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Cited by 113Cellular transport and secretionAutophagy in Disease and TherapyCalcium signaling and nucleotide metabolism

The DOI matches its Crossref record; 70 of the 70 references it cites were found in a registry or an index; 57 of the 72 sources its claims cite were checked for support.

Endosomal PI(3)P regulation by the COMMD/CCDC22/CCDC93 (CCC) complex controls membrane protein recycling

Open access
Singla, Amika, Fedoseienko, Alina, Giridharan, Sai S. P., Overlee, Brittany L., Lopez, Adam, Jia, Da, Song, Jie, Huff-Hardy, Kayci, Weisman, Lois, Burstein, Ezra, Billadeau, Daniel D.·2019·Nature Communications
DOI 10.1038/s41467-019-12221-6PMC PMC6753146
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Abstract

Protein recycling through the endolysosomal system relies on molecular assemblies that interact with cargo proteins, membranes, and effector molecules. Among them, the COMMD/CCDC22/CCDC93 (CCC) complex plays a critical role in recycling events. While CCC is closely associated with retriever, a cargo recognition complex, its mechanism of action remains unexplained. Herein we show that CCC and retriever are closely linked through sharing a common subunit (VPS35L), yet the integrity of CCC, but not retriever, is required to maintain normal endosomal levels of phosphatidylinositol-3-phosphate (PI(3)P). CCC complex depletion leads to elevated PI(3)P levels, enhanced recruitment and activation of WASH (an actin nucleation promoting factor), excess endosomal F-actin and trapping of internalized receptors. Mechanistically, we find that CCC regulates the phosphorylation and endosomal recruitment of the PI(3)P phosphatase MTMR2. Taken together, we show that the regulation of PI(3)P levels by the CCC complex is critical to protein recycling in the endosomal compartment.

Overview

AI snapshot

Written from the paper’s full text.

TL;DR

The CCC complex is a distinct ~700 kDa assembly from retriever (~250 kDa) that shares VPS35L and negatively regulates WASH-dependent endosomal F-actin by controlling PI(3)P turnover via MTMR2. CCC loss elevates endosomal PI(3)P, recruits/activates WASH, traps recycling receptors, and VPS34 inhibition or phosphorylation-resistant MTMR2 S58A rescues these phenotypes.

Key findings

  • CCC and retriever are separable: blue native gel analysis places CCC components COMMD1/CCDC22 near 700 kDa, while VPS35L migrates in both ~700 kDa and ~250 kDa complexes; retriever is ~250 kDa, and VPS35L is a shared subunit. CCC loss collapses the 700 kDa complex, whereas VPS26C loss affects the lower complex.
  • CCC depletion, but not VPS26C or FAM45A depletion, increases endosomal PI(3)P: a dsRed-EEA1-FYVE reporter shows ~2.5-fold higher PI(3)P in COMMD3 and CCDC93 KO cells, and HPLC shows ~3.2-fold higher total PI(3)P in COMMD3 KO cells.
  • CCC loss increases WASH recruitment/activity and endosomal F-actin: WASH, F-actin, cortactin, and ARPC2 accumulate on endosomal compartments; WASH knockdown eliminates excess endosomal F-actin, and Commd1 depletion increases WASH actin-nucleating activity.
  • Mechanistically, CCDC22 binds MTMR2; COMMD1 deficiency elevates phosphorylated MTMR2, MTMR2 knockdown phenocopies CCC loss, and endosomally localized MTMR2 S58A rescues F-actin accumulation in COMMD3 KO cells. VPS34 inhibition or siRNA reduces PI(3)P, WASH, F-actin, and restores integrin recycling defects.

Methods

  • CRISPR/Cas9 knockout HeLa lines for CCDC93, VPS26C, COMMD3, VPS35L, and FAM45A, with lentiviral rescue; HEK293T cells, mouse embryonic fibroblasts, and patient CCDC22 p.T17A fibroblasts; siRNA/shRNA for VPS34, MTMR2, WASH, and COMMD1.
  • Proteomics by HA-CCDC93/HA-VPS26C immunoprecipitation and LC-MS/MS; blue native PAGE; co-immunoprecipitation/immunoblotting; confocal immunofluorescence; pyrene-actin assembly assays; HPLC for phosphoinositides; VPS34 in vitro kinase ELISA; integrin endocytosis/recycling assays.
  • Statistics: mean ± SEM; one-way ANOVA with Dunnett’s test or unpaired two-tailed t test; P < 0.05 considered significant.

Limitations

  • Stated: the precise mechanisms by which endosomal PI(3)P controls WASH recruitment/activation remain to be fully elucidated; how CCC controls MTMR2 phosphorylation and how PI(3)P impacts WASH need future study; the unique contributions of each COMMD protein remain poorly understood.
  • Evident: mechanistic conclusions rely largely on cultured HeLa, HEK293T, mouse embryonic fibroblast, and patient fibroblast systems, with in vivo disease relevance inferred from prior models; the proteomic interactome was n = 1 per group; no direct MTMR2 phosphatase activity assay is shown, only phosphorylation, recruitment, and genetic rescue data.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/s41467-019-12221-6

Cites

70 of 70 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Verification

Claims the paper makes

57 of 72 cited sources checked · 18 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    CCC deficiency in human and mouse cells causes defective recycling of SNX17-retriever cargoes such as β1 integrin, LDLR, and LRP1.

    “CCC deficiency in human and mouse cells causes defective recycling of SNX17-retriever cargoes such as β1 integrin, LDLR, and LRP120,31,32”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [20] McNally, K. E. et al. Retriever is a multiprotein complex for retromer-independent endosomal cargo recycling. Nat. Cell Biol. 30, 3481 (2017). source

      The Results section on CCC depletion (page not provided) reports that suppressing CCC components reduced recycling of β1 integrin. The proteomic-identification Results section describes LDLR recycling as CCC-dependent, citing earlier work; another Results section finds that LRP1 cell-surface levels depend on SNX17 and retriever, but does not establish the effect of CCC deficiency on LRP1. The passages found do not report the claimed defect in mouse cells. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [31] Bartuzi, P. et al. CCC- and WASH-mediated endosomal sorting of LDLR is required for normal clearance of circulating LDL. Nat. Commun. 7, 10961 (2016). source

      The Results section, “COMMD1 and WASH facilitate LDLR trafficking” (page unavailable), reports mislocalized LDLR and markedly reduced surface LDLR in COMMD1-deficient mouse cells. The Discussion describes SNX17 involvement in LDLR trafficking as a hypothesis, not an established CCC–SNX17-retriever pathway. Our copy does not show defective recycling of β1 integrin or LRP1 caused by CCC deficiency, or demonstrate the claimed cargo defects in human cells. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [32] Fedoseienko, A. et al. The COMMD family regulates plasma LDL levels and attenuates satherosclerosis through stabilizing the CCC complex in endosomal LDLR trafficking. Circ. Res. 122, 1648–1660 (2018). source

      The Discussion (page unavailable) reports that CCC inactivation affects LDLR and LRP1 function and reduces their surface levels in mouse liver, likely through impaired endosomal trafficking. It describes their recycling through the SNX17–CCC–WASH pathway as a speculation, not an established retriever-cargo result. The passages do not establish defective β1-integrin recycling or the claimed recycling defect in human cells. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
  • Findingcitation

    Interaction experiments by unbiased screens in mammalian cells and protein-protein interaction studies in Drosophila produced interactome data that in aggregate includes all the proteins identified experimentally in our mass spectrometry data, including FAM45A as a CCC and retriever binding protein.

