Bilateral BBSRC-SFI: Structure-function relationships in the ciliary transition zone
Lead Research Organisation:
University of Leeds
Department Name: School of Medicine
Abstract
Cilia are small 'antennae-like' structures which protrude from the surface of most animal cells. Like antennae, they receive and transduce signals from other cells and their surroundings in order to coordinate appropriate cell behaviours. This is especially important in development, and defects in cilia lead to a range of human developmental diseases called "ciliopathies". These conditions range from severe, lethal conditions that involve complex defects in multiple organs including the brain, to relatively mild and organ-specific conditions such as retinitis pigmentosa, which is a form of hereditary, progressive sight loss. Scientists still do not fully understand how cilia help to control brain and retina development. Although these conditions are individually rare, collectively they are a common cause of morbidity and mortality in babies and young children but remain difficult to diagnose and treat.
This research proposes to identify genes that contribute individually, or within groups (pathways), to the formation of a sub-structure of the cilium called the transition zone. The transition zone at the base of the cilium is thought to function as a type of 'gate', controlling which signal transduction molecules are allowed to enter and exit the cilium. Many of the genes that cause ciliopathies are thought to function in the transition zone and regulate its 'gating' function. To achieve our aims, we will take advantage of recent exciting advances in genetic technology that allow us to evaluate the contribution of every gene to cilia and transition zone formation ("reverse genetics screen"). We are uniquely placed to do this work and the team of investigators have a proven track record of success in this field: we have formed excellent research partnerships with other workers in the field to participate in gene identification studies; we have the appropriate state-of-the-art technology, image analysis tools and experience; and we have already produced and validated large data-sets from existing work that we now wish to exploit more extensively in the present research proposal. We will study key genes ("screen hits") and their contributions to cilia and transition zone formation, and, in particular we will use specialised cell model systems in combination with a versatile animal model (a nematode roundworm).
The identification and characterisation of new genes required for the structure and function of the cilium and the transition zone 'gate' provides a number of major benefits. Firstly, important and often unexpected scientific insights are made into disease processes and into the normal function of the disease gene that can lead to new treatments. Secondly, new ciliary genes often enable accurate genetic testing for patients and families with ciliopathies, which improve diagnosis and genetic counselling. Thirdly, our proposed work has a wider biological relevance because cilia have recently been shown to control metabolism, which may link to neurodegenerative diseases and metabolic disorders such as insulin resistance. Thus, a better understanding of cilium biology may provide opportunities for developing drugs or new treatments to prevent the progression of more common ailments (e.g., diabetes. obesity) that present in some ciliopathy patients. Finally, we expect that our work will provide new insights into how cilia disease proteins are arranged relative to one another within the transition zone, and how this molecular organisation facilitates the gating function of this discrete region of the cilium. This new knowledge will have important implications for molecular 'gates' that exist elsewhere in the cell, and serve to expand the impact of our work to scientists working on related questions.
This research proposes to identify genes that contribute individually, or within groups (pathways), to the formation of a sub-structure of the cilium called the transition zone. The transition zone at the base of the cilium is thought to function as a type of 'gate', controlling which signal transduction molecules are allowed to enter and exit the cilium. Many of the genes that cause ciliopathies are thought to function in the transition zone and regulate its 'gating' function. To achieve our aims, we will take advantage of recent exciting advances in genetic technology that allow us to evaluate the contribution of every gene to cilia and transition zone formation ("reverse genetics screen"). We are uniquely placed to do this work and the team of investigators have a proven track record of success in this field: we have formed excellent research partnerships with other workers in the field to participate in gene identification studies; we have the appropriate state-of-the-art technology, image analysis tools and experience; and we have already produced and validated large data-sets from existing work that we now wish to exploit more extensively in the present research proposal. We will study key genes ("screen hits") and their contributions to cilia and transition zone formation, and, in particular we will use specialised cell model systems in combination with a versatile animal model (a nematode roundworm).
