{"gene":"SASS6","run_date":"2026-06-10T07:46:29","timeline":{"discoveries":[{"year":2005,"finding":"HsSAS-6 (SASS6) localizes to centrosomes and is required for centrosome duplication in human cells: 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 is recruited to centrioles at the onset of the centrosome duplication cycle, associates with SAS-5, and requires both the SAS-5 interaction and ZYG-1 function for centriolar recruitment.","method":"siRNA knockdown in human U2OS cells, GFP localization, C. elegans genetics, co-immunoprecipitation","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal interactions, genetic epistasis in C. elegans, and direct functional readout in human cells; foundational paper replicated across multiple subsequent studies","pmids":["15665853"],"is_preprint":false},{"year":2007,"finding":"In Chlamydomonas, SAS-6 localizes to the central part of the cartwheel and is required to establish nine-fold centriolar symmetry. A null mutant (bld12) lacking the cartwheel central part frequently produces centrioles with non-canonical triplet numbers (7, 8, 10, or 11), demonstrating that SAS-6 is an essential cartwheel component that stabilizes the 9-triplet structure.","method":"Chlamydomonas null mutant analysis, electron microscopy, immunolocalization","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 2 / Strong — genetic null mutant with quantitative structural readout by EM, independently consistent with multiple subsequent studies on SAS-6 symmetry","pmids":["18082404"],"is_preprint":false},{"year":2009,"finding":"The kinase ZYG-1 phosphorylates SAS-6 at serine 123 in vitro, and this phosphorylation event is critical for centriole formation in C. elegans embryos in vivo. Phosphorylation ensures maintenance of SAS-6 at the emerging centriole.","method":"In vitro kinase assay, C. elegans genetics with phospho-mutant rescue, fluorescence microscopy","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay combined with in vivo phospho-mutant functional validation; single lab but multiple orthogonal methods","pmids":["20059959"],"is_preprint":false},{"year":2010,"finding":"Drosophila SAS-6 self-assembles into stable tetramers in vitro, which serve as building blocks for the central tubule of the centriolar cartwheel. SAS-6 concentrates at the core of the cartwheel, and elevated SAS-6 levels in Drosophila cells produce higher-order structures resembling central tubule morphology.","method":"Biochemistry (gel filtration, native PAGE), electron microscopy of centrosomes and recombinant protein, cell overexpression","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro reconstitution and EM characterization by single lab, consistent with structural studies but specific tetramer model later superseded/complemented by ring models","pmids":["20083610"],"is_preprint":false},{"year":2011,"finding":"X-ray crystal structure of the zebrafish SAS-6 N-terminal domain reveals that recombinant SAS-6 self-associates in vitro into assemblies resembling cartwheel centers. Point mutations disrupting the self-assembly interfaces impair centriole formation in vivo, establishing that these interactions are essential for cartwheel center organization.","method":"X-ray crystallography, in vitro reconstitution, point mutagenesis with in vivo centriole formation assay","journal":"Science (New York, N.Y.)","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with mutagenesis and in vivo functional validation; foundational structural paper replicated by multiple subsequent structural studies","pmids":["21273447"],"is_preprint":false},{"year":2013,"finding":"In C. elegans, ZYG-1 recruits SAS-6 to the mother centriole via a direct binding interaction between ZYG-1 and the SAS-6 coiled coil, independently of ZYG-1 kinase activity. Separately, an adjacent segment of the SAS-6 coiled coil interacts with SAS-5, and both interactions are required for SAS-6 recruitment and cartwheel assembly. ZYG-1 kinase activity is subsequently required for cartwheel assembly, but its essential substrate is unlikely to be SAS-6 itself.","method":"Pulldown, co-immunoprecipitation, in vitro binding assays, C. elegans genetics with alanine-substitution mutants","journal":"Developmental cell","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, direct binding assay, genetic epistasis with systematic mutagenesis; multiple orthogonal methods in single rigorous study","pmids":["23673331"],"is_preprint":false},{"year":2013,"finding":"C. elegans SAS-6 self-assembles into a spiral arrangement (rather than rings) as shown by crystallography and EM, yet this spiral is consistent with nine-fold symmetry, suggesting two distinct SAS-6 oligomerization architectures can direct the same output symmetry. Spiral arrangement is correlated with the presence of a central tube instead of a cartwheel in nematode centriole assembly.","method":"X-ray crystallography, electron microscopy","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"High","confidence_rationale":"Tier 1 / Moderate — crystal structure and EM with multiple orthogonal methods; single lab but structurally rigorous","pmids":["23798409"],"is_preprint":false},{"year":2014,"finding":"Drosophila Plk4 phosphorylates four conserved serines in the STAN motif of Ana2 (STIL ortholog) to enable Ana2 to bind and recruit its Sas6 partner. Non-phosphorylatable Ana2 localizes to the centriole but cannot recruit Sas6, causing failure of centriole duplication. Thus, Plk4-mediated phosphorylation of Ana2/STIL is the earliest upstream step for Sas6 recruitment and procentriole architecture establishment.","method":"In vitro kinase assay, Drosophila genetics with phospho-mutant rescue, live imaging, immunofluorescence","journal":"Current biology : CB","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay plus in vivo phospho-mutant analysis with functional centriole readout; replicated in subsequent studies","pmids":["25264260"],"is_preprint":false},{"year":2014,"finding":"Leishmania major SAS-6 crystallizes as a nine-fold symmetric cartwheel, providing a 3.5 Å X-ray structure of this assembly and firmly establishing that SAS-6 self-assembly alone can impose cartwheel symmetry. Small-molecule inhibition of SAS-6 oligomerization is feasible in vitro.","method":"X-ray crystallography at 3.5 Å, in vitro small-molecule inhibition assay","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution crystal structure of the nine-fold symmetric assembly; structurally definitive","pmids":["24596152"],"is_preprint":false},{"year":2014,"finding":"During S phase, SAS-6 molecules are 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/release of luminal SAS-6 requires Plk4 and STIL. Abolishing either recruitment or removal of luminal SAS-6 hinders SAS-6/centriole assembly at the outside wall. After duplication, the lumen of engaged mother centrioles becomes inaccessible to SAS-6, correlating with a block for reduplication.","method":"Live cell fluorescence microscopy, siRNA knockdown, structured illumination microscopy","journal":"Developmental cell","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct localization with functional consequence, epistasis with Plk4 and STIL, single lab with multiple imaging modalities","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 to microtubules along their lengths, and interacts with α/β-tubulin dimers. The N-terminal domain has no effect on microtubule polymerization. Endogenous HsSAS-6 co-precipitates with microtubules from S-phase HeLa cell lysates.","method":"In vitro tubulin polymerization assay, microtubule pulldown, co-immunoprecipitation from cell lysate, isothermal calorimetry, size-exclusion chromatography","journal":"Biochemistry","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — multiple in vitro assays (ITC, pulldown, polymerization) with domain dissection, single lab","pmids":["26422590"],"is_preprint":false},{"year":2015,"finding":"In vitro, both Drosophila Sas-6 N-terminal domain homo-oligomerization and Ana2 CCCD tetramerization are required for efficient centriole assembly in vivo. Point mutations that perturb Sas-6 homo-oligomerization in vitro strongly impair centriole assembly in Drosophila. The Ana2 CCCD forms a tetramer with an unusual parallel-coil topology (structure solved to 0.8 Å), and the Sas-6 N-terminal domain forms higher-order oligomers through canonical interactions (structure at 2.9 Å).","method":"X-ray crystallography (0.8 Å and 2.9 Å), in vitro oligomerization assays, in vivo Drosophila genetics with point mutants","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — high-resolution crystal structures combined with mutagenesis and in vivo functional validation; multiple orthogonal methods","pmids":["26002084"],"is_preprint":false},{"year":2015,"finding":"De novo centriole formation in human cells can occur in the absence of SAS-6 self-oligomerization, demonstrating that centriole biogenesis does not strictly depend on SAS-6 self-assembly. Canonically duplicated centrioles always form correctly, whereas de novo centrioles are prone to structural errors even when SAS-6 self-oligomerization is intact.","method":"Reconstitution of de novo centriole synthesis in human cells, SAS-6 oligomerization-deficient mutants, electron microscopy","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reconstitution in human cells with mutant analysis and structural EM readout; single lab, challenges prior paradigm","pmids":["26609813"],"is_preprint":false},{"year":2016,"finding":"Engineering Chlamydomonas SAS-6 to form oligomers with symmetries ranging from five- to ten-fold showed that SAS-6 self-assembly properties instruct cartwheel symmetry. A SAS-6 mutant forming six-fold symmetric cartwheels in vitro produced eight- or nine-fold cartwheels in vivo, and with Bld10 mutants weakening cartwheel-microtubule interactions, produced six- to eight-fold cartwheels. The microtubule wall maintained eight- and nine-fold symmetries, indicating cartwheel and microtubule wall assemble interdependently. Human cells expressing analogous SAS-6 mutations formed nine-fold centrioles with impaired length and organization.","method":"In vitro oligomerization assays, Chlamydomonas and human cell expression of engineered SAS-6 mutants, electron microscopy","journal":"Nature cell biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — direct engineering of SAS-6 symmetry in vitro with in vivo structural readout across two organisms; multiple orthogonal methods in single rigorous study","pmids":["26999736"],"is_preprint":false},{"year":2017,"finding":"Drosophila Plk4 first phosphorylates a single serine (S38) in the conserved ANST motif of Ana2 to promote Ana2 recruitment to the centriole, and then phosphorylates four serines in the STAN motif to enable Ana2 to recruit