    “interaction experiments by unbiased screens in mammalian cells46, as well as protein–protein interaction studies in Drosophila47–49, produced interactome data that in aggregate includes all the proteins identified experimentally in our mass spectrometry data, including the identification of FAM45A as a CCC and retriever binding protein”
    Quote in the paperNot a substantial claim3 of 4 sources checked
    • cites [46] Huttlin, E. L. et al. Architecture of the human interactome defines protein communities and disease networks. Nature 545, 505–509 (2017). source

      Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [47] Giot, L. et al. A protein interaction map of Drosophila melanogaster. Science 302, 1727–1736 (2003). source
    • cites [48] Guruharsha, K. G. et al. A protein complex network of Drosophila melanogaster. Cell 147, 690–703 (2011). source
    • cites [49] Formstecher, E. et al. Protein interaction mapping: a Drosophila case study. Genome Res. 15, 376–384 (2005). source
  • Premisecitation

    CCC recruitment to endosomal membranes is dependent on the WASH complex.

    “CCC recruitment to endosomal membranes is dependent on the pentameric Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complex27”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [27] Phillips-Krawczak, C. A. et al. COMMD1 is linked to the WASH complex and regulates endosomal trafficking of the copper transporter ATP7A. Mol. Biol. Cell 26, 91–103 (2015). source

      The Results section, “FAM21 is required for endosomal recruitment of the CCC complex” (page unavailable), concludes that the WASH-complex subunit FAM21 is required for CCC recruitment to endosomes. But it also reports that loss of Wash did not abolish COMMD1 endosomal localization, so dependence on the WASH complex as a whole is broader than the demonstrated finding. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Premisecitation

    VPS34 is the main lipid kinase involved in the generation of endosomal PI(3)P.

    “VPS34, the main lipid kinase involved in the generation of endosomal PI(3)P2–4”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [2] Volinia, S. et al. A human phosphatidylinositol 3-kinase complex related to the yeast Vps34p-Vps15p protein sorting system. EMBO J. 14, 3339–3348 (1995). source

      The Introduction identifies yeast Vps34p as a PtdIns 3-kinase, and the paper reports that the human homologue produces PtdIns3P. The discussion proposes a role in intracellular protein trafficking, but does not establish that VPS34 is the main source of endosomal PI(3)P. Page numbers are unavailable in our copy. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [3] Christoforidis, S. et al. Phosphatidylinositol-3-OH kinases are Rab5 effectors. Nat. Cell Biol. 1, 249–252 (1999). source
    • cites [4] Murray, J. T., Panaretou, C., Stenmark, H., Miaczynska, M. & Backer, J. M. Role of Rab5 in the recruitment of hVps34/p150 to the early endosome. Traffic 3, 416–427 (2002). source
  • Premisecitation

    Two independent high-throughput proteomics screens identified that MTMR2 may interact with CCDC22.

    “two independent high-throughput proteomics screens identified that MTMR2 may interact with CCDC2246,57”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [46] Huttlin, E. L. et al. Architecture of the human interactome defines protein communities and disease networks. Nature 545, 505–509 (2017). source

      Our copy describes the BioPlex high-throughput protein-interaction screen (Affinity-Purification Mass Spectrometry methods; page unavailable), but the retrieved passages do not identify an MTMR2–CCDC22 interaction. Searches for “MTMR2 CCDC22 interaction,” “MTMR2,” and “CCDC22” found no passage addressing that protein pair, so our copy cannot verify this source's contribution to the claim about two screens. (LLM, conf=0.72)

      Experimental — accuracy not yet measured
    • cites [57] St-Denis, N. et al. Phenotypic and interaction profiling of the human phosphatases identifies diverse mitotic regulators. Cell Rep. 17, 2488–2501 (2016). source

      The supplied passage has no section or page label and contains only URLs, not findings about protein interactions. Searches of our copy for “MTMR2 CCDC22,” each protein name separately, and interaction proteomics found no passage stating that MTMR2 may interact with CCDC22. (LLM, conf=0.75)

      Experimental — accuracy not yet measured
  • Probably not a substantial claim. 29
  • It may state two separate claims as one. 47

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    CCC shows close co-evolution with the WASH complex.

    “the CCC complex shows close co-evolution with the WASH complex20,36”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [20] McNally, K. E. et al. Retriever is a multiprotein complex for retromer-independent endosomal cargo recycling. Nat. Cell Biol. 30, 3481 (2017). source
    • cites [36] Li, Y., Calvo, S. E., Gutman, R., Liu, J. S. & Mootha, V. K. Expansion of biological pathways based on evolutionary inference. Cell 158, 213–225 (2014). source

      In Results, under “Exploring the evolutionary modularity of 1025 canonical pathways and complexes” (page not provided), two genes, CCDC93 and CCDC22, score highly in the expansion of a WASH evolutionary module. That supports shared evolutionary history for those genes and WASH components, but the passage does not identify them as the CCC complex or establish close co-evolution of the complexes as wholes. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim.

    Premisecitation

    The CCC protein complex consists of CCDC22, CCDC93, and any of the 10 COMMD proteins.

    “the CCC protein complex, which consists of CCDC22, CCDC93, and any of the 10 COMMD proteins”
    Quote in the paperSubstantial claim5 of 6 sources checked
    • cites [27] Phillips-Krawczak, C. A. et al. COMMD1 is linked to the WASH complex and regulates endosomal trafficking of the copper transporter ATP7A. Mol. Biol. Cell 26, 91–103 (2015). source

      The Results ('Identification of the CCC complex') report that all 10 COMMD proteins interact with CCDC93, and the Discussion identifies COMMD proteins, CCDC22, and CCDC93 as core components. But the Discussion also includes C16orf62 in the core composition, so the claim's apparently complete list omits a component. Page numbers are not provided in our copy's passages. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [28] Burstein, E. COMMD proteins, a novel family of structural and functional homologs of MURR1. J. Biol. Chem. 280, 22222–22232 (2005). source

      The “COMMD1 Can Associate with Other COMMD Proteins” section identifies 10 COMMD proteins and reports that COMMD1 interacts with the others (page unavailable). It does not identify a CCC complex or establish that CCDC22 and CCDC93 form a complex with any of those proteins; searches for those names and the CCC complex found no such passage. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [29] van de Sluis, B., Rothuizen, J., Pearson, P. L., van Oost, B. A. & Wijmenga, C. Identification of a new copper metabolism gene by positional cloning in a purebred dog population. Hum. Mol. Genet. 11, 165–173 (2002). source
    • cites [30] Starokadomskyy, P. et al. CCDC22 deficiency in humans blunts activation of proinflammatory NF-κB signaling. J. Clin. Invest. 123, 2244–2256 (2013). source

      In “COMMD proteins associate with CCDC22, a broadly expressed gene,” the paper reports that all 10 COMMD proteins interact with CCDC22. The Discussion proposes distinct CCDC22–COMMD complexes rather than one containing all 10 at once. Our copy does not establish CCDC93 as a component or describe the three-part CCC complex claimed here (searched: CCDC93, CCC complex). (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [31] Bartuzi, P. et al. CCC- and WASH-mediated endosomal sorting of LDLR is required for normal clearance of circulating LDL. Nat. Commun. 7, 10961 (2016). source

      The paper states that all 10 COMMD family members can engage in the CCC complex (Discussion), supporting the claim's 'any of the 10' phrasing. Its description of the complex's composition also includes C16orf62 alongside CCDC22, CCDC93, and COMMD proteins (opening section); the citing sentence omits that component. Page numbers are not available in the supplied copy. (LLM, conf=0.91)

      Experimental — accuracy not yet measured
    • cites [32] Fedoseienko, A. et al. The COMMD family regulates plasma LDL levels and attenuates satherosclerosis through stabilizing the CCC complex in endosomal LDLR trafficking. Circ. Res. 122, 1648–1660 (2018). source

      The section on COMMD protein stability identifies CCDC22 and CCDC93 as CCC core components and describes their association with COMMD proteins. But it repeatedly identifies C16orf62 as a third CCC core component, which the claimed description of what the complex consists of omits. Our copy also describes all 10 COMMD proteins as assembled into CCC complexes, rather than specifying that a complex contains any one of them. Page numbers are not provided for these passages. (LLM, conf=0.90)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim.