The identification and characterisation of new genes required for the structure and function of the cilium and the transition zone 'gate' provides a number of major benefits. Firstly, important and often unexpected scientific insights are made into disease processes and into the normal function of the disease gene that can lead to new treatments. Secondly, new ciliary genes often enable accurate genetic testing for patients and families with ciliopathies, which improve diagnosis and genetic counselling. Thirdly, our proposed work has a wider biological relevance because cilia have recently been shown to control metabolism, which may link to neurodegenerative diseases and metabolic disorders such as insulin resistance. Thus, a better understanding of cilium biology may provide opportunities for developing drugs or new treatments to prevent the progression of more common ailments (e.g., diabetes. obesity) that present in some ciliopathy patients. Finally, we expect that our work will provide new insights into how cilia disease proteins are arranged relative to one another within the transition zone, and how this molecular organisation facilitates the gating function of this discrete region of the cilium. This new knowledge will have important implications for molecular 'gates' that exist elsewhere in the cell, and serve to expand the impact of our work to scientists working on related questions.
Technical Summary
This research programme builds on the applicants' combined expertise in reverse genetics screening and in vivo modelling of transition zone (TZ) and ciliary function in cell and C. elegans animal models. The proposal impetus, and an example of our collaboration, was our 2015 study on TMEM107. Using high-throughput siRNA technology in mammalian cells (Workpackage 1; WP1) and C. elegans mutant alleles (WP2), we will identify novel candidate TZ genes. The resulting functional genomics data-sets will be integrated and common 'hits' validated and functionally interrogated in both systems (including knockout cell lines) to define new TZ proteins (WP3). Specifically, we will dissect their roles in cilium-based signalling, transport, composition, 'gating', and relationship to known or novel functional modules. In WP4, both models will integrate new and known TZ proteins into the emerging architectural framework of the ciliary gate, and provide new insights into structure-function relationships within the ciliary apparatus such as the TZ permeability barrier. These experiments will involve advanced confocal imaging to visualise protein-protein interactions in living cells (FRET; BioID; BiDC) and worms (BiFC), and complementary super-resolution microscopy approaches (STED, dSTORM), combined with multiplexed imaging using artificial binding proteins ("Adhirons"), to enable unprecedented spatial and temporal resolution of TZ proteins. We provide preliminary data on exemplar projects to show the exciting discoveries that rapidly arise from functional genomics to guide hypothesis generation. In vitro high content imaging and in vivo genetic complementation assays is a cost and time effective approach to complex combinatorial and multiplexed experimentation, compared to more conventional approaches. The research will significantly improve the functional annotation of genes contributing to TZ formation and ciliogenesis, and will enhance our understanding of basic cilium biology.
Planned Impact
Although ciliopathies are a heterogenous group of rare inherited conditions, collectively they are a common cause of perinatal morbidity and mortality (1 in 500 births) and remain difficult to diagnose and treat. In the UK, about 4000 patients require renal replacement therapy (dialysis and transplantation) as a result of cystic kidney disease leading to kidney failure. The identification of new disease gene functions and pathways related to the ciliary transition zone (TZ) may enable the future rational design of preventative treatments or new therapeutic interventions for cystic kidney disease, retinal degeneration or ciliopathy disease progression, or approaches to improve the long-term outlook of patients with these conditions. For example, by understanding how the ciliary TZ and associated ciliopathy proteins regulate signalling via diffusion barriers, our work could lead to the design of small molecules that can advantageously increase or decrease permeability at the ciliary base, and thereby alleviate some of the progressive post-natal ciliopathy pathologies. Also, since these conditions result from absence of normal TZ proteins, they could in principle be corrected by gene replacement, therapeutic approaches now undergoing Phase III clinical trials.
It is likely that new TZ genes that we discover in this project will subsequently link to ciliary disease. Patients in whom new mutations are found can therefore be given a clearer prognosis and prioritised for any emerging new treatments, making inherited disease a top priority for further characterization. Recent advances have improved the molecular diagnosis of children with a known or suspected ciliopathy, and reduced the burden and disruption of a "diagnostic odyssey" for families. For clinicians, new gene identification can also reduce misdiagnosis or late diagnosis, inform the establishment of proper care pathways for ciliopathy patients, and prioritize patients that can most benefit from future targeted therapies. In families with a ciliopathy, the identification of a novel disease gene immediately permits genetic testing to at-risk relatives. Carrier tests and prenatal diagnosis can also encourage informed reproductive choices for families.
Recently, cilia have been shown to be essential for normal autophagic processes and have a broader role in metabolic control through mTOR signalling. This has wider medical relevance because autophagy is implicated in many human pathological processes, including neurodegenerative diseases and human metabolic disorders. The understanding of these pathophysiological processes poses some of the most significant challenges to modern biomedical research, and the regulatory relationships that control cilia function could therefore provide a new appreciation of their potential medical relevance in older age. The identification of new ciliary genes and pathways provides insights into normal human physiology and development, as well as the pathophysiology of common conditions (such as such as insulin resistance, obesity and cystic kidney disease) thus benefiting the wider population.
