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A complex containing lysine-acetylated actin inhibits the formin INF2



Inverted formin 2 (INF2) is a member of the formin family of actin assembly factors. Dominant missense mutations in INF2 are linked to two diseases: focal segmental glomerulosclerosis, a kidney disease, and Charcot–Marie–Tooth disease, a neuropathy. All of the disease mutations map to the autoinhibitory diaphanous inhibitory domain. Interestingly, purified INF2 is not autoinhibited, suggesting the existence of other cellular inhibitors. Here, we purified an INF2 inhibitor from mouse brain tissue, and identified it as a complex of lysine-acetylated actin (KAc-actin) and cyclase-associated protein (CAP). Inhibition of INF2 by CAP–KAc-actin is dependent on the INF2 diaphanous inhibitory domain (DID). Treatment of CAP–KAc-actin-inhibited INF2 with histone deacetylase 6 releases INF2 inhibition, whereas inhibitors of histone deacetylase 6 block the activation of cellular INF2. Disease-associated INF2 mutants are poorly inhibited by CAP–KAc-actin, suggesting that focal segmental glomerulosclerosis and Charcot–Marie–Tooth disease result from reduced CAP–KAc-actin binding. These findings reveal a role for KAc-actin in the regulation of an actin assembly factor by a mechanism that we call facilitated autoinhibition.

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Data availability

The mass-spectrometry proteomics data (Supplementary Tables 1, 2 and 3) have been deposited to the ProteomeXchange Consortium70 through the PRIDE partner repository under PX accession number PXD010484. Raw numerical data for the results in Figs. 4b, 5g–j and 6e are provided in Supplementary Table 4. Unprocessed blots and gels for all relevant figures are in Supplementary Fig. 7.

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  1. 1.

    Pufall, M. A. & Graves, B. J. Autoinhibitory domains: modular effectors of cellular regulation. Annu. Rev. Cell Dev. Biol. 18, 421–462 (2002).

  2. 2.

    Torres, E. & Rosen, M. K. Contingent phosphorylation/dephosphorylation provides a mechanism of molecular memory in WASP. Mol. Cell 11, 1215–1227 (2003).

  3. 3.

    Campellone, K. G. & Welch, M. D. A nucleator arms race: cellular control of actin assembly. Nat. Rev. Mol. Cell Biol. 11, 237–251 (2010).

  4. 4.

    Goode, B. L. & Eck, M. J. Mechanism and function of formins in control of actin assembly. Annu. Rev. Biochem. 76, 593–627 (2007).

  5. 5.

    Higgs, H. N. Formin proteins: a domain-based approach. Trends Biochem. Sci. 30, 342–353 (2005).

  6. 6.

    Higgs, H. N. & Peterson, K. J. Phylogenetic analysis of the formin homology 2 domain. Mol. Biol. Cell 16, 1–13 (2005).

  7. 7.

    Pruyne, D. Revisiting the phylogeny of the animal formins: two new subtypes, relationships with multiple wing hairs proteins, and a lost human formin. PLoS ONE 11, e0164067 (2016).

  8. 8.

    Li, F. & Higgs, H. N. The mouse formin mDia1 is a potent actin nucleation factor regulated by autoinhibition. Curr. Biol. 13, 1335–1340 (2003).

  9. 9.

    Otomo, T., Otomo, C., Tomchick, D. R., Machius, M. & Rosen, M. K. Structural basis of Rho GTPase-mediated activation of the formin mDia1. Mol. Cell 18, 273–281 (2005).

  10. 10.

    Lammers, M., Rose, R., Scrima, A. & Wittinghofer, A. The regulation of mDia1 by autoinhibition and its release by Rho•GTP. EMBO J. 24, 4176–4187 (2005).

  11. 11.

    Chhabra, E. S. & Higgs, H. N. INF2 is a WASP homology 2 motif-containing formin that severs actin filaments and accelerates both polymerization and depolymerization. J. Biol. Chem. 281, 26754–26767 (2006).

  12. 12.

