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. 2012 Oct 5;287(41):34234-45.
doi: 10.1074/jbc.M112.365122. Epub 2012 Aug 9.

Mutations to the formin homology 2 domain of INF2 protein have unexpected effects on actin polymerization and severing

Vinay Ramabhadran  1 , Pinar S Gurel, Henry N Higgs
Affiliations

Mutations to the formin homology 2 domain of INF2 protein have unexpected effects on actin polymerization and severing

Vinay Ramabhadran et al. J Biol Chem. .

Abstract

INF2 (inverted formin 2) is a formin protein with unusual biochemical characteristics. As with other formins, the formin homology 2 (FH2) domain of INF2 accelerates actin filament assembly and remains at the barbed end, modulating elongation. The unique feature of INF2 is its ability to sever filaments and enhance depolymerization, which requires the C-terminal region. Physiologically, INF2 acts in the secretory pathway and is mutated in two human diseases, focal and segmental glomerulosclerosis and Charcot-Marie-Tooth disease. In this study, we investigate the effects of mutating two FH2 residues found to be key in other formins: Ile-643 and Lys-792. Surprisingly, neither mutation abolishes barbed end binding, as judged by pyrene-actin and total internal reflection (TIRF) microscopy elongation assays. The I643A mutation causes tight capping of a subset of filaments, whereas K792A causes slow elongation of all filaments. The I643A mutation has a minor inhibitory effect on polymerization activity but causes almost complete abolition of severing and depolymerization activity. The K792A mutation has relatively small effects on polymerization, severing, and depolymerization. In cells, the K792A mutant causes actin accumulation around the endoplasmic reticulum to a similar extent as wild type, whereas the I643A mutant causes no measurable polymerization. The inability of I643A to induce actin polymerization in cells is explained by its inability to promote robust actin polymerization in the presence of capping protein. These results highlight an important point: it is dangerous to assume that mutation of conserved FH2 residues will have equivalent effects in all formins. The work also suggests that both mutations have effects on the mechanism of processive elongation.

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Figures

FIGURE 1.
FIGURE 1.
FH2 mutations do not abolish INF2-mediated elongation inhibition. A, domain architecture of full-length human INF2 (CAAX) and the FFC and FF constructs used in this study. The approximate locations of the I643A and K792A mutations are shown. B, elongation of 0.5 μm actin monomers (25% pyrene) from phalloidin-stabilized seeds in the presence of 10 nm of the indicated FF construct. The percentage of inhibition is shown on the side of the graph. C, concentration dependence of filament elongation inhibition by FH2 mutants. The slopes were taken at 10% completion of elongation reactions and were converted to elongation rates. D, traces of individual filaments from TIRF assays with various mutants. 1 μm actin monomers (20% Alexa 488-labeled) and 1 nm of the formin were used for WT and the K792A mutant. 10 nm was used for the I643A mutant. Arrows indicate points where the elongation rate changes, suggesting that the formin is binding to or releasing from the barbed end. E, quantification of elongation rates for the various INF2-FF constructs shown in D. The error bars represent standard deviation (numbers on the bars represent n).
FIGURE 2.
FIGURE 2.
FH2 mutations have varying effects on the polymerization activity of INF2-FF. A, pyrene-actin polymerization assays containing 1 μm actin monomers (5% pyrene) with 200 nm of the indicated FF construct of INF2. B, plot showing time to 50% completion (T½) versus formin concentration for polymerization assays conducted with various concentrations of INF2 (data not shown). The EC50 values for WT and K792A and I643A mutants are 13.9, 20.5, and >165 nm, respectively. C, plot showing barbed end concentration versus formin concentration. D, expanded view of C, showing barbed end concentration versus formin concentration in greater detail for the I643A mutant.
FIGURE 3.
FIGURE 3.
FH2 mutations have varying effects on the polymerization ability of INF2-FFC. A, pyrene-actin polymerization assays containing 1 μm actin monomers (5% pyrene) with 200 nm of the indicated FFC construct of INF2. B, pyrene-actin polymerization assays containing 1 μm actin monomers (5% pyrene) with 200 nm of the indicated FFC construct of INF2 in the presence of 10 mm sodium phosphate. C, plot showing time to 50% completion (T½) versus formin concentration for polymerization assays conducted various concentrations of INF2 (data not shown). The EC50 values for WT and I643A, K792A, double (I643A/K792A), and WH2 mutants are 2, 43, 1.2, 87, and 7.7 nm, respectively.
FIGURE 4.
FIGURE 4.
Depolymerization assays in the presence of INF2-FFC constructs. A, actin monomers (1.05 μm, 5% pyrene-labeled) were polymerized 16 h at 23 °C in polymerization buffer and then diluted to 1 μm in the same buffer containing 400 nm of the indicated FFC construct; the filament depolymerization rate was measured by the decrease in pyrene fluorescence intensity with time. A.U., arbitrary units. B, depolymerization rates (derived from initial slopes of depolymerization curves) for WT and K792A mutant plotted as a function of FFC concentration.
FIGURE 5.
FIGURE 5.
Severing assays in the presence of FFC constructs. A–G, severing assays were performed using 2 μm polymerized actin and 200 nm of various INF2 constructs for 2 min. Scale bar, 5 μm. H, bar graph shows average actin filament lengths (μm) in the presence 200 nm of various INF2 constructs. At least 200 filaments were measured for each construct in two independent experiments. The error bars represent standard deviation. I, graph showing average actin filament length measured from severing assays using 200 nm of INF2-FFC-WT or K792A. The protein was incubated with filaments for 1, 2, or 5 min prior to mounting and imaging. The error bars represent standard error. J, graph showing average actin filament length measured from severing assays using various concentrations of INF2-FFC-WT or K792A. Average length of actin filaments without any added protein was 5.2 ± 0.17 μm. The protein was incubated with filaments for 2 min prior to mounting and imaging. The error bars represent standard error.
FIGURE 6.
FIGURE 6.
The INF2-FFC I643A mutant does not polymerize actin in cells. U2OS cells were co-transfected with CFP-Sec61β (blue) and various GFP-tagged INF2 mutants (green). 18–24 h post-transfection, the cells were fixed and stained with rhodamine-phalloidin (red) to visualize actin filaments. ER-Green was used as a negative control. The images show small sections of single Z-slices of confocal images. The entire cell is shown in supplemental Fig. S1. The arrowheads indicate regions where the GFP and Sec61β signals co-localize. Intensity line scans along the length of the arrow are shown to the right. Asterisks indicate a peak on the Sec61β intensity profile that corresponds to an ER tubule. Scale bar, 5 μm.
FIGURE 7.
FIGURE 7.
Pyrene-actin polymerization assays with various INF2 FFC constructs in the presence of profilin and capping protein. 3 μm actin (5% pyrene labeled), 9 μm profilin, 20 nm capping protein, and 25 nm of each formin construct was used was used in these assays. A, with profilin. B, with profilin and capping protein (CP).
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