{"gene":"SASS6","run_date":"2026-04-28T20:42:07","timeline":{"discoveries":[{"year":2005,"finding":"HsSAS-6 (human SASS6) localizes to centrosomes and is required for centrosome duplication: siRNA-mediated inactivation in U2OS cells abrogates centrosome overduplication following aphidicolin treatment and interferes with the normal centrosome duplication cycle. In C. elegans, SAS-6 and SAS-5 associate, and this interaction as well as ZYG-1 function is required for SAS-6 centriolar recruitment.","method":"siRNA knockdown, GFP localization, co-immunoprecipitation, genetic analysis in C. elegans","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 — multiple orthogonal methods (siRNA, GFP localization, co-IP, genetic epistasis) across two organisms; foundational paper with 329 citations","pmids":["15665853"],"is_preprint":false},{"year":2007,"finding":"SAS-6 localizes to the central part of the centriolar cartwheel and is required to establish the 9-fold symmetry of centrioles; null mutants in Chlamydomonas (bld12) lack the central cartwheel hub and frequently produce centrioles with aberrant triplet numbers (7, 8, 10, or 11 instead of 9).","method":"Immunolocalization, genetic null mutant analysis, electron microscopy","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 — direct localization plus functional null mutant with defined structural phenotype; 217 citations","pmids":["18082404"],"is_preprint":false},{"year":2009,"finding":"ZYG-1 kinase phosphorylates SAS-6 at serine 123 in vitro, and this phosphorylation is required for centriole formation in C. elegans embryos in vivo; phosphorylation maintains SAS-6 at the emerging centriole.","method":"In vitro kinase assay, phospho-site mutagenesis, in vivo centriole formation assay in C. elegans","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay combined with in vivo mutagenesis and functional readout","pmids":["20059959"],"is_preprint":false},{"year":2010,"finding":"Drosophila SAS-6 self-assembles into stable tetramers in vitro that serve as building blocks for the cartwheel central tubule; EM of centrosomes identifies SAS-6 concentrating at the core of the cartwheel.","method":"Biochemistry (size exclusion chromatography), electron microscopy, recombinant protein assembly assay","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 1 — in vitro reconstitution of self-assembly combined with EM structural validation","pmids":["20083610"],"is_preprint":false},{"year":2011,"finding":"The X-ray crystal structure of the SAS-6 N-terminal domain from zebrafish reveals that SAS-6 self-associates in vitro into assemblies resembling cartwheel centers; point mutations at the self-assembly interfaces block centriole formation in vivo, establishing these interactions as essential for cartwheel organization.","method":"X-ray crystallography, in vitro self-assembly assay, point mutagenesis with in vivo functional validation","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus mutagenesis plus in vivo functional validation; 263 citations","pmids":["21273447"],"is_preprint":false},{"year":2013,"finding":"ZYG-1 directly binds the SAS-6 coiled coil and recruits SAS-6 to the mother centriole independently of its kinase activity; ZYG-1 kinase activity is subsequently required for cartwheel assembly. The SAS-6 coiled coil also interacts with SAS-5, and both interactions are required for SAS-6 centriolar recruitment.","method":"Direct binding assay, reciprocal co-immunoprecipitation, mutagenesis, in vivo recruitment assay in C. elegans","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 — direct binding demonstrated, reciprocal interaction validated, combined with in vivo epistasis","pmids":["23673331"],"is_preprint":false},{"year":2013,"finding":"C. elegans SAS-6 self-assembles into a spiral arrangement (rather than a ring) in vitro, as revealed by X-ray crystallography and EM; the spiral is consistent with 9-fold symmetry and provides a structural basis for the central tube found in nematode centriole assembly.","method":"X-ray crystallography, electron microscopy, structural analysis","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus EM reconstitution","pmids":["23798409"],"is_preprint":false},{"year":2014,"finding":"Leishmania major SAS-6 crystallizes as a 9-fold symmetric cartwheel; the X-ray structure demonstrates that SAS-6 self-assembly alone can dictate cartwheel symmetry. SAS-6 oligomerization can be inhibited by a small molecule in vitro.","method":"X-ray crystallography (3.5 Å resolution), small-molecule inhibition of oligomerization in vitro","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 — high-resolution crystal structure of full cartwheel plus biochemical inhibition assay","pmids":["24596152"],"is_preprint":false},{"year":2014,"finding":"In Drosophila, PLK4 phosphorylates four conserved serines in the STAN motif of Ana2 (the SAS-6 recruitment factor) to enable Ana2 to bind and recruit Sas6 to the procentriole; non-phosphorylatable Ana2 localizes to the centriole but cannot recruit Sas6, blocking centriole duplication.","method":"In vitro kinase assay, phospho-site mutagenesis, in vivo centriole duplication assay, co-immunoprecipitation","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay combined with mutagenesis and in vivo functional phenotype","pmids":["25264260"],"is_preprint":false},{"year":2014,"finding":"During S phase, SAS-6 is first recruited to the proximal lumen of the mother centriole, adopting a cartwheel-like organization through interactions with the luminal wall rather than via self-oligomerization; removal of luminal SAS-6 requires PLK4 and STIL, and abolishing either recruitment or removal hinders SAS-6 assembly at the outer wall.","method":"Live-cell imaging, FRAP, siRNA knockdown, structured illumination microscopy","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 — live imaging with functional epistasis (PLK4 and STIL dependence) and multiple orthogonal methods","pmids":["25017693"],"is_preprint":false},{"year":2015,"finding":"The C-terminal tail of human SAS-6 (residues 470–657) nucleates and promotes microtubule polymerization in vitro, binds microtubules and tubulin dimers directly, and co-precipitates with microtubules from S-phase synchronized HeLa cell lysates; the N-terminal domain does not affect microtubule polymerization.","method":"In vitro microtubule polymerization assay, microtubule pulldown, co-immunoprecipitation, isothermal titration calorimetry, size exclusion chromatography","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1 — multiple in vitro reconstitution assays plus ITC and co-IP from cell lysates","pmids":["26422590"],"is_preprint":false},{"year":2015,"finding":"De novo centriole formation in human cells can occur in the absence of SAS-6 self-oligomerization, whereas canonically duplicated centrioles always form correctly; de novo centrioles are structurally error-prone even with SAS-6 self-oligomerization, indicating a template/mother-centriole requirement for structural fidelity.","method":"Reconstitution of de novo centriole synthesis in human cells, mutagenesis of SAS-6 self-oligomerization interface, EM structural analysis","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 — reconstitution in human cells with oligomerization-dead mutants and structural readout","pmids":["26609813"],"is_preprint":false},{"year":2015,"finding":"Drosophila Sas-6 N-terminal domain forms higher-order oligomers through canonical interactions (crystal structure at 2.9 Å); point mutations that perturb Sas-6 homo-oligomerization in vitro strongly impair centriole assembly in vivo.","method":"X-ray crystallography, in vitro oligomerization assay, in vivo centriole assembly assay with point mutants","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus mutagenesis with in vitro and in vivo validation","pmids":["26002084"],"is_preprint":false},{"year":2016,"finding":"Engineering Chlamydomonas SAS-6 to form different symmetry oligomers in vitro reveals that SAS-6 self-assembly properties instruct cartwheel symmetry, and that the cartwheel and microtubule wall assemble interdependently. Expressing analogous SAS-6 mutations in human cells produced 9-fold symmetric centrioles with impaired length and organization.","method":"SAS-6 mutagenesis, in vitro oligomerization assay, in vivo centriole symmetry analysis by EM, human cell expression","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1 — engineered mutants with defined symmetry tested in vitro and in vivo with structural readout","pmids":["26999736"],"is_preprint":false},{"year":2017,"finding":"In Drosophila, PLK4 performs two-step phosphorylation of Ana2: first at S38 in the ANST motif to promote Ana2 centriolar recruitment, then at four serines in the STAN motif to enable Ana2 to recruit Sas6. Mutation of S38 to alanine blocks Ana2 loading and centriole duplication.","method":"In vitro kinase assay, phospho-site mutagenesis, in vivo centriole duplication assay, live-cell imaging","journal":"Open biology","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay with mutagenesis and in vivo functional validation","pmids":["29263250"],"is_preprint":false},{"year":2017,"finding":"CDC6, a DNA replication licensing factor, is recruited to the proximal centriole via cyclin A and inhibits centrosome duplication by binding SAS-6 and preventing formation of a stable SAS-6–STIL complex; PLK4 phosphorylates CDC6 to disrupt the CDC6–SAS-6 interaction, relieving inhibition.","method":"Co-immunoprecipitation, centrosome localization assay, overexpression/knockdown experiments, phosphorylation assay","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP with functional rescue, but single-lab study","pmids":["28447620"],"is_preprint":false},{"year":2018,"finding":"High-speed photothermal off-resonance AFM (PORT) reveals the kinetics of SAS-6 ring formation in real time and demonstrates that distinct biogenesis routes (different assembly intermediates) can converge to produce a 9-fold symmetric ring structure.","method":"High-speed atomic force microscopy (PORT), real-time self-assembly monitoring","journal":"Nature nanotechnology","confidence":"High","confidence_rationale":"Tier 1 — direct real-time structural visualization of assembly with kinetic analysis","pmids":["29784964"],"is_preprint":false},{"year":2019,"finding":"Pericentrin (Pcp1) interacts with and recruits SAS-6 to the pericentriolar material; this interaction is conserved between fission yeast SPB and animal centrosomes and is important for centriole assembly, particularly elongation. The calmodulin-binding region of pericentrin is critical for SAS-6 interaction.","method":"Yeast two-hybrid, co-immunoprecipitation, ectopic expression in fission yeast, functional centriole elongation assay","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 — reciprocal interaction validated across organisms with functional consequence, single lab","pmids":["31182187"],"is_preprint":false},{"year":2020,"finding":"Human SAS-6 C-terminus interacts with the γ-tubulin ring complex (γ-TuRC) at the centrosome; deletion of the SAS-6 C-terminus disrupts microtubule formation in daughter centrioles causing failure to form new centrioles. High-resolution microscopy reveals γ-tubulin localizing as multiple lobes surrounding the SAS-6-containing central hub.","method":"Co-immunoprecipitation, deletion mutagenesis, super-resolution microscopy, siRNA knockdown with phenotypic readout","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 — co-IP, deletion mutagenesis, and super-resolution imaging with defined functional phenotype, multiple orthogonal approaches","pmids":["32442461"],"is_preprint":false},{"year":2020,"finding":"The Drosophila Golgi protein Gorab binds as a monomer to the parallel coiled-coil dimer of Sas6, forming a stable heterotrimer visualized by EM; mutation of a single leucine in Sas6's Gorab-binding domain reduces affinity 16-fold and abolishes centriole duplication, revealing a functional Sas6–Gorab interaction required for duplication.","method":"Hydrogen-deuterium exchange mass spectrometry, electron microscopy, mutagenesis, in vivo centriole duplication assay","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 — HDX-MS structural definition of interaction interface plus EM visualization plus in vivo mutagenesis","pmids":["33704067"],"is_preprint":false},{"year":2020,"finding":"HDX-MS of the Drosophila Ana2–Sas6 complex identifies short critical regions in the C-terminal parts of both proteins as the interaction interface; phosphomimetic Ana2 (Ana2-4D) forms a complex with Sas6 in vitro and in vivo, and mutations in these regions disrupt the Ana2–Sas6 interaction. The Sas6 site for Ana2 binding is distinct from the Gorab-binding site, allowing simultaneous binding.","method":"Hydrogen-deuterium exchange mass spectrometry, co-immunoprecipitation in vitro and in vivo, mutagenesis","journal":"Open biology","confidence":"High","confidence_rationale":"Tier 1 — HDX-MS interaction interface mapping combined with mutagenesis and co-IP validation","pmids":["33171067"],"is_preprint":false},{"year":2021,"finding":"Monobodies developed against Chlamydomonas SAS-6 (characterized by X-ray crystallography, AFM, and cryo-EM) reveal distinct interaction modes; one monobody (MBCRS6-15) induces a conformational change causing SAS-6 to form a helix instead of a ring, and expression of this monobody in human cells impairs centriole biogenesis.","method":"X-ray crystallography, atomic force microscopy, cryo-EM, human cell centriole biogenesis assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 — multiple structural methods plus in vivo functional validation in human cells","pmids":["34155202"],"is_preprint":false},{"year":2022,"finding":"Crystal structures of Chlamydomonas SAS-6 coiled-coil complexes reveal an asymmetric homo-oligomerization interaction that imparts polarity to the cartwheel; cryo-EM reconstitution assay demonstrates that amino acid substitutions disrupting this asymmetric association impair SAS-6 ring stacking.","method":"X-ray crystallography, cryo-EM reconstitution assay, mutagenesis of coiled-coil","journal":"Structure (London, England : 1993)","confidence":"High","confidence_rationale":"Tier 1 — crystal structure plus cryo-EM functional validation with mutagenesis","pmids":["35240058"],"is_preprint":false},{"year":2024,"finding":"In mouse embryos, Sass6 (SASS6) is essential for centriole formation; Sass6-mutant embryos lack centrioles, activate the mitotic surveillance cell death pathway, and arrest at mid-gestation. In mouse embryonic stem cells (mESCs), SAS-6 is not required for centriole formation but is essential for centriole architecture maintenance; elevated PLK4 activity in mESCs enables a SAS-6-independent centriole biogenesis pathway.","method":"Genetic knockout in mouse, embryo phenotypic analysis, mESC culture, PLK4 activity manipulation, centrosome/centriole imaging","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 — clean genetic knockout in mouse with defined cellular and developmental phenotypes, mechanistic epistasis with PLK4","pmids":["38407237"],"is_preprint":false},{"year":2025,"finding":"FBXW7 E3 ubiquitin ligase mediates degradation of the STIL–SAS6 cartwheel assembly in a PLK4 kinase activity-dependent manner; PLK4 phosphorylation of STIL drives both STIL–SAS6 interaction (promoting new centriole assembly) and FBXW7 binding to STIL (promoting STIL–SAS6 degradation), creating an opposing mechanism that prevents centriole overduplication.","method":"siRNA knockdown of FBXW7, co-immunoprecipitation, ubiquitination assay, PLK4 kinase inhibition","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — co-IP with functional assay, but single recent paper; dual role mechanistic model is plausible","pmids":["41453690"],"is_preprint":false},{"year":2025,"finding":"SAS-6 levels are controlled by APC/C-Cdh1-targeted degradation at the end of mitosis/G1; expression of a non-degradable SAS-6 mutant (SAS-6ND) increases ciliation and cell invasion through activation of the YAP/TAZ pathway and nuclear YAP translocation leading to TEAD-dependent transcription. SAS-6-mediated invasion is blocked by YAP downregulation or by blocking ciliogenesis.","method":"Non-degradable SAS-6 mutant expression, siRNA knockdown, invasion assay, YAP/TAZ pathway analysis, ciliogenesis assay","journal":"Life science alliance","confidence":"Medium","confidence_rationale":"Tier 2 — multiple orthogonal assays in single lab; mechanistic pathway placement via rescue experiments","pmids":["40825584"],"is_preprint":false},{"year":2025,"finding":"SAS-6 undergoes phase separation in vitro; CDK-1 directly phosphorylates SAS-6 at its C-terminus in cells and in vivo, and this phosphorylation inhibits SAS-6 phase separation and weakens interactions between centriolar proteins. Phospho-mimetic