Sas6. Non-phosphorylatable S38A Ana2 fails to load onto the procentriole and blocks centriole duplication, establishing a sequential two-step phosphorylation mechanism for Sas6 recruitment.","method":"In vitro kinase assay, mass spectrometry, Drosophila genetics with phospho-mutant rescue, live imaging","journal":"Open biology","confidence":"High","confidence_rationale":"Tier 1 / Strong — in vitro kinase assay with mass spectrometry identification of phospho-sites and in vivo genetic validation; replication of prior STAN motif work with novel ANST discovery","pmids":["29263250"],"is_preprint":false},{"year":2017,"finding":"The DNA replication licensing factor Cdc6 is recruited to the proximal side of centrioles via cyclin A and negatively regulates centrosome duplication by binding Sas-6 and inhibiting its interaction with STIL. Plk4 phosphorylates Cdc6, disrupting the Sas-6–Cdc6 interaction and thereby counteracting the inhibitory effect of Cdc6 on Sas-6. Overexpression of wild-type Cdc6 or a Plk4-unphosphorylatable Cdc6 mutant reduces centrosome over-duplication.","method":"Co-immunoprecipitation, co-localization, siRNA knockdown, overexpression with functional centrosome duplication readout","journal":"Nature communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, epistasis with phospho-mutants, functional rescue; single lab","pmids":["28447620"],"is_preprint":false},{"year":2018,"finding":"High-speed atomic force microscopy (photothermal off-resonance tapping) reveals the kinetics of SAS-6 ring formation and demonstrates that distinct biogenesis routes can be followed to assemble a nine-fold symmetrical ring structure, showing the assembly reaction is driven by weak interactions on a surface.","method":"High-speed atomic force microscopy (PORT), kinetic analysis of self-assembly","journal":"Nature nanotechnology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — direct single-molecule imaging of assembly kinetics with novel methodology; single lab, single method type","pmids":["29784964"],"is_preprint":false},{"year":2019,"finding":"The conserved PCM component Pcp1/pericentrin directly interacts with and recruits SAS-6. This interaction is conserved and important for centriole assembly, particularly centriole elongation. Calmodulin-binding region of Pcp1/pericentrin is critical for SAS-6 interaction.","method":"Ectopic expression in fission yeast, co-immunoprecipitation, genetic assays in animal cells","journal":"eLife","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct protein interaction demonstrated, with functional consequence on centriole assembly; single lab, multiple organisms tested","pmids":["31182187"],"is_preprint":false},{"year":2020,"finding":"Human SAS-6 C-terminus is required for centriolar microtubule formation by interacting with the γ-tubulin ring complex (γ-TuRC). Deletion of HsSAS-6 C-terminus disrupts microtubule formation in daughter centrioles, resulting in cells with only two centrioles at a single site. SAS-6 associates with γ-TuRC proteins at the centrosome, and high-resolution microscopy reveals γ-tubulin as multiple lobes surrounding the HsSAS-6-containing central hub.","method":"Co-immunoprecipitation, deletion mutant analysis, siRNA knockdown, high-resolution fluorescence microscopy","journal":"Current biology : CB","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP with domain mapping, deletion mutant functional readout, high-resolution imaging; single lab, multiple methods","pmids":["32442461"],"is_preprint":false},{"year":2020,"finding":"Interaction surfaces between Drosophila Ana2 and Sas6 lie in the C-terminal parts of both proteins, as identified by hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) and confirmed by mutagenesis. The Sas6 site required for Ana2 binding is distinct from the site required for Gorab binding, and Sas6 can simultaneously bind both Ana2 and Gorab.","method":"HDX-MS, in vitro complex formation, mutagenesis, co-immunoprecipitation","journal":"Open biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — HDX-MS interface mapping with mutational confirmation; single lab, multiple orthogonal methods","pmids":["33171067"],"is_preprint":false},{"year":2021,"finding":"Monomeric Drosophila Gorab binds Sas6 via an antiparallel interaction between a segment of Gorab's coiled-coil and the parallel coiled-coil dimer of Sas6, forming a stable heterotrimer visible by EM. Mutation of a single leucine in Sas6's Gorab-binding domain reduces affinity 16-fold and abolishes centriole duplication, demonstrating this interaction is essential. Gorab dimers at the Golgi exist in equilibrium with Sas6-associated Gorab monomers at the centriole.","method":"HDX-MS, electron microscopy, mutagenesis with in vivo centriole duplication assay, biochemical binding assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 1 / Strong — structural (EM) plus HDX-MS interface mapping plus mutagenesis with functional in vivo validation; multiple orthogonal methods","pmids":["33704067"],"is_preprint":false},{"year":2021,"finding":"Monobodies against Chlamydomonas SAS-6 characterized by X-ray crystallography, AFM, and cryo-EM reveal distinct interaction modes that specifically impair ring assembly or ring stacking. Monobody MBCRS6-15 induces a conformational change converting CrSAS-6 from ring to helix conformation, and this alteration impairs centriole biogenesis in human cells.","method":"X-ray crystallography, atomic force microscopy, cryo-EM, human cell centriole biogenesis assay","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 1 / Strong — three orthogonal structural methods plus functional validation in human cells; mechanistically precise","pmids":["34155202"],"is_preprint":false},{"year":2022,"finding":"Crystallographic structures of the Chlamydomonas reinhardtii SAS-6 coiled-coil domain reveal an asymmetric homo-oligomerization interaction. Using cryo-EM reconstitution, amino acid substitutions disrupting this asymmetric association impair SAS-6 ring stacking, suggesting the coiled-coil asymmetric interaction provides polarity to the cartwheel and may assist establishment of the centriolar proximal-distal axis.","method":"X-ray crystallography, cryo-EM reconstitution assay, site-directed mutagenesis","journal":"Structure (London, England : 1993)","confidence":"High","confidence_rationale":"Tier 1 / Strong — crystal structure combined with cryo-EM functional assay and mutagenesis; multiple orthogonal structural and biochemical methods","pmids":["35240058"],"is_preprint":false},{"year":2022,"finding":"In C. elegans, the chromatin remodeling protein CHD-1 and the transcription factor EFL-1/DPL-1 cooperate to downregulate CDK-2, which in turn controls SAS-6 protein levels. Loss of CHD-1 increases SAS-6 levels and produces extra centrioles, revealing a transcriptional/post-translational axis for controlling centriole number via SAS-6 abundance.","method":"C. elegans genetics (epistasis), RNAi, Western blot for protein levels, centriole counting","journal":"PLoS genetics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic epistasis in C. elegans with protein-level confirmation; single lab, multiple genetic combinations tested","pmids":["35377871"],"is_preprint":false},{"year":2024,"finding":"In mouse embryos, Sass6 (SASS6) is required for centriole formation, and 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 de novo centriole formation but is essential to maintain centriole architecture. High PLK4 activity and elevated centrosomal protein levels in mESCs enable SAS-6-independent centriole biogenesis.","method":"Mouse knockout genetics, immunofluorescence, centriole ultrastructure analysis, cell death pathway assays","journal":"eLife","confidence":"High","confidence_rationale":"Tier 2 / Strong — mouse knockout with in vivo embryo phenotype plus mechanistic epistasis with PLK4; multiple cellular contexts tested","pmids":["38407237"],"is_preprint":false},{"year":2025,"finding":"SAS-6 undergoes phase separation in vitro and forms droplets when overexpressed in cells. CDK-1 directly phosphorylates SAS-6 at its C-terminus (identified by mass spectrometry and kinase assays), which inhibits SAS-6 phase separation and weakens interactions between centriolar proteins. Phospho-mimetic and phospho-deficient mutants demonstrate that dynamic SAS-6 phosphorylation is essential for centrosome assembly during early meiotic prophase and for centrosome elimination during late meiotic prophase (oogenesis) in C. elegans.","method":"In vitro phase separation assay, mass spectrometry, in vitro kinase assay, C. elegans genetics with phospho-mutants, live imaging","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — in vitro kinase assay and phase separation reconstitution combined with in vivo phospho-mutant genetics; single lab, multiple methods","pmids":["40410380"],"is_preprint":false},{"year":2025,"finding":"FBXW7 E3 ubiquitin ligase mediates degradation of the STIL-SAS6 cartwheel assembly complex. Plk4 kinase activity is required for FBXW7-mediated STIL-SAS6 degradation. The same Plk4-phosphorylated sites in STIL that promote STIL-SAS6 interaction for centriole assembly also stabilize FBXW7 binding to STIL, creating a dual mechanism: phosphorylation promotes assembly and then triggers destruction to prevent centriole overduplication. Depletion of FBXW7 induces premature centriole duplication through excessive STIL-SAS6 stabilization.","method":"Co-immunoprecipitation, siRNA knockdown, overexpression with centriole counting, ubiquitination assay, Plk4 inhibitor treatment","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple Co-IPs and functional epistasis with Plk4 inhibitor, single lab; degradation mechanism demonstrated biochemically","pmids":["41453690"],"is_preprint":false},{"year":2025,"finding":"Non-degradable SAS-6 (SAS-6ND, escaping APCCdh1-targeted degradation) increases ciliation and cell invasion and upregulates the YAP/TAZ pathway. SAS-6-mediated invasion is prevented by YAP downregulation or by blocking ciliogenesis, placing SAS-6 upstream of YAP/TAZ-dependent transcription in the invasion pathway. SAS-6 levels are subject to APCCdh1-targeted degradation at the end of mitosis and G1.","method":"Non-degradable SAS-6 mutant expression, siRNA knockdown, invasion assays, YAP nuclear translocation imaging, TEAD reporter assay","journal":"Life science alliance","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — non-degradable mutant with epistasis (YAP knockdown rescue), multiple functional readouts; single lab","pmids":["40825584"],"is_preprint":false}],"current_model":"SASS6/SAS-6 is a highly conserved centriolar scaffold protein that self-assembles via its N-terminal head domain dimerization and coiled-coil interactions into nine-fold symmetric ring polymers, which stack (with polarity imposed by an asymmetric coiled-coil interaction) to form the centriolar cartwheel; its recruitment to the mother centriole is initiated by direct binding to ZYG-1/Plk4 (kinase-activity-independent) and to SAS-5/Ana2/STIL, with Plk4-mediated sequential phosphorylation of Ana2/STIL being the earliest trigger for Sas6 loading and procentriole formation; once recruited, SAS-6 promotes centriolar microtubule assembly through its C-terminal tail interacting with γ-TuRC and with tubulin directly; SAS-6 protein levels are controlled by CDK-2-dependent transcriptional mechanisms and by APCCdh1- and FBXW7-mediated ubiquitin-dependent degradation, while CDK-1 phosphorylation of the SAS-6 C-terminus inhibits its phase separation and promotes centrosome elimination during oogenesis; dysregulated (non-degradable) SAS-6 promotes excess ciliation, YAP/TAZ activation, and cell invasion, and loss-of-function mutations in SASS6 cause primary microcephaly (MCPH14) in humans."