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Cited by 14Protein Kinase Regulation and GTPase SignalingMicrotubule and mitosis dynamicsCellular Mechanics and Interactions

Structural basis of microtubule-mediated signal transduction

Open access
Choi, Sung Ryul, Blum, Thorsten B., Giono, Matteo, Roy, Bibhas, Vakonakis, Ioannis, Schmid, Dominic, Oelgarth, Nicole, Ranganathan, Apisha +4 more·2026·Cell
DOI 10.1016/j.cell.2025.11.011
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Abstract

Microtubules have long been recognized as upstream mediators of intracellular signaling, but the mechanisms underlying this fundamental function remain elusive. Here, we identify the structural basis by which microtubules regulate the guanine nucleotide exchange factor H1 (GEFH1), a key activator of the Ras homolog family member A (RhoA) pathway. We show that specific features of the microtubule lattice bind the C1 domain of GEFH1, leading to the sequestration and inactivation of this signaling protein. Targeted mutations in C1 residues disrupt this interaction, triggering GEFH1 release and activation of RhoA-dependent immune responses. Building on this sequestration-and-release mechanism, we identify microtubule-binding C1 domains in additional signaling proteins, including other guanine nucleotide exchange factors (GEFs), kinases, a GTPase-activating protein (GAP), and a tumor suppressor, and show that microtubule-mediated regulation via C1 domains is conserved in the Ras association domain-containing protein 1A (RASSF1A). Our findings establish a structural framework for understanding how microtubules can function as spatiotemporal signal sensors, integrating and processing diverse signaling pathways to control important cellular processes.

Verified 2026-11-02 · 2 references not checked yet

AI snapshot

Written from the paper’s full text.

TL;DR

This study identifies the structural basis by which microtubules directly bind and sequester the guanine nucleotide exchange factor GEFH1, holding it inactive until release triggers RhoA signaling. It extends this "microtubule sequestration-and-release" model to C1 domains in other signaling proteins, including the tumor suppressor RASSF1A.

Key findings

  • Full-length GEFH1 forms a coiled-coil–mediated dimer (SEC-MALS: 228.9 ± 6.8 kDa vs. 110.4 kDa calculated monomer) and binds microtubules directly; the C1 domain is the key determinant, with the C1-DH linker and PH domain playing supportive roles.
  • The NMR solution structure of the GEFH1 C1 domain (residues 35–89; backbone RMSD 0.09 ± 0.04 Å over 55 Cα) is an atypical C1 domain, and cryo-EM of the microtubule–C1 complex (~3.3 Å for tubulin, ~3.5 Å for C1; ~40% of lattice sites occupied) shows C1 binding via its positively charged face in an inter-protofilament cleft spanning two laterally associated αβ-tubulin heterodimers, with minimal conformational change (RMSD 0.6 Å over 60 Cα).
  • Disrupting this interface with the FL-α′β′ mutant (Lys58Ala, Arg78Ala) increased active RhoA 1.4-fold in HeLa cells (comparable to ansamitocin P3 treatment) and raised CD40, CD80, CD86, and MHC class II on mouse bone marrow-derived dendritic cells to levels seen with the microtubule-destabilizing agent.
  • C1-G4 variants of AKAP13, RASSF1A, RACGAP1, and RAF-1 bound microtubules (saturation near 10 μM tubulin at 1.25 μM protein, low-micromolar affinity), whereas VAV1, PKCζ, and p190RhoGEF did not; RASSF1A mutants (ΔN, C65R, R53A) lost microtubule co-localization and significantly increased HMF3a proliferation.

Methods

  • Combined structural, biophysical, biochemical, and cell-biology approach: SEC-MALS, circular dichroism, solution NMR (1,895 NOE-derived distance restraints), SAXS with ensemble optimization modeling, and cryo-EM of paclitaxel-stabilized microtubule–C1 complexes.
  • Microtubule pelleting assays (including subtilisin-treated microtubules and salt titrations) tested recombinant GEFH1 fragments and C1-G4 fusions; binding was quantified by densitometry.
  • Cell work used HMF3a and HeLa cells and mouse bone marrow-derived dendritic cells, with fluorescence microscopy (Manders' co-localization), RhoA G-LISA, EdU proliferation assays, and FACS for maturation markers.

Limitations

  • Stated by the authors: more high-resolution microtubule–C1 complex structures are needed; additional protein regions likely contribute to stable binding in cells and require characterization; and the roles of the microtubule lattice, post-translational modifications, and accessory factors need further study.
  • Evident in the text: the AKAP13 C1 cryo-EM density was low resolution (~6 Å for C1 vs. ~3 Å for tubulin), so its structure could not be unambiguously modeled, and the shifted binding site is inferred rather than fully resolved.

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

Integrity signals

Registry

Crossref · 10.1016/j.cell.2025.11.011

Cites

103 of 105 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

29 of 47 cited sources checked · 14 supported · 0 flagged · accuracy not yet measured

  • Premisecitation
    § Introduction

    Dynamic microtubules serve as upstream mediators in signaling pathways such as Rho, hedgehog, Wnt, Jnk, and G-protein.

    “However, it has been known for a long time that dynamic microtubules also serve as upstream mediators in signaling pathways, such as Ras homology (Rho), hedgehog, Wingless-related integration site (Wnt), c-Jun N-terminal kinase (Jnk), and G-protein.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [3] 3. Gundersen, G.G. ∙ Cook, T.A. Microtubules and signal transduction Curr. Opin. Cell Biol. 1999; 11:81-94 Crossref PubMed Google Scholar source

      The Conclusion says microtubules play important roles in Hedgehog, Wnt, and JNK signaling, and the G-proteins section describes microtubule interactions that can modulate receptor signaling. But the Wnt section says their role in localizing signaling is only suggested, while the actin-and-signaling section also describes Rho acting upstream to stabilize microtubules. The paper therefore does not establish that dynamic microtubules serve as upstream mediators in all five named pathways. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
  • Premisecitation
    § Results

    The GEFH1 C1 domain does not bind diacylglycerol.

