Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Jan 8;18(1):9-19.
doi: 10.1016/j.cub.2007.11.062. Epub 2007 Dec 20.

The role of the FH1 domain and profilin in formin-mediated actin-filament elongation and nucleation

Aditya S Paul  1 , Thomas D Pollard
Affiliations

The role of the FH1 domain and profilin in formin-mediated actin-filament elongation and nucleation

Aditya S Paul et al. Curr Biol. .

Erratum in

  • Curr Biol. 2008 Feb 12;18(3):233. Paul, Aditya [corrected to Paul, Aditya S]; Pollard, Thomas [corrected to Pollard, Thomas D]

Abstract

Background: Formin proteins nucleate actin filaments de novo and stay associated with the growing barbed end. Whereas the formin-homology (FH) 2 domains mediate processive association, the FH1 domains-in concert with the actin-monomer-binding protein profilin-increase the rate of barbed-end elongation. The mechanism by which this effect is achieved is not well understood.

Results: We used total internal reflection fluorescence microscopy to measure the effect of profilin on the elongation of single actin filaments associated with FH1FH2 constructs (derived from the formin Bni1p from S. cerevisiae) with FH1 domains containing one to eight profilin-binding polyproline tracks. Over a large range of profilin concentrations (0.5-25 microM), the rate of barbed-end elongation increases with the number of polyproline tracks in the FH1 domain. The binding of profilin-actin to the FH1 domain is the rate-limiting step (up to rates of at least 88 s(-1)) in FH1-mediated transfer of actin subunits to the barbed end. Dissociation of formins from barbed ends growing in the presence of profilin is proportional to the elongation rate. Profilin profoundly inhibits nucleation by FH2 and FH1FH2 constructs, but profilin-actin bound to FH1 might contribute weakly to nucleation.