    Gurel, P. S., Hatch, A. L. & Higgs, H. N. Connecting the cytoskeleton to the endoplasmic reticulum and Golgi. Curr. Biol. 24, R660–R672 (2014).

  13. 13.

    Andres-Delgado, L. et al. INF2 promotes the formation of detyrosinated microtubules necessary for centrosome reorientation in T cells. J. Cell Biol. 198, 1025–1037 (2012).

  14. 14.

    Bartolini, F. et al. An mDia1-INF2 formin activation cascade facilitated by IQGAP1 regulates stable microtubules in migrating cells. Mol. Biol. Cell 27, 1797–1808 (2016).

  15. 15.

    Fernandez-Barrera, J. et al. The actin-MRTF-SRF transcriptional circuit controls tubulin acetylation via α-TAT1 gene expression. J. Cell Biol. 217, 929–944 (2018).

  16. 16.

    Gurel, P. S. et al. INF2-mediated severing through actin filament encirclement and disruption. Curr. Biol. 24, 156–164 (2014).

  17. 17.

    Andres-Delgado, L., Anton, O. M., Madrid, R., Byrne, J. A. & Alonso, M. A. Formin INF2 regulates MAL-mediated transport of Lck to the plasma membrane of human T lymphocytes. Blood 116, 5919–5929 (2010).

  18. 18.

    Chakrabarti, R. et al. INF2-mediated actin polymerization at the ER stimulates mitochondrial calcium uptake, inner membrane constriction, and division. J. Cell Biol. 217, 251–268 (2018).

  19. 19.

    Korobova, F., Ramabhadran, V. & Higgs, H. N. An actin-dependent step in mitochondrial fission mediated by the ER-associated formin INF2. Science 339, 464–467 (2013).

  20. 20.

    Madrid, R. et al. The formin INF2 regulates basolateral-to-apical transcytosis and lumen formation in association with Cdc42 and MAL2. Dev. Cell 18, 814–827 (2010).

  21. 21.

    Ramabhadran, V., Korobova, F., Rahme, G. J. & Higgs, H. N. Splice variant-specific cellular function of the formin INF2 in maintenance of Golgi architecture. Mol. Biol. Cell 22, 4822–4833 (2011).

  22. 22.

    Brown, E. J. et al. Mutations in the formin gene INF2 cause focal segmental glomerulosclerosis. Nat. Genet. 42, 72–76 (2010).

  23. 23.

    Boyer, O. et al. INF2 mutations in Charcot–Marie–Tooth disease with glomerulopathy. N. Engl. J. Med. 365, 2377–2388 (2011).

  24. 24.

    Ramabhadran, V., Hatch, A. L. & Higgs, H. N. Actin monomers activate inverted formin 2 by competing with its autoinhibitory interaction. J. Biol. Chem. 288, 26847–26855 (2013).

  25. 25.

    Sun, H., Schlondorff, J. S., Brown, E. J., Higgs, H. N. & Pollak, M. R. Rho activation of mDia formins is modulated by an interaction with inverted formin 2 (INF2). Proc. Natl Acad. Sci. USA 108, 2933–2938 (2011).

  26. 26.

    Chaudhry, F. et al. Srv2/cyclase-associated protein forms hexameric shurikens that directly catalyze actin filament severing by cofilin. Mol. Biol. Cell 24, 31–41 (2013).

  27. 27.

    Jansen, S., Collins, A., Golden, L., Sokolova, O. & Goode, B. L. Structure and mechanism of mouse cyclase-associated protein (CAP1) in regulating actin dynamics. J. Biol. Chem. 289, 30732–30742 (2014).

  28. 28.

    Pollard, T. D., Blanchoin, L. & Mullins, R. D. Molecular mechanisms controlling actin filament dynamics in nonmuscle cells. Annu. Rev. Biophys. Biomol. Struct. 29, 545–576 (2000).

  29. 29.

    Makkonen, M., Bertling, E., Chebotareva, N. A., Baum, J. & Lappalainen, P. Mammalian and malaria parasite cyclase-associated proteins catalyze nucleotide exchange on G-actin through a conserved mechanism. J. Biol. Chem. 288, 984–994 (2013).