    “Prior studies indicate that the GEFH1 C1 domain does not bind diacylglycerol,21”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [21] 21. Aijaz, S. ∙ D'Atri, F. ∙ Citi, S. ... Binding of GEF-H1 to the tight junction-associated adaptor cingulin results in inhibition of Rho signaling and G1/S phase transition Dev. Cell. 2005; 8:777-786 Full Text Full Text (PDF) Scopus (183) PubMed Google Scholar source

      The Introduction describes GEF-H1’s C1 domain and an assay of its binding to the PH domain, but does not address whether C1 binds diacylglycerol. Searches for diacylglycerol, DAG, lipid binding, and phorbol esters found no passage resolving the claim; page numbers were unavailable in our copy. (LLM, conf=0.72)

      Experimental — accuracy not yet measured
    • cites [26] 26. Hurley, J.H. ∙ Newton, A.C. ∙ Parker, P.J. ... Taxonomy and function of C1 protein kinase C homology domains Protein Sci. 1997; 6:477-480 Crossref Scopus (334) PubMed Google Scholar source

      In the passage on Rho, Rac, and Cdc42 regulators (page unavailable), the paper classifies Lfc as an atypical C1-containing protein. Its classification discussion says atypical C1 domains do not bind diacylglycerol, but also says domains not directly tested were classified tentatively by sequence. The paper does not name GEFH1 or report a direct diacylglycerol-binding test of its C1 domain, so the categorical claim goes beyond the evidence in our copy. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
  • Premisecitation
    § Introduction

    GEFH1 is a critical microtubule-regulated activator of Rho signaling pathways.

    “GEFH1; also known as Arhgef2 or Lfc), a critical microtubule-regulated activator of Rho signaling pathways.”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [10] 10. Krendel, M. ∙ Zenke, F.T. ∙ Bokoch, G.M. Nucleotide exchange factor GEF-H1 mediates cross-talk between microtubules and the actin cytoskeleton Nat. Cell Biol. 2002; 4:294-301 Crossref Scopus (569) PubMed Google Scholar source
    • cites [11] 11. Birkenfeld, J. ∙ Nalbant, P. ∙ Yoon, S.H. ... Cellular functions of GEF-H1, a microtubule-regulated Rho-GEF: is altered GEF-H1 activity a crucial determinant of disease pathogenesis? Trends Cell Biol. 2008; 18:210-219 Full Text Full Text (PDF) Scopus (161) PubMed Google Scholar source
    • cites [12] 12. Azoitei, M.L. ∙ Noh, J. ∙ Marston, D.J. ... Spatiotemporal dynamics of GEF-H1 activation controlled by microtubule- and Src-mediated pathways J. Cell Biol. 2019; 218:3077-3097 Crossref Scopus (0) PubMed Google Scholar source
  • Premisecitation
    § Author contributions

    The level of active RhoA and subsequent F-actin reorganization is linked to p190RhoGEF-microtubule interaction.

    “Additionally, the level of active RhoA and subsequent F-actin reorganization was linked to p190RhoGEF-microtubule interaction.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [41] 41. van Horck, F.P. ∙ Ahmadian, M.R. ∙ Haeusler, L.C. ... Characterization of p190RhoGEF, a RhoA-specific guanine nucleotide exchange factor that interacts with microtubules J. Biol. Chem. 2001; 276:4948-4956 Full Text Full Text (PDF) Scopus (140) PubMed Google Scholar source

      The paper reports that p190RhoGEF interacts with microtubules and that its overexpression activates RhoA with subsequent F-actin reorganization (Abstract). But the Microtubule Dynamics and p190RhoGEF Activity section reports that disrupting microtubules did not change p190RhoGEF-mediated RhoA activation; the Discussion treats a regulatory link as a possibility requiring further study, not an established finding. (LLM, conf=0.89)

      Experimental — accuracy not yet measured
  • Premisecitation
    § Discussion

    Acetylation of Lys40 in α-tubulin facilitates GEFH1 release and activation of the RhoA pathway.

    “acetylation of Lys40 in α-tubulin facilitates GEFH1 release and activation of the RhoA pathway,13”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [13] 13. Seetharaman, S. ∙ Vianay, B. ∙ Roca, V. ... Microtubules tune mechanosensitive cell responses Nat. Mater. 2022; 21:366-377 Crossref Scopus (149) PubMed Google Scholar source
    • cites [49] 49. Roy, A.D. ∙ Gonzalez, C.S. ∙ Shahid, F. ... Optogenetically Induced Microtubule Acetylation Unveils the Molecular Dynamics of Actin-Microtubule Crosstalk in Directed Cell Migration Preprint at bioRxiv. 2024;, 2024.12.01.626286 Crossref Scopus (0) Google Scholar source
  • May combine two claims. 44
  • Classified as probably not a substantial claim. 27
  • The quote only partly states this claim. 5

  • May combine two claims.

    Interpretationcitation
    § Results

    The inter-protofilament binding mode of the GEFH1 C1 domain resembles that of the CAMSAP1 CKK domain, which also spans two neighboring αβ-tubulin heterodimers, suggesting competition for the same binding site.

    “the inter-protofilament binding mode of the GEFH1 C1 domain resembles that of the CKK domain of calmodulin-regulated spectrin-associated protein 1 (CAMSAP1), which also spans two neighboring αβ-tubulin heterodimers”
    • Also stated in: § Results · hedged
      “Like GEFH1 C1, CAMSAP1 CKK engages α-tubulin (H11-helix, B5-H5-loop), α′-tubulin (H3- and H11-helices), β-tubulin (H10-helix), and β′-tubulin (H4-B5-loop), suggesting competition for the same binding site.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [37] 37. Atherton, J. ∙ Luo, Y. ∙ Xiang, S. ... Structural determinants of microtubule minus end preference in CAMSAP CKK domains Nat. Commun. 2019; 10:5236 Crossref Scopus (30) PubMed Google Scholar source

  • May combine two claims.

    Premisecitation
    § Introduction

    Microtubules sequester GEFH1, rendering it inactive, and depolymerization releases GEFH1, activating RhoA.

    “Microtubules sequester GEFH1, rendering it inactive, and depolymerization releases GEFH1, activating RhoA.”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [10] 10. Krendel, M. ∙ Zenke, F.T. ∙ Bokoch, G.M. Nucleotide exchange factor GEF-H1 mediates cross-talk between microtubules and the actin cytoskeleton Nat. Cell Biol. 2002; 4:294-301 Crossref Scopus (569) PubMed Google Scholar source
    • cites [18] 18. Glaven, J.A. ∙ Whitehead, I. ∙ Bagrodia, S. ... The Dbl-related protein, Lfc, localizes to microtubules and mediates the activation of Rac signaling pathways in cells J. Biol. Chem. 1999; 274:2279-2285 Full Text Full Text (PDF) Scopus (142) PubMed Google Scholar source

      The Cellular Localization of Lfc section reports that Lfc localizes to microtubules and that it stimulates RhoA nucleotide exchange in vitro. It does not identify Lfc as GEFH1 or show that microtubules render it inactive, that depolymerization releases it, or that release activates RhoA. No page number is available in our copy. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites [19] 19. Meiri, D. ∙ Marshall, C.B. ∙ Greeve, M.A. ... Mechanistic insight into the microtubule and actin cytoskeleton coupling through dynein-dependent RhoGEF inhibition Mol. Cell. 2012; 45:642-655 Full Text Full Text (PDF) Scopus (77) PubMed Google Scholar

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • Classified as probably not a substantial claim.

