{"gene":"CROCC","run_date":"2026-06-09T22:57:19","timeline":{"discoveries":[{"year":2002,"finding":"Rootletin is the major structural component of the ciliary rootlet; it forms detergent-insoluble filaments via its coiled-coil tail domain, assembles into parallel in-register homodimers and higher-order polymers through the tail domain alone, and its globular head domain is required for targeting to the basal body and binding to kinesin light chain. In retinal photoreceptors, rootlets anchor ER membranes along their length.","method":"Recombinant protein expression, detergent-insolubility assay, domain deletion analysis, immunoelectron microscopy, monoclonal antibody epitope mapping","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — reconstitution of filaments from recombinant protein, domain mutagenesis, structural imaging, replicated by subsequent studies","pmids":["12427867"],"is_preprint":false},{"year":2005,"finding":"Rootletin forms striking fibers emanating from the proximal ends of centrioles (shown by immunoelectron microscopy), interacts with C-Nap1, is phosphorylated by Nek2 kinase, and is displaced from centrosomes at mitosis onset. siRNA-mediated depletion of rootletin causes centrosome splitting, establishing its role in centrosome cohesion.","method":"Immunoelectron microscopy, Co-immunoprecipitation (C-Nap1 interaction), in vitro kinase assay (Nek2 phosphorylation), siRNA knockdown with centrosome splitting readout, overexpression fiber formation assay","journal":"The Journal of cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, kinase assay, immunoEM, siRNA phenotype), replicated across labs","pmids":["16203858"],"is_preprint":false},{"year":2005,"finding":"Rootletin interacts physically with C-Nap1 in vivo; they colocalize at basal bodies/centrioles and show coordinated cell-cycle-dependent centriole association. Rootletin fibers connect basal bodies in ciliated cells and span between centrioles in nonciliated cells (unlike C-Nap1 which is restricted to centriole ends). Expression of C-Nap1 fragments dissociates rootletin fibers from centrioles, causing centrosome separation in interphase.","method":"Co-immunoprecipitation, immunofluorescence co-localization, ultrastructural analysis, dominant-negative C-Nap1 fragment expression, overexpression phenotyping","journal":"Molecular biology of the cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal functional and localization evidence, dominant-negative dissection, consistent with independent study (PMID:16203858)","pmids":["16339073"],"is_preprint":false},{"year":2013,"finding":"C. elegans CHE-10/rootletin ortholog maintains ciliary integrity by modulating assembly, motility, and flux of IFT particles (axoneme length control) and by stabilizing ciliary transition zones and basal bodies. Loss of CHE-10/rootletin disrupts periciliary membrane organization, impairing delivery of basal body-associated and ciliary components and causing cilium degeneration.","method":"Genetic mutant analysis (che-10), in vivo IFT particle tracking, fluorescence imaging of transition zones and basal bodies, epistasis with IFT mutants","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Moderate — loss-of-function in intact organism with multiple orthogonal cellular phenotype readouts and IFT flux measurements","pmids":["24094853"],"is_preprint":false},{"year":2015,"finding":"Drosophila Rootletin assembles into rootlets of diverse lengths in sensory neurons, is required for basal body cohesion and sensory function (mechanosensation and chemosensation), and requires centrioles for normal rootlet assembly. The N-terminal conserved domain of Root is essential for its function in vivo. Rootletin localizes asymmetrically to mother centrosomes in neuroblasts and to mother centrioles in spermatocytes, both requiring Bld10.","method":"Drosophila genetic knockout, behavioral assays, immunofluorescence, domain deletion rescue experiments, Bld10 epistasis","journal":"The Journal of cell biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic loss-of-function with defined phenotypic readouts, domain mapping; single organism model","pmids":["26483560"],"is_preprint":false},{"year":2015,"finding":"Drosophila Rootletin knockdown results in loss of ciliary rootlet in chordotonal neurons and severe disruption of mechanosensory function, while cilium structure and protein localization appear largely normal, suggesting rootletin's role is in anchoring/mechanotransduction rather than ciliogenesis per se.","method":"RNAi knockdown in Drosophila, electron microscopy of cilia, electrophysiological/behavioral mechanosensory assays","journal":"Cilia","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with structural and functional readouts, single model organism","pmids":["26140210"],"is_preprint":false},{"year":2012,"finding":"siRNA depletion of rootletin or C-NAP1 increases radiation-induced centriole splitting, and rootletin or C-NAP1 knockdown reduces primary cilium formation, establishing that the centriole cohesion apparatus at the proximal end of centrioles facilitates ciliogenesis.","method":"siRNA knockdown, gamma irradiation, immunofluorescence scoring of centriole splitting and primary cilia formation","journal":"Cell cycle (Georgetown, Tex.)","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — loss-of-function with two defined phenotypic readouts, single lab","pmids":["23070519"],"is_preprint":false},{"year":2017,"finding":"Rootletin prevents VHL E3 ligase-mediated proteasomal degradation of Cep68: in the absence of rootletin, VHL ubiquitinates Cep68 both in vitro and in vivo, leading to Cep68 loss and centrosome