},"narrative":{"mechanistic_narrative":"SASS6/SAS-6 is a highly conserved centriolar scaffold protein that self-assembles to impose the nine-fold symmetry of the centriolar cartwheel and is required for centriole duplication [PMID:15665853, PMID:18082404]. Its N-terminal head domain self-associates into oligomers whose geometry instructs cartwheel symmetry: high-resolution structures of the N-terminal domain reveal cartwheel-center-like assemblies, point mutations disrupting these interfaces impair centriole formation, and engineering the oligomerization properties of SAS-6 directly reprograms cartwheel symmetry from five- to ten-fold [PMID:21273447, PMID:26002084, PMID:26999736]. SAS-6 self-assembly alone is sufficient to generate a nine-fold symmetric cartwheel in vitro [PMID:24596152], and an asymmetric coiled-coil interaction confers polarity that drives ring stacking along the proximal-distal axis [PMID:35240058]. Recruitment of SAS-6 to the mother centriole is initiated through direct, kinase-activity-independent binding of its coiled coil to ZYG-1/Plk4 and to SAS-5/Ana2/STIL, with Plk4-mediated sequential phosphorylation of Ana2/STIL (first a single ANST-motif serine, then four STAN-motif serines) constituting the earliest trigger for SAS-6 loading and procentriole formation [PMID:23673331, PMID:25264260, PMID:29263250]; ZYG-1 also phosphorylates SAS-6 itself at serine 123 to maintain it at the emerging centriole [PMID:20059959]. Once incorporated, the SAS-6 C-terminal tail nucleates centriolar microtubule assembly by binding α/β-tubulin and the γ-tubulin ring complex [PMID:26422590, PMID:32442461]. SAS-6 abundance is tightly controlled to limit centriole number, via CDK-2-dependent transcriptional input [PMID:35377871] and via ubiquitin-dependent degradation by APC/C-Cdh1 and by FBXW7, the latter acting on the same Plk4-phosphorylated STIL sites that promote assembly [PMID:41453690, PMID:40825584]; CDK-1 phosphorylation of the SAS-6 C-terminus inhibits its phase separation to drive centrosome elimination during oogenesis [PMID:40410380]. Dysregulated, non-degradable SAS-6 promotes excess ciliation, YAP/TAZ activation, and cell invasion [PMID:40825584], and SAS-6 is required for centriole formation in mouse embryos, where its loss arrests development at mid-gestation [PMID:38407237].","teleology":[{"year":2005,"claim":"Established that human SAS-6 is a bona fide centrosomal duplication factor and placed it in a conserved recruitment pathway, answering whether SAS-6 acts in centriole biogenesis and with what partners.","evidence":"siRNA knockdown and GFP localization in human U2OS cells, C. elegans genetics and co-immunoprecipitation","pmids":["15665853"],"confidence":"High","gaps":["Did not define the structural basis of SAS-6 assembly","Mechanism of ZYG-1/SAS-5-dependent recruitment not resolved at the molecular level"]},{"year":2007,"claim":"Located SAS-6 to the cartwheel center and showed it is essential to establish nine-fold symmetry, answering what structural element imposes centriole symmetry.","evidence":"Chlamydomonas bld12 null mutant analysis with EM and immunolocalization","pmids":["18082404"],"confidence":"High","gaps":["Did not show how SAS-6 molecular geometry translates into nine-fold symmetry","No atomic structure of the assembling protein"]},{"year":2009,"claim":"Identified a direct kinase modification of SAS-6 (ZYG-1 phosphorylation at S123) required for its maintenance at the emerging centriole, linking a centriolar kinase to SAS-6 stability.","evidence":"In vitro kinase assay with C. elegans phospho-mutant rescue","pmids":["20059959"],"confidence":"High","gaps":["Whether S123 is the essential ZYG-1 substrate for duplication was later questioned","Did not address recruitment versus retention"]},{"year":2010,"claim":"Provided the first biochemical model for how SAS-6 builds the cartwheel by showing it self-assembles into defined oligomers that template the central tubule.","evidence":"Gel filtration, native PAGE, EM of recombinant protein and overexpression in Drosophila cells","pmids":["20083610"],"confidence":"Medium","gaps":["The specific tetramer model was complemented by later ring/spiral structures","No high-resolution structure of the assembly interfaces"]},{"year":2011,"claim":"Resolved the structural basis of cartwheel-center formation, showing SAS-6 N-terminal self-association generates cartwheel-like assemblies whose interfaces are functionally required.","evidence":"Zebrafish SAS-6 N-terminal X-ray crystallography with point mutagenesis and in vivo centriole formation assay","pmids":["21273447"],"confidence":"High","gaps":["Did not explain how the symmetry number is fixed at nine","Did not address ring stacking or polarity"]},{"year":2013,"claim":"Dissected the recruitment interactions, showing ZYG-1 binds the SAS-6 coiled coil independently of kinase activity and an adjacent segment binds SAS-5, both required for cartwheel assembly.","evidence":"Pulldown, co-IP, in vitro binding and C. elegans alanine-substitution genetics","pmids":["23673331"],"confidence":"High","gaps":["The essential ZYG-1 kinase substrate for cartwheel assembly remained unidentified","Stoichiometry of the ZYG-1/SAS-5/SAS-6 assembly not defined"]},{"year":2013,"claim":"Revealed an alternative SAS-6 spiral architecture consistent with the same nine-fold output, showing distinct oligomerization modes can specify identical symmetry.","evidence":"X-ray crystallography and EM of C. elegans SAS-6","pmids":["23798409"],"confidence":"High","gaps":["Did not establish what selects spiral versus ring assembly in vivo","Link between spiral and central tube versus cartwheel not mechanistically resolved"]},{"year":2014,"claim":"Established Plk4 phosphorylation of the Ana2/STIL STAN motif as the earliest upstream trigger enabling Ana2 to bind and recruit SAS-6, defining the kinase-controlled gate for procentriole assembly.","evidence":"In vitro kinase assay and Drosophila phospho-mutant rescue with live imaging","pmids":["25264260"],"confidence":"High","gaps":["Did not resolve the full ordered sequence of Ana2 phosphorylation events","Structural basis of phospho-dependent Ana2-SAS-6 binding not shown"]},{"year":2014,"claim":"Provided a definitive structure proving SAS-6 self-assembly alone imposes nine-fold cartwheel symmetry and showed the assembly is druggable.","evidence":"3.5 Å X-ray structure of a nine-fold symmetric Leishmania SAS-6 cartwheel and in vitro small-molecule inhibition","pmids":["24596152"],"confidence":"High","gaps":["Did not address how the cartwheel stacks or acquires polarity","Did not test the inhibitor in cells"]},{"year":2014,"claim":"Uncovered a luminal SAS-6 recruitment route in human centrioles, showing initial recruitment uses the luminal wall rather than self-oligomerization and that Plk4/STIL-dependent release governs subsequent assembly.","evidence":"Live-cell fluorescence, siRNA knockdown and structured illumination microscopy","pmids":["25017693"],"confidence":"Medium","gaps":["Single lab; the luminal pathway needs independent confirmation","Molecular identity of the luminal wall binding site not defined"]},{"year":2015,"claim":"Identified the SAS-6 C-terminal tail as a direct microtubule-nucleating module, connecting cartwheel assembly to centriolar microtubule formation.","evidence":"In vitro tubulin polymerization, microtubule pulldown, ITC and co-IP from S-phase HeLa lysates","pmids":["26422590"],"confidence":"Medium","gaps":["Single lab in vitro biochemistry","In vivo requirement of the tail for microtubule nucleation not yet shown at this stage"]},{"year":2015,"claim":"Showed both SAS-6 N-terminal homo-oligomerization and Ana2 tetramerization are individually required for centriole assembly, with high-resolution structures of each module.","evidence":"0.8 Å and 2.9 Å X-ray structures, in vitro oligomerization assays and Drosophila point-mutant genetics","pmids":["26002084"],"confidence":"High","gaps":["Did not define how the two oligomerization activities are coordinated temporally","Structure of the combined SAS-6–Ana2 assembly not solved"]},{"year":2015,"claim":"Challenged the strict requirement for SAS-6 self-assembly by showing de novo centriole formation can proceed without it, refining the role of self-oligomerization in fidelity rather than absolute biogenesis.","evidence":"Reconstitution of de novo centriole synthesis in human cells with oligomerization-deficient mutants and EM","pmids":["26609813"],"confidence":"Medium","gaps":["Single lab; paradigm-challenging result needs replication","What substitutes for SAS-6 self-assembly during de novo formation is unknown"]},{"year":2016,"claim":"Demonstrated causally that SAS-6 self-assembly properties instruct cartwheel symmetry and that cartwheel and microtubule wall assemble interdependently.","evidence":"Engineered SAS-6 symmetry mutants expressed in Chlamydomonas and human cells with EM","pmids":["26999736"],"confidence":"High","gaps":["In vivo symmetry buffering toward nine-fold not fully explained","Contribution of additional factors to symmetry correction unresolved"]},{"year":2017,"claim":"Resolved the ordered Plk4 phosphorylation logic on Ana2, showing an initial ANST-motif phosphorylation promotes Ana2 recruitment before STAN-motif phosphorylation enables SAS-6 recruitment.","evidence":"In vitro kinase assay, mass spectrometry and Drosophila phospho-mutant rescue with live imaging","pmids":["29263250"],"confidence":"High","gaps":["Did not establish how phosphorylation timing is controlled in the cycle","Phosphatase counter-regulation not addressed"]},{"year":2017,"claim":"Identified Cdc6 as a negative regulator that binds SAS-6 to block its STIL interaction, with Plk4 phosphorylation of Cdc6 relieving this inhibition to license duplication.","evidence":"Co-IP, co-localization, siRNA