and phospho-deficient SAS-6 mutants demonstrate that dynamic phosphorylation controls centrosome assembly during oogenesis and timely centriole disassembly in C. elegans.","method":"In vitro phase separation assay, mass spectrometry, in vitro kinase assay, phospho-site mutagenesis, in vivo oogenesis phenotype analysis in C. elegans","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 1 — in vitro kinase assay plus phase separation reconstitution plus mutagenesis with in vivo functional validation","pmids":["40410380"],"is_preprint":false}],"current_model":"SASS6/SAS-6 is an evolutionarily conserved centriolar scaffold protein that self-assembles via its N-terminal globular domain (dimerization) and coiled-coil domain (ring stacking) into 9-fold symmetric ring polymers that stack to form the cartwheel central hub of nascent centrioles; it is recruited to the mother centriole through direct binding to the ZYG-1/PLK4 kinase and its cofactor SAS-5/Ana2/STIL (whose PLK4-mediated phosphorylation in the STAN motif is required for SAS-6 binding), its C-terminal domain anchors the γ-tubulin ring complex to nucleate centriolar microtubules, its levels are controlled by APC/C-Cdh1 degradation and by CDK-1 phosphorylation that inhibits phase separation and promotes centriole disassembly during oogenesis, and FBXW7-mediated degradation of the STIL–SAS-6 complex prevents centriole overduplication—with pericentrin providing an additional PCM-based recruitment route and Gorab/FBXW7 providing regulatory interactions at the centriole."},"narrative":{"teleology":[{"year":2005,"claim":"Establishing SAS-6 as a conserved centrosome duplication factor resolved the question of which proteins are essential for new centriole formation: SAS-6 is required for centrosome duplication in human cells and C. elegans, and its centriolar recruitment depends on ZYG-1 and SAS-5.","evidence":"siRNA knockdown in U2OS cells, co-IP and genetic epistasis in C. elegans","pmids":["15665853"],"confidence":"High","gaps":["Structural role of SAS-6 at the centriole was unknown","Mechanism of SAS-6 self-organization not addressed","Whether SAS-6 dictates centriole symmetry was untested"]},{"year":2007,"claim":"Determining that SAS-6 localizes specifically to the cartwheel hub and is required for 9-fold symmetry answered the long-standing question of what specifies centriole radial architecture: loss of SAS-6 produces centrioles with variable triplet numbers.","evidence":"Immunolocalization and EM of Chlamydomonas bld12 null mutants","pmids":["18082404"],"confidence":"High","gaps":["Molecular basis of 9-fold symmetry specification unknown","Whether SAS-6 alone is sufficient for symmetry establishment untested"]},{"year":2009,"claim":"Identifying ZYG-1 phosphorylation of SAS-6 at S123 as essential for centriole formation established that kinase signaling directly regulates the cartwheel scaffold, not just its upstream recruitment factors.","evidence":"In vitro kinase assay with phospho-site mutagenesis and in vivo centriole formation in C. elegans","pmids":["20059959"],"confidence":"High","gaps":["Whether phosphorylation affects SAS-6 self-assembly or protein interactions was unclear","Mammalian equivalent phosphosite not identified"]},{"year":2010,"claim":"Reconstituting SAS-6 self-assembly into tetramers in vitro demonstrated that SAS-6 is an intrinsic building block of the cartwheel, shifting the model from a passively recruited scaffold to a self-organizing polymer.","evidence":"Recombinant Drosophila SAS-6 size-exclusion chromatography and EM","pmids":["20083610"],"confidence":"High","gaps":["Atomic-resolution structure of self-assembly interface not yet available","Whether 9-fold ring forms spontaneously in vitro was unknown"]},{"year":2011,"claim":"Solving the crystal structure of the SAS-6 N-terminal domain and showing that mutations at the dimerization interface block centriole formation provided the first atomic-level explanation of how SAS-6 self-assembly builds the cartwheel.","evidence":"X-ray crystallography of zebrafish SAS-6 with in vitro assembly and in vivo mutagenesis","pmids":["21273447"],"confidence":"High","gaps":["Full-ring structure not yet solved","Coiled-coil contribution to stacking unknown","Species-specific variations in symmetry not addressed"]},{"year":2013,"claim":"Demonstrating that ZYG-1 directly binds the SAS-6 coiled coil independently of kinase activity, and that SAS-5 binds the same domain, resolved how SAS-6 is physically delivered to the mother centriole through a two-factor recruitment mechanism.","evidence":"Direct binding assays, reciprocal co-IP, and in vivo recruitment in C. elegans","pmids":["23673331"],"confidence":"High","gaps":["Whether PLK4 uses the same binding mode in mammals was untested","Stoichiometry of the recruitment complex unknown"]},{"year":2013,"claim":"Solving the C. elegans SAS-6 structure as a spiral rather than a planar ring revealed species-specific assembly variants consistent with the nematode central tube architecture, broadening the structural paradigm.","evidence":"X-ray crystallography and EM of C. elegans SAS-6","pmids":["23798409"],"confidence":"High","gaps":["How the spiral converts to an in vivo-functional central tube was unclear","Relationship between spiral and ring assembly pathways unresolved"]},{"year":2014,"claim":"Crystallizing the Leishmania SAS-6 as a complete 9-fold symmetric cartwheel proved that SAS-6 self-assembly alone can dictate cartwheel symmetry, and identification of a small-molecule oligomerization inhibitor opened a pharmacological approach.","evidence":"3.5 Å X-ray crystal structure plus in vitro small-molecule inhibition","pmids":["24596152"],"confidence":"High","gaps":["In vivo efficacy of the small-molecule inhibitor not tested","Whether mammalian SAS-6 forms equivalent complete rings in vitro was unknown"]},{"year":2014,"claim":"Showing that PLK4 phosphorylation of Ana2/STIL's STAN motif is the switch enabling SAS-6 recruitment defined the upstream signaling event that licenses cartwheel initiation at the procentriole.","evidence":"In vitro kinase assay, phospho-site mutagenesis, in vivo duplication assay in Drosophila; complemented by human S-phase SAS-6 dynamics","pmids":["25264260","25017693"],"confidence":"High","gaps":["Order of Ana2 phosphorylation events relative to SAS-6 ring assembly not resolved","Whether SAS-6 luminal recruitment and outer-wall assembly involve different SAS-6 pools was unclear"]},{"year":2015,"claim":"Identifying that the SAS-6 C-terminal tail directly binds tubulin and nucleates microtubule polymerization revealed a second functional domain beyond the cartwheel-forming N-terminus, linking the cartwheel scaffold to centriolar microtubule wall formation.","evidence":"In vitro microtubule polymerization, ITC, co-IP from HeLa lysates","pmids":["26422590"],"confidence":"High","gaps":["Structural basis of C-tail–tubulin interaction not resolved","Whether γ-TuRC or free tubulin is the physiological target was unclear"]},{"year":2015,"claim":"Demonstrating that centrioles can form de novo without SAS-6 self-oligomerization but with frequent structural errors, while template-directed duplication always preserves 9-fold symmetry, established that the mother centriole provides an essential fidelity mechanism beyond SAS-6 intrinsic self-assembly.","evidence":"Reconstitution of de novo centriole synthesis in human cells with oligomerization-dead SAS-6 mutants and EM analysis","pmids":["26609813"],"confidence":"High","gaps":["Nature of the template-based error-correction mechanism unknown","Whether other cartwheel components compensate in the absence of SAS-6 oligomerization unclear"]},{"year":2016,"claim":"Engineering SAS-6 variants with altered symmetry properties demonstrated that SAS-6 self-assembly directly instructs cartwheel symmetry and that cartwheel and microtubule wall assembly are interdependent, resolving whether symmetry is SAS-6-intrinsic or templated.","evidence":"Symmetry-altering SAS-6 mutations tested in vitro and in vivo in Chlamydomonas and human cells","pmids":["26999736"],"confidence":"High","gaps":["How the microtubule wall feeds back on cartwheel symmetry is mechanistically unclear"]},{"year":2017,"claim":"Identifying CDC6 as a negative regulator that binds SAS-6 and prevents STIL–SAS-6 complex formation until PLK4 phosphorylates CDC6 established a licensing checkpoint coupling DNA replication and centrosome