splitting. Co-silencing of rootletin and VHL rescues Cep68 levels and centrosome cohesion. A Cep68 mutant that cannot bind the VHL β-domain also suppresses centrosome splitting caused by rootletin depletion.","method":"In vitro ubiquitination assay, siRNA knockdown of rootletin and VHL (single and double), Cep68 ubiquitination-resistant mutants, immunofluorescence centrosome cohesion assay","journal":"Biochimica et biophysica acta. Molecular cell research","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro ubiquitination reconstitution plus in vivo genetic epistasis (double knockdown rescue) and mutagenesis","pmids":["28089774"],"is_preprint":false},{"year":2018,"finding":"STED nanoscopy revealed that C-Nap1 forms a ring at each centriole's proximal end that organizes a rootletin ring and multiple rootletin/CEP68 fibers. Rootletin/CEP68 fibers from the two centrosomes form an interdigitating network. Rootletin molecules within filaments are staggered N-to-N and C-to-C at 75-nm intervals; rootletin binds CEP68 via its C-terminal spectrin repeat-containing region at 75-nm intervals; estimated N-to-C rootletin length is ~35–40 nm, minimal rootletin length ~110 nm. CEP68 is required to form rootletin filaments branching from centrioles and modulates fiber thickness.","method":"STED super-resolution microscopy, protein interaction mapping (binding domain identification), siRNA depletion of CEP68 with filament phenotype readout","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Strong — super-resolution structural imaging plus domain-level binding experiments and loss-of-function validation; multiple orthogonal approaches in one study","pmids":["29463719"],"is_preprint":false},{"year":2020,"finding":"Cep44 localizes to the proximal end of centrioles, associates with rootletin, and regulates rootletin's stability and localization to the centrosome. Cep44 ablation leads to loss of centrosome cohesion; this is independent of C-Nap1, LRRC45, and Cep215 stability/recruitment.","method":"siRNA knockdown, co-immunoprecipitation (Cep44–rootletin interaction), immunofluorescence localization, protein stability assays","journal":"Journal of cell science","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP plus loss-of-function phenotype with multiple controls, single lab","pmids":["31974111"],"is_preprint":false},{"year":2021,"finding":"Rootletin, ELMOD2, and ARL2 act in a common pathway to suppress spurious ciliation and maintain centrosome cohesion. Rootletin deletion phenotypes (increased ciliation, multiciliation, loss of centrosome cohesion) are rescued by increasing ARL2 activity but not ELMOD2 overexpression. Epistasis analysis places this pathway downstream of TTBK2 and upstream of CP110, preventing spurious CP110 release and regulating ciliary vesicle docking.","method":"Mouse embryonic fibroblast genetic deletion (Rootletin KO), ARL2 overexpression rescue, epistasis with TTBK2/CP110 markers, immunofluorescence for ciliation and cohesion markers","journal":"Molecular biology of the cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic deletion with defined pathway epistasis and rescue experiments, single lab","pmids":["33596093"],"is_preprint":false},{"year":2024,"finding":"Rootletin molecules in mouse hippocampal neurons exhibit highly organized intracellular distributions, and formation of ciliary rootlets precedes that of primary cilia, as revealed by label-free second harmonic generation imaging.","method":"Second harmonic generation microscopy combined with coherent anti-Stokes Raman scattering imaging in mouse brain tissue (label-free)","journal":"bioRxiv","confidence":"Low","confidence_rationale":"Tier 3 / Weak — preprint, single imaging method, no functional manipulation; descriptive localization study","pmids":["bio_10.1101_2024.06.19.597702"],"is_preprint":true}],"current_model":"Rootletin (CROCC) is a coiled-coil protein that polymerizes via its tail domain into homopolymeric filaments anchored at the proximal ends of centrioles/basal bodies through C-Nap1 rings; these filaments form an interdigitating web (organized by CEP68 at 75-nm intervals and stabilized by Cep44) that holds duplicated centrosomes together during interphase, is dissolved at mitosis entry via Nek2-mediated phosphorylation, and is also required for ciliary rootlet formation, ciliogenesis, IFT regulation, and transition zone integrity—partly by protecting Cep68 from VHL-mediated proteasomal degradation."},"narrative":{"mechanistic_narrative":"Rootletin (CROCC) is a coiled-coil protein that serves as the principal structural building block of both the centrosome cohesion apparatus and the ciliary rootlet [PMID:12427867, PMID:16203858]. It self-assembles via its coiled-coil tail domain into parallel in-register homodimers and detergent-insoluble higher-order filaments, while its globular head domain targets the protein to the basal body/centriole and binds kinesin light chain [PMID:12427867]. At the proximal ends of duplicated centrioles, rootletin forms fibers anchored through interaction with C-Nap1, generating the cohesive link that holds centrosomes together during interphase; depletion causes centrosome splitting, and expression of dominant-negative C-Nap1 fragments dissociates rootletin fibers from centrioles [PMID:16203858, PMID:16339073]. Super-resolution imaging resolved this as a C-Nap1 ring organizing a rootletin ring and multiple rootletin/CEP68 fibers that interdigitate between the two centrosomes, with rootletin staggered N-to-N and C-to-C at 75-nm intervals and binding CEP68 through its C-terminal spectrin-repeat region [PMID:29463719]. Cohesion is dynamically