To understand ciliary TZ structure/function regulation, we will use functional genomics strategies to evaluate the contribution of large data-sets of genes. This is a proven strategy for identifying new and unexpected disease genes, mechanisms and pathways. We will integrate functional genomics with advanced cell biology methodologies in complementary physiologically-relevant cell and animal mutant models to derive cutting edge knowledge of the TZ system. Our work also aims to demonstrate the clinical utility and validity of integrated "systems medicine" annotation and its ability to make relevant predictions about ciliopathy disease causality. The development of a strong interface between gene discovery, functional annotation and diagnostic or translational development work is therefore an indirect but important added benefit of this research.
It is likely that new TZ genes that we discover in this project will subsequently link to ciliary disease. Patients in whom new mutations are found can therefore be given a clearer prognosis and prioritised for any emerging new treatments, making inherited disease a top priority for further characterization. Recent advances have improved the molecular diagnosis of children with a known or suspected ciliopathy, and reduced the burden and disruption of a "diagnostic odyssey" for families. For clinicians, new gene identification can also reduce misdiagnosis or late diagnosis, inform the establishment of proper care pathways for ciliopathy patients, and prioritize patients that can most benefit from future targeted therapies. In families with a ciliopathy, the identification of a novel disease gene immediately permits genetic testing to at-risk relatives. Carrier tests and prenatal diagnosis can also encourage informed reproductive choices for families.
Recently, cilia have been shown to be essential for normal autophagic processes and have a broader role in metabolic control through mTOR signalling. This has wider medical relevance because autophagy is implicated in many human pathological processes, including neurodegenerative diseases and human metabolic disorders. The understanding of these pathophysiological processes poses some of the most significant challenges to modern biomedical research, and the regulatory relationships that control cilia function could therefore provide a new appreciation of their potential medical relevance in older age. The identification of new ciliary genes and pathways provides insights into normal human physiology and development, as well as the pathophysiology of common conditions (such as such as insulin resistance, obesity and cystic kidney disease) thus benefiting the wider population.
To understand ciliary TZ structure/function regulation, we will use functional genomics strategies to evaluate the contribution of large data-sets of genes. This is a proven strategy for identifying new and unexpected disease genes, mechanisms and pathways. We will integrate functional genomics with advanced cell biology methodologies in complementary physiologically-relevant cell and animal mutant models to derive cutting edge knowledge of the TZ system. Our work also aims to demonstrate the clinical utility and validity of integrated "systems medicine" annotation and its ability to make relevant predictions about ciliopathy disease causality. The development of a strong interface between gene discovery, functional annotation and diagnostic or translational development work is therefore an indirect but important added benefit of this research.
Organisations
- University of Leeds (Lead Research Organisation)
- Eberhard Karls University of Tübingen (Collaboration)
- Radboud University Nijmegen Medical Center (Collaboration)
- University of Sheffield (Collaboration)
- University College Dublin (Collaboration)
- Newcastle University (Collaboration)
- University of California, San Diego (UCSD) (Collaboration)
- UNIVERSITY OF LEEDS (Collaboration)
- Johannes Gutenberg University of Mainz (Collaboration)
Publications
Abdelhamed ZA
(2019)
The ciliary Frizzled-like receptor Tmem67 regulates canonical Wnt/ß-catenin signalling in the developing cerebellum via Hoxb5.
in Scientific reports
Basu B
(2024)
Racgap1 knockdown results in cells with multiple cilia due to cytokinesis failure.
in Annals of human genetics
Buskin A
(2018)
Disrupted alternative splicing for genes implicated in splicing and ciliogenesis causes PRPF31 retinitis pigmentosa.
in Nature communications
Hartill VL
(2018)
DNAAF1 links heart laterality with the AAA+ ATPase RUVBL1 and ciliary intraflagellar transport.
in Human molecular genetics
Johnson C
(2019)
Obituary: Jarema Malicki (1965-2019)
in Development
Johnson CA
(2019)
The Nuclear Arsenal of Cilia.
in Developmental cell
Lange KI
(2022)
Interpreting ciliopathy-associated missense variants of uncertain significance (VUS) in Caenorhabditis elegans.