duplication through SAS-6.","evidence":"Co-IP, centrosome localization, overexpression/knockdown, PLK4 phosphorylation assay in human cells","pmids":["28447620"],"confidence":"Medium","gaps":["Single-lab study; independent validation needed","Structural basis of CDC6–SAS-6 interaction unknown","Whether CDC6 regulation is conserved beyond mammals untested"]},{"year":2018,"claim":"Real-time visualization of SAS-6 ring assembly by high-speed AFM revealed that multiple kinetically distinct assembly intermediates converge on 9-fold symmetric rings, demonstrating robust self-assembly through parallel pathways.","evidence":"Photothermal off-resonance AFM with kinetic analysis of Chlamydomonas SAS-6","pmids":["29784964"],"confidence":"High","gaps":["In vivo relevance of alternative assembly routes not established","Whether accessory factors constrain assembly pathways in cells unknown"]},{"year":2019,"claim":"Identifying pericentrin as a direct SAS-6-binding partner that recruits SAS-6 to the pericentriolar material revealed an additional, PCM-based route for SAS-6 centriolar loading conserved from fission yeast to animals.","evidence":"Yeast two-hybrid, co-IP, ectopic expression in fission yeast, centriole elongation assay","pmids":["31182187"],"confidence":"Medium","gaps":["Relative contribution of pericentrin- versus STIL-mediated recruitment unclear","Single-lab finding; independent confirmation in mammalian cells needed"]},{"year":2020,"claim":"Mapping the SAS-6 C-terminus–γ-TuRC interaction and showing that C-terminal deletion prevents daughter centriole microtubule nucleation unified the cartwheel scaffold and microtubule nucleation functions of SAS-6, while HDX-MS of Sas6–Gorab and Sas6–Ana2 complexes defined separable binding interfaces on the coiled-coil allowing simultaneous partner engagement.","evidence":"Co-IP, deletion mutagenesis, super-resolution microscopy (human cells); HDX-MS and EM of Drosophila complexes","pmids":["32442461","33704067","33171067"],"confidence":"High","gaps":["Atomic structure of SAS-6 C-tail–γ-TuRC complex not determined","Whether Gorab interaction is conserved in mammals unknown"]},{"year":2022,"claim":"Solving crystal structures of SAS-6 coiled-coil complexes revealed an asymmetric homo-oligomerization that imparts polarity to the cartwheel stack, explaining how directional growth of the cartwheel is encoded in SAS-6 itself.","evidence":"X-ray crystallography and cryo-EM reconstitution of Chlamydomonas SAS-6 coiled-coil with mutagenesis","pmids":["35240058"],"confidence":"High","gaps":["How polarity information is read out by downstream assembly factors is unknown","In vivo consequences of polarity disruption not tested in animal cells"]},{"year":2024,"claim":"Genetic knockout in mouse demonstrated that SAS-6 is essential for centriole formation in embryos (causing mid-gestation arrest) but dispensable in mESCs where elevated PLK4 enables a SAS-6-independent pathway, revealing context-dependent essentiality.","evidence":"Mouse genetic knockout, embryo phenotyping, mESC culture with PLK4 manipulation","pmids":["38407237"],"confidence":"High","gaps":["Mechanism of PLK4-dependent SAS-6-independent centriole assembly unknown","Whether SAS-6-independent pathway operates in any normal physiological context untested"]},{"year":2025,"claim":"Identifying FBXW7-mediated degradation of the STIL–SAS-6 complex as a PLK4-dependent opposing mechanism and APC/C-Cdh1 as a cell-cycle-timed SAS-6 degradation pathway, together with CDK-1 phosphorylation that inhibits SAS-6 phase separation, defined three layers of post-translational control ensuring centriole number fidelity and developmental centriole disassembly.","evidence":"FBXW7 siRNA/co-IP/ubiquitination assays in human cells; non-degradable SAS-6 mutant with YAP/TAZ pathway analysis; in vitro phase separation plus CDK-1 kinase assay and C. elegans oogenesis phenotypes","pmids":["41453690","40825584","40410380"],"confidence":"High","gaps":["Structural basis of APC/C-Cdh1 recognition of SAS-6 not defined","How CDK-1 phosphorylation coordinates with FBXW7 and APC/C pathways is unknown","Whether SAS-6 phase separation occurs in somatic mammalian cells in vivo remains untested"]},{"year":null,"claim":"Key unresolved questions include the complete atomic structure of a stacked mammalian cartwheel in situ, the mechanism by which the mother centriole templates structural fidelity, and how the PLK4-dependent SAS-6-independent centriole assembly pathway operates at the molecular level.","evidence":"","pmids":[],"confidence":"High","gaps":["No in situ atomic-resolution structure of the assembled mammalian cartwheel","Template-based error-correction mechanism during canonical duplication undefined","SAS-6-independent centriole biogenesis pathway molecularly uncharacterized"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[1,3,4,6,7,12,13,22]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[10,18]}],"localization":[{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[0,1,9,17,18]}],"pathway":[{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[0,1,11,13,23]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[9,15,24,25]}],"complexes":["cartwheel","STIL-SAS-6 complex"],"partners":["STIL","PLK4","PCNT","TUBG1","GORAB","FBXW7","CDC6"],"other_free_text":[]},"mechanistic_narrative":"SASS6 (SAS-6) is the master structural scaffold of the centriolar cartwheel, whose self-assembly into 9-fold symmetric ring polymers establishes the fundamental radial symmetry of centrioles and is essential for centriole biogenesis across eukaryotes. The N-terminal globular domain mediates head-to-head dimerization that drives ring formation, while the coiled-coil domain enables asymmetric homo-oligomerization that imparts polarity and supports ring stacking into the cartwheel hub; point mutations at either interface abolish centriole assembly in vivo [PMID:21273447, PMID:35240058, PMID:26002084]. SAS-6 is recruited to the mother centriole through direct binding of the ZYG-1/PLK4 kinase and PLK4-phosphorylated SAS-5/Ana2/STIL to its coiled-coil region, and its C-terminal tail anchors the γ-tubulin ring complex to nucleate centriolar microtubules [PMID:23673331, PMID:25264260, PMID:32442461]. SAS-6 protein levels are tightly controlled by APC/C-Cdh1 degradation in late mitosis/G1 and FBXW7-mediated ubiquitination of the STIL–SAS-6 complex to prevent centriole overduplication, while CDK-1 phosphorylation of its C-terminus inhibits phase separation to promote timely centriole disassembly during oogenesis [PMID:40825584, PMID:41453690, PMID:40410380]."},"prefetch_data":{"uniprot":{"accession":"Q6UVJ0","full_name":"Spindle assembly abnormal protein 6 homolog","aliases":["Spindle assembly defective protein 6"],"length_aa":657,"mass_kda":74.4,"function":"Central scaffolding component of the centrioles ensuring their 9-fold symmetry (By similarity). Required for centrosome biogenesis and duplication: required both for mother-centriole-dependent centriole duplication and deuterosome-dependent centriole amplification in multiciliated cells (PubMed:15665853, PubMed:16244668, PubMed:17681131). Not required for centriole formation in embryonic stem cells but necessary to maintain centriole architecture (By similarity). Required for the recruitment of STIL to the procentriole and for STIL-mediated centriole amplification (PubMed:22020124). Overexpression results in excess foci-bearing centriolar markers (PubMed:15665853)","subcellular_location":"Cytoplasm, cytoskeleton, microtubule organizing center, centrosome; Cytoplasm, cytoskeleton, microtubule organizing center, centrosome, centriole","url":"https://www.uniprot.org/uniprotkb/Q6UVJ0/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":true,"resolved_as":"","url":"https://depmap.org/portal/gene/SASS6","classification":"Common Essential","n_dependent_lines":1102,"n_total_lines":1208,"dependency_fraction":0.9122516556291391},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/SASS6","total_profiled":1310},"omim":[{"mim_id":"620676","title":"COILED-COIL DOMAIN-CONTAINING PROTEIN 61; CCDC61","url":"https://www.omim.org/entry/620676"},{"mim_id":"620142","title":"CENTROSOMAL AT-AC SPLICING FACTOR; 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In C. elegans, SAS-6 and SAS-5 associate, and this interaction as well as ZYG-1 function is required for SAS-6 centriolar recruitment.