle development, and atrioventricular septa formation. In this review, we summarize the role of motile and non-motile (primary cilia) in cardiac asymmetry establishment and later events during heart development.
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Affiliation(s)
- Wasay Mohiuddin Shaikh Qureshi
- Division of Evolution, Infection and Genomics, School of Biological Sciences, Faculty of Biology, Medicine, and Health, Manchester Academic Health Science CentreUniversity of ManchesterManchesterUK
| | - Kathryn E. Hentges
- Division of Evolution, Infection and Genomics, School of Biological Sciences, Faculty of Biology, Medicine, and Health, Manchester Academic Health Science CentreUniversity of ManchesterManchesterUK
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35
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Lee H, Lee J, Shin M, Park S. ANKS1A-Deficiency Aberrantly Increases the Entry of the Protein Transport Machinery into the Ependymal Cilia. Mol Cells 2023; 46:757-763. [PMID: 38052491 PMCID: PMC10701301 DOI: 10.14348/molcells.2023.0153] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2023] [Revised: 10/13/2023] [Accepted: 10/22/2023] [Indexed: 12/07/2023] Open
Abstract
In this study, we examine whether a change in the protein levels for FOP in Ankyrin repeat and SAM domain-containing protein 1A (ANKS1A)-deficient ependymal cells affects the intraflagellar transport (IFT) protein transport system in the multicilia. Three distinct abnormalities are observed in the multicilia of ANKS1A-deficient ependymal cells. First, there were a greater number of IFT88-positive trains along the cilia from ANKS1A deficiency. The results are similar to each isolated cilium as well. Second, each isolated cilium contains a significant increase in the number of extracellular vesicles (ECVs) due to the lack of ANKS1A. Third, Van Gogh-like 2 (Vangl2), a ciliary membrane protein, is abundantly detected along the cilia and in the ECVs attached to them for ANKS1A-deficient cells. We also use primary ependymal culture systems to obtain the ECVs released from the multicilia. Consequently, we find that ECVs from ANKS1A-deficient cells contain more IFT machinery and Vangl2. These results indicate that ANKS1A deficiency increases the entry of the protein transport machinery into the multicilia and as a result of these abnormal protein transports, excessive ECVs form along the cilia. We conclude that ependymal cells make use of the ECV-based disposal system in order to eliminate excessively transported proteins from basal bodies.
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Affiliation(s)
- Haeryung Lee
- Department of Biological Sciences, Sookmyung Women’s University, Seoul 04310, Korea
| | - Jiyeon Lee
- Department of Biological Sciences, Sookmyung Women’s University, Seoul 04310, Korea
| | - Miram Shin
- Department of Biological Sciences, Sookmyung Women’s University, Seoul 04310, Korea
| | - Soochul Park
- Department of Biological Sciences, Sookmyung Women’s University, Seoul 04310, Korea
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36
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Syu JJ, Chang CH, Chang PY, Liu CH, Yu CJ, Jou TS. Lipid raft interacting galectin 8 regulates primary ciliogenesis. FASEB J 2023; 37:e23300. [PMID: 37997673 DOI: 10.1096/fj.202301943r] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2023] [Revised: 10/23/2023] [Accepted: 10/26/2023] [Indexed: 11/25/2023]
Abstract
Primary cilium is a specialized sensory organelle that transmits environmental information into cells. Its length is tightly controlled by various mechanisms such as the frequency or the cargo size of the intraflagellar transport trains which deliver the building materials such as tubulin subunits essential for the growing cilia. Here, we show the sialoglycan interacting galectin 8 regulates the process of primary ciliogenesis. As the epithelia become polarized, there are more galectin 8 being apically secreted and these extracellular galectin 8 molecules apparently bind to a lipid raft enriched domain at the base of the primary cilia through interacting with lipid raft components, such as GD3 ganglioside and scaffold protein caveolin 1. Furthermore, the binding of galectin 8 at this critical region triggers rapid growth of primary cilia by perturbing the barrier function of the transition zone (TZ). Our study also demonstrates the functionality of this barrier depends on intact organization of lipid rafts at the cilia as genetically knockout of Cav1 and pharmacologically inhibition of lipid raft both phenocopy the effect of apical addition of recombinant galectin 8; that is, rapid elongation of primary cilia and redistribution of cilia proteins from TZ to the growing axoneme. Indeed, as cilia elongated, endogenous galectin 8, caveolin 1, and TZ component, TMEM231, also transited from the TZ to the growing axoneme. We also noted that the interaction between caveolin 1 and TMEM231 could be perturbed by exogenous galectin 8. Taken together, we proposed that galectin 8 promoted primary cilia elongation through impeding the barrier function of the TZ by interfering with the interaction between caveolin 1 and TMEM231.