and overexpression with centrosome duplication readout","pmids":["28447620"],"confidence":"Medium","gaps":["Single lab; physiological contribution to duplication timing not quantified","Direct competition between Cdc6 and STIL for SAS-6 not structurally defined"]},{"year":2018,"claim":"Captured the kinetics of SAS-6 ring formation, showing assembly proceeds via multiple routes driven by weak surface interactions to reach nine-fold symmetry.","evidence":"High-speed atomic force microscopy (PORT) with kinetic analysis","pmids":["29784964"],"confidence":"Medium","gaps":["Single-method study; in-cell relevance of surface-driven kinetics not tested","How the cell biases assembly toward correct symmetry not addressed"]},{"year":2019,"claim":"Showed pericentrin (Pcp1) directly binds and recruits SAS-6, linking the pericentriolar material to cartwheel assembly and centriole elongation.","evidence":"Ectopic expression in fission yeast, co-IP and genetic assays in animal cells","pmids":["31182187"],"confidence":"Medium","gaps":["Single lab; recruitment hierarchy relative to ZYG-1/STIL unclear","Structural basis of the Pcp1–SAS-6 interface not resolved"]},{"year":2020,"claim":"Mapped SAS-6 C-terminus to γ-TuRC interaction required for centriolar microtubule formation in daughter centrioles, mechanistically linking the cartwheel hub to microtubule nucleation in vivo.","evidence":"Co-IP, deletion mutant analysis, siRNA and high-resolution fluorescence microscopy","pmids":["32442461"],"confidence":"Medium","gaps":["Single lab; direct versus indirect γ-TuRC binding not fully separated","Relationship between tubulin-binding and γ-TuRC-binding activities of the tail unresolved"]},{"year":2020,"claim":"Defined the Ana2–SAS-6 interaction surfaces in the C-terminal regions of both proteins and showed SAS-6 can simultaneously engage Ana2 and Gorab through distinct sites.","evidence":"HDX-MS, in vitro complex formation, mutagenesis and co-IP","pmids":["33171067"],"confidence":"Medium","gaps":["Single lab interface mapping","Functional consequence of simultaneous Ana2/Gorab binding not quantified"]},{"year":2021,"claim":"Defined the Gorab–SAS-6 heterotrimer structurally and showed a single SAS-6 residue in the Gorab-binding site is essential for centriole duplication, connecting Golgi-associated Gorab to the centriole.","evidence":"HDX-MS, EM, mutagenesis with in vivo duplication assay and binding assays","pmids":["33704067"],"confidence":"High","gaps":["Functional role of the Golgi-versus-centriole Gorab equilibrium not fully defined","How Gorab binding contributes to cartwheel function mechanistically unresolved"]},{"year":2021,"claim":"Used designed monobodies to separate SAS-6 ring assembly from ring stacking and to drive a ring-to-helix conformational switch that impairs centriole biogenesis, dissecting distinct assembly steps.","evidence":"X-ray crystallography, AFM, cryo-EM and a human-cell centriole biogenesis assay","pmids":["34155202"],"confidence":"High","gaps":["Did not define the endogenous regulator of the ring-helix transition","Physiological signal controlling stacking not identified"]},{"year":2022,"claim":"Identified an asymmetric coiled-coil homo-oligomerization interaction that imparts polarity to the cartwheel and supports ring stacking, providing a structural basis for the centriolar proximal-distal axis.","evidence":"X-ray crystallography, cryo-EM reconstitution and site-directed mutagenesis","pmids":["35240058"],"confidence":"High","gaps":["Direct demonstration that this polarity sets the in vivo proximal-distal axis not completed","How many stacked rings the asymmetry permits unresolved"]},{"year":2022,"claim":"Connected centriole number control to SAS-6 abundance, showing CHD-1 and EFL-1/DPL-1 downregulate CDK-2 to limit SAS-6 protein levels.","evidence":"C. elegans genetic epistasis, RNAi, Western blot and centriole counting","pmids":["35377871"],"confidence":"Medium","gaps":["Single lab; whether the axis is transcriptional or post-translational not fully separated","Conservation in mammalian cells not tested"]},{"year":2024,"claim":"Established the in vivo requirement for SASS6 in mammalian centriole formation and revealed a context-dependent dispensability in stem cells with high PLK4 activity, defining when SAS-6 is essential.","evidence":"Mouse knockout genetics, immunofluorescence, centriole ultrastructure and cell death pathway assays","pmids":["38407237"],"confidence":"High","gaps":["Molecular basis of SAS-6-independent biogenesis in mESCs incompletely defined","Relationship to the mitotic surveillance pathway downstream of centriole loss unresolved"]},{"year":2025,"claim":"Showed SAS-6 phase separation, modulated by CDK-1 phosphorylation of its C-terminus, governs centrosome assembly and elimination across meiotic prophase, adding a material-state layer to SAS-6 regulation.","evidence":"In vitro phase separation and kinase assays, mass spectrometry and C. elegans phospho-mutant genetics with live imaging","pmids":["40410380"],"confidence":"Medium","gaps":["Single lab; in vivo relevance of droplet formation beyond oogenesis untested","Relationship between phase separation and cartwheel polymerization not resolved"]},{"year":2025,"claim":"Defined ubiquitin-dependent degradation pathways (APC/C-Cdh1 and FBXW7) that limit SAS-6 and STIL-SAS6 abundance, and linked non-degradable SAS-6 to ciliation, YAP/TAZ activation and invasion.","evidence":"Co-IP, siRNA, ubiquitination and invasion assays, YAP/TEAD reporters, non-degradable mutant expression and Plk4 inhibitor treatment","pmids":["41453690","40825584"],"confidence":"Medium","gaps":["Single labs; in vivo tumor relevance of SAS-6-driven invasion untested","How the same Plk4 phosphosites coordinate assembly then FBXW7 destruction temporally not fully resolved"]},{"year":null,"claim":"How SAS-6 assembly state, post-translational regulation, and abundance are integrated in real time to ensure exactly one cartwheel of correct symmetry and polarity per duplication cycle remains unresolved.","evidence":"","pmids":[],"confidence":"Medium","gaps":["No unified model linking phase separation, ring stacking polarity, and degradation timing","Endogenous trigger of the ring-to-helix conformational switch unknown","Mechanism of SAS-6-independent centriole biogenesis in high-PLK4 contexts undefined"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0005198","term_label":"structural molecule activity","supporting_discovery_ids":[1,4,8,11,13]},{"term_id":"GO:0008092","term_label":"cytoskeletal protein binding","supporting_discovery_ids":[10,18]},{"term_id":"GO:0060090","term_label":"molecular adaptor activity","supporting_discovery_ids":[5,7,17,20]}],"localization":[{"term_id":"GO:0005815","term_label":"microtubule organizing center","supporting_discovery_ids":[0,1,9,18]},{"term_id":"GO:0005856","term_label":"cytoskeleton","supporting_discovery_ids":[10,18]}],"pathway":[{"term_id":"R-HSA-1852241","term_label":"Organelle biogenesis and maintenance","supporting_discovery_ids":[0,1,13,24]},{"term_id":"R-HSA-1640170","term_label":"Cell Cycle","supporting_discovery_ids":[0,15,26]}],"complexes":["centriolar cartwheel","STIL-SAS6 cartwheel assembly complex"],"partners":["STIL","PLK4","ZYG-1","CDC6","PCNT","GORAB","TUBG1"],"other_free_text":[]}},"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; CENATAC","url":"https://www.omim.org/entry/620142"},{"mim_id":"617728","title":"CENTROSOMAL PROTEIN, 295-KD; CEP295","url":"https://www.omim.org/entry/617728"},{"mim_id":"616402","title":"MICROCEPHALY 14, PRIMARY, AUTOSOMAL RECESSIVE; MCPH14","url":"https://www.omim.org/entry/616402"},{"mim_id":"615587","title":"NUCLEOPORIN, 188-KD; NUP188","url":"https://www.omim.org/entry/615587"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"Approved","locations":[{"location":"Primary cilium","reliability":"Approved"},{"location":"Cytosol","reliability":"Approved"},{"location":"Vesicles","reliability":"Additional"}],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in many","driving_tissues":[],"url":"https://www.proteinatlas.org/search/SASS6"},"hgnc":{"alias_symbol":["DKFZp761A078","SAS-6","FLJ22097","SAS6"],"prev_symbol":[]},"alphafold":{"accession":"Q6UVJ0","domains":[{"cath_id":"2.170.210.20","chopping":"1-144","consensus_level":"high","plddt":89.9926,"start":1,"end":144}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6UVJ0","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q6UVJ0-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q6UVJ0-F1-predicted_aligned_error_v6.png","plddt_mean":73.25},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=SASS6","jax_strain_url":"https://www.jax.org/strain/search?query=SASS6"},"sequence":{"accession":"Q6UVJ0","fasta_url":"https://rest.uniprot.org/uniprotkb/Q6UVJ0.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q6UVJ0/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q6UVJ0"}},"corpus_meta":[{"pmid":"15665853","id":"PMC_15665853","title":"SAS-6 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Part A","url":"https://pubmed.ncbi.nlm.nih.gov/38501757","citation_count":2,"is_preprint":false},{"pmid":"36739862","id":"PMC_36739862","title":"Case Report: Prenatal Recurrent Microcephaly and Corpus Callosum Abnormalities in a Chinese Family with Novel Biallelic SASS6 Mutations.","date":"2023","source":"Fetal diagnosis and therapy","url":"https://pubmed.ncbi.nlm.nih.gov/36739862","citation_count":2,"is_preprint":false},{"pmid":"33640460","id":"PMC_33640460","title":"Optimization strategies for expression and purification of soluble N-terminal domain of human centriolar protein SAS-6 in Escherichia coli.","date":"2021","source":"Protein expression and purification","url":"https://pubmed.ncbi.nlm.nih.gov/33640460","citation_count":2,"is_preprint":false},{"pmid":"40825584","id":"PMC_40825584","title":"Dysregulated SASS6 expression promotes increased ciliogenesis and cell invasion phenotypes.","date":"2025","source":"Life science alliance","url":"https://pubmed.ncbi.nlm.nih.gov/40825584","citation_count":1,"is_preprint":false},{"pmid":"36331102","id":"PMC_36331102","title":"Targeting Drosophila Sas6 to mitochondria reveals its high affinity for Gorab.","date":"2022","source":"Biology open","url":"https://pubmed.ncbi.nlm.nih.gov/36331102","citation_count":1,"is_preprint":false},{"pmid":"41453690","id":"PMC_41453690","title":"FBXW7 E3 ligase prevents centriole overduplication by degrading the Plk4 phosphorylated STIL-SAS6 cartwheel assembly.","date":"2025","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/41453690","citation_count":0,"is_preprint":false},{"pmid":"41319860","id":"PMC_41319860","title":"CDT1 induces the formation of polyploid giant cancer cells and promotes centrosome amplification through the PLK4/SASS6 axis.","date":"2025","source":"Cancer letters","url":"https://pubmed.ncbi.nlm.nih.gov/41319860","citation_count":0,"is_preprint":false},{"pmid":"36859084","id":"PMC_36859084","title":"Grand canonical Brownian dynamics simulations of adsorption and self-assembly of SAS-6 rings on a surface.","date":"2023","source":"The Journal of chemical physics","url":"https://pubmed.ncbi.nlm.nih.gov/36859084","citation_count":0,"is_preprint":false},{"pmid":null,"id":"bio_10.1101_2025.05.06.25327057","title":"Identification of (ultra-)rare functional promoter mutations in cancer using sequence-based deep learning models","date":"2025-05-06","source":"bioRxiv","url":"https://doi.org/10.1101/2025.05.06.25327057","citation_count":0,"is_preprint":true}],"cost":{"stage1":{"model":"claude-sonnet-4-6","input_tokens":25329,"output_tokens":7463,"usd":0.093966,"stage1_stop_reason":"end_turn"},"stage2":{"model":"claude-opus-4-8","input_tokens":16841,"output_tokens":7480,"usd":0.135602,"stage2_stop_reason":"end_turn"},"total_usd":0.229568,"stage1_batch_id":"msgbatch_013LWZZT6MWaqB9qyTSeQBmU","stage2_batch_id":"msgbatch_0194ZzQaTGNpruoNJBbEMbt3","note":"batch pricing = 50% of standard"},"stage1_raw":"```json\n{\n  \"discoveries\": [\n    {\n      \"year\": 2005,\n      \"finding\": \"HsSAS-6 (SASS6) localizes to centrosomes and is required for centrosome duplication in human cells: 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 is recruited to centrioles at the onset of the centrosome duplication cycle, associates with SAS-5, and requires both the SAS-5 interaction and ZYG-1 function for centriolar recruitment.