    Premisenone given
    § Introduction

    The mechanism by which microtubules bind and regulate GEFH1, and consequently RhoA-mediated signaling, remains elusive.

    “The mechanism by which microtubules bind and regulate the activity of GEFH1, and consequently RhoA-mediated signaling, remains elusive.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [19] 19. Meiri, D. ∙ Marshall, C.B. ∙ Greeve, M.A. ... Mechanistic insight into the microtubule and actin cytoskeleton coupling through dynein-dependent RhoGEF inhibition Mol. Cell. 2012; 45:642-655 Full Text Full Text (PDF) Scopus (77) PubMed Google Scholar

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [23] 23. Siesser, P.F. ∙ Motolese, M. ∙ Walker, M.P. ... FAM123A binds to microtubules and inhibits the guanine nucleotide exchange factor ARHGEF2 to decrease actomyosin contractility Sci. Signal. 2012; 5:ra64 Crossref Scopus (15) PubMed Google Scholar source

      The Discussion (page unavailable) says that the precise mechanism by which FAM123A inhibits GEF-H1 remains to be established and that more work is needed to understand how FAM123A’s effect on microtubule dynamics reaches the actomyosin network through GEF-H1. It also links GEF-H1 to RhoA signaling. It does not specifically say that the mechanism by which microtubules bind and regulate GEF-H1 remains elusive; its stated uncertainty concerns FAM123A. (LLM, conf=0.78)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Results

    The C1 domain is part of a group of zinc-finger proteins homologous to the conserved region 1 of protein kinase C.

    “The C1 domain, also referred to as phorbol esters/diacylglycerol-binding domain, is part of a group of zinc-finger proteins homologous to the conserved region 1 of protein kinase C.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [26] 26. Hurley, J.H. ∙ Newton, A.C. ∙ Parker, P.J. ... Taxonomy and function of C1 protein kinase C homology domains Protein Sci. 1997; 6:477-480 Crossref Scopus (334) PubMed Google Scholar source

  • Classified as probably not a substantial claim.

    Premisecitation
    § Results

    Proteins in the related family of GEFs often regulate enzymatic activity through autoinhibitory interactions with regions flanking their DH-PH domains.

    “Proteins in the related family of GEFs often regulate enzymatic activity through autoinhibitory interactions with regions flanking their DH-PH domains.32”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [32] 32. Yohe, M.E. ∙ Rossman, K.L. ∙ Gardner, O.S. ... Auto-inhibition of the Dbl family protein Tim by an N-terminal helical motif J. Biol. Chem. 2007; 282:13813-13823 Full Text Full Text (PDF) Scopus (40) PubMed Google Scholar source

      The Abstract and Discussion show that Tim’s exchange activity is inhibited by a helix immediately N-terminal to its DH domain, and the Discussion describes a similar interaction in Vav proteins. The Abstract says truncation findings in many Dbl-family proteins led to a proposal of autoinhibition, while the Discussion calls similar regulation in Tim’s subgroup likely. The source therefore supports the mechanism and examples, but not the claim that such interactions are established as occurring often across the family. (LLM, conf=0.78)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data
    § Results

    The AlphaFold-predicted structure of GEFH1 Nter shows no intramolecular interactions between the C1 domain and the compact DH-PH domain and fits the envelope better, with a χ value of 4.6.

    “the AlphaFold-predicted structure of GEFH1 Nter shows no intramolecular interactions between the C1 domain and the compact DH-PH domain and fits the envelope better, with a χ value of 4.6”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [34] 34. Jumper, J. ∙ Evans, R. ∙ Pritzel, A. ... Highly accurate protein structure prediction with AlphaFold Nature. 2021; 596:583-589 Crossref Scopus (33174) PubMed Google Scholar source

      The Main section describes AlphaFold’s structure predictions, but does not discuss GEFH1 Nter. Searches for GEFH1, its C1 and DH-PH domains, and an envelope-fit χ value of 4.6 found no passage addressing the claimed structure or fit. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data
    § Results

    AlphaFold models of C1 domains from AKAP13, p190RhoGEF, RASSF1A, RACGAP1, RAF-1, and PKCζ compared with the GEFH1 C1 NMR structure revealed well-preserved structural folds, but only partial conservation of the microtubule-binding residues identified in GEFH1's C1 domain.

    “Structural models of the C1 domains of these proteins, predicted using AlphaFold, were compared against the NMR structure of GEFH1 C1 (Figure 2A). This comparison revealed well-preserved structural folds”
    • Also stated in: § Results · hedged
      “the corresponding structure-based sequence alignment suggested only partial conservation of the microtubule-binding residues identified in GEFH1’s C1 domain”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [34] 34. Jumper, J. ∙ Evans, R. ∙ Pritzel, A. ... Highly accurate protein structure prediction with AlphaFold Nature. 2021; 596:583-589 Crossref Scopus (33174) PubMed Google Scholar 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

  • Classified as probably not a substantial claim.

    Premisecitation
    § Introduction

    The mechanisms by which microtubules regulate signaling proteins and their diverse pathways remain poorly understood.

    “However, the mechanisms by which microtubules regulate them and their diverse pathways remain poorly understood.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [3] 3. Gundersen, G.G. ∙ Cook, T.A. Microtubules and signal transduction Curr. Opin. Cell Biol. 1999; 11:81-94 Crossref PubMed Google Scholar source
    • cites [4] 4. Moujaber, O. ∙ Stochaj, U. The Cytoskeleton as Regulator of Cell Signaling Pathways Trends Biochem. Sci. 2020; 45:96-107 Full Text Full Text (PDF) Scopus (135) PubMed Google Scholar source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § Results

    Human GEFH1 is a multidomain protein belonging to the Dbl homology family of Rho-GTPase activators.

    “Human GEFH1 is a multidomain protein belonging to the diffuse B cell lymphoma (Dbl) homology family of Rho-GTPase activators.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [11] 11. Birkenfeld, J. ∙ Nalbant, P. ∙ Yoon, S.H. ... Cellular functions of GEF-H1, a microtubule-regulated Rho-GEF: is altered GEF-H1 activity a crucial determinant of disease pathogenesis? Trends Cell Biol. 2008; 18:210-219 Full Text Full Text (PDF) Scopus (161) PubMed Google Scholar source
    • cites [25] 25. Joo, E. ∙ Olson, M.F. Regulation and functions of the RhoA regulatory guanine nucleotide exchange factor GEF-H1 Small GTPases. 2021; 12:358-371 Crossref Scopus (40) PubMed Google Scholar source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Results

    In the Rho GEF proto-oncogene VAV1, autoinhibition is mediated by interactions involving its atypical C1 and calponin homology (CH) domains.