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Affiliation(s)
- Jhan-Jhang Syu
- Graduate Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan
| | - Chieh-Hsiang Chang
- Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan
| | - Pei-Yu Chang
- Graduate Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan
| | - Chia-Hsiung Liu
- Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan
- Department of Surgery, College of Medicine, National Taiwan University, Taipei, Taiwan
| | - Chia-Jung Yu
- Department of Cell and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan, Taiwan
- Department of Thoracic Medicine, Chang Gung Memorial Hospital, Taoyuan, Taiwan
| | - Tzuu-Shuh Jou
- Graduate Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan
- Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan
- Center of Precision Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan
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37
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Gonçalves-Santos F, De-Castro ARG, Rodrigues DRM, De-Castro MJG, Gassmann R, Abreu CMC, Dantas TJ. Hot-wiring dynein-2 establishes roles for IFT-A in retrograde train assembly and motility. Cell Rep 2023; 42:113337. [PMID: 37883232 DOI: 10.1016/j.celrep.2023.113337] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2023] [Revised: 08/08/2023] [Accepted: 10/09/2023] [Indexed: 10/28/2023] Open
Abstract
Intraflagellar transport (IFT) trains, built around IFT-A and IFT-B complexes, are carried by opposing motors to import and export ciliary cargo. While transported by kinesin-2 on anterograde IFT trains, the dynein-2 motor adopts an autoinhibitory conformation until it needs to be activated at the ciliary tip to power retrograde IFT. Growing evidence has linked the IFT-A complex to retrograde IFT; however, its roles in this process remain unknown. Here, we use CRISPR-Cas9-mediated genome editing to disable the dynein-2 autoinhibition mechanism in Caenorhabditis elegans and assess its impact on IFT with high-resolution live imaging and photobleaching analyses. Remarkably, this dynein-2 "hot-wiring" approach reignites retrograde motility inside IFT-A-deficient cilia without triggering tug-of-war events. In addition to providing functional evidence that multiple mechanisms maintain dynein-2 inhibited during anterograde IFT, our data establish key roles for IFT-A in mediating motor-train coupling during IFT turnaround, promoting retrograde IFT initiation, and modulating dynein-2 retrograde motility.
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Affiliation(s)
- Francisco Gonçalves-Santos
- i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal
| | - Ana R G De-Castro
- i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal; ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal
| | - Diogo R M Rodrigues
- i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal; ICBAS - Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal
| | - Maria J G De-Castro
- i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal
| | - Reto Gassmann
- i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal
| | - Carla M C Abreu
- i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal.
| | - Tiago J Dantas
- i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal; IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal.
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Hoffmann F, Bolz S, Junger K, Klose F, Stehle IF, Ueffing M, Boldt K, Beyer T. Paralog-specific TTC30 regulation of Sonic hedgehog signaling. Front Mol Biosci 2023; 10:1268722. [PMID: 38074101 PMCID: PMC10701685 DOI: 10.3389/fmolb.2023.1268722] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2023] [Accepted: 11/09/2023] [Indexed: 02/12/2024] Open
Abstract
The intraflagellar transport (IFT) machinery is essential for cilia assembly, maintenance, and trans-localization of signaling proteins. The IFT machinery consists of two large multiprotein complexes, one of which is the IFT-B. TTC30A and TTC30B are integral components of this complex and were previously shown to have redundant functions in the context of IFT, preventing the disruption of IFT-B and, thus, having a severe ciliogenesis defect upon loss of one paralog. In this study, we re-analyzed the paralog-specific protein complexes and discovered a potential involvement of TTC30A or TTC30B in ciliary signaling. Specifically, we investigated a TTC30A-specific interaction with protein kinase A catalytic subunit α, a negative regulator of Sonic hedgehog (Shh) signaling. Defects in this ciliary signaling pathway are often correlated to synpolydactyly, which, intriguingly, is also linked to a rare TTC30 variant. For an in-depth analysis of this unique interaction and the influence on Shh, TTC30A or B single- and double-knockout hTERT-RPE1 were employed, as well as rescue cells harboring wildtype TTC30 or the corresponding mutation. We could show that mutant TTC30A inhibits the ciliary localization of Smoothened. This observed effect is independent of Patched1 but associated with a distinct phosphorylated PKA substrate accumulation upon treatment with forskolin. This rather prominent phenotype was attenuated in mutant TTC30B. Mass spectrometry analysis of wildtype versus mutated TTC30A or TTC30B uncovered differences in protein complex patterns and identified an impaired TTC30A-IFT57 interaction as the possible link leading to synpolydactyly. We could observe no impact on cilia assembly, leading to the hypothesis that a slight decrease in IFT-B binding can be compensated, but mild phenotypes, like synpolydactyly, can be induced by subtle signaling changes. Our systematic approach revealed the paralog-specific influence of TTC30A KO and mutated TTC30A on the activity of PRKACA and the uptake of Smoothened into the cilium, resulting in a downregulation of Shh. This downregulation, combined with interactome alterations, suggests a potential mechanism of how mutant TTC30A is linked to synpolydactyly.
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Affiliation(s)
- Felix Hoffmann
- Institute for Ophthalmic Research, Eberhard Karls University Tübingen, Tübingen, Germany
| | | | | | | | | | | | | | - Tina Beyer
- *Correspondence: Felix Hoffmann, ; Tina Beyer,
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39
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Gopalakrishnan J, Feistel K, Friedrich BM, Grapin‐Botton A, Jurisch‐Yaksi N, Mass E, Mick DU, Müller R, May‐Simera H, Schermer B, Schmidts M, Walentek P, Wachten D. Emerging principles of primary cilia dynamics in controlling tissue organization and function. EMBO J 2023; 42:e113891. [PMID: 37743763 PMCID: PMC10620770 DOI: 10.15252/embj.2023113891] [Citation(s) in RCA: 19] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2023] [Revised: 08/07/2023] [Accepted: 09/08/2023] [Indexed: 09/26/2023] Open
Abstract
Primary cilia project from the surface of most vertebrate cells and are key in sensing extracellular signals and locally transducing this information into a cellular response. Recent findings show that primary cilia are not merely static organelles with a distinct lipid and protein composition. Instead, the function of primary cilia relies on the dynamic composition of molecules within the cilium, the context-dependent sensing and processing of extracellular stimuli, and cycles of assembly and disassembly in a cell- and tissue-specific manner. Thereby, primary cilia dynamically integrate different cellular inputs and control cell fate and function during tissue development. Here, we review the recently emerging concept of primary cilia dynamics in tissue development, organization, remodeling, and function.