regulated: rootletin is phosphorylated by Nek2 and displaced from centrosomes at mitotic onset [PMID:16203858], and its stability and localization are governed by Cep44 [PMID:31974111] and by its capacity to shield CEP68 from VHL E3 ligase-mediated proteasomal degradation, loss of which causes CEP68 destruction and centrosome splitting [PMID:28089774]. Beyond cohesion, rootletin is required for ciliary rootlet formation and ciliogenesis [PMID:23070519], and acts in an ELMOD2/ARL2 pathway downstream of TTBK2 and upstream of CP110 to suppress spurious ciliation [PMID:33596093]. Conserved orthologs maintain ciliary integrity by controlling IFT particle flux, axoneme length, and transition zone/basal body stability, and underpin sensory rootlet anchoring and mechanotransduction [PMID:24094853, PMID:26483560, PMID:26140210].","teleology":[{"year":2002,"claim":"Established the molecular identity and self-assembly logic of the ciliary rootlet, answering what protein builds this structure and how.","evidence":"Recombinant protein expression, detergent-insolubility filament assays, domain deletion, and immunoelectron microscopy in retinal photoreceptors","pmids":["12427867"],"confidence":"High","gaps":["Mechanism of head-domain basal body targeting not resolved at residue level","Functional consequence of ER anchoring along rootlets untested"]},{"year":2005,"claim":"Connected rootletin to centrosome biology by showing it forms proximal-end fibers, binds C-Nap1, is Nek2-regulated, and is required for centrosome cohesion.","evidence":"Immunoelectron microscopy, Co-IP, in vitro Nek2 kinase assay, and siRNA depletion with centrosome splitting readout; reciprocal C-Nap1 localization and dominant-negative fragment dissection","pmids":["16203858","16339073"],"confidence":"High","gaps":["Phosphosites mediating mitotic displacement not mapped","How C-Nap1 anchors rootletin fibers structurally unresolved at the time"]},{"year":2012,"claim":"Linked the cohesion apparatus to ciliogenesis, showing that loss of rootletin or C-NAP1 both destabilizes centriole engagement and reduces primary cilium formation.","evidence":"siRNA knockdown with gamma irradiation and immunofluorescence scoring of splitting and cilia","pmids":["23070519"],"confidence":"Medium","gaps":["Mechanism linking cohesion to cilium assembly not defined","Single-lab phenotype"]},{"year":2013,"claim":"Demonstrated in an intact organism that the rootletin ortholog maintains cilium integrity through IFT flux control and transition zone/basal body stabilization, extending its role beyond cohesion.","evidence":"C. elegans che-10 mutant analysis, in vivo IFT particle tracking, transition zone imaging, and IFT epistasis","pmids":["24094853"],"confidence":"High","gaps":["Direct molecular partners in IFT regulation not identified","Relationship to mammalian rootletin function inferred by orthology"]},{"year":2015,"claim":"Defined rootletin's conserved domain requirements and its predominantly anchoring/mechanotransduction role in sensory neurons, distinguishing rootlet function from ciliogenesis per se.","evidence":"Drosophila genetic knockout and RNAi, behavioral/electrophysiological mechanosensory assays, EM, and domain rescue with Bld10 epistasis","pmids":["26483560","26140210"],"confidence":"Medium","gaps":["Mechanism of asymmetric mother-centriole localization unclear","Mammalian relevance of mechanosensory role untested"]},{"year":2017,"claim":"Revealed a stability-based mechanism for cohesion: rootletin protects CEP68 from VHL-mediated degradation, so its loss destroys CEP68 and splits centrosomes.","evidence":"In vitro ubiquitination reconstitution, single/double siRNA epistasis, and VHL-binding-deficient CEP68 mutants with cohesion readout","pmids":["28089774"],"confidence":"High","gaps":["How rootletin physically occludes the VHL-CEP68 interaction not shown","Regulation of this protection across the cell cycle unknown"]},{"year":2018,"claim":"Resolved the nanoscale architecture of the cohesion linker, showing the C-Nap1 ring organizes staggered rootletin/CEP68 fibers at defined 75-nm periodicity.","evidence":"STED super-resolution microscopy, binding-domain mapping, and CEP68 siRNA filament phenotyping","pmids":["29463719"],"confidence":"High","gaps":["Dynamics of fiber assembly/disassembly not captured","Stoichiometry of the full interdigitating network undefined"]},{"year":2020,"claim":"Identified Cep44 as a regulator of rootletin stability and localization acting independently of C-Nap1/LRRC45/Cep215, adding a parallel control input to cohesion.","evidence":"siRNA knockdown, Cep44–rootletin Co-IP, localization, and protein stability assays","pmids":["31974111"],"confidence":"Medium","gaps":["Whether Cep44 acts directly or via a complex unresolved","Single-lab evidence"]},{"year":2021,"claim":"Placed rootletin in an ELMOD2/ARL2 pathway downstream of TTBK2 and upstream of CP110 that suppresses spurious ciliation and gates ciliary vesicle docking.","evidence":"Rootletin-KO mouse embryonic fibroblasts, ARL2 overexpression rescue, and TTBK2/CP110 marker epistasis","pmids":["33596093"],"confidence":"Medium","gaps":["Biochemical link between rootletin and ARL2 not established","Single-lab pathway placement"]},{"year":2024,"claim":"Provided label-free in situ evidence that rootlet formation temporally precedes primary cilium formation in neurons.","evidence":"Second harmonic generation and CARS microscopy in mouse brain tissue (preprint)","pmids":["bio_10.1101_2024.06.19.597702"],"confidence":"Low","gaps":["Preprint, single descriptive imaging method with no functional manipulation","Causality of rootlet-before-cilium ordering