  30. 30.

    Drazic, A., Myklebust, L. M., Ree, R. & Arnesen, T. The world of protein acetylation. Biochim. Biophys. Acta 1864, 1372–1401 (2016).

  31. 31.

    Ji, W. K., Hatch, A. L., Merrill, R. A., Strack, S. & Higgs, H. N. Actin filaments target the oligomeric maturation of the dynamin GTPase Drp1 to mitochondrial fission sites. eLife 4, e11553 (2015).

  32. 32.

    Shao, X., Li, Q., Mogilner, A., Bershadsky, A. D. & Shivashankar, G. V. Mechanical stimulation induces formin-dependent assembly of a perinuclear actin rim. Proc. Natl Acad. Sci. USA 112, E2595–E2601 (2015).

  33. 33.

    Wales, P. et al. Calcium-mediated actin reset (CaAR) mediates acute cell adaptations. eLife 5, e19850 (2016).

  34. 34.

    Terman, J. R. & Kashina, A. Post-translational modification and regulation of actin. Curr. Opin. Cell Biol. 25, 30–38 (2013).

  35. 35.

    Arnesen, T., Marmorstein, R. & Dominguez, R. Actin’s N-terminal acetyltransferase uncovered. Cytoskeleton 75, 318–322 (2018).

  36. 36.

    Zhang, X. et al. HDAC6 modulates cell motility by altering the acetylation level of cortactin. Mol. Cell 27, 197–213 (2007).

  37. 37.

    Li, X. et al. Histone deacetylase 6 regulates cytokinesis and erythrocyte enucleation through deacetylation of formin protein mDia2. Haematologica 102, 984–994 (2017).

  38. 38.

    Destaing, O. et al. A novel Rho-mDia2-HDAC6 pathway controls podosome patterning through microtubule acetylation in osteoclasts. J. Cell Sci. 118, 2901–2911 (2005).

  39. 39.

    Thurston, S. F., Kulacz, W. A., Shaikh, S., Lee, J. M. & Copeland, J. W. The ability to induce microtubule acetylation is a general feature of formin proteins. PLoS ONE 7, e48041 (2012).

  40. 40.

    Colicelli, J. et al. Mutational mapping of RAS-responsive domains of the Saccharomyces cerevisiae adenylyl cyclase. Mol. Cell. Biol. 10, 2539–2543 (1990).

  41. 41.

    Fedor-Chaiken, M., Deschenes, R. J. & Broach, J. R. SRV2, a gene required for RAS activation of adenylate cyclase in yeast. Cell 61, 329–340 (1990).

  42. 42.

    Balcer, H. I. et al. Coordinated regulation of actin filament turnover by a high-molecular-weight Srv2/CAP complex, cofilin, profilin, and Aip1. Curr. Biol. 13, 2159–2169 (2003).

  43. 43.

    Gottwald, U., Brokamp, R., Karakesisoglou, I., Schleicher, M. & Noegel, A. A. Identification of a cyclase-associated protein (CAP) homologue in Dictyostelium discoideum and characterization of its interaction with actin. Mol. Biol. Cell 7, 261–272 (1996).

  44. 44.

    Moriyama, K. & Yahara, I. Human CAP1 is a key factor in the recycling of cofilin and actin for rapid actin turnover. J. Cell Sci. 115, 1591–1601 (2002).

  45. 45.

    Normoyle, K. P. & Brieher, W. M. Cyclase-associated protein (CAP) acts directly on F-actin to accelerate cofilin-mediated actin severing across the range of physiological pH. J. Biol. Chem. 287, 35722–35732 (2012).

  46. 46.

    Johnston, A. B., Collins, A. & Goode, B. L. High-speed depolymerization at actin filament ends jointly catalysed by Twinfilin and Srv2/CAP. Nat. Cell Biol. 17, 1504–1511 (2015).

  47. 47.

    Bertling, E. et al. Cyclase-associated protein 1 (CAP1) promotes cofilin-induced actin dynamics in mammalian nonmuscle cells. Mol. Biol. Cell 15, 2324–2334 (2004).