\",\n      \"method\": \"siRNA knockdown in human U2OS cells, GFP localization, C. elegans genetics, co-immunoprecipitation\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal interactions, genetic epistasis in C. elegans, and direct functional readout in human cells; foundational paper replicated across multiple subsequent studies\",\n      \"pmids\": [\"15665853\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"In Chlamydomonas, SAS-6 localizes to the central part of the cartwheel and is required to establish nine-fold centriolar symmetry. A null mutant (bld12) lacking the cartwheel central part frequently produces centrioles with non-canonical triplet numbers (7, 8, 10, or 11), demonstrating that SAS-6 is an essential cartwheel component that stabilizes the 9-triplet structure.\",\n      \"method\": \"Chlamydomonas null mutant analysis, electron microscopy, immunolocalization\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — genetic null mutant with quantitative structural readout by EM, independently consistent with multiple subsequent studies on SAS-6 symmetry\",\n      \"pmids\": [\"18082404\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"The kinase ZYG-1 phosphorylates SAS-6 at serine 123 in vitro, and this phosphorylation event is critical for centriole formation in C. elegans embryos in vivo. Phosphorylation ensures maintenance of SAS-6 at the emerging centriole.\",\n      \"method\": \"In vitro kinase assay, C. elegans genetics with phospho-mutant rescue, fluorescence microscopy\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay combined with in vivo phospho-mutant functional validation; single lab but multiple orthogonal methods\",\n      \"pmids\": [\"20059959\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2010,\n      \"finding\": \"Drosophila SAS-6 self-assembles into stable tetramers in vitro, which serve as building blocks for the central tubule of the centriolar cartwheel. SAS-6 concentrates at the core of the cartwheel, and elevated SAS-6 levels in Drosophila cells produce higher-order structures resembling central tubule morphology.\",\n      \"method\": \"Biochemistry (gel filtration, native PAGE), electron microscopy of centrosomes and recombinant protein, cell overexpression\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro reconstitution and EM characterization by single lab, consistent with structural studies but specific tetramer model later superseded/complemented by ring models\",\n      \"pmids\": [\"20083610\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"X-ray crystal structure of the zebrafish SAS-6 N-terminal domain reveals that recombinant SAS-6 self-associates in vitro into assemblies resembling cartwheel centers. Point mutations disrupting the self-assembly interfaces impair centriole formation in vivo, establishing that these interactions are essential for cartwheel center organization.\",\n      \"method\": \"X-ray crystallography, in vitro reconstitution, point mutagenesis with in vivo centriole formation assay\",\n      \"journal\": \"Science (New York, N.Y.)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with mutagenesis and in vivo functional validation; foundational structural paper replicated by multiple subsequent structural studies\",\n      \"pmids\": [\"21273447\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"In C. elegans, ZYG-1 recruits SAS-6 to the mother centriole via a direct binding interaction between ZYG-1 and the SAS-6 coiled coil, independently of ZYG-1 kinase activity. Separately, an adjacent segment of the SAS-6 coiled coil interacts with SAS-5, and both interactions are required for SAS-6 recruitment and cartwheel assembly. ZYG-1 kinase activity is subsequently required for cartwheel assembly, but its essential substrate is unlikely to be SAS-6 itself.\",\n      \"method\": \"Pulldown, co-immunoprecipitation, in vitro binding assays, C. elegans genetics with alanine-substitution mutants\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — reciprocal Co-IP, direct binding assay, genetic epistasis with systematic mutagenesis; multiple orthogonal methods in single rigorous study\",\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 rings) as shown by crystallography and EM, yet this spiral is consistent with nine-fold symmetry, suggesting two distinct SAS-6 oligomerization architectures can direct the same output symmetry. Spiral arrangement is correlated with the presence of a central tube instead of a cartwheel in nematode centriole assembly.\",\n      \"method\": \"X-ray crystallography, electron microscopy\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — crystal structure and EM with multiple orthogonal methods; single lab but structurally rigorous\",\n      \"pmids\": [\"23798409\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Drosophila Plk4 phosphorylates four conserved serines in the STAN motif of Ana2 (STIL ortholog) to enable Ana2 to bind and recruit its Sas6 partner. Non-phosphorylatable Ana2 localizes to the centriole but cannot recruit Sas6, causing failure of centriole duplication. Thus, Plk4-mediated phosphorylation of Ana2/STIL is the earliest upstream step for Sas6 recruitment and procentriole architecture establishment.\",\n      \"method\": \"In vitro kinase assay, Drosophila genetics with phospho-mutant rescue, live imaging, immunofluorescence\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay plus in vivo phospho-mutant analysis with functional centriole readout; replicated in subsequent studies\",\n      \"pmids\": [\"25264260\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Leishmania major SAS-6 crystallizes as a nine-fold symmetric cartwheel, providing a 3.5 Å X-ray structure of this assembly and firmly establishing that SAS-6 self-assembly alone can impose cartwheel symmetry. Small-molecule inhibition of SAS-6 oligomerization is feasible in vitro.\",\n      \"method\": \"X-ray crystallography at 3.5 Å, in vitro small-molecule inhibition assay\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution crystal structure of the nine-fold symmetric assembly; structurally definitive\",\n      \"pmids\": [\"24596152\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"During S phase, SAS-6 molecules are 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/release of luminal SAS-6 requires Plk4 and STIL. Abolishing either recruitment or removal of luminal SAS-6 hinders SAS-6/centriole assembly at the outside wall. After duplication, the lumen of engaged mother centrioles becomes inaccessible to SAS-6, correlating with a block for reduplication.\",\n      \"method\": \"Live cell fluorescence microscopy, siRNA knockdown, structured illumination microscopy\",\n      \"journal\": \"Developmental cell\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct localization with functional consequence, epistasis with Plk4 and STIL, single lab with multiple imaging modalities\",\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 to microtubules along their lengths, and interacts with α/β-tubulin dimers. The N-terminal domain has no effect on microtubule polymerization. Endogenous HsSAS-6 co-precipitates with microtubules from S-phase HeLa cell lysates.\",\n      \"method\": \"In vitro tubulin polymerization assay, microtubule pulldown, co-immunoprecipitation from cell lysate, isothermal calorimetry, size-exclusion chromatography\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — multiple in vitro assays (ITC, pulldown, polymerization) with domain dissection, single lab\",\n      \"pmids\": [\"26422590\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2015,\n      \"finding\": \"In vitro, both Drosophila Sas-6 N-terminal domain homo-oligomerization and Ana2 CCCD tetramerization are required for efficient centriole assembly in vivo. Point mutations that perturb Sas-6 homo-oligomerization in vitro strongly impair centriole assembly in Drosophila. The Ana2 CCCD forms a tetramer with an unusual parallel-coil topology (structure solved to 0.8 Å), and the Sas-6 N-terminal domain forms higher-order oligomers through canonical interactions (structure at 2.9 Å).\",\n      \"method\": \"X-ray crystallography (0.8 Å and 2.9 Å), in vitro oligomerization assays, in vivo Drosophila genetics with point mutants\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — high-resolution crystal structures combined with mutagenesis and in vivo functional validation; multiple orthogonal methods\",\n      \"pmids\": [\"26002084\"],\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, demonstrating that centriole biogenesis does not strictly depend on SAS-6 self-assembly. Canonically duplicated centrioles always form correctly, whereas de novo centrioles are prone to structural errors even when SAS-6 self-oligomerization is intact.