    Premisecitation

    WASH serves as an actin nucleation-promoting factor for the ARP2/3 complex, which drives the generation of branched F-actin filaments on endosomal membranes.

    “WASH serves as an actin nucleation-promoting factor for the ARP2/3 complex, which drives the generation of branched F-actin filaments on endosomal membranes37–40”
    Quote in the paperSubstantial claim4 of 4 sources checked
    • cites [37] Derivery, E. et al. The Arp2/3 activator WASH controls the fission of endosomes through a large multiprotein complex. Dev. Cell 17, 712–723 (2009). source
    • cites [38] Gomez, T. S. & Billadeau, D. D. A FAM21-containing WASH complex regulates retromer-dependent sorting. Dev. Cell 17, 699–711 (2009). source
    • cites [39] Gomez, T. S., Gorman, J. A., de Narvajas, A. A.-M., Koenig, A. O. & Billadeau, D. D. Trafficking defects in WASH-knockout fibroblasts originate from collapsed endosomal and lysosomal networks. Mol. Biol. Cell 23, 3215–3228 (2012). source

      The Introduction identifies WASH as a member of the WASP family of nucleation-promoting factors, which activate Arp2/3 to stimulate F-actin polymerization. The Results show that WASH loss eliminates endosomal F-actin and Arp2/3-rich puncta. Our copy does not specify that the resulting endosomal filaments are branched, so that detail is not supported. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [40] Jia, D. et al. WASH and WAVE actin regulators of the Wiskott-Aldrich syndrome protein (WASP) family are controlled by analogous structurally related complexes. Proc. Natl Acad. Sci. USA 107, 10442–10447 (2010). source

  • Probably not a substantial claim.

    Premisecitation

    Regulation of branched F-actin formation by WASH is needed for proper morphology of the endolysosomal system and endosomal trafficking of a broad range of cargoes.

    “Regulation of branched F-actin formation by WASH is needed for proper morphology of the endolysosomal system and endosomal trafficking of a broad range of cargoes37,39,41–44”
    Quote in the paperSubstantial claim6 of 6 sources checked
    • cites [37] Derivery, E. et al. The Arp2/3 activator WASH controls the fission of endosomes through a large multiprotein complex. Dev. Cell 17, 712–723 (2009). source

      The Results show that blocking WASH-mediated Arp2/3 activation causes endosomal tubulation, and that WASH depletion impairs transferrin recycling. They do not establish defective trafficking of a broad range of cargoes or a requirement for normal morphology across the entire endolysosomal system; the LDL experiments track WASH localization rather than a trafficking defect. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [39] Gomez, T. S., Gorman, J. A., de Narvajas, A. A.-M., Koenig, A. O. & Billadeau, D. D. Trafficking defects in WASH-knockout fibroblasts originate from collapsed endosomal and lysosomal networks. Mol. Biol. Cell 23, 3215–3228 (2012). source

      The Results show that loss of WASH-mediated F-actin causes endosomal collapse, and the Discussion reports altered lysosomal morphology. The paper limits trafficking defects to a subset of receptors: EGFR is affected, but transferrin-receptor recycling is unaffected. It therefore does not establish a requirement for trafficking a broad range of cargoes. The indexed copy provides section labels but no page numbers. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [41] Duleh, S. N. & Welch, M. D. WASH and the Arp2/3 complex regulate endosome shape and trafficking. Cytoskeleton (Hoboken) 67, 193–206 (2010). source

      The Results show that disrupting WASH, Arp2/3, or actin alters endosome shape, and that WASH depletion delays EGF transport to late endosomes (page not available in our copy). The Discussion also says WASH depletion did not affect transferrin uptake or recycling. Thus the paper supports a role in endosome morphology and trafficking of EGF, but not the claim that WASH is needed for trafficking a broad range of cargoes throughout the endolysosomal system. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [42] Carnell, M. et al. Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis. J. Cell Biol. 193, 831–839 (2011). source

      The Results and discussion show that WASH-driven actin polymerization is needed for V-ATPase retrieval and exocytosis, and that WASH-null cells develop enlarged vesicles. The Introduction also describes endosomal tubulation and two trafficking pathways in mammalian cells. These passages support a role in endolysosomal morphology and trafficking, but do not establish trafficking of a broad range of cargoes. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [43] Piotrowski, J. T., Gomez, T. S., Schoon, R. A., Mangalam, A. K. & Billadeau, D. D. WASH knockout T cells demonstrate defective receptor trafficking, proliferation, and effector function. Mol. Cell Biol 33, 958–973 (2013). source
    • cites [44] Ryder, P. V. et al. The WASH complex, an endosomal Arp2/3 activator, interacts with the Hermansky-Pudlak syndrome complex BLOC-1 and its cargo phosphatidylinositol-4-kinase type IIα. Mol. Biol. Cell 24, 2269–2284 (2013). source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Higher order COMMD hexamers may be possible through additional interactions via their shared helical N-terminal domains.

    “these studies suggest that higher order COMMD hexamers may be possible through additional interactions via their shared helical N-terminal domains.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [68] Healy, M. D. et al. Structural insights into the architecture and membrane interactions of the conserved COMMD proteins. Elife 7, pii: e35898 (2018). source

      The Discussion speculates that COMMD N-terminal domains may contribute to higher-order assemblies and notes that related proteins form hexamers through N-terminal interactions. It does not specifically propose a hexameric stoichiometry for COMMD proteins; it says the stoichiometries of their larger complexes remain undetermined. No page number is available in our copy. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • It may state two separate claims as one.

    Findingcitation

    FAM45A binds to CCC and retriever.

    “FAM45A, a DENN-domain containing protein of unknown function, binds to CCC and retriever, in agreement with high-throughput protein–protein interaction studies46,60.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [46] Huttlin, E. L. et al. Architecture of the human interactome defines protein communities and disease networks. Nature 545, 505–509 (2017). source

      The passages on poorly characterized proteins and WASH-complex interactions (Abstract; page not provided) do not identify FAM45A or report its binding to CCC or retriever. Searches for “FAM45A” and “retriever CCC complex” found no passage in our copy that addresses those interactions. (LLM, conf=0.72)

      Experimental — accuracy not yet measured
    • cites [60] Hein, M. Y. et al. A human interactome in three quantitative dimensions organized by stoichiometries and abundances. Cell 163, 712–723 (2015). source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    NCBI Gene collates data from three prior interactome studies.