untested"]},{"year":null,"claim":"How rootletin filament assembly, mitotic disassembly, and its degradation-protective and ciliation-suppressive functions are coordinately switched across the cell cycle remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No integrated model coupling Nek2 phosphorylation to CEP68 protection and ARL2 signaling","No high-resolution structure of the rootletin filament core"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[0,8]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[0]},{"term_id":"GO:0140313","term_label":"molecular sequestering activity","supporting_discovery_ids":[7]}],"localization":[{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[1,2,8]},{"term_id":"GO:0005929","term_label":"cilium","supporting_discovery_ids":[0,3]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[0]}],"pathway":[{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[1,2,8]},{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[6,10]}],"complexes":["centriole proximal-end cohesion apparatus (C-Nap1/rootletin/CEP68)","ciliary rootlet"],"partners":["CEP250","CEP68","NEK2","CEP44","VHL","ARL2","ELMOD2"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q5TZA2","full_name":"Rootletin","aliases":["Ciliary rootlet coiled-coil protein"],"length_aa":2017,"mass_kda":228.4,"function":"Major structural component of the ciliary rootlet, a cytoskeletal-like structure in ciliated cells which originates from the basal body at the proximal end of a cilium and extends proximally toward the cell nucleus (By similarity). Furthermore, is required for the correct positioning of the cilium basal body relative to the cell nucleus, to allow for ciliogenesis (PubMed:27623382). Contributes to centrosome cohesion before mitosis (PubMed:16203858)","subcellular_location":"Cytoplasm, cytoskeleton, microtubule organizing center, centrosome, centriole; Cytoplasm, cytoskeleton, cilium basal body; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome","url":"https://www.uniprot.org/uniprotkb/Q5TZA2/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CROCC","classification":"Not Classified","n_dependent_lines":107,"n_total_lines":1208,"dependency_fraction":0.08857615894039735},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/CROCC","total_profiled":1310},"omim":[{"mim_id":"621312","title":"LEUCINE-RICH REPEAT-CONTAINING PROTEIN 45; LRRC45","url":"https://www.omim.org/entry/621312"},{"mim_id":"621147","title":"COILED-COIL DOMAIN-CONTAINING PROTEIN 102B; CCDC102B","url":"https://www.omim.org/entry/621147"},{"mim_id":"620217","title":"CENTROSOMAL PROTEIN, 44-KD; CEP44","url":"https://www.omim.org/entry/620217"},{"mim_id":"616889","title":"CENTROSOMAL PROTEIN, 68-KD; CEP68","url":"https://www.omim.org/entry/616889"},{"mim_id":"615776","title":"CILIARY ROOTLET COILED-COIL PROTEIN; CROCC","url":"https://www.omim.org/entry/615776"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Supported","locations":[{"location":"Centrosome","reliability":"Supported"},{"location":"Basal body","reliability":"Supported"},{"location":"Plasma membrane","reliability":"Additional"},{"location":"Primary cilium transition zone","reliability":"Additional"},{"location":"Mid piece","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/CROCC"},"hgnc":{"alias_symbol":["rootletin","ROLT","CROCC1"],"prev_symbol":[]},"alphafold":{"accession":"Q5TZA2","domains":[{"cath_id":"-","chopping":"1338-1421","consensus_level":"medium","plddt":73.2682,"start":1338,"end":1421},{"cath_id":"-","chopping":"1595-1645_1667-1737_1763-1823","consensus_level":"medium","plddt":77.264,"start":1595,"end":1823},{"cath_id":"1.20.5","chopping":"1530-1560","consensus_level":"medium","plddt":71.4965,"start":1530,"end":1560}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5TZA2","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q5TZA2-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q5TZA2-F1-predicted_aligned_error_v6.png","plddt_mean":69.0},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CROCC","jax_strain_url":"https://www.jax.org/strain/search?query=CROCC"},"sequence":{"accession":"Q5TZA2","fasta_url":"https://rest.uniprot.org/uniprotkb/Q5TZA2.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q5TZA2/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q5TZA2"}},"corpus_meta":[{"pmid":"16203858","id":"PMC_16203858","title":"Rootletin forms centriole-associated filaments and functions in centrosome cohesion.","date":"2005","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/16203858","citation_count":268,"is_preprint":false},{"pmid":"12427867","id":"PMC_12427867","title":"Rootletin, a novel coiled-coil protein, is a structural component of the ciliary rootlet.","date":"2002","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/12427867","citation_count":199,"is_preprint":false},{"pmid":"16339073","id":"PMC_16339073","title":"Rootletin interacts with C-Nap1 and may function as a physical linker between the pair of centrioles/basal bodies in cells.","date":"2005","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/16339073","citation_count":130,"is_preprint":false},{"pmid":"29463719","id":"PMC_29463719","title":"STED nanoscopy of the centrosome linker reveals a CEP68-organized, periodic rootletin network anchored to a C-Nap1 ring at centrioles.","date":"2018","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/29463719","citation_count":53,"is_preprint":false},{"pmid":"24094853","id":"PMC_24094853","title":"Striated rootlet and nonfilamentous forms of rootletin maintain ciliary function.","date":"2013","source":"Current biology : CB","url":"https://pubmed.ncbi.nlm.nih.gov/24094853","citation_count":47,"is_preprint":false},{"pmid":"26483560","id":"PMC_26483560","title":"Rootletin organizes the ciliary rootlet to achieve neuron sensory function in Drosophila.","date":"2015","source":"The Journal of cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/26483560","citation_count":47,"is_preprint":false},{"pmid":"23070519","id":"PMC_23070519","title":"C-NAP1 and rootletin restrain DNA damage-induced centriole splitting and facilitate ciliogenesis.","date":"2012","source":"Cell cycle (Georgetown, Tex.)","url":"https://pubmed.ncbi.nlm.nih.gov/23070519","citation_count":35,"is_preprint":false},{"pmid":"26140210","id":"PMC_26140210","title":"The Drosophila homologue of Rootletin is required for mechanosensory function and ciliary rootlet formation in chordotonal sensory neurons.","date":"2015","source":"Cilia","url":"https://pubmed.ncbi.nlm.nih.gov/26140210","citation_count":28,"is_preprint":false},{"pmid":"33596093","id":"PMC_33596093","title":"Roles for ELMOD2 and Rootletin in ciliogenesis.","date":"2021","source":"Molecular biology of the cell","url":"https://pubmed.ncbi.nlm.nih.gov/33596093","citation_count":17,"is_preprint":false},{"pmid":"31974111","id":"PMC_31974111","title":"Cep44 functions in centrosome cohesion by stabilizing rootletin.","date":"2020","source":"Journal of cell science","url":"https://pubmed.ncbi.nlm.nih.gov/31974111","citation_count":14,"is_preprint":false},{"pmid":"28089774","id":"PMC_28089774","title":"Rootletin prevents Cep68 from VHL-mediated proteasomal degradation to maintain centrosome cohesion.","date":"2017","source":"Biochimica et biophysica acta. Molecular cell research","url":"https://pubmed.ncbi.nlm.nih.gov/28089774","citation_count":13,"is_preprint":false},{"pmid":"31799620","id":"PMC_31799620","title":"Up-regulated microRNA-33b inhibits epithelial-mesenchymal transition in gallbladder cancer through down-regulating CROCC.","date":"2020","source":"Bioscience reports","url":"https://pubmed.ncbi.nlm.nih.gov/31799620","citation_count":9,"is_preprint":false},{"pmid":"30852641","id":"PMC_30852641","title":"CROCC-mutated rhabdoid colorectal carcinoma showing in intercellular spaces lamellipodia and cellular projections revealed by electron microscopy.","date":"2019","source":"Virchows Archiv : an international journal of pathology","url":"https://pubmed.ncbi.nlm.nih.gov/30852641","citation_count":6,"is_preprint":false},{"pmid":"37239344","id":"PMC_37239344","title":"Loss of Primary Cilia Potentiates BRAF/MAPK Pathway Activation in Rhabdoid Colorectal Carcinoma: A Series of 21 Cases Showing Ciliary Rootlet CoiledCoil (CROCC) Alterations.","date":"2023","source":"Genes","url":"https://pubmed.ncbi.nlm.nih.gov/37239344","citation_count":4,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2024.06.19.597702","title":"Label-free visualization of ciliary rootlets in mouse brain","date":"2024-06-22","source":"bioRxiv","url":"https://doi.org/10.1101/2024.06.19.597702","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":8659,"output_tokens":3234,"usd":0.037243,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":10699,"output_tokens":3552,"usd":0.071148,"stage2_stop_reason":"end_turn"},"total_usd":0.108391,"stage1_batch_id":"msgbatch_01ACAxJQabDsht39JXeWG22d","stage2_batch_id":"msgbatch_01QEU22A9U5sbPTgsMzTmvSf","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2002,\n      \"finding\": \"Rootletin is the major structural component of the ciliary rootlet; it forms detergent-insoluble filaments via its coiled-coil tail domain, assembles into parallel in-register homodimers and higher-order polymers through the tail domain alone, and its globular head domain is required for targeting to the basal body and binding to kinesin light chain. In retinal photoreceptors, rootlets anchor ER membranes along their length.\",\n      \"method\": \"Recombinant protein expression, detergent-insolubility assay, domain deletion analysis, immunoelectron microscopy, monoclonal antibody epitope mapping\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — reconstitution of filaments from recombinant protein, domain mutagenesis, structural imaging, replicated by subsequent studies\",\n      \"pmids\": [\"12427867\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Rootletin forms striking fibers emanating from the proximal ends of centrioles (shown by immunoelectron microscopy), interacts with C-Nap1, is phosphorylated by Nek2 kinase, and is displaced from centrosomes at mitosis onset. siRNA-mediated depletion of rootletin causes centrosome splitting, establishing its role in centrosome cohesion.\",\n      \"method\": \"Immunoelectron microscopy, Co-immunoprecipitation (C-Nap1 interaction), in vitro kinase assay (Nek2 phosphorylation), siRNA knockdown with centrosome splitting readout, overexpression fiber formation assay\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, kinase assay, immunoEM, siRNA phenotype), replicated across labs\",\n      \"pmids\": [\"16203858\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Rootletin interacts physically with C-Nap1 in vivo; they colocalize at basal bodies/centrioles and show coordinated cell-cycle-dependent centriole association. Rootletin fibers connect basal bodies in ciliated cells and span between centrioles in nonciliated cells (unlike C-Nap1 which is restricted to centriole ends). Expression of C-Nap1 fragments dissociates rootletin fibers from centrioles, causing centrosome separation in interphase.