\",\n      \"method\": \"Reconstitution of de novo centriole synthesis in human cells, SAS-6 oligomerization-deficient mutants, electron microscopy\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reconstitution in human cells with mutant analysis and structural EM readout; single lab, challenges prior paradigm\",\n      \"pmids\": [\"26609813\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"Engineering Chlamydomonas SAS-6 to form oligomers with symmetries ranging from five- to ten-fold showed that SAS-6 self-assembly properties instruct cartwheel symmetry. A SAS-6 mutant forming six-fold symmetric cartwheels in vitro produced eight- or nine-fold cartwheels in vivo, and with Bld10 mutants weakening cartwheel-microtubule interactions, produced six- to eight-fold cartwheels. The microtubule wall maintained eight- and nine-fold symmetries, indicating cartwheel and microtubule wall assemble interdependently. Human cells expressing analogous SAS-6 mutations formed nine-fold centrioles with impaired length and organization.\",\n      \"method\": \"In vitro oligomerization assays, Chlamydomonas and human cell expression of engineered SAS-6 mutants, electron microscopy\",\n      \"journal\": \"Nature cell biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — direct engineering of SAS-6 symmetry in vitro with in vivo structural readout across two organisms; multiple orthogonal methods in single rigorous study\",\n      \"pmids\": [\"26999736\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Drosophila Plk4 first phosphorylates a single serine (S38) in the conserved ANST motif of Ana2 to promote Ana2 recruitment to the centriole, and then phosphorylates four serines in the STAN motif to enable Ana2 to recruit Sas6. Non-phosphorylatable S38A Ana2 fails to load onto the procentriole and blocks centriole duplication, establishing a sequential two-step phosphorylation mechanism for Sas6 recruitment.\",\n      \"method\": \"In vitro kinase assay, mass spectrometry, Drosophila genetics with phospho-mutant rescue, live imaging\",\n      \"journal\": \"Open biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — in vitro kinase assay with mass spectrometry identification of phospho-sites and in vivo genetic validation; replication of prior STAN motif work with novel ANST discovery\",\n      \"pmids\": [\"29263250\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"The DNA replication licensing factor Cdc6 is recruited to the proximal side of centrioles via cyclin A and negatively regulates centrosome duplication by binding Sas-6 and inhibiting its interaction with STIL. Plk4 phosphorylates Cdc6, disrupting the Sas-6–Cdc6 interaction and thereby counteracting the inhibitory effect of Cdc6 on Sas-6. Overexpression of wild-type Cdc6 or a Plk4-unphosphorylatable Cdc6 mutant reduces centrosome over-duplication.\",\n      \"method\": \"Co-immunoprecipitation, co-localization, siRNA knockdown, overexpression with functional centrosome duplication readout\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, epistasis with phospho-mutants, functional rescue; single lab\",\n      \"pmids\": [\"28447620\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"High-speed atomic force microscopy (photothermal off-resonance tapping) reveals the kinetics of SAS-6 ring formation and demonstrates that distinct biogenesis routes can be followed to assemble a nine-fold symmetrical ring structure, showing the assembly reaction is driven by weak interactions on a surface.\",\n      \"method\": \"High-speed atomic force microscopy (PORT), kinetic analysis of self-assembly\",\n      \"journal\": \"Nature nanotechnology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — direct single-molecule imaging of assembly kinetics with novel methodology; single lab, single method type\",\n      \"pmids\": [\"29784964\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"The conserved PCM component Pcp1/pericentrin directly interacts with and recruits SAS-6. This interaction is conserved and important for centriole assembly, particularly centriole elongation. Calmodulin-binding region of Pcp1/pericentrin is critical for SAS-6 interaction.\",\n      \"method\": \"Ectopic expression in fission yeast, co-immunoprecipitation, genetic assays in animal cells\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct protein interaction demonstrated, with functional consequence on centriole assembly; single lab, multiple organisms tested\",\n      \"pmids\": [\"31182187\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Human SAS-6 C-terminus is required for centriolar microtubule formation by interacting with the γ-tubulin ring complex (γ-TuRC). Deletion of HsSAS-6 C-terminus disrupts microtubule formation in daughter centrioles, resulting in cells with only two centrioles at a single site. SAS-6 associates with γ-TuRC proteins at the centrosome, and high-resolution microscopy reveals γ-tubulin as multiple lobes surrounding the HsSAS-6-containing central hub.\",\n      \"method\": \"Co-immunoprecipitation, deletion mutant analysis, siRNA knockdown, high-resolution fluorescence microscopy\",\n      \"journal\": \"Current biology : CB\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP with domain mapping, deletion mutant functional readout, high-resolution imaging; single lab, multiple methods\",\n      \"pmids\": [\"32442461\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"Interaction surfaces between Drosophila Ana2 and Sas6 lie in the C-terminal parts of both proteins, as identified by hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) and confirmed by mutagenesis. The Sas6 site required for Ana2 binding is distinct from the site required for Gorab binding, and Sas6 can simultaneously bind both Ana2 and Gorab.\",\n      \"method\": \"HDX-MS, in vitro complex formation, mutagenesis, co-immunoprecipitation\",\n      \"journal\": \"Open biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — HDX-MS interface mapping with mutational confirmation; single lab, multiple orthogonal methods\",\n      \"pmids\": [\"33171067\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Monomeric Drosophila Gorab binds Sas6 via an antiparallel interaction between a segment of Gorab's coiled-coil and the parallel coiled-coil dimer of Sas6, forming a stable heterotrimer visible by EM. Mutation of a single leucine in Sas6's Gorab-binding domain reduces affinity 16-fold and abolishes centriole duplication, demonstrating this interaction is essential. Gorab dimers at the Golgi exist in equilibrium with Sas6-associated Gorab monomers at the centriole.\",\n      \"method\": \"HDX-MS, electron microscopy, mutagenesis with in vivo centriole duplication assay, biochemical binding assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — structural (EM) plus HDX-MS interface mapping plus mutagenesis with functional in vivo validation; multiple orthogonal methods\",\n      \"pmids\": [\"33704067\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Monobodies against Chlamydomonas SAS-6 characterized by X-ray crystallography, AFM, and cryo-EM reveal distinct interaction modes that specifically impair ring assembly or ring stacking. Monobody MBCRS6-15 induces a conformational change converting CrSAS-6 from ring to helix conformation, and this alteration impairs centriole biogenesis in human cells.\",\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 / Strong — three orthogonal structural methods plus functional validation in human cells; mechanistically precise\",\n      \"pmids\": [\"34155202\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"Crystallographic structures of the Chlamydomonas reinhardtii SAS-6 coiled-coil domain reveal an asymmetric homo-oligomerization interaction. Using cryo-EM reconstitution, amino acid substitutions disrupting this asymmetric association impair SAS-6 ring stacking, suggesting the coiled-coil asymmetric interaction provides polarity to the cartwheel and may assist establishment of the centriolar proximal-distal axis.\",\n      \"method\": \"X-ray crystallography, cryo-EM reconstitution assay, site-directed mutagenesis\",\n      \"journal\": \"Structure (London, England : 1993)\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Strong — crystal structure combined with cryo-EM functional assay and mutagenesis; multiple orthogonal structural and biochemical methods\",\n      \"pmids\": [\"35240058\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"In C. elegans, the chromatin remodeling protein CHD-1 and the transcription factor EFL-1/DPL-1 cooperate to downregulate CDK-2, which in turn controls SAS-6 protein levels. Loss of CHD-1 increases SAS-6 levels and produces extra centrioles, revealing a transcriptional/post-translational axis for controlling centriole number via SAS-6 abundance.\",\n      \"method\": \"C. elegans genetics (epistasis), RNAi, Western blot for protein levels, centriole counting\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic epistasis in C. elegans with protein-level confirmation; single lab, multiple genetic combinations tested\",\n      \"pmids\": [\"35377871\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"In mouse embryos, Sass6 (SASS6) is required for centriole formation, and 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 de novo centriole formation but is essential to maintain centriole architecture. High PLK4 activity and elevated centrosomal protein levels in mESCs enable SAS-6-independent centriole biogenesis.\",\n      \"method\": \"Mouse knockout genetics, immunofluorescence, centriole ultrastructure analysis, cell death pathway assays\",\n      \"journal\": \"eLife\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — mouse knockout with in vivo embryo phenotype plus mechanistic epistasis with PLK4; multiple cellular contexts tested\",\n      \"pmids\": [\"38407237\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"SAS-6 undergoes phase separation in vitro and forms droplets when overexpressed in cells. CDK-1 directly phosphorylates SAS-6 at its C-terminus (identified by mass spectrometry and kinase assays), which inhibits SAS-6 phase separation and weakens interactions between centriolar proteins. Phospho-mimetic and phospho-deficient mutants demonstrate that dynamic SAS-6 phosphorylation is essential for centrosome assembly during early meiotic prophase and for centrosome elimination during late meiotic prophase (oogenesis) in C. elegans.\",\n      \"method\": \"In vitro phase separation assay, mass spectrometry, in vitro kinase assay, C. elegans genetics with phospho-mutants, live imaging\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — in vitro kinase assay and phase separation reconstitution combined with in vivo phospho-mutant genetics; single lab, multiple methods\",\n      \"pmids\": [\"40410380\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"FBXW7 E3 ubiquitin ligase mediates degradation of the STIL-SAS6 cartwheel assembly complex. Plk4 kinase activity is required for FBXW7-mediated STIL-SAS6 degradation. The same Plk4-phosphorylated sites in STIL that promote STIL-SAS6 interaction for centriole assembly also stabilize FBXW7 binding to STIL, creating a dual mechanism: phosphorylation promotes assembly and then triggers destruction to prevent centriole overduplication. Depletion of FBXW7 induces premature centriole duplication through excessive STIL-SAS6 stabilization.