    “Interacting partners in Drosophila melanogaster were identified in NCBI Gene, which collates data from three prior interactome studies47–49.”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [47] Giot, L. et al. A protein interaction map of Drosophila melanogaster. Science 302, 1727–1736 (2003). source
    • cites [48] Guruharsha, K. G. et al. A protein complex network of Drosophila melanogaster. Cell 147, 690–703 (2011). source

      The Introduction and Results (‘A Drosophila Protein Interaction Map’; page not provided) establish that this paper is a Drosophila interaction-mapping study, so it fits the citation’s identification of a prior interactome study. Our copy does not say that NCBI Gene collates its data or data from exactly three studies; a separate Results passage describes DroID as consolidating seven sources. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [49] Formstecher, E. et al. Protein interaction mapping: a Drosophila case study. Genome Res. 15, 376–384 (2005). source

      The Discussion identifies this paper as a Drosophila interactome study reporting 2,300 interactions, and the comparison section discusses three large-scale fly screens. But the paper does not say that NCBI Gene collates data from those studies; its Discussion instead names FlyBase and other repositories for its deposited interactions. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    The CCC complex has been recognized for its role in regulating endosomal recycling of various cargos.

    “The CCC complex is a molecular assembly that has been recognized for its role in regulating endosomal recycling of various cargos20,27,30–32,34”
    Quote in the paperNot a substantial claim6 of 6 sources checked
    • cites [20] McNally, K. E. et al. Retriever is a multiprotein complex for retromer-independent endosomal cargo recycling. Nat. Cell Biol. 30, 3481 (2017). source
    • cites [27] Phillips-Krawczak, C. A. et al. COMMD1 is linked to the WASH complex and regulates endosomal trafficking of the copper transporter ATP7A. Mol. Biol. Cell 26, 91–103 (2015). source
    • cites [30] Starokadomskyy, P. et al. CCDC22 deficiency in humans blunts activation of proinflammatory NF-κB signaling. J. Clin. Invest. 123, 2244–2256 (2013). source

      The section ‘COMMD proteins associate with CCDC22, a broadly expressed gene’ establishes that CCDC22 and COMMD proteins interact, and ‘CCDC22 regulates the cellular localization of COMMD proteins’ reports changes in their cellular distribution. Neither section addresses endosomal recycling of cargos; searches for ‘endosomal,’ ‘recycling,’ and cargo trafficking found no passage establishing the attributed role. Page numbers were not available in our copy. (LLM, conf=0.75)

      Experimental — accuracy not yet measured
    • cites [31] Bartuzi, P. et al. CCC- and WASH-mediated endosomal sorting of LDLR is required for normal clearance of circulating LDL. Nat. Commun. 7, 10961 (2016). source
    • cites [32] Fedoseienko, A. et al. The COMMD family regulates plasma LDL levels and attenuates satherosclerosis through stabilizing the CCC complex in endosomal LDLR trafficking. Circ. Res. 122, 1648–1660 (2018). source
    • cites [34] Li, H. et al. Endosomal sorting of Notch receptors through COMMD9-dependent pathways modulates Notch signaling. J. Cell Biol. 211, 605–617 (2015). source

  • It may state two separate claims as one.

    Premisecitation

    The spectrum of defects seen with CCC deficiency are far more profound, including neurologic disability and cardiovascular congenital anomalies.

    “the spectrum of defects seen with CCC deficiency are far more profound, including neurologic disability and cardiovascular congenital anomalies67”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [67] Voineagu, I. et al. CCDC22: a novel candidate gene for syndromic X-linked intellectual disability. Mol. Psychiatry 17, 4–7 (2012). source

      The phenotype passage (page unavailable) reports intellectual disability and congenital cardiac abnormalities in affected members of the IGOLD #586 family. The paper links this family to a CCDC22 variant and reduced CCDC22 expression, but does not establish CCC-complex deficiency or compare the severity of its defects with another condition. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    Both retromer and retriever are trimeric assemblies that share VPS29 and can utilize specific SNX proteins to identify cargo.

    “Both retromer and retriever are trimeric assemblies that share one common subunit (VPS29), and can utilize specific SNX proteins to identify cargo in the endosomal compartment14,15,20.”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [14] Burd, C. & Cullen, P. J. Retromer: a master conductor of endosome sorting. Cold Spring Harb. Perspect. Biol. 6, a016774–a016774 (2014). source
    • cites [15] Gallon, M. et al. A unique PDZ domain and arrestin-like fold interaction reveals mechanistic details of endocytic recycling by SNX27-retromer. Proc. Natl Acad. Sci. USA 111, E3604–E3613 (2014). source

      The Abstract identifies retromer as a VPS26–VPS29–VPS35 heterotrimer and describes SNX27 as an endosomal cargo adaptor for retromer. The paper does not address retriever, so it cannot establish that retriever is trimeric, shares VPS29, or uses SNX proteins to identify cargo. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [20] McNally, K. E. et al. Retriever is a multiprotein complex for retromer-independent endosomal cargo recycling. Nat. Cell Biol. 30, 3481 (2017). source

  • Probably not a substantial claim. It may state two separate claims as one.

    Premisecitation

    WASH is recruited to endosomes where it is necessary for branched F-actin deposition, a process essential to maintain endolysosomal network structure, promote fission of membrane tubules, and traffic cargo proteins.

    “WASH is recruited to endosomes where it is necessary for branched F-actin deposition, a process that is essential to maintain the structure of the endolysosomal network, to promote effective fission of membrane tubules and trafficking of cargo proteins37,39,41,43,44.”
    Quote in the paperSubstantial claim5 of 5 sources checked
    • cites [37] Derivery, E. et al. The Arp2/3 activator WASH controls the fission of endosomes through a large multiprotein complex. Dev. Cell 17, 712–723 (2009). source
    • cites [39] Gomez, T. S., Gorman, J. A., de Narvajas, A. A.-M., Koenig, A. O. & Billadeau, D. D. Trafficking defects in WASH-knockout fibroblasts originate from collapsed endosomal and lysosomal networks. Mol. Biol. Cell 23, 3215–3228 (2012). source

      The Introduction and Results support WASH recruitment to endosomes and its role in endosomal F-actin, endolysosomal network integrity, and cargo trafficking. The Discussion, however, says the knockout results do not establish a role in tubule scission; it treats that role as possible rather than showing that WASH-dependent actin is essential for fission. Page numbers are unavailable in our copy. (LLM, conf=0.89)

      Experimental — accuracy not yet measured
    • cites [41] Duleh, S. N. & Welch, M. D. WASH and the Arp2/3 complex regulate endosome shape and trafficking. Cytoskeleton (Hoboken) 67, 193–206 (2010). source

      The Introduction and Results report WASH on endosomes, a role for WASH and actin in maintaining endosome shape, and reduced EGF transport to late endosomes after WASH depletion. The Discussion says that actin assembly might sever endosomal tubules, but explicitly presents fission as speculation, not an established essential process. The paper also studies particular endosomal compartments and EGF trafficking rather than establishing a requirement across the entire endolysosomal network and cargo proteins generally. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [43] Piotrowski, J. T., Gomez, T. S., Schoon, R. A., Mangalam, A. K. & Billadeau, D. D. WASH knockout T cells demonstrate defective receptor trafficking, proliferation, and effector function. Mol. Cell Biol 33, 958–973 (2013). source

      The Introduction says WASH localizes to endomembranes and participates in trafficking through localized Arp2/3-dependent F-actin nucleation. The Results show collapsed endosomal and lysosomal compartments and defective cargo trafficking in WASH-deficient T cells. Our copy does not establish that this actin process promotes fission of membrane tubules; the References list an earlier study on endosome fission, but that title alone does not support the more specific claim. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [44] Ryder, P. V. et al. The WASH complex, an endosomal Arp2/3 activator, interacts with the Hermansky-Pudlak syndrome complex BLOC-1 and its cargo phosphatidylinositol-4-kinase type IIα. Mol. Biol. Cell 24, 2269–2284 (2013). source

      The Results and Discussion (page unavailable) place WASH and actin on early endosomes and report elongated endosomal tubules and cargo missorting after WASH depletion. The paper supports a role in endosomal morphology and cargo trafficking, but describes WASH-driven membrane scission as a hypothesis awaiting testing; it does not establish that this process is essential to maintain the structure of the endolysosomal network. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Several WASP family members contain PIP-interacting motifs that impact their localization and activation state.