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Affiliation(s)
- Jay Gopalakrishnan
- Institute for Human Genetics, Heinrich‐Heine‐UniversitätUniversitätsklinikum DüsseldorfDüsseldorfGermany
| | - Kerstin Feistel
- Department of Zoology, Institute of BiologyUniversity of HohenheimStuttgartGermany
| | | | - Anne Grapin‐Botton
- Cluster of Excellence Physics of Life, TU DresdenDresdenGermany
- Max Planck Institute of Molecular Cell Biology and GeneticsDresdenGermany
- Paul Langerhans Institute Dresden of the Helmholtz Center Munich at The University Hospital Carl Gustav Carus and Faculty of Medicine of the TU DresdenDresdenGermany
| | - Nathalie Jurisch‐Yaksi
- Department of Clinical and Molecular MedicineNorwegian University of Science and TechnologyTrondheimNorway
| | - Elvira Mass
- Life and Medical Sciences Institute, Developmental Biology of the Immune SystemUniversity of BonnBonnGermany
| | - David U Mick
- Center for Molecular Signaling (PZMS), Center of Human and Molecular Biology (ZHMB)Saarland School of MedicineHomburgGermany
| | - Roman‐Ulrich Müller
- Department II of Internal Medicine and Center for Molecular Medicine Cologne, Faculty of Medicine and University Hospital CologneUniversity of CologneCologneGermany
- Cologne Excellence Cluster on Cellular Stress Responses in Aging‐Associated Diseases (CECAD), Faculty of Medicine and University Hospital CologneUniversity of CologneCologneGermany
| | - Helen May‐Simera
- Institute of Molecular PhysiologyJohannes Gutenberg‐UniversityMainzGermany
| | - Bernhard Schermer
- Department II of Internal Medicine and Center for Molecular Medicine Cologne, Faculty of Medicine and University Hospital CologneUniversity of CologneCologneGermany
- Cologne Excellence Cluster on Cellular Stress Responses in Aging‐Associated Diseases (CECAD), Faculty of Medicine and University Hospital CologneUniversity of CologneCologneGermany
| | - Miriam Schmidts
- Pediatric Genetics Division, Center for Pediatrics and Adolescent MedicineUniversity Hospital FreiburgFreiburgGermany
- CIBSS‐Centre for Integrative Biological Signalling StudiesUniversity of FreiburgFreiburgGermany
| | - Peter Walentek
- CIBSS‐Centre for Integrative Biological Signalling StudiesUniversity of FreiburgFreiburgGermany
- Renal Division, Internal Medicine IV, Medical CenterUniversity of FreiburgFreiburgGermany
| | - Dagmar Wachten
- Institute of Innate Immunity, Biophysical Imaging, Medical FacultyUniversity of BonnBonnGermany
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Fassad MR, Rumman N, Junger K, Patel MP, Thompson J, Goggin P, Ueffing M, Beyer T, Boldt K, Lucas JS, Mitchison HM. Defective airway intraflagellar transport underlies a combined motile and primary ciliopathy syndrome caused by IFT74 mutations. Hum Mol Genet 2023; 32:3090-3104. [PMID: 37555648 PMCID: PMC10586200 DOI: 10.1093/hmg/ddad132] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2023] [Revised: 08/01/2023] [Indexed: 08/10/2023] Open
Abstract
Ciliopathies are inherited disorders caused by defective cilia. Mutations affecting motile cilia usually cause the chronic muco-obstructive sinopulmonary disease primary ciliary dyskinesia (PCD) and are associated with laterality defects, while a broad spectrum of early developmental as well as degenerative syndromes arise from mutations affecting signalling of primary (non-motile) cilia. Cilia assembly and functioning requires intraflagellar transport (IFT) of cargos assisted by IFT-B and IFT-A adaptor complexes. Within IFT-B, the N-termini of partner proteins IFT74 and IFT81 govern tubulin transport to build the ciliary microtubular cytoskeleton. We detected a homozygous 3-kb intragenic IFT74 deletion removing the exon 2 initiation codon and 40 N-terminal amino acids in two affected siblings. Both had clinical features of PCD with bronchiectasis, but no laterality defects. They also had retinal dysplasia and abnormal bone growth, with a narrowed thorax and short ribs, shortened long bones and digits, and abnormal skull shape. This resembles short-rib thoracic dysplasia, a skeletal ciliopathy previously linked to IFT defects in primary cilia, not motile cilia. Ciliated nasal epithelial cells collected from affected individuals had reduced numbers of shortened motile cilia with disarranged microtubules, some misorientation of the basal feet, and disrupted cilia structural and IFT protein distributions. No full-length IFT74 was expressed, only truncated forms that were consistent with N-terminal deletion and inframe translation from downstream initiation codons. In affinity purification mass spectrometry, exon 2-deleted IFT74 initiated from the nearest inframe downstream methionine 41 still interacts as part of the IFT-B complex, but only with reduced interaction levels and not with all its usual IFT-B partners. We propose that this is a hypomorphic mutation with some residual protein function retained, which gives rise to a primary skeletal ciliopathy combined with defective motile cilia and PCD.