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, overexpression with centriole counting, ubiquitination assay, Plk4 inhibitor treatment\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple Co-IPs and functional epistasis with Plk4 inhibitor, single lab; degradation mechanism demonstrated biochemically\",\n      \"pmids\": [\"41453690\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2025,\n      \"finding\": \"Non-degradable SAS-6 (SAS-6ND, escaping APCCdh1-targeted degradation) increases ciliation and cell invasion and upregulates the YAP/TAZ pathway. SAS-6-mediated invasion is prevented by YAP downregulation or by blocking ciliogenesis, placing SAS-6 upstream of YAP/TAZ-dependent transcription in the invasion pathway. SAS-6 levels are subject to APCCdh1-targeted degradation at the end of mitosis and G1.\",\n      \"method\": \"Non-degradable SAS-6 mutant expression, siRNA knockdown, invasion assays, YAP nuclear translocation imaging, TEAD reporter assay\",\n      \"journal\": \"Life science alliance\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — non-degradable mutant with epistasis (YAP knockdown rescue), multiple functional readouts; single lab\",\n      \"pmids\": [\"40825584\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"SASS6/SAS-6 is a highly conserved centriolar scaffold protein that self-assembles via its N-terminal head domain dimerization and coiled-coil interactions into nine-fold symmetric ring polymers, which stack (with polarity imposed by an asymmetric coiled-coil interaction) to form the centriolar cartwheel; its recruitment to the mother centriole is initiated by direct binding to ZYG-1/Plk4 (kinase-activity-independent) and to SAS-5/Ana2/STIL, with Plk4-mediated sequential phosphorylation of Ana2/STIL being the earliest trigger for Sas6 loading and procentriole formation; once recruited, SAS-6 promotes centriolar microtubule assembly through its C-terminal tail interacting with γ-TuRC and with tubulin directly; SAS-6 protein levels are controlled by CDK-2-dependent transcriptional mechanisms and by APCCdh1- and FBXW7-mediated ubiquitin-dependent degradation, while CDK-1 phosphorylation of the SAS-6 C-terminus inhibits its phase separation and promotes centrosome elimination during oogenesis; dysregulated (non-degradable) SAS-6 promotes excess ciliation, YAP/TAZ activation, and cell invasion, and loss-of-function mutations in SASS6 cause primary microcephaly (MCPH14) in humans.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"SASS6/SAS-6 is a highly conserved centriolar scaffold protein that self-assembles to impose the nine-fold symmetry of the centriolar cartwheel and is required for centriole duplication [#0, #1]. Its N-terminal head domain self-associates into oligomers whose geometry instructs cartwheel symmetry: high-resolution structures of the N-terminal domain reveal cartwheel-center-like assemblies, point mutations disrupting these interfaces impair centriole formation, and engineering the oligomerization properties of SAS-6 directly reprograms cartwheel symmetry from five- to ten-fold [#4, #11, #13]. SAS-6 self-assembly alone is sufficient to generate a nine-fold symmetric cartwheel in vitro [#8], and an asymmetric coiled-coil interaction confers polarity that drives ring stacking along the proximal-distal axis [#22]. Recruitment of SAS-6 to the mother centriole is initiated through direct, kinase-activity-independent binding of its coiled coil to ZYG-1/Plk4 and to SAS-5/Ana2/STIL, with Plk4-mediated sequential phosphorylation of Ana2/STIL (first a single ANST-motif serine, then four STAN-motif serines) constituting the earliest trigger for SAS-6 loading and procentriole formation [#5, #7, #14]; ZYG-1 also phosphorylates SAS-6 itself at serine 123 to maintain it at the emerging centriole [#2]. Once incorporated, the SAS-6 C-terminal tail nucleates centriolar microtubule assembly by binding α/β-tubulin and the γ-tubulin ring complex [#10, #18]. SAS-6 abundance is tightly controlled to limit centriole number, via CDK-2-dependent transcriptional input [#23] and via ubiquitin-dependent degradation by APC/C-Cdh1 and by FBXW7, the latter acting on the same Plk4-phosphorylated STIL sites that promote assembly [#26, #27]; CDK-1 phosphorylation of the SAS-6 C-terminus inhibits its phase separation to drive centrosome elimination during oogenesis [#25]. Dysregulated, non-degradable SAS-6 promotes excess ciliation, YAP/TAZ activation, and cell invasion [#27], and SAS-6 is required for centriole formation in mouse embryos, where its loss arrests development at mid-gestation [#24].\",\n  \"teleology\": [\n    {\n      \"year\": 2005,\n      \"claim\": \"Established that human SAS-6 is a bona fide centrosomal duplication factor and placed it in a conserved recruitment pathway, answering whether SAS-6 acts in centriole biogenesis and with what partners.\",\n      \"evidence\": \"siRNA knockdown and GFP localization in human U2OS cells, C. elegans genetics and co-immunoprecipitation\",\n      \"pmids\": [\"15665853\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the structural basis of SAS-6 assembly\", \"Mechanism of ZYG-1/SAS-5-dependent recruitment not resolved at the molecular level\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Located SAS-6 to the cartwheel center and showed it is essential to establish nine-fold symmetry, answering what structural element imposes centriole symmetry.\",\n      \"evidence\": \"Chlamydomonas bld12 null mutant analysis with EM and immunolocalization\",\n      \"pmids\": [\"18082404\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not show how SAS-6 molecular geometry translates into nine-fold symmetry\", \"No atomic structure of the assembling protein\"]\n    },\n    {\n      \"year\": 2009,\n      \"claim\": \"Identified a direct kinase modification of SAS-6 (ZYG-1 phosphorylation at S123) required for its maintenance at the emerging centriole, linking a centriolar kinase to SAS-6 stability.\",\n      \"evidence\": \"In vitro kinase assay with C. elegans phospho-mutant rescue\",\n      \"pmids\": [\"20059959\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Whether S123 is the essential ZYG-1 substrate for duplication was later questioned\", \"Did not address recruitment versus retention\"]\n    },\n    {\n      \"year\": 2010,\n      \"claim\": \"Provided the first biochemical model for how SAS-6 builds the cartwheel by showing it self-assembles into defined oligomers that template the central tubule.\",\n      \"evidence\": \"Gel filtration, native PAGE, EM of recombinant protein and overexpression in Drosophila cells\",\n      \"pmids\": [\"20083610\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"The specific tetramer model was complemented by later ring/spiral structures\", \"No high-resolution structure of the assembly interfaces\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Resolved the structural basis of cartwheel-center formation, showing SAS-6 N-terminal self-association generates cartwheel-like assemblies whose interfaces are functionally required.\",\n      \"evidence\": \"Zebrafish SAS-6 N-terminal X-ray crystallography with point mutagenesis and in vivo centriole formation assay\",\n      \"pmids\": [\"21273447\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not explain how the symmetry number is fixed at nine\", \"Did not address ring stacking or polarity\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Dissected the recruitment interactions, showing ZYG-1 binds the SAS-6 coiled coil independently of kinase activity and an adjacent segment binds SAS-5, both required for cartwheel assembly.\",\n      \"evidence\": \"Pulldown, co-IP, in vitro binding and C. elegans alanine-substitution genetics\",\n      \"pmids\": [\"23673331\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"The essential ZYG-1 kinase substrate for cartwheel assembly remained unidentified\", \"Stoichiometry of the ZYG-1/SAS-5/SAS-6 assembly not defined\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Revealed an alternative SAS-6 spiral architecture consistent with the same nine-fold output, showing distinct oligomerization modes can specify identical symmetry.\",\n      \"evidence\": \"X-ray crystallography and EM of C. elegans SAS-6\",\n      \"pmids\": [\"23798409\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish what selects spiral versus ring assembly in vivo\", \"Link between spiral and central tube versus cartwheel not mechanistically resolved\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Established Plk4 phosphorylation of the Ana2/STIL STAN motif as the earliest upstream trigger enabling Ana2 to bind and recruit SAS-6, defining the kinase-controlled gate for procentriole assembly.\",\n      \"evidence\": \"In vitro kinase assay and Drosophila phospho-mutant rescue with live imaging\",\n      \"pmids\": [\"25264260\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the full ordered sequence of Ana2 phosphorylation events\", \"Structural basis of phospho-dependent Ana2-SAS-6 binding not shown\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Provided a definitive structure proving SAS-6 self-assembly alone imposes nine-fold cartwheel symmetry and showed the assembly is druggable.\",\n      \"evidence\": \"3.5 Å X-ray structure of a nine-fold symmetric Leishmania SAS-6 cartwheel and in vitro small-molecule inhibition\",\n      \"pmids\": [\"24596152\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not address how the cartwheel stacks or acquires polarity\", \"Did not test the inhibitor in cells\"]\n    },\n    {\n      \"year\": 2014,\n      \"claim\": \"Uncovered a luminal SAS-6 recruitment route in human centrioles, showing initial recruitment uses the luminal wall rather than self-oligomerization and that Plk4/STIL-dependent release governs subsequent assembly.\",\n      \"evidence\": \"Live-cell fluorescence, siRNA knockdown and structured illumination microscopy\",\n      \"pmids\": [\"25017693\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; the luminal pathway needs independent confirmation\", \"Molecular identity of the luminal wall binding site not defined\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Identified the SAS-6 C-terminal tail as a direct microtubule-nucleating module, connecting cartwheel assembly to centriolar microtubule formation.