    “For instance, in the Rho GEF proto-oncogene VAV1, such autoinhibition is mediated by interactions involving its atypical C1 and calponin homology (CH) domains.33”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [33] 33. Yu, B. ∙ Martins, I.R.S. ∙ Li, P. ... Structural and energetic mechanisms of cooperative autoinhibition and activation of Vav1 Cell. 2010; 140:246-256 Full Text Full Text (PDF) Scopus (132) PubMed Google Scholar source

      The Introduction and Results (pages unavailable in our copy) show that CH-domain interactions strengthen Vav1 autoinhibition. But the Results describe the inhibitory contacts as involving the CH, Acidic, PH and DH regions, and explicitly state that the CH domain does not contact the zinc-finger domain; they do not establish the claimed involvement of an atypical C1 domain in those inhibitory interactions. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data
    § Results

    Wild-type GEFH1 FL co-localized and bundled microtubules, while the FL-C53R mutant and the FL-αβ, FL-α′β′, and FL-ββ′ mutants showed perturbed microtubule co-localization.

    “wild-type FL co-localized and bundled microtubules, consistent with its known microtubule-associated protein behavior”
    • Also stated in: § Results · plain
      “the FL-C53R mutant exhibited a markedly reduced co-localization with microtubules, consistent with previous findings.”
    • Also stated in: § Results · plain
      “All three mutants showed a perturbed co-localization with microtubules, similar to the FL-C53R mutant”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [10] 10. Krendel, M. ∙ Zenke, F.T. ∙ Bokoch, G.M. Nucleotide exchange factor GEF-H1 mediates cross-talk between microtubules and the actin cytoskeleton Nat. Cell Biol. 2002; 4:294-301 Crossref Scopus (569) PubMed Google Scholar source
    • cites [22] 22. Kashyap, A.S. ∙ Fernandez-Rodriguez, L. ∙ Zhao, Y. ... GEF-H1 Signaling upon Microtubule Destabilization Is Required for Dendritic Cell Activation and Specific Anti-tumor Responses Cell Rep. 2019; 28:3367-3380.e8 Full Text Full Text (PDF) Scopus (54) PubMed Google Scholar source

      The Results section (“Microtubule Destabilization by MDAs Releases and Activates GEF-H1”; page unavailable) reports GEF-H1 association with microtubules and binding by a purified GEF-H1-derived construct. It does not report that wild-type full-length GEF-H1 bundles microtubules or that the named mutants have perturbed co-localization. The verbatim citing sentence does not mention those mutants. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data
    § Results

    None of the EOM models showed a specific interaction between the C1 and DH-PH domains as seen in the autoinhibited VAV1 DH-PH-C1 structure, and SEC-MALS experiments did not find an interaction between C1 and DH-PH.

    “none of these models showed a specific interaction between the C1 and DH-PH domains, as seen in the autoinhibited VAV1 DH-PH-C1 structure.”
    • Also stated in: § Results · plain
      “we did not find an interaction between C1 and DH-PH in SEC-MALS experiments”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [33] 33. Yu, B. ∙ Martins, I.R.S. ∙ Li, P. ... Structural and energetic mechanisms of cooperative autoinhibition and activation of Vav1 Cell. 2010; 140:246-256 Full Text Full Text (PDF) Scopus (132) PubMed Google Scholar source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Author contributions

    p190RhoGEF directly binds microtubules both in vitro and in vivo.

    “p190 RhoGTPase guanine exchange factor (p190RhoGEF, also known as Arhgef28) is a member of the RhoGTPase activator family and has been identified to directly bind microtubules both in vitro and in vivo.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [41] 41. van Horck, F.P. ∙ Ahmadian, M.R. ∙ Haeusler, L.C. ... Characterization of p190RhoGEF, a RhoA-specific guanine nucleotide exchange factor that interacts with microtubules J. Biol. Chem. 2001; 276:4948-4956 Full Text Full Text (PDF) Scopus (140) PubMed Google Scholar source

  • May combine two claims.

    Premisecitation
    § Author contributions

    Pharmacologically perturbing microtubule dynamics resulted in changes of RAF-1 activation, which was further enhanced by mutations targeting the RAF-1 C1 domain.

    “Pharmacologically perturbing microtubule dynamics resulted in changes of RAF-1 activation, which was further enhanced by mutations targeting the RAF-1 C1 domain.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [93] 93. Zang, M. ∙ Waelde, C.A. ∙ Xiang, X. ... Microtubule integrity regulates Pak leading to Ras-independent activation of Raf-1. insights into mechanisms of Raf-1 activation J. Biol. Chem. 2001; 276:25157-25165 Full Text Full Text (PDF) Scopus (44) Google Scholar source
    • cites [94] 94. Blagosklonny, M.V. ∙ Giannakakou, P. ∙ el-Deiry, W.S. ... Raf-1/bcl-2 phosphorylation: a step from microtubule damage to cell death Cancer Res. 1997; 57:130-135 PubMed Google Scholar

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data
    § Discussion

    The findings provide a structural framework for understanding how microtubules modulate signaling proteins via a C1 domain-dependent mechanism and support a microtubule sequestration-and-release (MSR) model.

    “Our findings provide a structural framework for understanding how microtubules modulate signaling proteins via a C1 domain-dependent mechanism.”
    • Also stated in: § Discussion · plain
      “They support a “microtubule sequestration-and-release” (MSR) model3”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [3] 3. Gundersen, G.G. ∙ Cook, T.A. Microtubules and signal transduction Curr. Opin. Cell Biol. 1999; 11:81-94 Crossref PubMed Google Scholar source

      The section “Mechanisms for microtubule ‘signal transduction’” proposes sequestration and release of microtubule-bound signaling factors, supporting the model in broad terms. It does not describe a C1 domain-dependent mechanism or provide structural findings; the citing sentence calls those findings its own. The indexed passage has no page number. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § Author contributions

    RACGAP1 is a GAP that spatially and temporally activates Rac and RhoA to regulate Jnk and Yap signaling as well as cell division.

    “and a GAP that spatially and temporally activates Rac and RhoA to regulate Jnk and Yap signaling as well as cell division.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [90] 90. Zhao, W.M. ∙ Fang, G. MgcRacGAP controls the assembly of the contractile ring and the initiation of cytokinesis Proc. Natl. Acad. Sci. USA. 2005; 102:13158-13163 Crossref Scopus (178) PubMed Google Scholar source

      The Results and Discussion (page numbers unavailable) support a role for MgcRacGAP in RhoA localization and the initiation of cytokinesis. The Discussion describes ECT2—not MgcRacGAP directly—as a positive regulator of RhoA, and the cited passages do not support activation of Rac or regulation of Jnk or Yap signaling. (LLM, conf=0.89)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § Discussion

    Ansamitocin P3-induced dissociation of GEFH1 promotes dendritic cell maturation and antigen cross-presentation.