  48. 48.

    Swiston, J., Hubberstey, A., Yu, G. & Young, D. Differential expression of CAP and CAP2 in adult rat tissues. Gene 165, 273–277 (1995).

  49. 49.

    Ono, S. The role of cyclase-associated protein in regulating actin filament dynamics - more than a monomer-sequestration factor. J. Cell Sci. 126, 3249–3258 (2013).

  50. 50.

    Bor, B., Vizcarra, C. L., Phillips, M. L. & Quinlan, M. E. Autoinhibition of the formin Cappuccino in the absence of canonical autoinhibitory domains. Mol. Biol. Cell 23, 3801–3813 (2012).

  51. 51.

    Chesarone, M., Gould, C. J., Moseley, J. B. & Goode, B. L. Displacement of formins from growing barbed ends by bud14 is critical for actin cable architecture and function. Dev. Cell 16, 292–302 (2009).

  52. 52.

    Chesarone-Cataldo, M. et al. The myosin passenger protein Smy1 controls actin cable structure and dynamics by acting as a formin damper. Dev. Cell 21, 217–230 (2011).

  53. 53.

    Eisenmann, K. M. et al. Dia-interacting protein modulates formin-mediated actin assembly at the cell cortex. Curr. Biol. 17, 579–591 (2007).

  54. 54.

    Garabedian, M. V. et al. Integrated control of formin-mediated actin assembly by a stationary inhibitor and a mobile activator. J. Cell. Biol. 217, 3512–3530 (2018).

  55. 55.

    Graziano, B. R., Jonasson, E. M., Pullen, J. G., Gould, C. J. & Goode, B. L. Ligand-induced activation of a formin-NPF pair leads to collaborative actin nucleation. J. Cell Biol. 201, 595–611 (2013).

  56. 56.

    Quinlan, M. E., Hilgert, S., Bedrossian, A., Mullins, R. D. & Kerkhoff, E. Regulatory interactions between two actin nucleators, Spire and Cappuccino. J. Cell Biol. 179, 117–128 (2007).

  57. 57.

    Vizcarra, C. L. et al. Structure and function of the interacting domains of Spire and Fmn-family formins. Proc. Natl Acad. Sci. USA 108, 11884–11889 (2011).

  58. 58.

    Kotila, T. et al. Structural basis of actin monomer re-charging by cyclase-associated protein. Nat. Commun. 9, 1892 (2018).

  59. 59.

    Hatch, A. L., Ji, W. K., Merrill, R. A., Strack, S. & Higgs, H. N. Actin filaments as dynamic reservoirs for Drp1 recruitment. Mol. Biol. Cell 27, 3109–3121 (2016).

  60. 60.

    Denton, R. M. Regulation of mitochondrial dehydrogenases by calcium ions. Biochim. Biophys. Acta 1787, 1309–1316 (2009).

  61. 61.

    Menzies, K. J., Zhang, H., Katsyuba, E. & Auwerx, J. Protein acetylation in metabolism - metabolites and cofactors. Nat. Rev. Endocrinol. 12, 43–60 (2016).

  62. 62.

    Mishra, P. & Chan, D. C. Metabolic regulation of mitochondrial dynamics. J. Cell Biol. 212, 379–387 (2016).

  63. 63.

    Ran, J., Yang, Y., Li, D., Liu, M. & Zhou, J. Deacetylation of α-tubulin and cortactin is required for HDAC6 to trigger ciliary disassembly. Sci. Rep. 5, 12917 (2015).

  64. 64.

    Benoy, V. et al. HDAC6 is a therapeutic target in mutant GARS-induced Charcot-Marie-Tooth disease. Brain 141, 673–687 (2018).

  65. 65.

    d’Ydewalle, C. et al. HDAC6 inhibitors reverse axonal loss in a mouse model of mutant HSPB1-induced Charcot-Marie-Tooth disease. Nat. Med. 17, 968–974 (2011).

  66. 66.