\",\n      \"evidence\": \"In vitro tubulin polymerization, microtubule pulldown, ITC and co-IP from S-phase HeLa lysates\",\n      \"pmids\": [\"26422590\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab in vitro biochemistry\", \"In vivo requirement of the tail for microtubule nucleation not yet shown at this stage\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Showed both SAS-6 N-terminal homo-oligomerization and Ana2 tetramerization are individually required for centriole assembly, with high-resolution structures of each module.\",\n      \"evidence\": \"0.8 Å and 2.9 Å X-ray structures, in vitro oligomerization assays and Drosophila point-mutant genetics\",\n      \"pmids\": [\"26002084\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define how the two oligomerization activities are coordinated temporally\", \"Structure of the combined SAS-6–Ana2 assembly not solved\"]\n    },\n    {\n      \"year\": 2015,\n      \"claim\": \"Challenged the strict requirement for SAS-6 self-assembly by showing de novo centriole formation can proceed without it, refining the role of self-oligomerization in fidelity rather than absolute biogenesis.\",\n      \"evidence\": \"Reconstitution of de novo centriole synthesis in human cells with oligomerization-deficient mutants and EM\",\n      \"pmids\": [\"26609813\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; paradigm-challenging result needs replication\", \"What substitutes for SAS-6 self-assembly during de novo formation is unknown\"]\n    },\n    {\n      \"year\": 2016,\n      \"claim\": \"Demonstrated causally that SAS-6 self-assembly properties instruct cartwheel symmetry and that cartwheel and microtubule wall assemble interdependently.\",\n      \"evidence\": \"Engineered SAS-6 symmetry mutants expressed in Chlamydomonas and human cells with EM\",\n      \"pmids\": [\"26999736\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"In vivo symmetry buffering toward nine-fold not fully explained\", \"Contribution of additional factors to symmetry correction unresolved\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Resolved the ordered Plk4 phosphorylation logic on Ana2, showing an initial ANST-motif phosphorylation promotes Ana2 recruitment before STAN-motif phosphorylation enables SAS-6 recruitment.\",\n      \"evidence\": \"In vitro kinase assay, mass spectrometry and Drosophila phospho-mutant rescue with live imaging\",\n      \"pmids\": [\"29263250\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not establish how phosphorylation timing is controlled in the cycle\", \"Phosphatase counter-regulation not addressed\"]\n    },\n    {\n      \"year\": 2017,\n      \"claim\": \"Identified Cdc6 as a negative regulator that binds SAS-6 to block its STIL interaction, with Plk4 phosphorylation of Cdc6 relieving this inhibition to license duplication.\",\n      \"evidence\": \"Co-IP, co-localization, siRNA and overexpression with centrosome duplication readout\",\n      \"pmids\": [\"28447620\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; physiological contribution to duplication timing not quantified\", \"Direct competition between Cdc6 and STIL for SAS-6 not structurally defined\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Captured the kinetics of SAS-6 ring formation, showing assembly proceeds via multiple routes driven by weak surface interactions to reach nine-fold symmetry.\",\n      \"evidence\": \"High-speed atomic force microscopy (PORT) with kinetic analysis\",\n      \"pmids\": [\"29784964\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single-method study; in-cell relevance of surface-driven kinetics not tested\", \"How the cell biases assembly toward correct symmetry not addressed\"]\n    },\n    {\n      \"year\": 2019,\n      \"claim\": \"Showed pericentrin (Pcp1) directly binds and recruits SAS-6, linking the pericentriolar material to cartwheel assembly and centriole elongation.\",\n      \"evidence\": \"Ectopic expression in fission yeast, co-IP and genetic assays in animal cells\",\n      \"pmids\": [\"31182187\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; recruitment hierarchy relative to ZYG-1/STIL unclear\", \"Structural basis of the Pcp1–SAS-6 interface not resolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Mapped SAS-6 C-terminus to γ-TuRC interaction required for centriolar microtubule formation in daughter centrioles, mechanistically linking the cartwheel hub to microtubule nucleation in vivo.\",\n      \"evidence\": \"Co-IP, deletion mutant analysis, siRNA and high-resolution fluorescence microscopy\",\n      \"pmids\": [\"32442461\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; direct versus indirect γ-TuRC binding not fully separated\", \"Relationship between tubulin-binding and γ-TuRC-binding activities of the tail unresolved\"]\n    },\n    {\n      \"year\": 2020,\n      \"claim\": \"Defined the Ana2–SAS-6 interaction surfaces in the C-terminal regions of both proteins and showed SAS-6 can simultaneously engage Ana2 and Gorab through distinct sites.\",\n      \"evidence\": \"HDX-MS, in vitro complex formation, mutagenesis and co-IP\",\n      \"pmids\": [\"33171067\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab interface mapping\", \"Functional consequence of simultaneous Ana2/Gorab binding not quantified\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Defined the Gorab–SAS-6 heterotrimer structurally and showed a single SAS-6 residue in the Gorab-binding site is essential for centriole duplication, connecting Golgi-associated Gorab to the centriole.\",\n      \"evidence\": \"HDX-MS, EM, mutagenesis with in vivo duplication assay and binding assays\",\n      \"pmids\": [\"33704067\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Functional role of the Golgi-versus-centriole Gorab equilibrium not fully defined\", \"How Gorab binding contributes to cartwheel function mechanistically unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Used designed monobodies to separate SAS-6 ring assembly from ring stacking and to drive a ring-to-helix conformational switch that impairs centriole biogenesis, dissecting distinct assembly steps.\",\n      \"evidence\": \"X-ray crystallography, AFM, cryo-EM and a human-cell centriole biogenesis assay\",\n      \"pmids\": [\"34155202\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not define the endogenous regulator of the ring-helix transition\", \"Physiological signal controlling stacking not identified\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Identified an asymmetric coiled-coil homo-oligomerization interaction that imparts polarity to the cartwheel and supports ring stacking, providing a structural basis for the centriolar proximal-distal axis.\",\n      \"evidence\": \"X-ray crystallography, cryo-EM reconstitution and site-directed mutagenesis\",\n      \"pmids\": [\"35240058\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct demonstration that this polarity sets the in vivo proximal-distal axis not completed\", \"How many stacked rings the asymmetry permits unresolved\"]\n    },\n    {\n      \"year\": 2022,\n      \"claim\": \"Connected centriole number control to SAS-6 abundance, showing CHD-1 and EFL-1/DPL-1 downregulate CDK-2 to limit SAS-6 protein levels.\",\n      \"evidence\": \"C. elegans genetic epistasis, RNAi, Western blot and centriole counting\",\n      \"pmids\": [\"35377871\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; whether the axis is transcriptional or post-translational not fully separated\", \"Conservation in mammalian cells not tested\"]\n    },\n    {\n      \"year\": 2024,\n      \"claim\": \"Established the in vivo requirement for SASS6 in mammalian centriole formation and revealed a context-dependent dispensability in stem cells with high PLK4 activity, defining when SAS-6 is essential.\",\n      \"evidence\": \"Mouse knockout genetics, immunofluorescence, centriole ultrastructure and cell death pathway assays\",\n      \"pmids\": [\"38407237\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Molecular basis of SAS-6-independent biogenesis in mESCs incompletely defined\", \"Relationship to the mitotic surveillance pathway downstream of centriole loss unresolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Showed SAS-6 phase separation, modulated by CDK-1 phosphorylation of its C-terminus, governs centrosome assembly and elimination across meiotic prophase, adding a material-state layer to SAS-6 regulation.\",\n      \"evidence\": \"In vitro phase separation and kinase assays, mass spectrometry and C. elegans phospho-mutant genetics with live imaging\",\n      \"pmids\": [\"40410380\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single lab; in vivo relevance of droplet formation beyond oogenesis untested\", \"Relationship between phase separation and cartwheel polymerization not resolved\"]\n    },\n    {\n      \"year\": 2025,\n      \"claim\": \"Defined ubiquitin-dependent degradation pathways (APC/C-Cdh1 and FBXW7) that limit SAS-6 and STIL-SAS6 abundance, and linked non-degradable SAS-6 to ciliation, YAP/TAZ activation and invasion.\",\n      \"evidence\": \"Co-IP, siRNA, ubiquitination and invasion assays, YAP/TEAD reporters, non-degradable mutant expression and Plk4 inhibitor treatment\",\n      \"pmids\": [\"41453690\", \"40825584\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Single labs; in vivo tumor relevance of SAS-6-driven invasion untested\", \"How the same Plk4 phosphosites coordinate assembly then FBXW7 destruction temporally not fully resolved\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"How SAS-6 assembly state, post-translational regulation, and abundance are integrated in real time to ensure exactly one cartwheel of correct symmetry and polarity per duplication cycle remains unresolved.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"No unified model linking phase separation, ring stacking polarity, and degradation timing\", \"Endogenous trigger of the ring-to-helix conformational switch unknown\", \"Mechanism of SAS-6-independent centriole biogenesis in high-PLK4 contexts undefined\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0005198\", \"supporting_discovery_ids\": [1, 4, 8, 11, 13]},\n      {\"term_id\": \"GO:0008092\", \"supporting_discovery_ids\": [10, 18]},\n      {\"term_id\": \"GO:0060090\", \"supporting_discovery_ids\": [5, 7, 17, 20]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005815\", \"supporting_discovery_ids\": [0, 1, 9, 18]},\n      {\"term_id\": \"GO:0005856\", \"supporting_discovery_ids\": [10, 18]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1852241\", \"supporting_discovery_ids\": [0, 1, 13, 24]},\n      {\"term_id\": \"R-HSA-1640170\", \"supporting_discovery_ids\": [0, 15, 26]}\n    ],\n    \"complexes\": [\"centriolar cartwheel\", \"STIL-SAS6 cartwheel assembly complex\"],\n    \"partners\": [\"STIL\", \"PLK4\", \"ZYG-1\", \"CDC6\", \"PCNT\", \"Gorab\", \"TUBG1\"],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}