    “ansamitocin P3-induced dissociation of GEFH1 promotes dendritic cell maturation and antigen cross-presentation.22”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [22] 22. Kashyap, A.S. ∙ Fernandez-Rodriguez, L. ∙ Zhao, Y. ... GEF-H1 Signaling upon Microtubule Destabilization Is Required for Dendritic Cell Activation and Specific Anti-tumor Responses Cell Rep. 2019; 28:3367-3380.e8 Full Text Full Text (PDF) Scopus (54) PubMed Google Scholar source

  • May combine two claims.

    Premisecitation
    § Author contributions

    PKCζ co-localizes and co-immunoprecipitates with microtubules and tubulin, respectively, in cells.

    “It has been observed to co-localize and co-immunoprecipitate with microtubules and tubulin, respectively, in cells”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [42] 42. García-Rocha, M. ∙ Avila, J. ∙ Lozano, J. The zeta isozyme of protein kinase C binds to tubulin through the pseudosubstrate domain Exp. Cell Res. 1997; 230:1-8 Crossref Scopus (31) PubMed Google Scholar

      Not checked in the free run.

    • cites [95] 95. Yingying, X. ∙ Caijuan, W. ∙ Yenan, Y. ... The Effect of SIN1 and Microtubules on Insulin Induced PKC ζ Activation Med. Sci. Monit. 2017; 23:3666-3672 Crossref PubMed Google Scholar source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Author contributions

    RACGAP1 is a component of the centralspindlin complex.

    “Rac GTPase-activating protein 1 (RACGAP1, also known as MgcRacGAP/CYK4) is a component of the centralspindlin complex”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [90] 90. Zhao, W.M. ∙ Fang, G. MgcRacGAP controls the assembly of the contractile ring and the initiation of cytokinesis Proc. Natl. Acad. Sci. USA. 2005; 102:13158-13163 Crossref Scopus (178) PubMed Google Scholar source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § Author contributions

    PKCζ plays an important role in insulin stimulated glucose transport and insulin receptor signaling pathways.

    “Protein kinase C isoform zeta (PKCζ) plays an important role in insulin stimulated glucose transport and insulin receptor signaling pathways”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [95] 95. Yingying, X. ∙ Caijuan, W. ∙ Yenan, Y. ... The Effect of SIN1 and Microtubules on Insulin Induced PKC ζ Activation Med. Sci. Monit. 2017; 23:3666-3672 Crossref PubMed Google Scholar source

  • May combine two claims.

    Findingown data
    § Results

    Subtilisin-treated microtubules lacking C-terminal tails significantly reduced C1L-G4 binding compared with untreated controls, confirming the importance of the tubulin C-terminal tails in this interaction.

    “Further, the use of subtilisin-treated microtubules lacking C-terminal tails significantly reduced C1L-G4 binding compared with untreated controls, confirming the importance of the tubulin C-terminal tails in this interaction (Figure S1K).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data
    § Results

    Zinc-finger motif 2 comprises His40, Cys68, Cys71, and Cys86.

    “Motif 2 comprises His40 (N terminus), Cys68 (β4-strand), Cys71 (β4-β5-loop), and Cys86 (α2-helix).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Results

    GEFH1 C1 comprises two antiparallel β-sheets formed by three (β1, β4, and β5) and two (β2 and β3) β-strands, respectively, and two C-terminal α-helices (α1 and α2).

    “It comprises two antiparallel β-sheets formed by three (β1, β4, and β5) and two (β2 and β3) β-strands, respectively, and two C-terminal α-helices (α1 and α2; Figure 2B).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Results

    Binding curves indicated that AKAP13, RASSF1A, RACGAP1, and RAF-1 C1-G4 variants bind microtubules with low micromolar affinity, while VAV1, PKCζ, and p190RhoGEF C1-G4 variants did not exhibit binding profiles indicative of microtubule interaction under the conditions applied.

    “Binding curves of AKAP13, RASSF1A, RACGAP1, and RAF-1 indicated that, in the presence of 1.25 μM of a C1-G4 variant, saturation occurred at microtubule-incorporated tubulin concentrations of around 10 μM, suggesting affinities in the low micromolar range.”
    • Also stated in: § Results · plain
      “the C1-G4 variants of VAV1, PKCζ, and p190RhoGEF did not exhibit binding profiles indicative of microtubule interaction under the conditions applied”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    The refined cryo-EM model revealed that C1 binds to the microtubule via its positively charged face, occupies a cleft between two adjacent protofilaments, and interacts simultaneously with two laterally associated αβ-tubulin heterodimers.

    “The refined model shown in Figure 4B revealed that C1 binds to the microtubule via its positively charged face”
    • Also stated in: § Results · plain
      “It occupies a cleft between two adjacent protofilaments, interacting simultaneously with two laterally associated αβ-tubulin heterodimers.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    FL GEFH1 has a measured molecular weight of 228.9 ± 6.8 kDa, consistent with dimer formation.

    “Using size-exclusion chromatography paired with multi-angle light scattering (SEC-MALS), we measured the molecular weight of FL as 228.9 ± 6.8 kDa, consistent with dimer formation (calculated molecular weight for FL monomer, 110.4 kDa; Table S1; Figure S1A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Results

    The three variants containing the C1 domain (C1-PH, C1-G4, and C1-PH-G4) were all capable of microtubule binding, with G4 dimerization strengthening the interaction.

    “The results in Figure 1C indicate that the three variants containing the C1 domain (C1-PH, C1-G4, and C1-PH-G4) were all capable of microtubule binding, with G4 dimerization strengthening the interaction (Figure S1H).”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data
    § Results

    The model of the microtubule-GEFH1 C1 complex reveals that C1 interacts with four adjacent tubulin monomers (α, β, α′, and β′) via specific interactions.

    “The model of the microtubule-GEFH1 C1 complex structure reveals that C1 interacts with four adjacent tubulin monomers (denoted as α-, β-, α′-, and β′-tubulin).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    GEFH1 forms a coiled coil-mediated dimer that binds microtubules in vitro.

    “Collectively, these findings demonstrate that GEFH1 forms a coiled coil-mediated dimer that binds microtubules in vitro.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    HeLa cells expressing the FL-α′β′ mutant exhibited a 1.4-fold increase in active RhoA (RhoA-GTP) compared with cells expressing the wild type, and this level was comparable to wild-type cells treated with ansamitocin P3.