    “several WASP family members including WASP/N-WASP, WAVE proteins, and WHAMM, all contain PIP-interacting motifs that impact their localization and activation state62–66.”
    Quote in the paperNot a substantial claim4 of 5 sources checked
    • cites [62] Rohatgi, R., Nollau, P., Ho, H. Y., Kirschner, M. W. & Mayer, B. J. Nck and phosphatidylinositol 4,5-bisphosphate synergistically activate actin polymerization through the N-WASP-Arp2/3 pathway. J. Biol. Chem. 276, 26448–26452 (2001). source

      The Results and Discussion (page unavailable) show that PIP2 cooperates with Nck to activate N-WASP and report that PIP2 is an activation input for WASP. They do not identify PIP-interacting motifs or show that such motifs determine localization; the discussion instead describes Nck bringing N-WASP near membrane PIP2. This source does not address WAVE proteins or WHAMM. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [63] Higgs, H. N. & Pollard, T. D. Activation by Cdc42 and PIP(2) of Wiskott-Aldrich syndrome protein (WASp) stimulates actin nucleation by Arp2/3 complex. J. Cell Biol. 150, 1311–1320 (2000). source

      The Introduction reports that PIP2 stimulates WASp activation, and the Discussion proposes a role for PIP2-dependent membrane binding. But the Discussion explicitly says the PIP2-binding site on WASp is unknown, so this paper does not establish a PIP-interacting motif that controls localization. It also does not establish the claim for WAVE proteins or WHAMM. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [64] Oikawa, T. et al. PtdIns(3,4,5)P3 binding is necessary for WAVE2-induced formation of lamellipodia. Nat. Cell Biol. 6, 420–426 (2004). source
    • cites [65] Senju, Y. et al. Mechanistic principles underlying regulation of the actin cytoskeleton by phosphoinositides. Proc. Natl. Acad. Sci. USA 114, E8977–E8986 (2017). source

      The Membranes section reports that N-WASP’s polybasic motif binds PI(4,5)P2-containing membranes, and the Discussion suggests that high PI(4,5)P2 density may restrict its activation to particular cell regions. The passages do not establish the claimed PIP-interacting motifs or effects on localization and activation for WAVE proteins or WHAMM; the evidence for WASP itself is limited to a cited reference. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [66] Mathiowetz, A. J. et al. An Amish founder mutation disrupts a PI(3)P-WHAMM-Arp2/3 complex-driven autophagosomal remodeling pathway. Mol. Biol. Cell 28, 2492–2507 (2017). source

      The Results section (‘WHAMM is recruited to nascent autophagosomes by binding to PI(3)P’; page unavailable) identifies a phospholipid-binding motif in WHAMM and shows that mutations disrupting PI(3)P binding prevent its localization to autophagosomes. The paper does not show that this interaction changes WHAMM’s activation state, and it does not establish the motif claim for WASP/N-WASP or WAVE proteins. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • It may state two separate claims as one.

    Findingown data

    PI(3)P reporter signal revealed a ~2.5-fold increase in PI(3)P levels in endosomes from COMMD3 and CCDC93 KO cells, which was not seen in VPS26C KO cells.

    “The PI(3)P reporter signal revealed a ~2.5-fold increase in PI(3)P levels in endosomes from COMMD3 and CCDC93 KO cells, which was not seen in VPS26C KO cells”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    ITGβ1 protein levels were reduced in every instance when endosomal trapping was observed, and these reduced levels were restored upon treatment with Bafilomycin A.

    “ITGβ1 protein levels were reduced in every instance when endosomal trapping was observed, and these reduced levels were restored upon treatment with the lysosome protease inhibitor Bafilomycin A”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    CCC components (COMMD1 and CCDC22) migrated close to 700 kDa, whereas VPS35L migrated in two complexes, one of similar mass ~700 kDa and one of ~250 kDa.

    “CCC components (COMMD1 and CCDC22) migrated close to 700 kDa, whereas VPS35L migrated in two complexes, one of similar mass ~700 kDa and one of ~250 kDa”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Full-length MTMR2 is required for its interaction with CCDC22, and no single MTMR2 domain recapitulates binding.

    “full-length MTMR2 is required for its interaction with CCDC22 and no single domain was able to recapitulate binding (Supplementary Fig. 8a).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    Staining revealed accumulation of F-actin on WASH-positive endosomal foci in CCDC93, COMMD3, and VPS35L KO cells, but not in VPS26C and FAM45A KO cells.

    “The staining revealed accumulation of F-actin on WASH-positive endosomal foci in CCDC93, COMMD3, and VPS35L KO cells, but not in VPS26C and FAM45A KO cells”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Endosomal-associated WASH levels were increased in CCDC93, COMMD3, and VPS35L KO, but not in VPS26C KO and FAM45A KO cells.

    “endosomal-associated WASH levels were increased in CCDC93, COMMD3, and VPS35L KO, but not in VPS26C KO and FAM45A KO cells”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    VPS26C and FAM45A had minimal impact on CCC or retriever subunit expression, indicating that neither protein is required for the stability of these assemblies.

    “VPS26C and FAM45A had minimal impact on CCC or retriever subunit expression, indicating that neither protein is required for the stability of these assemblies”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    MTMR2 silencing leads to defective endosomal recycling of ITGβ1 and associated WASH accumulation on endosomes, both reversed by inhibition of PI(3)P synthesis.

    “MTMR2 silencing also led to defective endosomal recycling of ITGβ1, and associated WASH accumulation on endosomes, both of which were reversed by inhibition of PI(3)P synthesis (Fig. 7e, Supplementary Fig. 8f).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Endosomal EEA1 levels were significantly elevated in CCDC93 and COMMD3 KO cells, and only slightly elevated in VPS26C KO cells.

    “endosomal EEA1 levels were significantly elevated in CCDC93 and COMMD3 KO cells, and only slightly elevated in VPS26C KO cells”
    Quote in the paperSubstantial claim
    • Rests on an assumption: PI(3)P plays a central role in endosomal recruitment of effector proteins such as EEA1.

  • It may state two separate claims as one.

    Findingown data

    Neither VPS26C nor COMMD3 loss affected WASH or retromer mobility, indicating that these are separate assemblies from CCC or retriever.

    “neither VPS26C or COMMD3 loss affected WASH or retromer mobility, indicating that these are separate assemblies from CCC or retriever”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Depletion of WASH (shWASH) led to near complete loss of F-actin on endosomes of CCDC93 KO cells, whereas control shRNA continued to show enhanced F-actin accumulation.