\",\n      \"method\": \"siRNA knockdown, GFP localization, co-immunoprecipitation, genetic analysis in C. elegans\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal methods (siRNA, GFP localization, co-IP, genetic epistasis) across two organisms; foundational paper with 329 citations\",\n      \"pmids\": [\"15665853\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"SAS-6 localizes to the central part of the centriolar cartwheel and is required to establish the 9-fold symmetry of centrioles; null mutants in Chlamydomonas (bld12) lack the central cartwheel hub and frequently produce centrioles with aberrant triplet numbers (7, 8, 10, or 11 instead of 9).\",\n      \"method\": \"Immunolocalization, genetic null mutant analysis, electron microscopy\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct localization plus functional null mutant with defined structural phenotype; 217 citations\",\n      \"pmids\": [\"18082404\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"ZYG-1 kinase phosphorylates SAS-6 at serine 123 in vitro, and this phosphorylation is required for centriole formation in C. elegans embryos in vivo; phosphorylation maintains SAS-6 at the emerging centriole.\",\n      \"method\": \"In vitro kinase assay, phospho-site mutagenesis, in vivo centriole formation assay in C. elegans\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay combined with in vivo mutagenesis and functional readout\",\n      \"pmids\": [\"20059959\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Drosophila SAS-6 self-assembles into stable tetramers in vitro that serve as building blocks for the cartwheel central tubule; EM of centrosomes identifies SAS-6 concentrating at the core of the cartwheel.\",\n      \"method\": \"Biochemistry (size exclusion chromatography), electron microscopy, recombinant protein assembly assay\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro reconstitution of self-assembly combined with EM structural validation\",\n      \"pmids\": [\"20083610\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"The X-ray crystal structure of the SAS-6 N-terminal domain from zebrafish reveals that SAS-6 self-associates in vitro into assemblies resembling cartwheel centers; point mutations at the self-assembly interfaces block centriole formation in vivo, establishing these interactions as essential for cartwheel organization.\",\n      \"method\": \"X-ray crystallography, in vitro self-assembly assay, point mutagenesis with in vivo functional validation\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus mutagenesis plus in vivo functional validation; 263 citations\",\n      \"pmids\": [\"21273447\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"ZYG-1 directly binds the SAS-6 coiled coil and recruits SAS-6 to the mother centriole independently of its kinase activity; ZYG-1 kinase activity is subsequently required for cartwheel assembly. The SAS-6 coiled coil also interacts with SAS-5, and both interactions are required for SAS-6 centriolar recruitment.\",\n      \"method\": \"Direct binding assay, reciprocal co-immunoprecipitation, mutagenesis, in vivo recruitment assay in C. elegans\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — direct binding demonstrated, reciprocal interaction validated, combined with in vivo epistasis\",\n      \"pmids\": [\"23673331\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"C. elegans SAS-6 self-assembles into a spiral arrangement (rather than a ring) in vitro, as revealed by X-ray crystallography and EM; the spiral is consistent with 9-fold symmetry and provides a structural basis for the central tube found in nematode centriole assembly.\",\n      \"method\": \"X-ray crystallography, electron microscopy, structural analysis\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus EM reconstitution\",\n      \"pmids\": [\"23798409\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Leishmania major SAS-6 crystallizes as a 9-fold symmetric cartwheel; the X-ray structure demonstrates that SAS-6 self-assembly alone can dictate cartwheel symmetry. SAS-6 oligomerization can be inhibited by a small molecule in vitro.\",\n      \"method\": \"X-ray crystallography (3.5 Å resolution), small-molecule inhibition of oligomerization in vitro\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — high-resolution crystal structure of full cartwheel plus biochemical inhibition assay\",\n      \"pmids\": [\"24596152\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"In Drosophila, PLK4 phosphorylates four conserved serines in the STAN motif of Ana2 (the SAS-6 recruitment factor) to enable Ana2 to bind and recruit Sas6 to the procentriole; non-phosphorylatable Ana2 localizes to the centriole but cannot recruit Sas6, blocking centriole duplication.\",\n      \"method\": \"In vitro kinase assay, phospho-site mutagenesis, in vivo centriole duplication assay, co-immunoprecipitation\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay combined with mutagenesis and in vivo functional phenotype\",\n      \"pmids\": [\"25264260\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"During S phase, SAS-6 is first recruited to the proximal lumen of the mother centriole, adopting a cartwheel-like organization through interactions with the luminal wall rather than via self-oligomerization; removal of luminal SAS-6 requires PLK4 and STIL, and abolishing either recruitment or removal hinders SAS-6 assembly at the outer wall.\",\n      \"method\": \"Live-cell imaging, FRAP, siRNA knockdown, structured illumination microscopy\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — live imaging with functional epistasis (PLK4 and STIL dependence) and multiple orthogonal methods\",\n      \"pmids\": [\"25017693\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"The C-terminal tail of human SAS-6 (residues 470–657) nucleates and promotes microtubule polymerization in vitro, binds microtubules and tubulin dimers directly, and co-precipitates with microtubules from S-phase synchronized HeLa cell lysates; the N-terminal domain does not affect microtubule polymerization.\",\n      \"method\": \"In vitro microtubule polymerization assay, microtubule pulldown, co-immunoprecipitation, isothermal titration calorimetry, size exclusion chromatography\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple in vitro reconstitution assays plus ITC and co-IP from cell lysates\",\n      \"pmids\": [\"26422590\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"De novo centriole formation in human cells can occur in the absence of SAS-6 self-oligomerization, whereas canonically duplicated centrioles always form correctly; de novo centrioles are structurally error-prone even with SAS-6 self-oligomerization, indicating a template/mother-centriole requirement for structural fidelity.\",\n      \"method\": \"Reconstitution of de novo centriole synthesis in human cells, mutagenesis of SAS-6 self-oligomerization interface, EM structural analysis\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — reconstitution in human cells with oligomerization-dead mutants and structural readout\",\n      \"pmids\": [\"26609813\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"Drosophila Sas-6 N-terminal domain forms higher-order oligomers through canonical interactions (crystal structure at 2.9 Å); point mutations that perturb Sas-6 homo-oligomerization in vitro strongly impair centriole assembly in vivo.\",\n      \"method\": \"X-ray crystallography, in vitro oligomerization assay, in vivo centriole assembly assay with point mutants\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus mutagenesis with in vitro and in vivo validation\",\n      \"pmids\": [\"26002084\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Engineering Chlamydomonas SAS-6 to form different symmetry oligomers in vitro reveals that SAS-6 self-assembly properties instruct cartwheel symmetry, and that the cartwheel and microtubule wall assemble interdependently. Expressing analogous SAS-6 mutations in human cells produced 9-fold symmetric centrioles with impaired length and organization.\",\n      \"method\": \"SAS-6 mutagenesis, in vitro oligomerization assay, in vivo centriole symmetry analysis by EM, human cell expression\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — engineered mutants with defined symmetry tested in vitro and in vivo with structural readout\",\n      \"pmids\": [\"26999736\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"In Drosophila, PLK4 performs two-step phosphorylation of Ana2: first at S38 in the ANST motif to promote Ana2 centriolar recruitment, then at four serines in the STAN motif to enable Ana2 to recruit Sas6. Mutation of S38 to alanine blocks Ana2 loading and centriole duplication.