    “HeLa cells expressing the FL-α′β′ mutant exhibited a 1.4-fold increase in active RhoA (RhoA-GTP) compared with cells expressing the wild type”
    • Also stated in: § Results · plain
      “the RhoA activation level in FL-α′β′-expressing cells was comparable to that in cells transfected with the wild-type protein and treated with the microtubule-destabilizing agent ansamitocin P3”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    The cryo-EM structure enabled identification of several surface residues on the C1 domain involved in microtubule binding, and a multiple sequence alignment of GEFH1 orthologs revealed that these residues are highly conserved across animal species.

    “The cryo-EM structure of the microtubule-GEFH1 C1 domain complex enabled us to identify several surface residues on the C1 domain involved in microtubule binding.”
    • Also stated in: § Results · plain
      “A multiple sequence alignment of GEFH1 orthologs revealed that these residues are highly conserved across animal species”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    The surface of C1 features two distinct faces: a hydrophobic face near zinc-finger motif 2, the central β-sheet, and the β1-β2-loop, and a positively charged face formed by basic residues surrounding motif 1, the β3-β4-loop, and the α1-helix.

    “The surface of C1 features two distinct faces: a hydrophobic face near zinc-finger motif 2, the central β-sheet, and the β1-β2-loop, and a positively charged face formed by basic residues surrounding motif 1, the β3-β4-loop, and the α1-helix (Figure 2C).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Results

    The β-sheet regions of C1, particularly the β1-β2 and β3-β4 loops, extend deeply into the inter-protofilament cleft and form specific interactions with the microtubule lattice, and microtubule-bound C1 closely matches its solution NMR structure with an RMSD of 0.6 Å over 60 Cα atoms.

    “The β-sheet regions of C1, particularly the β1-β2 and β3-β4 loops, corresponding to the ligand-binding site in typical C1 domains (Figures 2D and 2E), extend deeply into the inter-protofilament cleft, forming specific interactions with the microtubule lattice.”
    • Also stated in: § Results · plain
      “The conformation of microtubule-bound C1 closely matches its solution NMR structure, with an RMSD of 0.6 Å over 60 Cα atoms, indicating minimal structural rearrangement upon binding.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    While the C1 domain is central to microtubule binding, flanking regions such as the C1-DH linker and the PH domain play supportive roles for this binding.

    “They also highlight that while the C1 domain is central to this interaction, flanking regions such as the C1-DH linker and the PH domain play supportive roles for this binding.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    The G4-containing variants C1-PH-G4, C1-G4, and PH-G4 form dimers, whereas C1-PH is monomeric.

    “SEC-MALS analysis showed that the three G4-containing variants, C1-PH-G4, C1-G4, and PH-G4, form dimers, whereas C1-PH is monomeric (Table S1; Figure S1G).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    SAXS P(r) analysis yielded Rg values of 38.5 Å for GEFH1 Nter and 27.7 Å for NterΔC1, and Dmax values of 13.4 nm and 8.0 nm, respectively.

    “The pair distribution function, P(r), for Nter and NterΔC1 revealed radius of gyration (Rg) values of 38.5 and 27.7 Å and maximal particle dimensions (Dmax) of 13.4 and 8.0 nm, respectively”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    Cryo-EM analysis showed that the 13-protofilament microtubule population was predominant, about 40% of available lattice binding sites were occupied by C1, and the density map had resolution of about 3.3 Å for the two αβ-tubulin heterodimers and about 3.5 Å for C1.

    “Three-dimensional (3D) classification of microtubule particles revealed that the 13-protofilament microtubule population was predominant.”
    • Also stated in: § Results · plain
      “Analysis of single particles, comprising two laterally associated rows of βαβ-tubulin monomers, showed that ∼40% of the available lattice binding sites were occupied by C1.”
    • Also stated in: § Results · plain
      “The resolution for the two αβ-tubulin heterodimers is ∼3.3 Å, while that for C1 is ∼3.5 Å.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    FL GEFH1 alone largely remained in the supernatant after centrifugation but pelleted when incubated with microtubules, indicating complex formation.

    “As shown in Figure 1B, FL alone largely remained in the supernatant after centrifugation, but it pelleted when incubated with microtubules, indicating complex formation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    EOM best-fit models favored more compact conformations than the non-selected pool, with Rg values between 31 and 35 Å representing 20%-40% of all models.

    “The best-fit model pool, across all linker characteristics, favored more compact conformations than the non-selected pool, with Rg values between 31 and 35 Å, representing 20%–40% of all models”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data
    § Results

    Cryo-EM validated microtubule binding by the C1-G4 variants of AKAP13, RASSF1A, RACGAP1, and RAF-1, showing a hairy appearance, 4 nm and 8 nm layer lines, and 8 nm repeating structures consistent with one C1 domain binding per tubulin heterodimer.

    “Cryo-EM analysis further validated microtubule binding by the C1-G4 variants of AKAP13, RASSF1A, RACGAP1, and RAF-1.”
    • Also stated in: § Results · plain
      “The electron micrographs showed a “hairy” appearance on the outer microtubule surfaces, distinct from undecorated microtubules”
    • Also stated in: § Results · plain
      “line scan analysis of filtered microtubules co-incubated with the various C1-G4 variants revealed repeating structures at 8 nm intervals (Figure S7B), aligning with a binding mode where one C1 domain binds per tubulin heterodimer, consistent with the cryo-EM structure of the microtubule-GEFH1 C1 complex”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    Nter is monomeric, while Cter and CC form dimers.

    “SEC-MALS analysis demonstrated that Nter is monomeric, while both Cter and CC form dimers (Table S1; Figures S1B–S1D).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § Results

    The AKAP13 C1 domain exhibits an intra-tubulin dimer and inter-protofilament binding mode, but its binding site is shifted to a site defined by the two α-tubulin monomers from adjacent αβ-tubulin heterodimers; low resolution prevented unambiguous modeling of its structure.

    “similar to the GEFH1 C1 domain, the AKAP13 C1 domain exhibits an intra-tubulin dimer and inter-protofilament binding mode.”
    • Also stated in: § Results · plain
      “However, its binding site is shifted: rather than occupying the interface formed by four tubulin monomers, it localizes to a site defined by the two α-tubulin monomers from adjacent αβ-tubulin heterodimers.”
    • Also stated in: § Results · plain
      “Due to the low resolution obtained for the C1 domain (∼6 Å, compared with ∼3 Å for the tubulin dimer), we were unable to unambiguously model its structure within the cryo-EM density map.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data
    § Discussion

    The reduction in GEFH1-microtubule binding by C1 domain mutagenesis resulted in increased RhoA activity in mammalian cells.

    “resulting in increased RhoA activity in mammalian cells.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data
    § Author contributions

    Final SAXS envelopes for Nter and NterΔC1 were calculated by averaging 22 models, resulting in NSD values of 0.66 ± 0.04 and 0.78 ± 0.09, respectively.