in Human molecular genetics
Description | Our current research has uncovered an unexpected series of genetics interactions between key proteins in the primary cilium, the sensory "antenae" of the cell. The ciliary proteins include an transmembrane receptor (TMEM67), a fundamental organizing protein of ciliary architecture (RPGRIP1L) and a protein that mediates trafficking of components within the cilium (IFT88). The genetic interactions were identified using "crispant" cells, carrying mutations engineered by CRISPR-Cas9 genome editing, in combination with high content imaging (a type of automated microscopy imaging). More recently, the crispant cells have been used to validate the effect of a repurposed drug on cilia formation. We identified this drug from a screen of known drugs and clinical development compounds using high content imaging. |
Exploitation Route | Our characterized crispant cell-lines will be useful to other researchers in the cilia biology field for similar high content experiments including cell-based drug screening. The repurposed drug forms the basis of new pre-clinical studies in stem cell-derived tissues (renal organoids) which are currently submitted as grant applications to LifeArc and Action Medical Research. |
Sectors | Healthcare Pharmaceuticals and Medical Biotechnology |
Description | development and characterization of of novel non-antibody based protein binding reagents ("Affimers") against ciliary proteins (post-translationally modified tubulin, CEP290); these reagents have potential as detection reagents in (for example in super-resolution microscopy) and for modulation of ciliary processes; identification of a potential repurposed drug that could be used in pre-clinical studies, and that could form the basis of more extensive "hit derivation" work. (There are several hundred derivatives available as clinical-development compounds). The identification of a new repurposed medical or new derivative compounds with defined mechanisms of action are very likely to result in patentable or commercially exploitable outcomes. |
First Year Of Impact | 2017 |
Sector | Pharmaceuticals and Medical Biotechnology |
Impact Types | Economic |
Description | Deciphering the function of intrinsically disordered protein regions in a cellular context |
Amount | £4,267,285 (GBP) |
Funding ID | BB/V003577/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 01/2021 |
End | 10/2023 |
Description | Elucidating splicing factor function and retinal splicing programmes: developing new therapeutic strategies for splicing factor retinitis pigmentosa |
Amount | £1,456,000 (GBP) |
Funding ID | MR/T017503/1 |
Organisation | Medical Research Council (MRC) |
Sector | Public |
Country | United Kingdom |
Start | 01/2020 |
End | 12/2024 |
Description | Pre-clinical testing of ROCK2 inhibition as a new therapeutic treatment for cystic kidney diseases |
Amount | £249,459 (GBP) |
Funding ID | PKD_RP_006_20211124 |
Organisation | Kidney Research UK |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 05/2022 |
End | 05/2025 |
Description | Pre-clinical testing of new therapeutic treatments for cystic kidney disease |
Amount | £200,000 (GBP) |
Funding ID | GN2628 |
Organisation | University of Leeds |
Sector | Academic/University |
Country | United Kingdom |
Start | 06/2018 |
End | 06/2021 |
Description | Wellcome Trust multi-user equipment grant |
Amount | £348,000 (GBP) |
Funding ID | 221542/Z/20/Z |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 09/2020 |
End | 09/2025 |
Description | multi-user equipment grant |
Amount | £429,000 (GBP) |
Funding ID | 208395/Z/17/Z |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 08/2017 |
End | 08/2022 |
Description | project grant |
Amount | £200,000 (GBP) |
Funding ID | GN2628 |
Organisation | Action Medical Research |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 03/2018 |
End | 02/2021 |
Title | Affimers against ciliary proteins |
Description | Affimer artificial non-antibody affinity and binding reagents against ciliary proteins for pull-down adn localization studies |
Type Of Material | Technology assay or reagent |
Year Produced | 2018 |
Provided To Others? | Yes |
Impact | development of better localization precision reagents in supper resolution microscopy |
Title | genome-edited cell-lines |
Description | a panel of isogenic knock-out cell-lines for key ciliary genes (validated by sequencing, western blotting and assays of ciliary function) to be used in reverse genetics screening, drug screening and as model systems of ciliopathies. New crispant cell-lines include null & missense alleles in CEP290 that cause frame-shifting or exon skipping alternative splicing events |
Type Of Material | Cell line |
Year Produced | 2017 |
Provided To Others? | Yes |