    “depletion of WASH (shWASH) led to near complete loss of F-actin on endosomes of these cells, whereas the CCDC93-deficient cell line infected with a control shRNA lentivirus continued to show enhanced F-actin accumulation on endosomes”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Wild-type MTMR2 is largely cytosolic and its presence in endosomes is hard to detect by imaging in WT cells.

    “the WT protein was largely cytosolic and its presence in endosomes was hard to detect by imaging in WT cells (Fig. 7g, top row)”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    In CCC-deficient cells, WASH, FAM21, and VPS35 were present at the tips of cortactin tails, and ITGα5 with FAM21 at the tips of F-actin tails.

    “Closer observation of actin endosomal structures in CCC-deficient cells revealed the presence of WASH, FAM21, and VPS35 at the tips of cortactin tails, and ITGα5 with FAM21 at the tips of F-actin tails”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    COMMD3 deficiency had a lesser impact on retriever complex stability, with a smaller effect on VPS35L and VPS26C protein expression.

    “COMMD3 deficiency had a lesser impact on retriever complex stability by BN gel electrophoresis, this corresponded to a smaller effect on VPS35L and VPS26C protein expression”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    COMMD3 loss led to collapse of the 700 kDa complex, without loss of the 250 kDa retriever complex.

    “COMMD3 loss led to the collapse of the 700 kDa complex, best noted by the change in COMMD1 mobility and the disappearance of a 700 kDa band of VPS35L, without the loss of the 250 kDa retriever complex”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    VPS34 silencing in COMMD3-deficient cells partially restores endosomal morphology and increases the cellular area of WASH-positive vesicles.

    “VPS34 silencing in COMMD3-deficient led to partial restoration of endosomal morphology and increased the cellular area of WASH-positive vesicles (Supplementary Fig. 6e).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    The central region of CCDC22 (amino acids 321-447) does not bind COMMD1 and does not support CCC complex assembly.

    “this region does not bind to COMMD1 and thus does not support CCC complex assembly”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    COMMD3 deficiency resulted in a ~3.2-fold increase in total PI(3)P levels; other PIs were also elevated but to a lesser extent.

    “COMMD3 deficiency resulted in a ~3.2-fold increase in total PI(3)P levels; other PIs were also elevated but to a lesser extent”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    VPS35L deletion led to reduced expression of CCC subunits (CCDC22 and COMMD3 in particular) as well as retriever subunits (VPS26C).

    “VPS35L deletion led to reduced expression of CCC subunits (CCDC22 and COMMD3 in particular) as well as retriever subunits (VPS26C)”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    siRNA depletion of VPS34 in COMMD3-deficient cells diminishes endosomal WASH recruitment and largely eliminates endosomal trapping of internalized ITGα5.

    “siRNA depletion of VPS34 in COMMD3-deficient cells diminished endosomal WASH recruitment and most significantly, this largely eliminated the endosomal trapping of internalized ITGα5 (Fig. 6d–f).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    The retriever complex is of slightly lower mass in VPS26C-deficient cells compared with rescue cells, indicating that this is the main complex containing VPS26C.

    “the retriever complex is of slightly lower mass in VPS26C-deficient cells compared with rescue cells, indicating that this is the main complex containing VPS26C”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    ARPC2 foci were substantially larger in COMMD3-deficient cells, where they still contained internalized ITGα5.

    “ARPC2 foci were substantially larger in COMMD3-deficient cells, where they still contained internalized ITGα5”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Endogenous immunoprecipitation of the CCC complex led to limited or undetectable VPS26C and VPS29 co-precipitation, whereas retriever precipitation led to reliable precipitation of other retriever components.

    “endogenous immunoprecipitation of the CCC complex (by CCDC93 or CCDC22 antibodies) led to limited or undetectable VPS26C and VPS29 co-precipitation, whereas retriever precipitation (by VPS35L antibodies) led to reliable precipitation of other retriever components”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    VPS34 inhibition reduces endosomal EEA1 and WASH complex recruitment and prevents excessive endosomal F-actin accumulation in CCDC93 and COMMD3 KO cells.

    “VPS34 inhibition significantly reduced endosomal EEA1 and WASH complex recruitment, and prevented excessive endosomal F-actin accumulation in CCDC93 and COMMD3 KO cells (Fig. 6c and Supplementary Fig. 6a–c).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    WASH-positive vesicles in CCDC93−, COMMD3−, and VPS35L-deficient cells appeared substantially enlarged and accumulated in a tighter perinuclear area, resulting in a smaller area of the cytosol being occupied by WASH-positive vesicles.

    “WASH-positive vesicles in CCDC93−, COMMD3−, and VPS35L-deficient cells appeared substantially enlarged and accumulated in a tighter perinuclear area (Fig. 4b), thus resulting in a smaller area of the cytosol being occupied by WASH-positive vesicles”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Increased endosomal recruitment of the WASH complex in CCC-deficient cells is directly correlated to an increased activity state for this complex and to F-actin accumulation.

    “The increased endosomal recruitment of the WASH complex in CCC-deficient cells is directly correlated to an increased activity state for this complex, and to F-actin accumulation.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingown data

    Altered endosomal morphology, F-actin, WASH accumulation, and PI(3)P accumulation are unique to CCC complex deletion and not seen in VPS26C- or FAM45A-deleted cell lines.

    “altered endosomal morphology, F-actin, and WASH accumulation, as well as PI(3)P accumulation, are unique to CCC complex deletion and not seen in VPS26C- or FAM45A-deleted cell lines.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Findingnone given

    In cell lysates, both retriever and retromer have similar mobility, larger than the predicted trimeric assembly of their principal subunits (~150-170 kDa).

    “in cell lysates both retriever and retromer have similar mobility, which in both cases is larger than the predicted trimeric assembly of their principal subunits, predicted at ~150–170 kDa.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingown data

    The CCC complex and retriever are two separable assemblies of different sizes (~700 kDa and 250 kDa, respectively) that share VPS35L as a common subunit.

    “data presented here indicate that the CCC complex and retriever are two separable assemblies of different sizes (~700 kDa and 250 kDa, respectively), which share VPS35L as a common subunit.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim.

    Premisecitation

    PI(3)P accumulation on endosomes facilitates the spatial and temporal recruitment of numerous proteins that contain PI(3)P-binding domains such as sorting nexins (SNX).

    “PI(3)P accumulation on endosomes facilitates the spatial and temporal recruitment of numerous proteins that contain PI(3)P-binding domains such as sorting nexins (SNX)5”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [5] Teasdale, R. D. & Collins, B. M. Insights into the PX (phox-homology) domain and SNX (sorting nexin) protein families: structures, functions and roles in disease. Biochem. J. 441, 39–59 (2012). source

  • It may state two separate claims as one.

    Interpretationown data

    The integrity of the CCC complex, but not retriever, is required to maintain normal endosomal levels of phosphatidylinositol-3-phosphate (PI(3)P).

    “yet the integrity of CCC, but not retriever, is required to maintain normal endosomal levels of phosphatidylinositol-3-phosphate (PI(3)P)”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Myotubularins regulate PI(3)P levels.

    “myotubularins56, which also regulate PI(3)P levels”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [56] Raess, M. A., Friant, S., Cowling, B. S. & Laporte, J. WANTED—Dead or alive: myotubularins, a large disease-associated protein family. Adv. Biol. Regul. 63, 49–58 (2017). source

  • It may state two separate claims as one.