    Mo, Z. et al. Aberrant GlyRS-HDAC6 interaction linked to axonal transport deficits in Charcot-Marie-Tooth neuropathy. Nat. Commun. 9, 1007 (2018).

  67. 67.

    Hulme, E. C. & Trevethick, M. A. Ligand binding assays at equilibrium: validation and interpretation. Br. J. Pharmacol. 161, 1219–1237 (2010).

  68. 68.

    Eng, J. K., Jahan, T. A. & Hoopmann, M. R. Comet: an open-source MS/MS sequence database search tool. Proteomics 13, 22–24 (2013).

  69. 69.

    Valot, B., Langella, O., Nano, E. & Zivy, M. MassChroQ: a versatile tool for mass spectrometry quantification. Proteomics 11, 3572–3577 (2011).

  70. 70.

    Vizcaino, J. A. et al. ProteomeXchange provides globally coordinated proteomics data submission and dissemination. Nat. Biotechnol. 32, 223–226 (2014).

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We thank A. Hatch for conducting the initial pilot studies on BIF purification, Z. Svindrich and A. Lavanway for help with imaging and image analysis, T. Y. Chang for supplying neurons, W. Wickner for advice on protein purification, M. Pollak for input on FSGS, J. McLellan and M. Ragusa for comments and support, and C. Detteyala for the initial notion of actin post-translational modification. This work was supported by NIH R01 GM069818 and R35 GM122545 to H.N.H., R01 DK088826 to M. Pollak (HNH sub-contract), R35 GM119455 to A.N.K., and P20 GM113132 to the BioMT COBRE.

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The authors declare no competing interests.

Correspondence to Henry N. Higgs.

Integrated supplementary information

Supplementary Figure 1 Quantification of cytosolic actin polymerization by exogenously expressed INF2.

Confocal microscopy of U2OS cells transfected with GFP alone (vector), GFP-INF2-nonCAAX wildtype (WT) or A149D mutant. Fixed cells stained with TRITC-phalloidin (actin filaments). (a) Schematic of confocal imaging plane used. (b) Representative GFP and TRITC-phalloidin images. Arrows represent cytosolic regions to be quantified. Scale bar, 10 µm. (c) Normalized fluorescence intensity of TRITC-phalloidin. Lines represent mean (Vector: n = 31 cells, mean = 1.35, STD = 0.72; WT: n = 31 cells, mean = 1.19, STD = 1.02; A149D: n = 34 cells, mean = 2.48, STD = 2.14). p values determined by one-sided student’s t-test. N.S., no significant difference.

Supplementary Figure 2 Fractionation of INF2 inhibitory activity by column chromatography.

All panels conducted once, except panel a (First SourceQ step), which was conducted three times. (a) Graph showing protein elution profile (OD280 nm, grey) from first SourceQ step (pH 7.4) as well as the effect of each fraction on the activities of 20 nM INF2-FL (red) or FFC (green) in pyrene-actin assays. Activity of INF2-FL (blue), INF2-FFC (pink), or actin alone (black) are points on the left of graph. Fractions in purple box are those pooled for subsequent chromatography. Below graph are anti-CAP1 and CAP2 western blots of selected fractions. (b) Graph showing protein elution and activity profiles for the Superdex200 size exclusion step. Elution positions of standard proteins (thyroglobulin, ferritin, and γ-globulin) are indicated. Below graph are anti-CAP1 and CAP2 western blots of selected fractions. (c) Graph showing protein elution and activity profiles from the second SourceQ (pH 8.8). (d) Pyrene actin polymerization assay (2 µM actin monomer, 5% pyrene) testing effect of inhibitor (fraction #8 in fig. 2e) on 10 nM full-length INF2 or 10 nM mDia1 FFC. (e) Test of GFP-INF2 proteolysis in the presence of inhibitory fractions. Fractions were taken from pyrene-actin assays conducted in Fig. 2c, after completion of assay (1200 sec), and probed for INF2 by western blotting. (f) Pyrene actin polymerization assays (2 µM actin monomer, 5% pyrene) containing 20 nM INF2-FL alone (green) or with an inhibitory fraction from final purification step (fraction #8 in Fig. 2e) that was either boiled (black) or not boiled (grey). Actin alone curve in red.