    “Final envelopes for both Nter and NterΔC1 were calculated by averaging 22 models resulting in NSD values of 0.66 ± 0.04 and 0.78 ± 0.09, respectively.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

    Findingcitation
    § Author contributions

    The C1-DH linker of GEFH1 Nter was predicted to be disordered using DISOPRED3.

    “The C1-DH linker was predicted to be disordered using DISOPRED3.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data
    § Author contributions

    Additional analysis of the structural bundle after CYANA revealed seventeen hydrogen bonds, each present in more than six structures.

    “Additional analysis of the structural bundle after the CYANA calculation revealed seventeen hydrogen bonds (each present in more than six structures)”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data
    § Results

    FL RASSF1A co-localized with microtubules, while the ΔN, FL-C65R, and FL-R53A mutants exhibited markedly reduced co-localization compared with wild-type FL, and all three FL mutants significantly increased proliferation in transfected HMF3a cells.

    “FL co-localized with microtubules, consistent with a previous report.”
    • Also stated in: § Results · plain
      “the ΔN, FL-C65R, and FL-R53A mutants exhibited markedly reduced co-localization compared with wild-type FL”
    • Also stated in: § Results · plain
      “all three FL mutants significantly increased proliferation in transfected HMF3a cells.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [45] 45. Vos, M.D. ∙ Martinez, A. ∙ Elam, C. ... A role for the RASSF1A tumor suppressor in the regulation of tubulin polymerization and genomic stability Cancer Res. 2004; 64:4244-4250 Crossref Google Scholar source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data
    § Author contributions

    Ramachandran statistics for the structural bundle were 59.1% favored, 39.6% allowed, and 1.3% additionally allowed.

    “the following Ramachandran statistics: 59.1, 39.6, and 1.3% of residues in favored, allowed, and additionally allowed regions, respectively.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data
    § Author contributions

    Sample degradation of GEFH1 C1 was apparent after three days even in the presence of protease inhibitors.

    “Since sample degradation was apparent after three days even in the presence of protease inhibitors (Figure S2B), time-shared experiments were recorded where possible and fresh samples were used for every series of experiments.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data
    § Results

    A subset of C1 domains function as microtubule-binding domains across various signaling proteins, with variations in the exact microtubule binding mode among these C1 domains.

    “these results demonstrate that a subset of C1 domains function as microtubule-binding domains across various signaling proteins.”
    • Also stated in: § Results · hedged
      “Sequence analyses and structural information suggest variations in the exact microtubule binding mode among these C1 domains”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data
    § Results

    Unlike VAV1, where the C1 domain binds the DH domain and contributes to autoinhibition, the C1 domain of GEFH1 does not tightly associate with the PH domain.

    “This suggests that unlike VAV1, where the C1 domain binds the DH domain and contributes to autoinhibition, the C1 domain of GEFH1 does not tightly associate with the PH domain.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data
    § Results

    The C1 domain is the key determinant of GEFH1's direct interaction with microtubules, a mechanism that regulates RhoA signaling and immune cell activation, and a rationally engineered GEFH1 mutant deficient in microtubule binding phenocopies RhoA-mediated cellular effects of microtubule-destabilizing agents.

    “these findings demonstrate that the C1 domain is the key determinant of GEFH1’s direct interaction with microtubules, a mechanism that plays a critical role in regulating RhoA signaling and immune cell activation.”
    • Also stated in: § Results · plain
      “we established a rationally engineered GEFH1 mutant deficient in microtubule binding, which phenocopies RhoA-mediated cellular effects of microtubule-destabilizing agents.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data
    § Results

    CD analysis showed that the C53R mutation causes unfolding of the isolated C1 domain, while the α′β′ mutation did not significantly change its fold, indicating that reduced microtubule binding of the GEFH1 FL mutants was due to disruption of specific key interactions rather than protein misfolding.

    “CD analysis demonstrated that the C53R mutation in the context of the isolated C1 domain (C1-C53R) causes unfolding of the domain, as evidenced by a spectrum indicative of a random coil structure, unlike the well-folded wild-type C1 domain”
    • Also stated in: § Results · plain
      “CD analysis confirmed that the α′β′ mutation in the isolated C1 domain (C1-α′β′) did not introduce significant changes in its fold”
    • Also stated in: § Results · hedged
      “This indicates that the reduced microtubule binding of the GEFH1 FL mutants was due to disruption of specific key interactions rather than protein misfolding, as is the case for the FL-C53R mutant.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The αβ and ββ′ mutations in the GEFH1 C1 domain do not cause misfolding of the domain.

  • May combine two claims.

    Interpretationown data
    § Results

    The catalytic DH-PH domain of GEFH1 adopts a compact quaternary structure reminiscent of VAV1, but unlike VAV1, the C1 domain does not form a stable intramolecular interaction with DH-PH to establish a defined autoinhibited conformation and appears to function as an autonomous module.

    “Together, these findings indicate that the catalytic DH-PH domain of GEFH1 adopts a compact quaternary structure, reminiscent of that observed in VAV1.”
    • Also stated in: § Results · hedged
      “they suggest that unlike in VAV1, the C1 domain of GEFH1 does not form a stable intramolecular interaction with the DH-PH domain to establish a defined autoinhibited conformation. Instead, the C1 domain appears to function as an autonomous module.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

    Study design and conductown data
    § Author contributions

    Resonance assignments for GEFH1 C1 were determined to 96% completeness.

    “Resonance assignments were determined with the program CARA (www.cara.nmr.ch) to 96% completeness.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given
    § Author contributions

    As controls, paclitaxel-stabilized microtubules or GEFH1 variants alone were processed the same way.

    “As controls, paclitaxel-stabilized microtubules or GEFH1 variants alone were processed the same way.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given
    § Author contributions

    Microtubule or protein-alone controls were included in each analysis.

    “Microtubule or protein-alone controls were included in each analysis.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductown data
    § Author contributions

    Automatically picked peak lists were manually curated and resulted in 3477 peaks converted by CYANA to 1895 unambiguous distance restraints.

    “Automatically picked peak lists were manually curated and resulted in a set of 3477 peaks that were converted by CYANA to 1895 unambiguous distance restraints.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data
    § Author contributions

    The G-LISA assay was repeated independently three times.

    “Assay was repeated independently three times in the experiment.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data
    § Discussion

    The C1 domain serves as an autonomous module primarily mediating the direct and specific interaction between GEFH1 and microtubules; coiled-coil-mediated dimerization enhances the microtubule-binding capacity of C1, and GEFH1 operates through an MSR mechanism in which the microtubule lattice functions as an inhibitory platform for its catalytic activity.