    Interpretationown data

    The CCC complex provides a link between the retriever and WASH complexes, and this link is required for endosomal recruitment of retriever.

    “the CCC complex provides a link between the retriever and WASH complexes, and this link is required for endosomal recruitment of retriever”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationown data

    CCC and retriever are closely linked but distinct assemblies.

    “these data suggested that CCC and retriever are closely linked but distinct assemblies”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationown data

    CCC deficiency, but not retriever deficiency, alters specific PI lipids and has the largest impact on endosomal PI(3)P levels.

    “CCC deficiency, but not retriever deficiency, alters specific PI lipids and has the largest impact on endosomal PI(3)P levels”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationown data

    The 700 kDa complex likely represents the CCC complex and VPS35L is a shared subunit present in both CCC and retriever.

    “we surmised that the 700 kDa complex likely represents the CCC complex and that VPS35L is a shared subunit present in both CCC and retriever”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Study design and conductown data

    Results for individual cells are plotted, along with the mean and s.e.m. for each group, and statistical significance was assessed with one-way ANOVA and Dunnett's test to control.

    “Results for individual cells are plotted, along with the mean and s.e.m. for each group (n = 62 cells for WT, 45 for CCDC93 KO, 48 for COMMD3 KO, 50 for VPS35L KO, 43 for VPS26C KO, 48 for FAM45A KO); ***P < 0.0001 (one-way ANOVA and Dunnett’s test to control).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Interpretationown data

    PIKfyve activity is intact in CCC-deficient cells, as conversion of PI3P into PI(3,5)P2 is not impaired.

    “PIKfyve activity seemed intact as conversion of PI3P into PI(3,5)P2 in CCC-deficient cells was not impaired judging by the noted increase in PI(3,5)P2 (Supplementary Fig. 5b).”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationown data

    MTMR2 interacts with the CCC complex and its deficiency phenocopies the endosomal-recycling defect seen in CCC-deficient cells.

    “our data indicated that MTMR2 not only interacts with the CCC complex but its deficiency phenocopies the endosomal-recycling defect seen in CCC-deficient cells.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationown data

    The CCC complex functions as a negative regulator of WASH-dependent F-actin deposition, and this negative regulation is required for optimal cargo sorting from endosomes.

    “the CCC complex functions as a negative regulator of WASH-dependent F-actin deposition, and suggests that this negative regulation is required for optimal cargo sorting from endosomes”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationown data

    CCC and retriever are distinct entities that share VPS35L as common subunit.

    “these results are consistent with CCC and retriever being distinct entities that share VPS35L as common subunit”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim.

    Study design and conductown data

    Three independent VPS34 siRNAs were used to control for off-target effects of the VPS34 inhibitor.

    “To ensure that these results were not due to unintended off-target effects of the VPS34 inhibitor, we used three independent VPS34 siRNAs (Supplementary Fig. 6d).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductown data

    Quantification after normalization by the loading control (Actin) is presented as % compared with the parental control (representative of at least three separate iterations).

    “quantification after normalization by the loading control (Actin), is also presented as % compared with the parental control (representative of at least three separate iterations)”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductown data

    Representative images are shown from two or three independent experiments.

    “Representative images of three independent experiments are shown.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductnone given

    The study protocols for patient-related studies were reviewed and approved by the human research ethics committee of the Women's and Children's Health Network (Adelaide, South Australia, Australia).

    “after review and approval of the study protocols by the human research ethics committee of the Women’s and Children’s Health Network (Adelaide, South Australia, Australia)”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductnone given

    All patient-related studies were performed with the written consent of the participants or their legal guardians.

    “All patient-related studies were performed with the written consent of the participants or their legal guardians”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductnone given

    Murine studies were approved by the UT Southwestern Institutional Animal Care and Use Committee under study number APN 102011.

    “Murine studies were approved by the UT Southwestern Institutional Animal Care and Use Committee under study number APN 102011”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Interpretationown data

    The main defect in PI(3)P accumulation in CCC-deficient states is in PI(3)P conversion to PI owing to MTMR2 functional defects, specifically its phosphorylation status that controls recruitment to endosomes.

    “the aggregate of the data included here indicate that the main defect is in PI(3)P conversion to PI owing to MTMR2 functional defects, specifically its phosphorylation status that controls recruitment to endosomes.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Study design and conductnone given

    To control for protein recovery, samples were eluted in 4 × sodium dodecyl sulfate (SDS) sample buffer by boiling for 5 min, and subsequently loaded onto SDS-PAGE gels for immunoblotting.

    “To control for protein recovery, samples were eluted in 4 × sodium dodecyl sulfate (SDS) sample buffer by boiling for 5 min, and subsequently loaded onto SDS–PAGE gels for immunoblotting, as described above.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductreasoning

    These protocols authorized the preparation of mouse embryo fibroblast cell lines utilized in the studies presented here.

    “These protocols authorized the preparation of mouse embryo fibroblast cell lines utilized in the studies presented here.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductnone given

    Both protein and peptide false discovery rates (FDR) were set to 1% in the proteomics analysis.

    “Both protein and peptide FDR were set to 1%.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductnone given

    An additional requirement of two unique peptide sequences per protein was used for protein identification.

    “An additional requirement of two unique peptide sequences per protein was used for protein identification.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Interpretationreasoning

    PI(3)P turnover is necessary for the loss of WASH complex from endosomes and the termination of F-actin nucleation.

    “PI(3)P turnover is necessary for the loss of WASH complex from endosomes and the termination of F-actin nucleation”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductnone given

    Murine studies were approved by the Mayo Clinic Institutional Animal Care and Use Committee under study number A21314.

    “and by the Mayo Clinic Institutional Animal Care and Use Committee under study number A21314”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim.

    Study design and conductnone given

    As a cutoff, proteins with a minimum enrichment of 10-fold and whose spectral indexes were within one order of magnitude of the spectral index of the bait were selected as putative interacting proteins.

    “As a cutoff, proteins with a minimum enrichment of 10-fold and whose spectral indexes were within one order of magnitude of the spectral index of the bait were selected as putative interacting proteins.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Interpretationreasoning

    VPS35L deletion shares properties with CCC-deficient states, consistent with its presence in both retriever and the CCC complexes.

    “VPS35L deletion shares properties with CCC-deficient states consistent with its presence in both retriever and the CCC complexes.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Inhibition of PIKfyve kinase activity can result in increased F-actin on endosomes.

    “inhibition of PIKfyve kinase activity, which prevents PI(3)P conversion to PI(3,5) P2 can also result in increased F-actin on endosomes53.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [53] Hong, N. H., Qi, A. & Weaver, A. M. PI(3,5)P2 controls endosomal branched actin dynamics by regulating cortactin-actin interactions. J. Cell Biol. 210, 753–769 (2015). source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Study design and conductnone given

    The protocol authorized the derivation of dermal fibroblasts from patients with CCDC22 mutations.

    “This protocol authorized the derivation of dermal fibroblasts from patients with CCDC22 mutations.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    FAM21 has two basic regions within its extended C-terminal tail that interact with PI(3)P and other PIs.