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Affiliation(s)
- Mahmoud R Fassad
- Genetics and Genomic Medicine Research and Teaching Department, University College London, UCL Great Ormond Street Institute of Child Health, 30 Guilford Street, London WC1N 1EH, United Kingdom
- Department of Human Genetics, Medical Research Institute, Alexandria University, 22 El-Guish Road, El-Shatby, Alexandria 21526, Egypt
| | - Nisreen Rumman
- Department of Pediatrics, Faculty of Medicine, Makassed Hospital and Al-Quds University, East Jerusalem 91220, Palestine
- Section of Pulmonary, Critical Care and Sleep Medicine, Department of Internal Medicine, Yale University School of Medicine, 300 Cedar St #441, New Haven, CT 06520, United States
| | - Katrin Junger
- Institute for Ophthalmic Research, Eberhard Karl University of Tübingen, Elfreide-Alhorn-Strasse 5-7, Tübingen 72076, Germany
| | - Mitali P Patel
- Genetics and Genomic Medicine Research and Teaching Department, University College London, UCL Great Ormond Street Institute of Child Health, 30 Guilford Street, London WC1N 1EH, United Kingdom
- MRC Prion Unit at UCL, Institute of Prion Diseases, University College London, 33 Cleveland Street, London W1W 7FF, United Kingdom
| | - James Thompson
- Primary Ciliary Dyskinesia Centre, NIHR Biomedical Research Centre, University Hospital Southampton NHS Foundation Trust, Southampton SO16 6YD, United Kingdom
- School of Clinical and Experimental Sciences, University of Southampton Faculty of Medicine, University Road, Southampton SO17 1BJ, United Kingdom
- Biomedical Imaging Unit, University of Southampton Faculty of Medicine, University Road, Southampton SO17 1BJ, United Kingdom
| | - Patricia Goggin
- Primary Ciliary Dyskinesia Centre, NIHR Biomedical Research Centre, University Hospital Southampton NHS Foundation Trust, Southampton SO16 6YD, United Kingdom
- Biomedical Imaging Unit, University of Southampton Faculty of Medicine, University Road, Southampton SO17 1BJ, United Kingdom
| | - Marius Ueffing
- Institute for Ophthalmic Research, Eberhard Karl University of Tübingen, Elfreide-Alhorn-Strasse 5-7, Tübingen 72076, Germany
| | - Tina Beyer
- Institute for Ophthalmic Research, Eberhard Karl University of Tübingen, Elfreide-Alhorn-Strasse 5-7, Tübingen 72076, Germany
| | - Karsten Boldt
- Institute for Ophthalmic Research, Eberhard Karl University of Tübingen, Elfreide-Alhorn-Strasse 5-7, Tübingen 72076, Germany
| | - Jane S Lucas
- Primary Ciliary Dyskinesia Centre, NIHR Biomedical Research Centre, University Hospital Southampton NHS Foundation Trust, Southampton SO16 6YD, United Kingdom
- School of Clinical and Experimental Sciences, University of Southampton Faculty of Medicine, University Road, Southampton SO17 1BJ, United Kingdom
| | - Hannah M Mitchison
- Genetics and Genomic Medicine Research and Teaching Department, University College London, UCL Great Ormond Street Institute of Child Health, 30 Guilford Street, London WC1N 1EH, United Kingdom
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Masek M, Bachmann-Gagescu R. Control of protein and lipid composition of photoreceptor outer segments-Implications for retinal disease. Curr Top Dev Biol 2023; 155:165-225. [PMID: 38043951 DOI: 10.1016/bs.ctdb.2023.09.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/05/2023]
Abstract
Vision is arguably our most important sense, and its loss brings substantial limitations to daily life for affected individuals. Light is perceived in retinal photoreceptors (PRs), which are highly specialized neurons subdivided into several compartments with distinct functions. The outer segments (OSs) of photoreceptors represent highly specialized primary ciliary compartments hosting the phototransduction cascade, which transforms incoming light into a neuronal signal. Retinal disease can result from various pathomechanisms originating in distinct subcompartments of the PR cell, or in the retinal pigment epithelium which supports the PRs. Dysfunction of primary cilia causes human disorders known as "ciliopathies", in which retinal disease is a common feature. This chapter focuses on PR OSs, discussing the mechanisms controlling their complex structure and composition. A sequence of tightly regulated sorting and trafficking events, both upstream of and within this ciliary compartment, ensures the establishment and maintenance of the adequate proteome and lipidome required for signaling in response to light. We discuss in particular our current understanding of the role of ciliopathy proteins involved in multi-protein complexes at the ciliary transition zone (CC2D2A) or BBSome (BBS1) and how their dysfunction causes retinal disease. While the loss of CC2D2A prevents the fusion of vesicles and delivery of the photopigment rhodopsin to the ciliary base, leading to early OS ultrastructural defects, BBS1 deficiency results in precocious accumulation of cholesterol in mutant OSs and decreased visual function preceding morphological changes. These distinct pathomechanisms underscore the central role of ciliary proteins involved in multiple processes controlling OS protein and lipid composition.