    “Our findings establish that the C1 domain serves as an autonomous module, primarily mediating the direct and specific interaction between GEFH1 and microtubules.”
    • Also stated in: § Discussion · plain
      “This dimerization, when coupled with the basic C1-DH linker and the PH domain, significantly enhances the microtubule-binding capacity of the C1 domain.”
    • Also stated in: § Discussion · hedged
      “These findings suggest that GEFH1 operates through an MSR mechanism in which the microtubule lattice functions as an inhibitory platform for its catalytic activity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data
    § Results

    The function of RASSF1A is linked to microtubule co-localization in a C1 domain-dependent manner, and RASSF1A acts as a tumor suppressor when bound to microtubules.

    “these findings suggest that, like GEFH1, the function of RASSF1A is linked to microtubule co-localization in a C1 domain-dependent manner.”
    • Also stated in: § Results · hedged
      “They indicate that RASSF1A acts as a tumor suppressor when bound to microtubules.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Author contributions

    PKCζ co-localizes to the mitotic spindle during mitosis.

    “as well as co-localizing to the mitotic spindle during mitosis.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [96] 96. Liu, X.F. ∙ Xie, X. ∙ Miki, T. Inhibition of protein kinase C zeta blocks the attachment of stable microtubules to kinetochores leading to abnormal chromosome alignment Cell. Signal. 2006; 18:2314-2323 Crossref Scopus (14) PubMed Google Scholar source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given
    § Author contributions

    Binding assays were repeated in three independent experiments.

    “Binding assays were repeated in three independent experiments.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § Author contributions

    AKAP13 is a Rho-family GEF that shares the same C1-DH-PH domain organization as GEFH1.

    “A-kinase anchoring protein 13 (AKAP13, also known as AKAP-Lbc) is a Rho-family GEF that shares the same C1-DH-PH domain organization as GEFH1.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [88] 88. Rossman, K.L. ∙ Der, C.J. ∙ Sondek, J. GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors Nat. Rev. Mol. Cell Biol. 2005; 6:167-180 Crossref Scopus (1441) PubMed Google Scholar source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data
    § Author contributions

    The purities and identities of GEFH1 variants were assessed by Coomassie-stained SDS-PAGE and mass spectral analyses.

    “Their purities and identities were assessed by Coomassie-stained SDS-PAGE and mass spectral analyses, respectively.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § Author contributions

    RAF-1 is a serine/threonine protein kinase that acts as a regulatory link between membrane-associated Ras GTPases and the Ras-Raf-MEK-Erk MAPK pathway.

    “Rapidly accelerated fibrosarcoma 1 (RAF-1, also known as c-Raf) is a serine/threonine protein kinase that acts as a regulatory link between the membrane-associated Ras GTPases and the Ras-Raf-MEK-Erk mitogen activated protein kinase (MAPK) pathway.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [92] 92. Avruch, J. ∙ Zhang, X.F. ∙ Kyriakis, J.M. Raf meets Ras: completing the framework of a signal transduction pathway Trends Biochem. Sci. 1994; 19:279-283 Full Text (PDF) Scopus (573) PubMed Google Scholar source
66 of 66

  • The paper relies on this without support.

    The αβ and ββ′ mutations in the GEFH1 C1 domain do not cause misfolding of the domain.

    “CD analysis confirmed that the α′β′ mutation in the isolated C1 domain (C1-α′β′) did not introduce significant changes in its fold”

  • The paper relies on this without support.

    The rationally engineered GEFH1 mutant deficient in microtubule binding affects RhoA activity specifically through loss of microtubule binding.

    “Remarkably, we were able to replicate this phenotype using our rationally engineered GEFH1 mutant deficient in microtubule binding, thereby suggesting that microtubule lattices directly regulate RhoA-mediated cellular processes.”

  • The authors state this as an assumption.

    Whether AKAP13 interacts with microtubules remains unknown.

    “AKAP13 plays a role in stress fiber formation; however, whether it interacts with microtubules remains unknown.”
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?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • NMR sample described as both tag-free and His-tagged

    Major

    The results say the labeled GEFH1 C1 sample used for structural studies was tag-free, whereas the NMR methods describe the sample as 6xHis-tagged.

  • Reported binding-curve saturation exceeds the stated titration range

    Minor

    The results place saturation at around 10 μM microtubule-incorporated tubulin, but the methods say the microtubule titration extended only to 8 μM. The text does not identify the 10 μM value as an extrapolation.

  • Six G4-fused variants claimed, but only five constructs listed

    Minor

    The cloning methods introduce six variants fused to G4, then name only five constructs. One of those, C1-PH, is listed without G4.

  • AKAP13 microtubule association described as both reported and unknown

    Minor

    The discussion includes AKAP13 among proteins reported to associate with microtubules, but the methods say whether AKAP13 interacts with microtubules remains unknown.

4 of 4

Cites

Flagged first, then by citations

Research works printed but not linked: 1

  1. Blagosklonny, 1997, Raf-1/bcl-2 phosphorylation: a step from microtubule damage to cell death, Cancer Res., 57, 130

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Authors
Choi, S. R., Blum, T. B., Giono, M., Roy, B., Vakonakis, I., Schmid, D., Oelgarth, N., Ranganathan, A., Gossert, A. D., Shivashankar, G. V., Zippelius, A., & Steinmetz, M. O.
Affiliations
  • Paul Scherrer Institute (CH)
  • University of Oxford (GB)
  • University of Basel (CH)
  • ETH Zurich (CH)
Corresponding author
  • Choi, Sung Ryul
  • Steinmetz, Michel O.
ORCID
Venue
Cell
Publisher
Elsevier BV
Published
2026-01
Volume
189
Issue
2
Pages
461-477.e16
ISSN
0092-8674 (ISSN-L)
DOI
10.1016/j.cell.2025.11.011
OpenAlex
W4417122962
Cited by
14
References
93
Citations by year
2026: 13 · 2025: 1
FWCI
4.18
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DORA PSI (Paul Scherrer Institute) (submittedVersion, cc-by)
  • Repository for Publications and Research Data (ETH Zurich) (submittedVersion, cc-by)
  • ETH Zürich Research Collection (cc-by)
Funders
Swiss National Science Foundation (31003A_166608)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Microtubule and mitosis dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular Mechanics and Interactions
Keywords
microtubules, signal transduction, C1 domain, RhoA, microtubule lattice, protein sequestration, guanine nucleotide exchange factor, RASSF1A, GTPase-activating protein, immune response, spatiotemporal signaling
MeSH terms
Protein Domains, Animals, Humans, Microtubules, Protein Binding, Signal Transduction, Guanine Nucleotide Exchange Factors, rhoA GTP-Binding Protein, Tumor Suppressor Proteins, Rho Guanine Nucleotide Exchange Factors

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.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-11-02.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-11-02.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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