\",\n      \"method\": \"In vitro kinase assay, phospho-site mutagenesis, in vivo centriole duplication assay, live-cell imaging\",\n      \"journal\": \"Open biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay with mutagenesis and in vivo functional validation\",\n      \"pmids\": [\"29263250\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"CDC6, a DNA replication licensing factor, is recruited to the proximal centriole via cyclin A and inhibits centrosome duplication by binding SAS-6 and preventing formation of a stable SAS-6–STIL complex; PLK4 phosphorylates CDC6 to disrupt the CDC6–SAS-6 interaction, relieving inhibition.\",\n      \"method\": \"Co-immunoprecipitation, centrosome localization assay, overexpression/knockdown experiments, phosphorylation assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP with functional rescue, but single-lab study\",\n      \"pmids\": [\"28447620\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"High-speed photothermal off-resonance AFM (PORT) reveals the kinetics of SAS-6 ring formation in real time and demonstrates that distinct biogenesis routes (different assembly intermediates) can converge to produce a 9-fold symmetric ring structure.\",\n      \"method\": \"High-speed atomic force microscopy (PORT), real-time self-assembly monitoring\",\n      \"journal\": \"Nature nanotechnology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — direct real-time structural visualization of assembly with kinetic analysis\",\n      \"pmids\": [\"29784964\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"Pericentrin (Pcp1) interacts with and recruits SAS-6 to the pericentriolar material; this interaction is conserved between fission yeast SPB and animal centrosomes and is important for centriole assembly, particularly elongation. The calmodulin-binding region of pericentrin is critical for SAS-6 interaction.\",\n      \"method\": \"Yeast two-hybrid, co-immunoprecipitation, ectopic expression in fission yeast, functional centriole elongation assay\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — reciprocal interaction validated across organisms with functional consequence, single lab\",\n      \"pmids\": [\"31182187\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Human SAS-6 C-terminus interacts with the γ-tubulin ring complex (γ-TuRC) at the centrosome; deletion of the SAS-6 C-terminus disrupts microtubule formation in daughter centrioles causing failure to form new centrioles. High-resolution microscopy reveals γ-tubulin localizing as multiple lobes surrounding the SAS-6-containing central hub.\",\n      \"method\": \"Co-immunoprecipitation, deletion mutagenesis, super-resolution microscopy, siRNA knockdown with phenotypic readout\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — co-IP, deletion mutagenesis, and super-resolution imaging with defined functional phenotype, multiple orthogonal approaches\",\n      \"pmids\": [\"32442461\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"The Drosophila Golgi protein Gorab binds as a monomer to the parallel coiled-coil dimer of Sas6, forming a stable heterotrimer visualized by EM; mutation of a single leucine in Sas6's Gorab-binding domain reduces affinity 16-fold and abolishes centriole duplication, revealing a functional Sas6–Gorab interaction required for duplication.\",\n      \"method\": \"Hydrogen-deuterium exchange mass spectrometry, electron microscopy, mutagenesis, in vivo centriole duplication assay\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — HDX-MS structural definition of interaction interface plus EM visualization plus in vivo mutagenesis\",\n      \"pmids\": [\"33704067\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"HDX-MS of the Drosophila Ana2–Sas6 complex identifies short critical regions in the C-terminal parts of both proteins as the interaction interface; phosphomimetic Ana2 (Ana2-4D) forms a complex with Sas6 in vitro and in vivo, and mutations in these regions disrupt the Ana2–Sas6 interaction. The Sas6 site for Ana2 binding is distinct from the Gorab-binding site, allowing simultaneous binding.\",\n      \"method\": \"Hydrogen-deuterium exchange mass spectrometry, co-immunoprecipitation in vitro and in vivo, mutagenesis\",\n      \"journal\": \"Open biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — HDX-MS interaction interface mapping combined with mutagenesis and co-IP validation\",\n      \"pmids\": [\"33171067\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Monobodies developed against Chlamydomonas SAS-6 (characterized by X-ray crystallography, AFM, and cryo-EM) reveal distinct interaction modes; one monobody (MBCRS6-15) induces a conformational change causing SAS-6 to form a helix instead of a ring, and expression of this monobody in human cells impairs centriole biogenesis.\",\n      \"method\": \"X-ray crystallography, atomic force microscopy, cryo-EM, human cell centriole biogenesis assay\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — multiple structural methods plus in vivo functional validation in human cells\",\n      \"pmids\": [\"34155202\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Crystal structures of Chlamydomonas SAS-6 coiled-coil complexes reveal an asymmetric homo-oligomerization interaction that imparts polarity to the cartwheel; cryo-EM reconstitution assay demonstrates that amino acid substitutions disrupting this asymmetric association impair SAS-6 ring stacking.\",\n      \"method\": \"X-ray crystallography, cryo-EM reconstitution assay, mutagenesis of coiled-coil\",\n      \"journal\": \"Structure (London, England : 1993)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — crystal structure plus cryo-EM functional validation with mutagenesis\",\n      \"pmids\": [\"35240058\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In mouse embryos, Sass6 (SASS6) is essential for centriole formation; Sass6-mutant embryos lack centrioles, activate the mitotic surveillance cell death pathway, and arrest at mid-gestation. In mouse embryonic stem cells (mESCs), SAS-6 is not required for centriole formation but is essential for centriole architecture maintenance; elevated PLK4 activity in mESCs enables a SAS-6-independent centriole biogenesis pathway.\",\n      \"method\": \"Genetic knockout in mouse, embryo phenotypic analysis, mESC culture, PLK4 activity manipulation, centrosome/centriole imaging\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 — clean genetic knockout in mouse with defined cellular and developmental phenotypes, mechanistic epistasis with PLK4\",\n      \"pmids\": [\"38407237\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FBXW7 E3 ubiquitin ligase mediates degradation of the STIL–SAS6 cartwheel assembly in a PLK4 kinase activity-dependent manner; PLK4 phosphorylation of STIL drives both STIL–SAS6 interaction (promoting new centriole assembly) and FBXW7 binding to STIL (promoting STIL–SAS6 degradation), creating an opposing mechanism that prevents centriole overduplication.\",\n      \"method\": \"siRNA knockdown of FBXW7, co-immunoprecipitation, ubiquitination assay, PLK4 kinase inhibition\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — co-IP with functional assay, but single recent paper; dual role mechanistic model is plausible\",\n      \"pmids\": [\"41453690\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SAS-6 levels are controlled by APC/C-Cdh1-targeted degradation at the end of mitosis/G1; expression of a non-degradable SAS-6 mutant (SAS-6ND) increases ciliation and cell invasion through activation of the YAP/TAZ pathway and nuclear YAP translocation leading to TEAD-dependent transcription. SAS-6-mediated invasion is blocked by YAP downregulation or by blocking ciliogenesis.\",\n      \"method\": \"Non-degradable SAS-6 mutant expression, siRNA knockdown, invasion assay, YAP/TAZ pathway analysis, ciliogenesis assay\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple orthogonal assays in single lab; mechanistic pathway placement via rescue experiments\",\n      \"pmids\": [\"40825584\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SAS-6 undergoes phase separation in vitro; CDK-1 directly phosphorylates SAS-6 at its C-terminus in cells and in vivo, and this phosphorylation inhibits SAS-6 phase separation and weakens interactions between centriolar proteins. Phospho-mimetic and phospho-deficient SAS-6 mutants demonstrate that dynamic phosphorylation controls centrosome assembly during oogenesis and timely centriole disassembly in C. elegans.