Supplementary Figure 3 Testing candidate proteins in the BIF for INF2 inhibition by immuno-depletion.

(a) Immuno-depletion of inhibitor candidates. An INF2 inhibitory fraction from mouse brain (Fraction #38–40 pool from SourceQ#1 (Supplementary Fig. 2a)) was incubated with either nonspecific antibody control (NS) or the following antibodies: (i) Vacuolar ATPase subunit A (VATA), (ii) Vacuolar ATPase subunit B2 (VATB2, partial depletion), or (iii) Heat shock cognate 71kDa protein (HSP7C). Experiment conducted once. (b) Pyrene-actin polymerization assay (2 µM actin monomer, 5% pyrene) containing 20 nM INF2-FL and supernatant fractions after immuno-depletion. IP/NS-Sup, NS depletion. IP/VATA-Sup, VATA depletion. IP/VATB2-Sup, VATB2 depletion. IP/HSP7C-Sup, HSP7C depletion. Pyrene-actin assays conducted once in duplicate.

Supplementary Figure 4 Characterization of recombinant CAP.

(a) Coomassie stained SDS-PAGE of CAP1 or CAP2 purified from HEK293 cells, either as GFP-fusions or cleaved versions. Experiment conducted 6, 2, 24, and 20 times for CAP1–GFP, CAP1, CAP2–GFP, and CAP2, respectively. (b) Superdex200 gel filtration chromatography of purified CAP1 or CAP2 (cleaved from GFP). Elution positions of standard proteins (thyroglobulin, ferritin, and γ-globulin) indicated. Experiment conducted two times for CAP1 and 10 times for CAP2, respectively. (c) Velocity analytical ultracentrifugation of 7 µM CAP2–GFP. Mass: 726 kDa (calculated from sedimentation), 83.7 kDa (calculated from sequence). Experiment conducted once. (d) Pyrene-actin polymerization assay (2 µM actin monomer, 5% pyrene) containing 20 nM INF2-FL in the presence or absence of 5 µM indicated purified CAP proteins. Experiment conducted two times. (e) Pyrene-actin polymerization assay (2 µM actin monomer, 5% pyrene) in the presence or absence of 5 µM indicated purified CAP proteins. Experiment conducted two times. (f) Pyrene actin polymerization assay (2 µM actin monomer, 5% pyrene) containing 20 nM GFP-INF2-FFC in the presence or absence of 1 µM purified CAP either without actin exchange or exchanged with chicken muscle actin (CAP/CKSA). Experiment conducted three times.

Supplementary Figure 5 Actin exchange on CAP, and acetylation analysis.