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Affiliation(s)
- Markus Masek
- Institute of Medical Genetics, University of Zurich, Zurich, Switzerland; Department of Molecular Life Sciences, University of Zurich, Zurich, Switzerland
| | - Ruxandra Bachmann-Gagescu
- Institute of Medical Genetics, University of Zurich, Zurich, Switzerland; Department of Molecular Life Sciences, University of Zurich, Zurich, Switzerland; University Research Priority Program AdaBD, University of Zurich, Zurich, Switzerland.
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42
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Luna LW, Williams LM, Duren K, Tyl R, Toews DPL, Avery JD. Whole genome assessment of a declining game bird reveals cryptic genetic structure and insights for population management. Mol Ecol 2023; 32:5498-5513. [PMID: 37688483 DOI: 10.1111/mec.17129] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2023] [Revised: 08/23/2023] [Accepted: 08/30/2023] [Indexed: 09/11/2023]
Abstract
Population genomics applied to game species conservation can help delineate management units, ensure appropriate harvest levels and identify populations needing genetic rescue to safeguard their adaptive potential. The ruffed grouse (Bonasa umbellus) is rapidly declining in much of the eastern USA due to a combination of forest maturation and habitat fragmentation. More recently, mortality from West Nile Virus may have affected connectivity of local populations; however, genetic approaches have never explicitly investigated this issue. In this study, we sequenced 54 individual low-coverage (~5X) grouse genomes to characterize population structure, assess migration rates across the landscape to detect potential barriers to gene flow and identify genomic regions with high differentiation. We identified two genomic clusters with no clear geographic correlation, with large blocks of genomic differentiation associated with chromosomes 4 and 20, likely due to chromosomal inversions. After excluding these putative inversions from the data set, we found weak but nonsignificant signals of population subdivision. Estimated gene flow revealed reduced rates of migration in areas with extensive habitat fragmentation and increased genetic connectivity in areas with less habitat fragmentation. Our findings provide a benchmark for wildlife managers to compare and scale the genetic diversity and structure of ruffed grouse populations in Pennsylvania and across the eastern USA, and we also reveal structural variation in the grouse genome that requires further study to understand its possible effects on individual fitness and population distribution.
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Affiliation(s)
- Leilton W Luna
- Department of Ecosystem Science and Management, Penn State University, University Park, Pennsylvania, USA
| | - Lisa M Williams
- Bureau of Wildlife Management, Pennsylvania Game Commission, Harrisburg, Pennsylvania, USA
| | - Kenneth Duren
- Bureau of Wildlife Management, Pennsylvania Game Commission, Harrisburg, Pennsylvania, USA
| | - Reina Tyl
- Bureau of Wildlife Management, Pennsylvania Game Commission, Harrisburg, Pennsylvania, USA
| | - David P L Toews
- Department of Biology, Penn State University, University Park, Pennsylvania, USA
| | - Julian D Avery
- Department of Ecosystem Science and Management, Penn State University, University Park, Pennsylvania, USA
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Sun Y, Jin Y. An intraflagellar transport dependent negative feedback regulates the MAPKKK DLK-1 to protect cilia from degeneration. Proc Natl Acad Sci U S A 2023; 120:e2302801120. [PMID: 37722038 PMCID: PMC10523469 DOI: 10.1073/pnas.2302801120] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2023] [Accepted: 08/15/2023] [Indexed: 09/20/2023] Open
Abstract
Primary cilia are specialized organelles supporting the development and function of cells and organisms. Intraflagellar transport (IFT) is essential for cilia formation, maintenance, and function. In C. elegans ciliated sensory neurons, IFT interacts with signaling molecules to generate distinct morphological and function features and also to maintain the integrity of cilia. Here, we report an IFT-dependent feedback control on the conserved MAPKKK DLK-1 in the ciliated sensory neurons. DLK proteins are widely known to act in synapse formation, axon regeneration, and degeneration, but their roles in other neuronal compartments are understudied. By forward genetic screening for altered expression of the endogenously tagged DLK-1 we identified multiple ift mutants showing increased DLK-1 accumulation in the defective sensory endings. We show that in response to acute IFT disruption, DLK-1 accumulates rapidly and reversibly. The expression levels of the transcription factor CEBP-1, known to act downstream of DLK-1 in the development and maintenance of synapses and axons, are also increased in the ciliated sensory neurons of ift mutants. Interestingly, the regulation of CEBP-1 expression shows sensory neuron-type dependency on DLK-1. Moreover, in the sensory neuron AWC, which has elaborate cilia morphology, up-regulated CEBP-1 represses DLK-1 at the transcription level, thereby dampening DLK-1 accumulation. Last, the IFT-dependent regulatory loop of DLK-1 and CEBP-1 offers neuroprotection in a cilia degeneration model. These findings uncover a surveillance mechanism in which tight control on the DLK-1 signaling protects cilia integrity in a context-specific manner.
