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. Author manuscript; available in PMC: 2019 Oct 8.
Published in final edited form as: Nat Cell Biol. 2019 Apr 8;21(5):592–602. doi: 10.1038/s41556-019-0307-4

Figure 6. INF2 disease mutants display decreased regulation by CAP/actin.

Figure 6.

(a) Pyrene-actin polymerization assay (2μM actin monomer, 5% pyrene) containing 20nM GFP-INF2 wildtype (WT) or mutants (FSGS mutant R218Q, CMTD mutant L77R) in presence or absence of 1μM CAP2/CSKA. Experiment conducted two times in triplicate. (b) Concentration dependence of CAP2/293A-T inhibition of INF2-FL and disease mutants, in pyrene-actin assays conducted as in panel a. Experiment conducted once (1000 nM CAP points conducted twice) in triplicate. (c) Representative GFP and TRITC-phalloidin confocal microscopy images showing cytosolic actin polymerization by exogenously expressed GFP-INF2 constructs (WT, L77R, or R218Q) in INF2-KO U2OS cells. Cells were fixed and stained with TRITC-phalloidin to label actin filaments. Images are maximum intensity projections of three confocal imaging planes in the middle z region of the cell. Bar, 10 μm. (d) Normalized fluorescence intensity of TRITC-phalloidin was quantified from images similar to (c). Lines represent mean (WT: n=31 cells, mean=1.19, STD=1.02; L77R: n=34 cells, mean=5.60, STD=3.40; R218Q: n=37 cells, mean=8.63, STD=5.47). p values determined by one-sided student’s t-test. (e) Time course of ionomycin-induced changes in actin filaments in INF2-KO U2OS cells expressing GFP-fusions of INF2 WT or the indicated mutants along with mApple-Ftractin. Mean and S.E.M. shown. N = 34, 35, and 33 cells for WT, L77R and R218Q, respectively. (f) Co-immunoprecipitation assay of endogenous CAP2 with transfected GFP-fusion proteins: GFP alone, GFP-INF2-FL, GFP-INF2FL L77R mutant, and GFP-INF2FL R218Q mutant. Top panel: anti-CAP2 western on precipitated samples (IgGH, IgG heavy chain from the IP). Middle panel: anti-GFP western showing GFP-INF2 in precipitated samples. Bottom panel: anti-CAP2 western of input samples. Experiment conducted once. (g) Schematic model of INF2 facilitated autoinhibition by CAP/KAc-actin, and activation by HDAC6-mediated actin de-acetylation. INF2 mutants linked with FSGS and CMTD are defective in CAP/KAc-actin binding, leading to constitutive activity.