\",\n      \"method\": \"Co-immunoprecipitation, immunofluorescence co-localization, ultrastructural analysis, dominant-negative C-Nap1 fragment expression, overexpression phenotyping\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal functional and localization evidence, dominant-negative dissection, consistent with independent study (PMID:16203858)\",\n      \"pmids\": [\"16339073\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"C. elegans CHE-10/rootletin ortholog maintains ciliary integrity by modulating assembly, motility, and flux of IFT particles (axoneme length control) and by stabilizing ciliary transition zones and basal bodies. Loss of CHE-10/rootletin disrupts periciliary membrane organization, impairing delivery of basal body-associated and ciliary components and causing cilium degeneration.\",\n      \"method\": \"Genetic mutant analysis (che-10), in vivo IFT particle tracking, fluorescence imaging of transition zones and basal bodies, epistasis with IFT mutants\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function in intact organism with multiple orthogonal cellular phenotype readouts and IFT flux measurements\",\n      \"pmids\": [\"24094853\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Drosophila Rootletin assembles into rootlets of diverse lengths in sensory neurons, is required for basal body cohesion and sensory function (mechanosensation and chemosensation), and requires centrioles for normal rootlet assembly. The N-terminal conserved domain of Root is essential for its function in vivo. Rootletin localizes asymmetrically to mother centrosomes in neuroblasts and to mother centrioles in spermatocytes, both requiring Bld10.\",\n      \"method\": \"Drosophila genetic knockout, behavioral assays, immunofluorescence, domain deletion rescue experiments, Bld10 epistasis\",\n      \"journal\": \"The Journal of cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic loss-of-function with defined phenotypic readouts, domain mapping; single organism model\",\n      \"pmids\": [\"26483560\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Drosophila Rootletin knockdown results in loss of ciliary rootlet in chordotonal neurons and severe disruption of mechanosensory function, while cilium structure and protein localization appear largely normal, suggesting rootletin's role is in anchoring/mechanotransduction rather than ciliogenesis per se.\",\n      \"method\": \"RNAi knockdown in Drosophila, electron microscopy of cilia, electrophysiological/behavioral mechanosensory assays\",\n      \"journal\": \"Cilia\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with structural and functional readouts, single model organism\",\n      \"pmids\": [\"26140210\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2012,\n      \"finding\": \"siRNA depletion of rootletin or C-NAP1 increases radiation-induced centriole splitting, and rootletin or C-NAP1 knockdown reduces primary cilium formation, establishing that the centriole cohesion apparatus at the proximal end of centrioles facilitates ciliogenesis.\",\n      \"method\": \"siRNA knockdown, gamma irradiation, immunofluorescence scoring of centriole splitting and primary cilia formation\",\n      \"journal\": \"Cell cycle (Georgetown, Tex.)\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — loss-of-function with two defined phenotypic readouts, single lab\",\n      \"pmids\": [\"23070519\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Rootletin prevents VHL E3 ligase-mediated proteasomal degradation of Cep68: in the absence of rootletin, VHL ubiquitinates Cep68 both in vitro and in vivo, leading to Cep68 loss and centrosome splitting. Co-silencing of rootletin and VHL rescues Cep68 levels and centrosome cohesion. A Cep68 mutant that cannot bind the VHL β-domain also suppresses centrosome splitting caused by rootletin depletion.\",\n      \"method\": \"In vitro ubiquitination assay, siRNA knockdown of rootletin and VHL (single and double), Cep68 ubiquitination-resistant mutants, immunofluorescence centrosome cohesion assay\",\n      \"journal\": \"Biochimica et biophysica acta. Molecular cell research\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro ubiquitination reconstitution plus in vivo genetic epistasis (double knockdown rescue) and mutagenesis\",\n      \"pmids\": [\"28089774\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"STED nanoscopy revealed that C-Nap1 forms a ring at each centriole's proximal end that organizes a rootletin ring and multiple rootletin/CEP68 fibers. Rootletin/CEP68 fibers from the two centrosomes form an interdigitating network. Rootletin molecules within filaments are staggered N-to-N and C-to-C at 75-nm intervals; rootletin binds CEP68 via its C-terminal spectrin repeat-containing region at 75-nm intervals; estimated N-to-C rootletin length is ~35–40 nm, minimal rootletin length ~110 nm. CEP68 is required to form rootletin filaments branching from centrioles and modulates fiber thickness.\",\n      \"method\": \"STED super-resolution microscopy, protein interaction mapping (binding domain identification), siRNA depletion of CEP68 with filament phenotype readout\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — super-resolution structural imaging plus domain-level binding experiments and loss-of-function validation; multiple orthogonal approaches in one study\",\n      \"pmids\": [\"29463719\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Cep44 localizes to the proximal end of centrioles, associates with rootletin, and regulates rootletin's stability and localization to the centrosome. Cep44 ablation leads to loss of centrosome cohesion; this is independent of C-Nap1, LRRC45, and Cep215 stability/recruitment.