Impact | The CEP290 knock-out cell-line is being used as a model system for renal cystic disease in preclinical studies that have arisen from drug screens of clinical development compounds; it provided preliminray data for a newly-funded Kidney Research UK research project (March 2023) |
Title | Reverse genetics siRNA-based visual phenotype screen datasets identifying genes involved in supernumerary primary cilium formation |
Description | High content siRNA screen carried out to identify genes which when knocked-down, cause the formation of multiple primary cilia in the mIMCD3 cell-line. The dataset includes data from both the primary and secondary screens as separate files. |
Type Of Material | Database/Collection of data |
Year Produced | 2021 |
Provided To Others? | Yes |
Impact | resource for research use |
URL | https://archive.researchdata.leeds.ac.uk/898/ |
Description | CILIAREN |
Organisation | Newcastle University |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Validated and characterised antisense oligonucleotides in CEP290-related disorders and established a GEMM-generated mouse model of a CEP290 nonsense mutation for further testing in Newcastle; we provide oligo design, data based on testing in iPSC-based kidney organoids and other reagents. |
Collaborator Contribution | Preclinical assessment of clinical efficacy of any AON-based therapies in the Cep290 mouse model. |
Impact | none formally published |
Start Year | 2024 |
Description | Polycystic Kidney Disease collaborative network in Yorkshire |
Organisation | University of Leeds |
Department | Astbury Centre for Structural Molecular Biology |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Generation of iPSC crispant lines for use as disease models of PKD with a view to understanding disease mechanism and in developing preclinical studies |
Collaborator Contribution | Access to structural biology modelling of PKD proteins, specifically fibrocystin, and effect of misssense mutations; access to clinical efficacy testing of candidate repurposed drugs using in vivo mouse models of PKD |
Impact | initial kick-off research meeting for the network (the "Yorkshire PKD Club") and initial PPIE activities which will be further developed throughout 2023 |
Start Year | 2022 |
Description | Polycystic Kidney Disease collaborative network in Yorkshire |
Organisation | University of Sheffield |
Department | Department of Biomedical Science |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Generation of iPSC crispant lines for use as disease models of PKD with a view to understanding disease mechanism and in developing preclinical studies |
Collaborator Contribution | Access to structural biology modelling of PKD proteins, specifically fibrocystin, and effect of misssense mutations; access to clinical efficacy testing of candidate repurposed drugs using in vivo mouse models of PKD |
Impact | initial kick-off research meeting for the network (the "Yorkshire PKD Club") and initial PPIE activities which will be further developed throughout 2023 |
Start Year | 2022 |
Description | functional characterization of ciliopathy proteins |
Organisation | Eberhard Karls University of Tübingen |
Department | Institute for Ophthalmic Research |
Country | Germany |
Sector | Academic/University |
PI Contribution | completed a reverse genetics screen to identify novel mediators of ciliogenesis and cilia maintenance |
Collaborator Contribution | partners contribution protein-protein interaction data from TAP-tagging experiments, key reagents (expression constructs) and antibodies, tissue samples or sections from ciliopthy animal models |
Impact | Wheway G, (2013) Aberrant Wnt signalling and cellular over-proliferation in a novel mouse model of Meckel-Gruber syndrome. Dev. Biol. 377:55-66 Abdelhamed Z et al. (2013). Variable expressivity of ciliopathy neurological phenotypes that encompass Meckel-Gruber syndrome and Joubert syndrome is caused by complex de-regulated ciliogenesis, Shh and Wnt signalling defects. Hum. Mol. Genet. 22: 1358-72 Chaki M, et al. (2012). Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to DNA damage response signaling Cell 150: 533-548 Elmehdawi F, et al. (2013) Human Homologue of Drosophila Ariadne (HHARI) is a marker of cellular proliferation associated with nuclear bodies. Exp. Cell Res. 319:161-172. |
Start Year | 2011 |
Description | functional characterization of ciliopathy proteins |
Organisation | Johannes Gutenberg University of Mainz |
Department | Department of Cell & Matrix Biology |
Country | Germany |
Sector | Academic/University |
PI Contribution | completed a reverse genetics screen to identify novel mediators of ciliogenesis and cilia maintenance |
Collaborator Contribution | partners contribution protein-protein interaction data from TAP-tagging experiments, key reagents (expression constructs) and antibodies, tissue samples or sections from ciliopthy animal models |