(a) Schematic of exchange process, in which column-bound CAP/293A is mixed with TMR-actin. CAP/actin eluted by HRV3C digestion. (b) StrepTrap bead chromatographic columns containing no protein (left), CAP1–GFP (middle) and GFP alone (right). Top photo: before mixing beads with TMR-actin. Bottom photo: after mixing with TMR-actin and washing. Coomassie gel shows similar actin content in the HRV3C-eluted complexes after exchange. Experiment conducted twice. (c) Coomassie gel of purified chicken muscle actin and brain actin. Experiment conducted twice. (d) Pyrene-actin polymerization assay (2 µM actin) containing 20 nM GFP-INF2-FL and 3 µM profilin in the presence or absence of 1 µM purified CAP2/CSKA. Experiment conducted twice. (e) Coomassie-stained SDS/PAGE of HDAC6 purified from HEK293 cells. Experiment conducted three times. (f) Western blot probing tubulin and Ac-tubulin after treatment of purified brain tubulin (10 μM) with purified HDAC6 (200 nM) or buffer for 1 hr, 37 °C. Experiment conducted three times. (g) Western blot probing tubulin and Ac-tubulin in 293 lysate after culturing in medium containing DMSO or 50 μM Tubastatin A for 1 hr before lysis. Experiment conducted three times. (h) Western blot probing actin and Ac-lysine in recombinant CAP2–GFP purified from 293 cells incubated with either DMSO or 50 μM Tubastatin A before lysis. Experiment conducted three times. (i) vAUC of GFP-INF2-FFC (1.8 µM), with or w/o 18 µM CAP2/293-D or CAP2/293-T. Mass of GFP-INF2-FFC: 218.3 kDa (sedimentation), 112.6 kDa (sequence). Left axis: 490 nm (GFP-INF2), right axis: 280 nm (CAP). Experiment conducted once. (j) Representative GFP and TRITC-phalloidin confocal microscopy images showing cytosolic actin polymerization induced by exogenously expressed GFP-INF2 constructs (WT, L77R, or R218Q) in INF2-KO U2OS cells. Cells treated with either DMSO or 50 μM Tubastatin A for 1 hr followed by fixation and staining. Images are maximum intensity projections of three confocal imaging planes in the middle z region of the cell. Bar, 10 µm. (k) Normalized fluorescence intensity of TRITC-phalloidin quantified from images similar to (j). Lines represent mean. (WT-DMSO: n = 23 cells, mean = 1.34, STD = 0.64; WT-TubA: n = 20 cells, mean = 1.25, STD = 0.74; L77R-DMSO: n = 23 cells, mean = 6.76, STD = 2.33; L77R-TubA: n = 20 cells, mean = 7.06, STD = 2.38; R218Q-DMSO: n = 25 cells, mean = 13.35, STD = 4.60; R218Q-TubA: n = 29 cells, mean = 16.55, STD = 6.09). p-values determined by one-sided student’s t-test.

Supplementary Figure 6 HDAC6-sensitive acetylation sites on actin.

Actin in grey with pointed end up in all panels. K50, purple; K68, green; K215, orange; K315, blue. Approximate position of lysine 50 (not resolved in the structure) indicated by purple arrow. Models constructed using Pymol Anaconda2. (a) Actin monomer (PDB 4PGK). Actin sub-domains indicated by I, II, III and IV. Structure to right is 180° rotated from the left-hand structure. ATP shown as blue stick figure. (b) Actin filament (PDB 3G37). (c) Actin bound to CARP domain of mouse CAP1 (PDB 6FM2). Structure to right is 180˚ rotated from the left-hand structure. ADP shown as blue stick figure. (d) Actin bound to FH2 domain of mouse FMNL3 (PDB 4EAH).

Supplementary Figure 7 Unprocessed scans of blots and gels.

Red boxes denote regions used in processed figure.

Supplementary information

Supplementary Information

Supplementary Figures 1–7 and Supplementary Table titles and legends

Reporting Summary

Supplementary Table 1

Protein identification in the BIF fraction.

Supplementary Table 2

Acetylated lysine sites identified on chicken skeletal muscle actin bound to CAP2.

Supplementary Table 3

Comparison of acetylation on specific lysines between CAP and actin samples.

Supplementary Table 4

Raw data for large data sets.

Supplementary Table 5

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Fig. 1: Constitutive activity of purified INF2, and inhibition by a brain factor.
Fig. 2: CAP–actin is an INF2 inhibitor.
Fig. 3: Actin-dependent differences in INF2 inhibition by CAP–actin.
Fig. 4: CAP inhibits INF2-mediated nucleation observed by TIRF microscopy.
Fig. 5: Acetylation-induced regulation of INF2 biochemically and in cells.
Fig. 6: Disease-associated INF2 mutants display decreased regulation by CAP–actin.
Supplementary Figure 1: Quantification of cytosolic actin polymerization by exogenously expressed INF2.
Supplementary Figure 2: Fractionation of INF2 inhibitory activity by column chromatography.
Supplementary Figure 3: Testing candidate proteins in the BIF for INF2 inhibition by immuno-depletion.
Supplementary Figure 4: Characterization of recombinant CAP.
Supplementary Figure 5: Actin exchange on CAP, and acetylation analysis.
Supplementary Figure 6: HDAC6-sensitive acetylation sites on actin.
Supplementary Figure 7: Unprocessed scans of blots and gels.