\",\n      \"method\": \"siRNA knockdown, co-immunoprecipitation (Cep44–rootletin interaction), immunofluorescence localization, protein stability assays\",\n      \"journal\": \"Journal of cell science\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP plus loss-of-function phenotype with multiple controls, single lab\",\n      \"pmids\": [\"31974111\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Rootletin, ELMOD2, and ARL2 act in a common pathway to suppress spurious ciliation and maintain centrosome cohesion. Rootletin deletion phenotypes (increased ciliation, multiciliation, loss of centrosome cohesion) are rescued by increasing ARL2 activity but not ELMOD2 overexpression. Epistasis analysis places this pathway downstream of TTBK2 and upstream of CP110, preventing spurious CP110 release and regulating ciliary vesicle docking.\",\n      \"method\": \"Mouse embryonic fibroblast genetic deletion (Rootletin KO), ARL2 overexpression rescue, epistasis with TTBK2/CP110 markers, immunofluorescence for ciliation and cohesion markers\",\n      \"journal\": \"Molecular biology of the cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic deletion with defined pathway epistasis and rescue experiments, single lab\",\n      \"pmids\": [\"33596093\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"Rootletin molecules in mouse hippocampal neurons exhibit highly organized intracellular distributions, and formation of ciliary rootlets precedes that of primary cilia, as revealed by label-free second harmonic generation imaging.\",\n      \"method\": \"Second harmonic generation microscopy combined with coherent anti-Stokes Raman scattering imaging in mouse brain tissue (label-free)\",\n      \"journal\": \"bioRxiv\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — preprint, single imaging method, no functional manipulation; descriptive localization study\",\n      \"pmids\": [\"bio_10.1101_2024.06.19.597702\"],\n      \"is_preprint\": true\n    }\n  ],\n  \"current_model\": \"Rootletin (CROCC) is a coiled-coil protein that polymerizes via its tail domain into homopolymeric filaments anchored at the proximal ends of centrioles/basal bodies through C-Nap1 rings; these filaments form an interdigitating web (organized by CEP68 at 75-nm intervals and stabilized by Cep44) that holds duplicated centrosomes together during interphase, is dissolved at mitosis entry via Nek2-mediated phosphorylation, and is also required for ciliary rootlet formation, ciliogenesis, IFT regulation, and transition zone integrity—partly by protecting Cep68 from VHL-mediated proteasomal degradation.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"Rootletin (CROCC) is a coiled-coil protein that serves as the principal structural building block of both the centrosome cohesion apparatus and the ciliary rootlet [#0, #1]. It self-assembles via its coiled-coil tail domain into parallel in-register homodimers and detergent-insoluble higher-order filaments, while its globular head domain targets the protein to the basal body/centriole and binds kinesin light chain [#0]. At the proximal ends of duplicated centrioles, rootletin forms fibers anchored through interaction with C-Nap1, generating the cohesive link that holds centrosomes together during interphase; depletion causes centrosome splitting, and expression of dominant-negative C-Nap1 fragments dissociates rootletin fibers from centrioles [#1, #2]. Super-resolution imaging resolved this as a C-Nap1 ring organizing a rootletin ring and multiple rootletin/CEP68 fibers that interdigitate between the two centrosomes, with rootletin staggered N-to-N and C-to-C at 75-nm intervals and binding CEP68 through its C-terminal spectrin-repeat region [#8]. Cohesion is dynamically regulated: rootletin is phosphorylated by Nek2 and displaced from centrosomes at mitotic onset [#1], and its stability and localization are governed by Cep44 [#9] and by its capacity to shield CEP68 from VHL E3 ligase-mediated proteasomal degradation, loss of which causes CEP68 destruction and centrosome splitting [#7]. Beyond cohesion, rootletin is required for ciliary rootlet formation and ciliogenesis [#6], and acts in an ELMOD2/ARL2 pathway downstream of TTBK2 and upstream of CP110 to suppress spurious ciliation [#10]. Conserved orthologs maintain ciliary integrity by controlling IFT particle flux, axoneme length, and transition zone/basal body stability, and underpin sensory rootlet anchoring and mechanotransduction [#3, #4, #5].\",\n  \"teleology\": [\n    {\n      \"year\": 2002,\n      \"claim\": \"Established the molecular identity and self-assembly logic of the ciliary rootlet, answering what protein builds this structure and how.\",\n      \"evidence\": \"Recombinant protein expression, detergent-insolubility filament assays, domain deletion, and immunoelectron microscopy in retinal photoreceptors\",\n      \"pmids\": [\"12427867\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of head-domain basal body targeting not resolved at residue level\", \"Functional consequence of ER anchoring along rootlets untested\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Connected rootletin to centrosome biology by showing it forms proximal-end fibers, binds C-Nap1, is Nek2-regulated, and is required for centrosome cohesion.\",\n      \"evidence\": \"Immunoelectron microscopy, Co-IP, in vitro Nek2 kinase assay, and siRNA depletion with centrosome splitting readout; reciprocal C-Nap1 localization and dominant-negative fragment dissection\",\n      \"pmids\": [\"16203858\", \"16339073\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Phosphosites mediating mitotic displacement not mapped\", \"How C-Nap1 anchors rootletin fibers structurally unresolved at the time\"]\n    },\n    {\n      \"year\": 2012,\n      \"claim\": \"Linked the cohesion apparatus to ciliogenesis, showing that loss of rootletin or C-NAP1 both destabilizes centriole engagement and reduces primary cilium formation.