Impact | Wheway G, (2013) Aberrant Wnt signalling and cellular over-proliferation in a novel mouse model of Meckel-Gruber syndrome. Dev. Biol. 377:55-66 Abdelhamed Z et al. (2013). Variable expressivity of ciliopathy neurological phenotypes that encompass Meckel-Gruber syndrome and Joubert syndrome is caused by complex de-regulated ciliogenesis, Shh and Wnt signalling defects. Hum. Mol. Genet. 22: 1358-72 Chaki M, et al. (2012). Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to DNA damage response signaling Cell 150: 533-548 Elmehdawi F, et al. (2013) Human Homologue of Drosophila Ariadne (HHARI) is a marker of cellular proliferation associated with nuclear bodies. Exp. Cell Res. 319:161-172. |
Start Year | 2011 |
Description | functional characterization of ciliopathy proteins |
Organisation | Radboud University Nijmegen Medical Center |
Country | Netherlands |
Sector | Academic/University |
PI Contribution | completed a reverse genetics screen to identify novel mediators of ciliogenesis and cilia maintenance |
Collaborator Contribution | partners contribution protein-protein interaction data from TAP-tagging experiments, key reagents (expression constructs) and antibodies, tissue samples or sections from ciliopthy animal models |
Impact | Wheway G, (2013) Aberrant Wnt signalling and cellular over-proliferation in a novel mouse model of Meckel-Gruber syndrome. Dev. Biol. 377:55-66 Abdelhamed Z et al. (2013). Variable expressivity of ciliopathy neurological phenotypes that encompass Meckel-Gruber syndrome and Joubert syndrome is caused by complex de-regulated ciliogenesis, Shh and Wnt signalling defects. Hum. Mol. Genet. 22: 1358-72 Chaki M, et al. (2012). Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to DNA damage response signaling Cell 150: 533-548 Elmehdawi F, et al. (2013) Human Homologue of Drosophila Ariadne (HHARI) is a marker of cellular proliferation associated with nuclear bodies. Exp. Cell Res. 319:161-172. |
Start Year | 2011 |
Description | functional characterization of ciliopathy proteins |
Organisation | University of California, San Diego (UCSD) |
Department | Institute for Genomic Medicine |
Country | United States |
Sector | Academic/University |
PI Contribution | completed a reverse genetics screen to identify novel mediators of ciliogenesis and cilia maintenance |
Collaborator Contribution | partners contribution protein-protein interaction data from TAP-tagging experiments, key reagents (expression constructs) and antibodies, tissue samples or sections from ciliopthy animal models |
Impact | Wheway G, (2013) Aberrant Wnt signalling and cellular over-proliferation in a novel mouse model of Meckel-Gruber syndrome. Dev. Biol. 377:55-66 Abdelhamed Z et al. (2013). Variable expressivity of ciliopathy neurological phenotypes that encompass Meckel-Gruber syndrome and Joubert syndrome is caused by complex de-regulated ciliogenesis, Shh and Wnt signalling defects. Hum. Mol. Genet. 22: 1358-72 Chaki M, et al. (2012). Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to DNA damage response signaling Cell 150: 533-548 Elmehdawi F, et al. (2013) Human Homologue of Drosophila Ariadne (HHARI) is a marker of cellular proliferation associated with nuclear bodies. Exp. Cell Res. 319:161-172. |
Start Year | 2011 |
Description | structural biology of ciliary proteins |
Organisation | University of Leeds |
Department | Astbury Centre for Structural Molecular Biology |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | protein purification of ciliary protein domains, production and charaterization of "Affimer" artifical binding proteins and their use as novel detection reagents in super resolution microscopy; optimization of cellular soft X-ray tomography of primary cilia using the SXT B24 beamline at Diamond optimization of methodologies for cryoCLEM, cryoEM and CryoFIB AlphaFold2 modelling of ciliary proteins & relevant missense mutations in TMEM67, fibrocystin and CEP290 |
Collaborator Contribution | protein production for Affimer generation and crysallography trials; optimization of novel analysis methods for super resolution microscopy (two colour dSTORM, STED); live cell imaging |
Impact | collation of preliminary data into a larger UKRI grant application and other charity grants (eg Kidney Research UK project grants) |
Start Year | 2018 |
Description | super-resolution microscopy of ciliary proteins |
Organisation | University College Dublin |
Department | UCD Conway Institute of Biomedical annd Biomolecular Research |
Country | Ireland |
Sector | Academic/University |
PI Contribution | We have developed a panel of antibodies specific for ciliary proteins that have been used for both PALM and STORM imaging. We are developing non-antibody binding proteins (Adhirons) for this application. |
Collaborator Contribution | Provided the facilities and technical expertise for both STORM and PALM image capture, processing and analysis. |