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Affiliation(s)
- Yue Sun
- Department of Neurobiology, School of Biological Sciences, University of California San Diego, La Jolla, CA92093
| | - Yishi Jin
- Department of Neurobiology, School of Biological Sciences, University of California San Diego, La Jolla, CA92093
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Boegholm N, Petriman NA, Loureiro‐López M, Wang J, Vela MIS, Liu B, Kanie T, Ng R, Jackson PK, Andersen JS, Lorentzen E. The IFT81-IFT74 complex acts as an unconventional RabL2 GTPase-activating protein during intraflagellar transport. EMBO J 2023; 42:e111807. [PMID: 37606072 PMCID: PMC10505919 DOI: 10.15252/embj.2022111807] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2022] [Revised: 07/24/2023] [Accepted: 08/03/2023] [Indexed: 08/23/2023] Open
Abstract
Cilia are important cellular organelles for signaling and motility and are constructed via intraflagellar transport (IFT). RabL2 is a small GTPase that localizes to the basal body of cilia via an interaction with the centriolar protein CEP19 before downstream association with the IFT machinery, which is followed by initiation of IFT. We reconstituted and purified RabL2 with CEP19 or IFT proteins to show that a reconstituted pentameric IFT complex containing IFT81/74 enhances the GTP hydrolysis rate of RabL2. The binding site on IFT81/74 that promotes GTP hydrolysis in RabL2 was mapped to a 70-amino-acid-long coiled-coil region of IFT81/74. We present structural models for RabL2-containing IFT complexes that we validate in vitro and in cellulo and demonstrate that Chlamydomonas IFT81/74 enhances GTP hydrolysis of human RabL2, suggesting an ancient evolutionarily conserved activity. Our results provide an architectural understanding of how RabL2 is incorporated into the IFT complex and a molecular rationale for why RabL2 dissociates from anterograde IFT trains soon after departure from the ciliary base.
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Affiliation(s)
- Niels Boegholm
- Department of Molecular Biology and GeneticsAarhus UniversityAarhus CDenmark
| | - Narcis A Petriman
- Department of Molecular Biology and GeneticsAarhus UniversityAarhus CDenmark
| | - Marta Loureiro‐López
- Department for Biochemistry and Molecular BiologyUniversity of Southern DenmarkOdense MDenmark
| | - Jiaolong Wang
- Department of Molecular Biology and GeneticsAarhus UniversityAarhus CDenmark
| | | | - Beibei Liu
- Department of Cell BiologyUniversity of Oklahoma Health Science CenterOklahomaOKUSA
| | - Tomoharu Kanie
- Department of Cell BiologyUniversity of Oklahoma Health Science CenterOklahomaOKUSA
- Baxter Laboratory, Department of Microbiology & ImmunologyStanford University School of MedicineStanfordCAUSA
| | - Roy Ng
- Baxter Laboratory, Department of Microbiology & ImmunologyStanford University School of MedicineStanfordCAUSA
| | - Peter K Jackson
- Baxter Laboratory, Department of Microbiology & ImmunologyStanford University School of MedicineStanfordCAUSA
- Department of PathologyStanford University School of MedicineStanfordCAUSA
| | - Jens S Andersen
- Department for Biochemistry and Molecular BiologyUniversity of Southern DenmarkOdense MDenmark
| | - Esben Lorentzen
- Department of Molecular Biology and GeneticsAarhus UniversityAarhus CDenmark
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45
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Tasaki K, Zhou Z, Ishida Y, Katoh Y, Nakayama K. Compound heterozygous IFT81 variations in a skeletal ciliopathy patient cause Bardet-Biedl syndrome-like ciliary defects. Hum Mol Genet 2023; 32:2887-2900. [PMID: 37427975 DOI: 10.1093/hmg/ddad112] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2023] [Revised: 07/04/2023] [Accepted: 07/04/2023] [Indexed: 07/11/2023] Open
Abstract
Owing to their crucial roles in development and homeostasis, defects in cilia cause ciliopathies with diverse clinical manifestations. The intraflagellar transport (IFT) machinery, containing the IFT-A and IFT-B complexes, mediates not only the intraciliary bidirectional trafficking but also import and export of ciliary proteins together with the kinesin-2 and dynein-2 motor complexes. The BBSome, containing eight subunits encoded by causative genes of Bardet-Biedl syndrome (BBS), connects the IFT machinery to ciliary membrane proteins to mediate their export from cilia. Although mutations in subunits of the IFT-A and dynein-2 complexes cause skeletal ciliopathies, mutations in some IFT-B subunits are also known to cause skeletal ciliopathies. We here show that compound heterozygous variations of an IFT-B subunit, IFT81, found in a patient with skeletal ciliopathy cause defects in its interactions with other IFT-B subunits, and in ciliogenesis and ciliary protein trafficking when one of the two variants was expressed in IFT81-knockout (KO) cells. Notably, we found that IFT81-KO cells expressing IFT81(Δ490-519), which lacks the binding site for the IFT25-IFT27 dimer, causes ciliary defects reminiscent of those found in BBS cells and those in IFT74-KO cells expressing a BBS variant of IFT74, which forms a heterodimer with IFT81. In addition, IFT81-KO cells expressing IFT81(Δ490-519) in combination with the other variant, IFT81 (L645*), which mimics the cellular conditions of the above skeletal ciliopathy patient, demonstrated essentially the same phenotype as those expressing only IFT81(Δ490-519). Thus, our data indicate that BBS-like defects can be caused by skeletal ciliopathy variants of IFT81.