    “FAM21 has two basic regions within its extended C-terminal tail that have been shown to interact with PI(3)P and other PIs40”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [40] Jia, D. et al. WASH and WAVE actin regulators of the Wiskott-Aldrich syndrome protein (WASP) family are controlled by analogous structurally related complexes. Proc. Natl Acad. Sci. USA 107, 10442–10447 (2010). source

      Checked before — its earlier verdict is held back until a new check runs.

71 of 71

  • The paper relies on this without support.

    PI(3)P plays a central role in endosomal recruitment of effector proteins such as EEA1.

    “As PI(3)P plays a central role in endosomal recruitment of effector proteins, such as early endosomal antigen 1 (EEA1), we assessed the levels of endosomally localized EEA1”

  • The paper relies on this without support.

    The phosphoserine antibody used recognizes phosphorylated MTMR2 at serine 58.

    “This residue is preceded by phenylalanine and upon phosphorylation may be recognized by a phosphoserine antibody raised against this motif (phosphoserine preceded by tyrosine, phenylalanine, or tryptophan).”

  • The paper relies on this without support.

    The dsRed-EEA1-FYVE reporter specifically reports endosomal PI(3)P levels.

    “cells transfected with a dsRed-EEA1-FYVE domain PI(3)P reporter plasmid”
3 of 3

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • dsRed-EEA1-FYVE potently and specifically binds PI(3)P.

    “We transfected rat hippocampal neurons with dsRed-EEA1-FYVE, a bioprobe that potently and specifically binds PI(3)P (Singla et al., 2019).”

    From Recruitment of the SNX17-Retriever recycling pathway regulates synaptic function and plasticity (2023) · cites this paper as Singla et al., 2019

    The Results section (‘CCC deficiency results in endosomal accumulation of PI(3)P’; page unavailable) identifies dsRed fused to the EEA1 FYVE domain as a PI(3)P reporter and shows its signal tracking PI(3)P levels. It does not establish that the probe binds PI(3)P potently or specifically. The rat-neuron transfection is the citing authors’ procedure, not a claim attributed to this source. (LLM, conf=0.84)

    Experimental — accuracy not yet measured

  • VPS35 is a marker of the Retromer complex, and both Retromer and Retriever pathways emerge from the same microdomains of VPS35-containing endosomes.

    “VPS35 is a marker of the Retromer complex, and both Retromer and Retriever pathways emerge from the same microdomains of VPS35-containing endosomes (McNally et al., 2017; Singla et al., 2019).”

    From Recruitment of the SNX17-Retriever recycling pathway regulates synaptic function and plasticity (2023) · cites this paper as Singla et al., 2019

    The Introduction identifies VPS35 as a retromer subunit, supporting its use as a marker. In Results (‘CCC links retriever to WASH-containing endosomal domains’), VPS35L puncta are reported near retromer-containing domains, but the paper does not establish that both pathways emerge from the same microdomains of VPS35-containing endosomes. Page numbers were not provided in our copy. (LLM, conf=0.87)

    Experimental — accuracy not yet measured

  • The 16th subunit of Commander is DENND10 (also called FAM45A).

    “The 16th subunit is DENND10 (differentially expressed in normal and neoplastic cells-containing protein 10, also called FAM45A)6,16,20,21,22”

    From Structure of the endosomal Commander complex linked to Ritscher-Schinzel syndrome (2023) · cites this paper as [21]

    The Results section (“Defining the CCC and retriever interactomes”; page unavailable) identifies FAM45A as an additional factor that binds components of both complexes. The paper does not call it Commander’s 16th subunit or identify it as DENND10; the Discussion describes it as a CCC- and retriever-binding protein rather than an established subunit. (LLM, conf=0.85)

    Experimental — accuracy not yet measured

  • Commander is composed of sixteen subunits.

    “Commander is composed of sixteen subunits, arranged in two sub-assemblies, the CCC and Retriever complexes.”

    From Structure of the endosomal Commander complex linked to Ritscher-Schinzel syndrome (2023) · cites this paper as [21]

    In Results (‘Defining the CCC and retriever interactomes’; page not specified), the paper identifies 15 components of CCC and Retriever and an additional protein, FAM45A, that binds to them. It does not identify FAM45A as a sixteenth subunit. The Discussion (page not specified) also describes CCC and Retriever as separable assemblies rather than one Commander complex. (LLM, conf=0.87)

    Experimental — accuracy not yet measured

  • The structure is consistent with truncation analyses that found N-terminal regions of CCDC22 and CCDC93 could interact with COMMD proteins but not Retriever.

    “The structure is also consistent with truncation analyses that found N-terminal regions of CCDC22 and CCDC93 could interact with COMMD proteins but not Retriever.21,65”

    From Structure of the endosomal Commander complex linked to Ritscher-Schinzel syndrome (2023) · cites this paper as [21]

    The Results sections ‘Defining the CCC and retriever interactomes’ and ‘CCC and retriever are closely linked but distinct complexes’ place CCDC22, CCDC93, and COMMD proteins in CCC and describe CCC and Retriever as distinct assemblies. They do not report truncation analyses or show that the N-terminal regions of CCDC22 and CCDC93 bind COMMD proteins but not Retriever; searches for truncation, N-terminal, and fragment interactions did not find that result. Page numbers are unavailable in our copy. (LLM, conf=0.76)

    Experimental — accuracy not yet measured

Citations

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Metadata

Authors
Singla, A., Fedoseienko, A., Giridharan, S. S. P., Overlee, B. L., Lopez, A., Jia, D., Song, J., Huff-Hardy, K., Weisman, L., Burstein, E., & Billadeau, D. D.
Affiliations
  • The University of Texas Southwestern Medical Center (US)
  • Mayo Clinic (US)
  • University of Michigan (US)
  • Sichuan University (CN)
Corresponding author
  • Burstein, Ezra
  • Billadeau, Daniel D.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2019-09-19
Volume
10
Issue
1
Pages
4271-4271
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-019-12221-6
PMC
PMC6753146
OpenAlex
W2974473258
Semantic Scholar
8f23c20a8c8de5f8b7a130b5cc6f7ed138ca3e2f
Cited by
113
References
70
Citations by year
2026: 11 · 2025: 22 · 2024: 15 · 2023: 18 · 2022: 20 · 2021: 14 · 2020: 12 · 2019: 1
FWCI
4.27
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
U.S. Department of Health & Human Services | National Institutes of Health (R01DK073639, RO1DK107733, R01NS064015, R01NS099340, K01DK106346)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Autophagy in Disease and Therapy
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Physiology › Calcium signaling and nucleotide metabolism
Keywords
protein recycling, endolysosomal system, WASH complex, MTMR2, cargo recognition, membrane protein recycling
MeSH terms
Actins, Animals, HeLa Cells, Humans, Lysosomes, Membrane Proteins, Microfilament Proteins, Phosphorylation, Proteins, Endosomes, Phosphatidylinositol Phosphates, Vesicular Transport Proteins, RNA Interference, RNA, Small Interfering, Cell Line, Tumor, Adaptor Proteins, Signal Transducing, Mice, Protein Tyrosine Phosphatases, Non-Receptor, HEK293 Cells

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

A language model drew the claims from the paper on 2026-11-08, under version 3 of the claim definition (model deepseek-v4.1-flash, prompt extract-v3).

A second model labelled each claim on 2026-11-08 (model jev-1.13-20260917, definition revision gold-guideline-v0.2).

Cited sources are checked with a language model’s help (gpt-6-sol).

Statistics and arithmetic are recomputed, not judged.

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