Impact | The collaboration has led to the publication: Lambacher NJ, et al. (2015). TMEM107 recruits ciliopathy proteins to anchored periodic subdomains of the ciliary transition zone membrane and is mutated in Joubert syndrome. Nat. Cell Biol. doi: 10.1038/ncb3273 New funding application have been made to the MRC and BBSRC/SFI joint funding stream |
Start Year | 2015 |
Description | super-resolution microscopy of ciliary proteins |
Organisation | University of Leeds |
Department | Faculty of Biological Sciences |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We have developed a panel of antibodies specific for ciliary proteins that have been used for both PALM and STORM imaging. We are developing non-antibody binding proteins (Adhirons) for this application. |
Collaborator Contribution | Provided the facilities and technical expertise for both STORM and PALM image capture, processing and analysis. |
Impact | The collaboration has led to the publication: Lambacher NJ, et al. (2015). TMEM107 recruits ciliopathy proteins to anchored periodic subdomains of the ciliary transition zone membrane and is mutated in Joubert syndrome. Nat. Cell Biol. doi: 10.1038/ncb3273 New funding application have been made to the MRC and BBSRC/SFI joint funding stream |
Start Year | 2015 |
Title | Pre-clinical testing of ROCK2 inhibition as a new therapeutic treatments for cystic kidney disease |
Description | Cilia are small structures that protrude from the surface of most animal cells like antennae. Like antennae, they receive signals from other cells and their surroundings to ensure that cells form tissues correctly. This is important in development, and defects in cilia cause inherited developmental disorders called "ciliopathies". These invariably have cystic kidney disease as a feature, and are a major cause of childhood morbidity and mortality, with about 5000 patients in the UK needing either |
Type | Therapeutic Intervention - Drug |
Current Stage Of Development | Initial development |
Year Development Stage Completed | 2019 |
Development Status | Actively seeking support |
Impact | Initial studies have leveraged further internal funding for development work, and we are activity seeking external funding for pre-clinical studies in disease models. Funding bodies include LifeArc, Action Medical Research, and Harrington UK Rare Disease scholarship. |
Description | BioJapan2017 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | presented current methodologies used for functional interpretation of mutations in rare Mendelian conditions using CRISPR-Cas9 genome and base editing; the presentation was to a large mixed audience of industry, big pharma and basic reseachers followed by a debate on ethic issues |
Year(s) Of Engagement Activity | 2017 |
Description | Cafe Scientifique |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Patients, carers and/or patient groups |
Results and Impact | About 50 attendees to the Cafe Scientifique meeting in Feb 2020; I presented on recent advances in the diagnosis and treatment of inherited blindness, and was able to answer questions and speak to patients and supporters after the formal presentation. |
Year(s) Of Engagement Activity | 2020 |
Description | FASEB "Polycystic Kidney Disease" meeting |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | to facillitate and establish research collabirations, with critique of current research |
Year(s) Of Engagement Activity | 2017 |
Description | Johnson Lab Twitter feed |
Form Of Engagement Activity | Engagement focused website, blog or social media channel |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Twitter feed for the lab group which reports on our professional activities and events |
Year(s) Of Engagement Activity | 2020 |
URL | https://twitter.com/johnsoncilialab |
Description | PEGS Boston 2018 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | presented current methodologies used for functional interpretation of mutations in rare Mendelian conditions using CRISPR-Cas9 genome and base editing; the presentation was to a large mixed audience of industry, big pharma and basic reseachers followed by a debate on ethic issues |
Year(s) Of Engagement Activity | 2018 |
Description | UK Cilia Club |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Professional Practitioners |
Results and Impact | informal research club for reseachers in the north of England and Scotland; first meeting was in Sheffield Sept 2014, with on-going meetings planned for every 6 months in Leeds, Newcastle adn Edinburgh new collaboration with University of Newcastle |
Year(s) Of Engagement Activity | 2014,2015,2016,2017,2018 |
Description | school out-reach activities |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | discussed careers in medical research with about 60 Year 12 and 13 students on a visit and presentation at their school; 10 had a follow-on visit to the lab to discuss research methods |
Year(s) Of Engagement Activity | 2015,2016,2017,2018 |