\",\n      \"method\": \"In vitro phase separation assay, mass spectrometry, in vitro kinase assay, phospho-site mutagenesis, in vivo oogenesis phenotype analysis in C. elegans\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 — in vitro kinase assay plus phase separation reconstitution plus mutagenesis with in vivo functional validation\",\n      \"pmids\": [\"40410380\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SASS6/SAS-6 is an evolutionarily conserved centriolar scaffold protein that self-assembles via its N-terminal globular domain (dimerization) and coiled-coil domain (ring stacking) into 9-fold symmetric ring polymers that stack to form the cartwheel central hub of nascent centrioles; it is recruited to the mother centriole through direct binding to the ZYG-1/PLK4 kinase and its cofactor SAS-5/Ana2/STIL (whose PLK4-mediated phosphorylation in the STAN motif is required for SAS-6 binding), its C-terminal domain anchors the γ-tubulin ring complex to nucleate centriolar microtubules, its levels are controlled by APC/C-Cdh1 degradation and by CDK-1 phosphorylation that inhibits phase separation and promotes centriole disassembly during oogenesis, and FBXW7-mediated degradation of the STIL–SAS-6 complex prevents centriole overduplication—with pericentrin providing an additional PCM-based recruitment route and Gorab/FBXW7 providing regulatory interactions at the centriole.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"SASS6 (SAS-6) is the master structural scaffold of the centriolar cartwheel, whose self-assembly into 9-fold symmetric ring polymers establishes the fundamental radial symmetry of centrioles and is essential for centriole biogenesis across eukaryotes. The N-terminal globular domain mediates head-to-head dimerization that drives ring formation, while the coiled-coil domain enables asymmetric homo-oligomerization that imparts polarity and supports ring stacking into the cartwheel hub; point mutations at either interface abolish centriole assembly in vivo [PMID:21273447, PMID:35240058, PMID:26002084]. SAS-6 is recruited to the mother centriole through direct binding of the ZYG-1/PLK4 kinase and PLK4-phosphorylated SAS-5/Ana2/STIL to its coiled-coil region, and its C-terminal tail anchors the γ-tubulin ring complex to nucleate centriolar microtubules [PMID:23673331, PMID:25264260, PMID:32442461]. SAS-6 protein levels are tightly controlled by APC/C-Cdh1 degradation in late mitosis/G1 and FBXW7-mediated ubiquitination of the STIL–SAS-6 complex to prevent centriole overduplication, while CDK-1 phosphorylation of its C-terminus inhibits phase separation to promote timely centriole disassembly during oogenesis [PMID:40825584, PMID:41453690, PMID:40410380].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Establishing SAS-6 as a conserved centrosome duplication factor resolved the question of which proteins are essential for new centriole formation: SAS-6 is required for centrosome duplication in human cells and C. elegans, and its centriolar recruitment depends on ZYG-1 and SAS-5.\",\n      \"evidence\": \"siRNA knockdown in U2OS cells, co-IP and genetic epistasis in C. elegans\",\n      \"pmids\": [\"15665853\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural role of SAS-6 at the centriole was unknown\", \"Mechanism of SAS-6 self-organization not addressed\", \"Whether SAS-6 dictates centriole symmetry was untested\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Determining that SAS-6 localizes specifically to the cartwheel hub and is required for 9-fold symmetry answered the long-standing question of what specifies centriole radial architecture: loss of SAS-6 produces centrioles with variable triplet numbers.\",\n      \"evidence\": \"Immunolocalization and EM of Chlamydomonas bld12 null mutants\",\n      \"pmids\": [\"18082404\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of 9-fold symmetry specification unknown\", \"Whether SAS-6 alone is sufficient for symmetry establishment untested\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identifying ZYG-1 phosphorylation of SAS-6 at S123 as essential for centriole formation established that kinase signaling directly regulates the cartwheel scaffold, not just its upstream recruitment factors.\",\n      \"evidence\": \"In vitro kinase assay with phospho-site mutagenesis and in vivo centriole formation in C. elegans\",\n      \"pmids\": [\"20059959\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether phosphorylation affects SAS-6 self-assembly or protein interactions was unclear\", \"Mammalian equivalent phosphosite not identified\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Reconstituting SAS-6 self-assembly into tetramers in vitro demonstrated that SAS-6 is an intrinsic building block of the cartwheel, shifting the model from a passively recruited scaffold to a self-organizing polymer.\",\n      \"evidence\": \"Recombinant Drosophila SAS-6 size-exclusion chromatography and EM\",\n      \"pmids\": [\"20083610\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic-resolution structure of self-assembly interface not yet available\", \"Whether 9-fold ring forms spontaneously in vitro was unknown\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Solving the crystal structure of the SAS-6 N-terminal domain and showing that mutations at the dimerization interface block centriole formation provided the first atomic-level explanation of how SAS-6 self-assembly builds the cartwheel.\",\n      \"evidence\": \"X-ray crystallography of zebrafish SAS-6 with in vitro assembly and in vivo mutagenesis\",\n      \"pmids\": [\"21273447\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Full-ring structure not yet solved\", \"Coiled-coil contribution to stacking unknown\", \"Species-specific variations in symmetry not addressed\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Demonstrating that ZYG-1 directly binds the SAS-6 coiled coil independently of kinase activity, and that SAS-5 binds the same domain, resolved how SAS-6 is physically delivered to the mother centriole through a two-factor recruitment mechanism.\",\n      \"evidence\": \"Direct binding assays, reciprocal co-IP, and in vivo recruitment in C. elegans\",\n      \"pmids\": [\"23673331\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether PLK4 uses the same binding mode in mammals was untested\", \"Stoichiometry of the recruitment complex unknown\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Solving the C. elegans SAS-6 structure as a spiral rather than a planar ring revealed species-specific assembly variants consistent with the nematode central tube architecture, broadening the structural paradigm.\",\n      \"evidence\": \"X-ray crystallography and EM of C. elegans SAS-6\",\n      \"pmids\": [\"23798409\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the spiral converts to an in vivo-functional central tube was unclear\", \"Relationship between spiral and ring assembly pathways unresolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Crystallizing the Leishmania SAS-6 as a complete 9-fold symmetric cartwheel proved that SAS-6 self-assembly alone can dictate cartwheel symmetry, and identification of a small-molecule oligomerization inhibitor opened a pharmacological approach.\",\n      \"evidence\": \"3.5 Å X-ray crystal structure plus in vitro small-molecule inhibition\",\n      \"pmids\": [\"24596152\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo efficacy of the small-molecule inhibitor not tested\", \"Whether mammalian SAS-6 forms equivalent complete rings in vitro was unknown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Showing that PLK4 phosphorylation of Ana2/STIL's STAN motif is the switch enabling SAS-6 recruitment defined the upstream signaling event that licenses cartwheel initiation at the procentriole.