\",\n      \"evidence\": \"siRNA knockdown with gamma irradiation and immunofluorescence scoring of splitting and cilia\",\n      \"pmids\": [\"23070519\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism linking cohesion to cilium assembly not defined\", \"Single-lab phenotype\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstrated in an intact organism that the rootletin ortholog maintains cilium integrity through IFT flux control and transition zone/basal body stabilization, extending its role beyond cohesion.\",\n      \"evidence\": \"C. elegans che-10 mutant analysis, in vivo IFT particle tracking, transition zone imaging, and IFT epistasis\",\n      \"pmids\": [\"24094853\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct molecular partners in IFT regulation not identified\", \"Relationship to mammalian rootletin function inferred by orthology\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Defined rootletin's conserved domain requirements and its predominantly anchoring/mechanotransduction role in sensory neurons, distinguishing rootlet function from ciliogenesis per se.\",\n      \"evidence\": \"Drosophila genetic knockout and RNAi, behavioral/electrophysiological mechanosensory assays, EM, and domain rescue with Bld10 epistasis\",\n      \"pmids\": [\"26483560\", \"26140210\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism of asymmetric mother-centriole localization unclear\", \"Mammalian relevance of mechanosensory role untested\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Revealed a stability-based mechanism for cohesion: rootletin protects CEP68 from VHL-mediated degradation, so its loss destroys CEP68 and splits centrosomes.\",\n      \"evidence\": \"In vitro ubiquitination reconstitution, single/double siRNA epistasis, and VHL-binding-deficient CEP68 mutants with cohesion readout\",\n      \"pmids\": [\"28089774\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How rootletin physically occludes the VHL-CEP68 interaction not shown\", \"Regulation of this protection across the cell cycle unknown\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Resolved the nanoscale architecture of the cohesion linker, showing the C-Nap1 ring organizes staggered rootletin/CEP68 fibers at defined 75-nm periodicity.\",\n      \"evidence\": \"STED super-resolution microscopy, binding-domain mapping, and CEP68 siRNA filament phenotyping\",\n      \"pmids\": [\"29463719\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Dynamics of fiber assembly/disassembly not captured\", \"Stoichiometry of the full interdigitating network undefined\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Identified Cep44 as a regulator of rootletin stability and localization acting independently of C-Nap1/LRRC45/Cep215, adding a parallel control input to cohesion.\",\n      \"evidence\": \"siRNA knockdown, Cep44–rootletin Co-IP, localization, and protein stability assays\",\n      \"pmids\": [\"31974111\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether Cep44 acts directly or via a complex unresolved\", \"Single-lab evidence\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Placed rootletin in an ELMOD2/ARL2 pathway downstream of TTBK2 and upstream of CP110 that suppresses spurious ciliation and gates ciliary vesicle docking.\",\n      \"evidence\": \"Rootletin-KO mouse embryonic fibroblasts, ARL2 overexpression rescue, and TTBK2/CP110 marker epistasis\",\n      \"pmids\": [\"33596093\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Biochemical link between rootletin and ARL2 not established\", \"Single-lab pathway placement\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Provided label-free in situ evidence that rootlet formation temporally precedes primary cilium formation in neurons.\",\n      \"evidence\": \"Second harmonic generation and CARS microscopy in mouse brain tissue (preprint)\",\n      \"pmids\": [\"bio_10.1101_2024.06.19.597702\"],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Preprint, single descriptive imaging method with no functional manipulation\", \"Causality of rootlet-before-cilium ordering untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How rootletin filament assembly, mitotic disassembly, and its degradation-protective and ciliation-suppressive functions are coordinately switched across the cell cycle remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No integrated model coupling Nek2 phosphorylation to CEP68 protection and ARL2 signaling\", \"No high-resolution structure of the rootletin filament core\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [0, 8]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [0]},\n      {\"term_id\": \"GO:0140313\", \"supporting_discovery_ids\": [7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [1, 2, 8]},\n      {\"term_id\": \"GO:0005929\", \"supporting_discovery_ids\": [0, 3]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [0]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [1, 2, 8]},\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [6, 10]}\n    ],\n    \"complexes\": [\n      \"centriole proximal-end cohesion apparatus (C-Nap1/rootletin/CEP68)\",\n      \"ciliary rootlet\"\n    ],\n    \"partners\": [\n      \"CEP250\",\n      \"CEP68\",\n      \"NEK2\",\n      \"CEP44\",\n      \"VHL\",\n      \"ARL2\",\n      \"ELMOD2\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":6,"faith_total":7,"faith_pct":85.71428571428571}}