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Affiliation(s)
- Koshi Tasaki
- Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan
| | - Zhuang Zhou
- Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan
| | - Yamato Ishida
- Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan
| | - Yohei Katoh
- Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan
| | - Kazuhisa Nakayama
- Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan
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Albisetti AC, Douglas RL, Welch MD. FAZ assembly in bloodstream form Trypanosoma brucei requires kinesin KIN-E. Mol Biol Cell 2023; 34:ar103. [PMID: 37531263 PMCID: PMC10551704 DOI: 10.1091/mbc.e23-01-0022] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2023] [Revised: 07/18/2023] [Accepted: 07/26/2023] [Indexed: 08/04/2023] Open
Abstract
Trypanosoma brucei, the causative agent of African sleeping sickness, uses its flagellum for movement, cell division, and signaling. The flagellum is anchored to the cell body membrane via the flagellum attachment zone (FAZ), a complex of proteins, filaments, and microtubules that spans two membranes with elements on both flagellum and cell body sides. How FAZ components are carried into place to form this complex is poorly understood. Here, we show that the trypanosome-specific kinesin KIN-E is required for building the FAZ in bloodstream-form parasites. KIN-E is localized along the flagellum with a concentration at its distal tip. Depletion of KIN-E by RNAi rapidly inhibits flagellum attachment and leads to cell death. A detailed analysis reveals that KIN-E depletion phenotypes include failure in cytokinesis completion, kinetoplast DNA missegregation, and transport vesicle accumulation. Together with previously published results in procyclic form parasites, these data suggest KIN-E plays a critical role in FAZ assembly in T. brucei.
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Affiliation(s)
- Anna C. Albisetti
- Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720
| | - Robert L. Douglas
- Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720
| | - Matthew D. Welch
- Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720
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47
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Hiyamizu S, Qiu H, Tsurumi Y, Hamada Y, Katoh Y, Nakayama K. Dynein-2-driven intraciliary retrograde trafficking indirectly requires multiple interactions of IFT54 in the IFT-B complex with the dynein-2 complex. Biol Open 2023; 12:bio059976. [PMID: 37309605 PMCID: PMC10320715 DOI: 10.1242/bio.059976] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2023] [Accepted: 06/05/2023] [Indexed: 06/14/2023] Open
Abstract
Within cilia, the dynein-2 complex needs to be transported as an anterograde cargo to achieve its role as a motor to drive retrograde trafficking of the intraflagellar transport (IFT) machinery containing IFT-A and IFT-B complexes. We previously showed that interactions of WDR60 and the DYNC2H1-DYNC2LI1 dimer of dynein-2 with multiple IFT-B subunits, including IFT54, are required for the trafficking of dynein-2 as an IFT cargo. However, specific deletion of the IFT54-binding site from WDR60 demonstrated only a minor effect on dynein-2 trafficking and function. We here show that the C-terminal coiled-coil region of IFT54, which participates in its interaction with the DYNC2H1-DYNC2LI1 dimer of dynein-2 and with IFT20 of the IFT-B complex, is essential for IFT-B function, and suggest that the IFT54 middle linker region between the N-terminal WDR60-binding region and the C-terminal coiled-coil is required for ciliary retrograde trafficking, probably by mediating the effective binding of IFT-B to the dynein-2 complex, and thereby ensuring dynein-2 loading onto the anterograde IFT trains. The results presented here agree with the notion predicted from the previous structural models that the dynein-2 loading onto the anterograde IFT train relies on intricate, multivalent interactions between the dynein-2 and IFT-B complexes.
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Affiliation(s)
- Shunya Hiyamizu
- Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
| | - Hantian Qiu
- Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
| | - Yuta Tsurumi
- Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
| | - Yuki Hamada
- Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
| | - Yohei Katoh
- Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
| | - Kazuhisa Nakayama
- Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan
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48
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Mill P, Christensen ST, Pedersen LB. Primary cilia as dynamic and diverse signalling hubs in development and disease. Nat Rev Genet 2023; 24:421-441. [PMID: 37072495 PMCID: PMC7615029 DOI: 10.1038/s41576-023-00587-9] [Citation(s) in RCA: 155] [Impact Index Per Article: 77.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/16/2023] [Indexed: 04/20/2023]
Abstract
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.
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Affiliation(s)
- Pleasantine Mill
- MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, Scotland
| | | | - Lotte B Pedersen
- Department of Biology, University of Copenhagen, Copenhagen, Denmark.
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Lee MS, Han HJ, Choi TI, Lee KH, Baasankhuu A, Kim HT, Kim CH. IFT46 gene promoter-driven ciliopathy disease model in zebrafish. Front Cell Dev Biol 2023; 11:1200599. [PMID: 37363725 PMCID: PMC10285392 DOI: 10.3389/fcell.2023.1200599] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2023] [Accepted: 05/26/2023] [Indexed: 06/28/2023] Open
Abstract
Ciliopathies are human genetic disorders caused by abnormal formation and dysfunction of cellular cilia. Cilia are microtubule-based organelles that project into the extracellular space and transduce molecular and chemical signals from the extracellular environment or neighboring cells. Intraflagellar transport (IFT) proteins are required for the assembly and maintenance of cilia by transporting proteins along the axoneme which consists of complexes A and B. IFT46, a core IFT-B protein complex, is required for cilium formation and maintenance during vertebrate embryonic development. Here, we introduce transgenic zebrafish lines under the control of ciliated cell-specific IFT46 promoter to recapitulate human ciliopathy-like phenotypes. We generated a Tg(IFT46:GAL4-VP16) line to temporo-spatially control the expression of effectors including fluorescent reporters or nitroreductase based on the GAL4/UAS system, which expresses GAL4-VP16 chimeric transcription factors in most ciliated tissues during embryonic development. To analyze the function of IFT46-expressing ciliated cells during zebrafish development, we generated the Tg(IFT46:GAL4-VP16;UAS;nfsb-mCherry) line, a ciliated cell-specific injury model induced by nitroreductase (NTR)/metrodinazole (MTZ). Conditionally, controlled ablation of ciliated cells in transgenic animals exhibited ciliopathy-like phenotypes including cystic kidneys and pericardial and periorbital edema. Altogether, we established a zebrafish NTR/MTZ-mediated ciliated cell injury model that recapitulates ciliopathy-like phenotypes and may be a vertebrate animal model to further investigate the etiology and therapeutic approaches to human ciliopathies.