\",\n      \"evidence\": \"In vitro kinase assay, phospho-site mutagenesis, in vivo duplication assay in Drosophila; complemented by human S-phase SAS-6 dynamics\",\n      \"pmids\": [\"25264260\", \"25017693\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Order of Ana2 phosphorylation events relative to SAS-6 ring assembly not resolved\", \"Whether SAS-6 luminal recruitment and outer-wall assembly involve different SAS-6 pools was unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identifying that the SAS-6 C-terminal tail directly binds tubulin and nucleates microtubule polymerization revealed a second functional domain beyond the cartwheel-forming N-terminus, linking the cartwheel scaffold to centriolar microtubule wall formation.\",\n      \"evidence\": \"In vitro microtubule polymerization, ITC, co-IP from HeLa lysates\",\n      \"pmids\": [\"26422590\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of C-tail–tubulin interaction not resolved\", \"Whether γ-TuRC or free tubulin is the physiological target was unclear\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Demonstrating that centrioles can form de novo without SAS-6 self-oligomerization but with frequent structural errors, while template-directed duplication always preserves 9-fold symmetry, established that the mother centriole provides an essential fidelity mechanism beyond SAS-6 intrinsic self-assembly.\",\n      \"evidence\": \"Reconstitution of de novo centriole synthesis in human cells with oligomerization-dead SAS-6 mutants and EM analysis\",\n      \"pmids\": [\"26609813\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Nature of the template-based error-correction mechanism unknown\", \"Whether other cartwheel components compensate in the absence of SAS-6 oligomerization unclear\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Engineering SAS-6 variants with altered symmetry properties demonstrated that SAS-6 self-assembly directly instructs cartwheel symmetry and that cartwheel and microtubule wall assembly are interdependent, resolving whether symmetry is SAS-6-intrinsic or templated.\",\n      \"evidence\": \"Symmetry-altering SAS-6 mutations tested in vitro and in vivo in Chlamydomonas and human cells\",\n      \"pmids\": [\"26999736\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How the microtubule wall feeds back on cartwheel symmetry is mechanistically unclear\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identifying CDC6 as a negative regulator that binds SAS-6 and prevents STIL–SAS-6 complex formation until PLK4 phosphorylates CDC6 established a licensing checkpoint coupling DNA replication and centrosome duplication through SAS-6.\",\n      \"evidence\": \"Co-IP, centrosome localization, overexpression/knockdown, PLK4 phosphorylation assay in human cells\",\n      \"pmids\": [\"28447620\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-lab study; independent validation needed\", \"Structural basis of CDC6–SAS-6 interaction unknown\", \"Whether CDC6 regulation is conserved beyond mammals untested\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Real-time visualization of SAS-6 ring assembly by high-speed AFM revealed that multiple kinetically distinct assembly intermediates converge on 9-fold symmetric rings, demonstrating robust self-assembly through parallel pathways.\",\n      \"evidence\": \"Photothermal off-resonance AFM with kinetic analysis of Chlamydomonas SAS-6\",\n      \"pmids\": [\"29784964\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo relevance of alternative assembly routes not established\", \"Whether accessory factors constrain assembly pathways in cells unknown\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Identifying pericentrin as a direct SAS-6-binding partner that recruits SAS-6 to the pericentriolar material revealed an additional, PCM-based route for SAS-6 centriolar loading conserved from fission yeast to animals.\",\n      \"evidence\": \"Yeast two-hybrid, co-IP, ectopic expression in fission yeast, centriole elongation assay\",\n      \"pmids\": [\"31182187\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Relative contribution of pericentrin- versus STIL-mediated recruitment unclear\", \"Single-lab finding; independent confirmation in mammalian cells needed\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Mapping the SAS-6 C-terminus–γ-TuRC interaction and showing that C-terminal deletion prevents daughter centriole microtubule nucleation unified the cartwheel scaffold and microtubule nucleation functions of SAS-6, while HDX-MS of Sas6–Gorab and Sas6–Ana2 complexes defined separable binding interfaces on the coiled-coil allowing simultaneous partner engagement.\",\n      \"evidence\": \"Co-IP, deletion mutagenesis, super-resolution microscopy (human cells); HDX-MS and EM of Drosophila complexes\",\n      \"pmids\": [\"32442461\", \"33704067\", \"33171067\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Atomic structure of SAS-6 C-tail–γ-TuRC complex not determined\", \"Whether Gorab interaction is conserved in mammals unknown\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Solving crystal structures of SAS-6 coiled-coil complexes revealed an asymmetric homo-oligomerization that imparts polarity to the cartwheel stack, explaining how directional growth of the cartwheel is encoded in SAS-6 itself.\",\n      \"evidence\": \"X-ray crystallography and cryo-EM reconstitution of Chlamydomonas SAS-6 coiled-coil with mutagenesis\",\n      \"pmids\": [\"35240058\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"How polarity information is read out by downstream assembly factors is unknown\", \"In vivo consequences of polarity disruption not tested in animal cells\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Genetic knockout in mouse demonstrated that SAS-6 is essential for centriole formation in embryos (causing mid-gestation arrest) but dispensable in mESCs where elevated PLK4 enables a SAS-6-independent pathway, revealing context-dependent essentiality.\",\n      \"evidence\": \"Mouse genetic knockout, embryo phenotyping, mESC culture with PLK4 manipulation\",\n      \"pmids\": [\"38407237\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Mechanism of PLK4-dependent SAS-6-independent centriole assembly unknown\", \"Whether SAS-6-independent pathway operates in any normal physiological context untested\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Identifying FBXW7-mediated degradation of the STIL–SAS-6 complex as a PLK4-dependent opposing mechanism and APC/C-Cdh1 as a cell-cycle-timed SAS-6 degradation pathway, together with CDK-1 phosphorylation that inhibits SAS-6 phase separation, defined three layers of post-translational control ensuring centriole number fidelity and developmental centriole disassembly.\",\n      \"evidence\": \"FBXW7 siRNA/co-IP/ubiquitination assays in human cells; non-degradable SAS-6 mutant with YAP/TAZ pathway analysis; in vitro phase separation plus CDK-1 kinase assay and C. elegans oogenesis phenotypes\",\n      \"pmids\": [\"41453690\", \"40825584\", \"40410380\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural basis of APC/C-Cdh1 recognition of SAS-6 not defined\", \"How CDK-1 phosphorylation coordinates with FBXW7 and APC/C pathways is unknown\", \"Whether SAS-6 phase separation occurs in somatic mammalian cells in vivo remains untested\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the complete atomic structure of a stacked mammalian cartwheel in situ, the mechanism by which the mother centriole templates structural fidelity, and how the PLK4-dependent SAS-6-independent centriole assembly pathway operates at the molecular level.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"High\",\n      \"gaps\": [\"No in situ atomic-resolution structure of the assembled mammalian cartwheel\", \"Template-based error-correction mechanism during canonical duplication undefined\", \"SAS-6-independent centriole biogenesis pathway molecularly uncharacterized\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [1, 3, 4, 6, 7, 12, 13, 22]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [10, 18]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [0, 1, 9, 17, 18]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [0, 1, 11, 13, 23]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [9, 15, 24, 25]}\n    ],\n    \"complexes\": [\n      \"cartwheel\",\n      \"STIL-SAS-6 complex\"\n    ],\n    \"partners\": [\n      \"STIL\",\n      \"PLK4\",\n      \"PCNT\",\n      \"TUBG1\",\n      \"GORAB\",\n      \"FBXW7\",\n      \"CDC6\"\n    ],\n    \"other_free_text\": []\n  }\n}\n```"}