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Affiliation(s)
- Mi-Sun Lee
- Department of Biology, Chungnam National University, Daejeon, Republic of Korea
- Michigan Neuroscience Institute (MNI), University of Michigan, Ann Arbor, MI, United States
| | - Hye-Jeong Han
- Soonchunhyang Institute of Medi-Bio Science (SIMS), Soonchunhyang University, Cheonan-Si, Republic of Korea
- Department of Integrated Biomedical Science, Soonchunhyang University, Cheonan-Si, Republic of Korea
| | - Tae-Ik Choi
- Department of Biology, Chungnam National University, Daejeon, Republic of Korea
| | - Kang-Han Lee
- Department of Biology, Chungnam National University, Daejeon, Republic of Korea
| | - Amartuvshin Baasankhuu
- Soonchunhyang Institute of Medi-Bio Science (SIMS), Soonchunhyang University, Cheonan-Si, Republic of Korea
- Department of Integrated Biomedical Science, Soonchunhyang University, Cheonan-Si, Republic of Korea
| | - Hyun-Taek Kim
- Soonchunhyang Institute of Medi-Bio Science (SIMS), Soonchunhyang University, Cheonan-Si, Republic of Korea
- Department of Integrated Biomedical Science, Soonchunhyang University, Cheonan-Si, Republic of Korea
| | - Cheol-Hee Kim
- Department of Biology, Chungnam National University, Daejeon, Republic of Korea
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Ritter J, Lisec K, Klinner M, Heinrich M, von Schweinitz D, Kappler R, Hubertus J. Genetic Disruption of Cilia-Associated Signaling Pathways in Patients with VACTERL Association. CHILDREN (BASEL, SWITZERLAND) 2023; 10:children10050882. [PMID: 37238430 DOI: 10.3390/children10050882] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/17/2023] [Revised: 05/05/2023] [Accepted: 05/13/2023] [Indexed: 05/28/2023]
Abstract
VACTERL association is a rare malformation complex consisting of vertebral defects, anorectal malformation, cardiovascular defects, tracheoesophageal fistulae with esophageal atresia, renal malformation, and limb anomalies. According to current knowledge, VACTERL is based on a multifactorial pathogenesis including genomic alterations. This study aimed to improve the understanding of the genetic mechanisms in the development of VACTERL by investigating the genetic background with a focus on signaling pathways and cilia function. The study was designed as genetic association study. For this, whole-exome sequencing with subsequent functional enrichment analyses was performed for 21 patients with VACTERL or a VACTERL-like phenotype. In addition, whole-exome sequencing was performed for three pairs of parents and Sanger-sequencing was performed for ten pairs of parents. Analysis of the WES-data revealed genetic alteration in the Shh- and Wnt-signaling pathways. Additional performed functional enrichment analysis identified an overrepresentation of the cilia, including 47 affected ciliary genes with clustering in the DNAH gene family and the IFT-complex. The examination of the parents showed that most of the genetic changes were inherited. In summary, this study indicates three genetically determined damage mechanisms for VACTERL with the potential to influence each other, namely Shh- and Wnt-signaling pathway disruption, structural cilia defects and disruption of the ciliary signal transduction.
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Affiliation(s)
- Jessica Ritter
- Department of Pediatric Surgery, Dr. von Hauner Children's Hospital, LMU Munich University, 80337 Munich, Germany
- Department of Diagnostic and Interventional Radiology, Klinikum Rechts der Isar, Technical University of Munich, Ismaninger Straße 22, 81675 Munich, Germany
| | - Kristina Lisec
- Department of Pediatric Surgery, Dr. von Hauner Children's Hospital, LMU Munich University, 80337 Munich, Germany
| | - Marina Klinner
- Department of Pediatric Surgery, Dr. von Hauner Children's Hospital, LMU Munich University, 80337 Munich, Germany
| | - Martina Heinrich
- Department of Pediatric Surgery, Dr. von Hauner Children's Hospital, LMU Munich University, 80337 Munich, Germany
| | - Dietrich von Schweinitz
- Department of Pediatric Surgery, Dr. von Hauner Children's Hospital, LMU Munich University, 80337 Munich, Germany
| | - Roland Kappler
- Department of Pediatric Surgery, Dr. von Hauner Children's Hospital, LMU Munich University, 80337 Munich, Germany
| | - Jochen Hubertus
- Department of Pediatric Surgery, Dr. von Hauner Children's Hospital, LMU Munich University, 80337 Munich, Germany
- Department of Pediatric Surgery, Marien Hospital Witten, Ruhr-University Bochum, 58452 Witten, Germany
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