Conclusions: To achieve fast elongation, formin FH1 domains bind profilin-actin complexes and deliver them rapidly to the barbed end associated with the FH2 domain. Because subunit addition promotes dissociation of FH2 domains from growing barbed ends, FH2 domains must pass through a state that is prone to dissociation during each cycle of actin subunit addition coupled to formin translocation.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Domain Maps of Bni1p FH1 Variants Used in this Study
Residue numbers are shown to mark domain boundaries and polyproline tracks pPA, pPB, pPC, and pPD. The amino acid sequence of the Bni1p FH1 domain (residues 1228–1347) is given at the bottom.
Figure 2
Figure 2. Time-Lapse TIRFM of Actin Polymerization in the Presence of Bni1(FH2)p or Bni1(pP8FH2)p with Profilin
Conditions: 1.5 μM actin monomers (33% Oregon green) and 5 μM profilin in microscopy buffer (9.6 mM imidazole, pH 7.0, 48 mM KCl, 0.96 mM MgCl2, 0.96 mM EGTA, 96 mM DTT, 1.92 mM ATP, 50 mM CaCl2, 14.4 mM glucose, 19.2 mg/ml catalase, 96 mg/ml glucose oxidase, 0.48% methylcellulose [4000 cP at 2%], 0.19% BSA) with (A) 10 nM Bni1(FH2)p or (B) 1 nM Bni1(pP8FH2)p in PF buffer. The time series of images shows growth of free barbed ends (red wedges) and formin-associated barbed ends (green wedges) in the same field. Triangles mark pointed ends. Formin-associated barbed ends grew at an average rate of 32.6 subunits/s for Bni1(pP8FH2)p and 4.5 subunits/s for Bni1(FH2)p. Free barbed ends in the same viewing fields grew at 13.1 subunits/s for Bni1(pP8FH2)p and 11.7 subunits/s for Bni1(FH2)p.
Figure 3
Figure 3. The Effect of the Number of the FH1 Polyproline Tracks on Barbed-End Elongation with Profilin
Conditions: 1.5 μM actin (33% Oregon green) in microscopy buffer with varying concentrations of profilin. All data were collected with TIRFM. All Bni1(pPnFH2)p-associated rates are normalized to that of Bni1(FH2)p (equation 1). (A) The dependence of the elongation rate of barbed ends on the concentration of profilin. Rates for ends associated with Bni1(pP2FH2)p and Bni1(pP8FH2)p are shown as representative plots for the Bni1p FH1 variants. The rates for free barbed ends and ends associated with Bni1(FH2)p were measured in the same viewing fields. (B and C) Plots are split into upper and lower panels for clarity. Gray vertical lines indicate polyproline track number of Bni1(FH1FH2)p on the x axis. (B) The dependence of the rate of elongation of barbed ends associated with Bni1(pPnFH2)p constructs on the total number of polyproline tracks in the FH1 domain for a range of profilin concentrations (μM). (C) The barbed-end rate per polyproline track (equation 2) versus the total number of FH1 polyproline tracks (per formin subunit) at indicated micromolar concentrations of profilin.
Figure 4
Figure 4. Dependence of the Elongation Rate of Barbed Ends Associated with Bni1(pP8FH2)p, Bni1(pP1FH2)p, or Bni1(FH2)p on the Concentration of Profilin-Actin
Conditions: Varying concentrations of actin monomers (33%Oregon Green) with 5 μM profiling in microscopy buffer. All data were collected with TIRFM. The dependence of the rates of elongation with profilin on the bulk actin-monomer concentration. Determination of the profilin-actin transfer rate constant for Bni1(pP1FH2)p and Bni1(pP8FH2)p. The differences in the rates of elongation mediated by Bni1(pPnFH2)p and Bni1(FH2)p (ΔpPn-FH2) versus the profilin-actin concentration (calculated from the profilin and actin concentrations and the Kd of their interaction [32]) are shown. The slopes yield apparent second-order association rate constants of 9.9 μM−1 s−1 for Bni1(pP1FH2)p (R2 = 0.91) and 39 μM−1 s−1 for Bni1(pP8FH2)p (R2 = 0.99).
Figure 5
Figure 5. Dissociation of Bni1(pP8FH2)p from Barbed Ends
Conditions as in Figure 4. Time course of dissociation of Bni1(pP8FH2)p from growing barbed ends in 5 μM profilin at three actin concentrations (μM). Straight lines are fit to the first part of the time course of each reaction. R2 values for fits at the various actin concentrations were >0.70. The dependence of the initial rate of dissociation of Bni1(pP8FH2)p from growing barbed ends (slopes from [A]) on the average barbed-end elongation rate of the filaments (from Figure 4A).
Figure 6
Figure 6. The Bni1p FH1 Domain Does Not Influence Nucleation of Filaments of Free Actin by FH2
Conditions: 4 μM actin monomers (20% pyrene) in polymerization buffer (10 mM imidazole, pH 7.0, 50 mM KCl, 1 mM EGTA, 1 mM MgCl2, supplemented with formin buffer). (A) Representative time courses of spontaneous polymerization measured by fluorescence enhancement of pyrenyl-actin (λex = 362 nm; λem = 407 nm) in the presence of indicated nanomolar concentrations of Bni1(FH2)p. (B) Dependence of the concentration of ends nucleated (calculated from the elongation rate and the slope of the pyrenyl-fluorescence enhancement when half of the monomers were polymerized [equation 3]) on the concentrations of Bni1(FH2)p or Bni1(pPnFH2)p. The reported values of concentration of ends created by formin were obtained by subtraction of the value for the concentration of ends created through self-nucleation of actin monomers (typically ∼0.2 nM; calculated from time courses of assembly of actin alone) from the total concentration of ends. For Bni1(pP1FH2)p, the data point at 50 nM formin appears anomalous and is therefore not connected by a line to the rest of the data. Inset: Dependence of the concentration of ends created per formin dimer (calculated from the data in the main plot) on the formin concentration (nM). Data for Bni1(FH2)p are shown as a representative plot.
Figure 7
Figure 7. The Influence of Profilin on Actin-Filament Nucleation by Bni1(FH2)p or Bni1(FH1FH2)p
Conditions as in Figure 6, except pH = 7.5. (A) Representative time courses of the polymerization of 4 μM actin monomers in the presence of 5 nM Bni1(FH2)p and indicated micromolar concentrations of profilin. (B) Ratios of simulated to experimental half-times for the assembly of pyrenyl-actin (4 μM, 20% pyrene) with Bni1(FH1FH2)p (upper panel) or Bni1(FH2)p (lower panel) over a range of profilin concentrations. For each profilin concentration and formin construct tested as in (A), the half-time (the point in the assembly time course at which half the pyrenyl-actin had polymerized) of simulated assembly was divided by the half-time of the experimental measurements. For Bni1(FH1FH2)p, simulated to experimental half-time ratios are shown for simulated assembly with a mechanism having no reaction for profilin-actin nucleation (filled circles) or a reaction for nucleation of FH1-bound profilin-actin (reaction 14 [forward], Figure S1) with a rate constant of either 3 × 10−2 μM−2 s−1(open circles) or 1 × 10−1 μM−2 s−1 (open triangles). For each condition tested in simulated assembly, simulated to experimental half-time ratios are shown for two sets of experimental measurements. (C)The dependence on profilin concentration of the initial rates of nucleation by FH1FH2 from free actin monomers (filled circles) or profilin-actin (open circles, open triangles) in the simulated polymerization reactions in (B). The reported values are the instantaneous rates at 15 s after the initiation of polymerization of reaction 5 (nucleation from free actin) or 14 (nucleation from profilin-actin) in Figure S1. This time point was chosen because in simulated polymerization it is near the outset of the polymerization reaction but far enough into the reaction to allow the individual pools of profilin, actin, and profilin-actin to equilibrate. For the plots of rates of nucleation from profilin-actin, the legend indicates the rate constant of the forward Reaction 14 tested in the simulations in (B). The initial rate of nucleation from free actin is identical for each simulated reaction with FH1FH2 in (B).
Figure 8
Figure 8. Model for Translocation of a Formin FH2 Domain Coupled to Actin Subunit Addition to the Barbed End
(A) The mechanical cycle of subunit addition coupled to translocation of formin is schematically illustrated from the upper left in clockwise order as a series of states 1-5. (Figure S4 shows this cycle with space-filling images derived from PDB files.) To proceed from state X to state Y, formin must pass through step X,Y indicated above the reaction arrows. For each state, the upper image displays the filament at a view normal to the filament axis, with the barbed end pointing down. The actin filament is shown with gray subunits along one long-pitch strand and blue subunits in the other strand. The lower images are views of the barbed end. The helical twist along the short-pitch helix of the three terminal barbed-end subunits is indicated for each state and is colored either red to indicate a conformation that prevents subunit addition (closed state) or green to indicate one that permits addition (open state). (Note that the 180° helical twist in closed states 1 and 5 does not propagate into the filament past the third subunit from the barbed end.) One subunit of the FH2 dimer is shown in green, the other in magenta. Each FH2 subunit has two sites to interact with actin: the knob (K) and the post (P). These sites are indicated in each image, and their states of association with the filament are indicated as (+) for bound and (‒) for unbound in barbed-end-on views. The flexible linkers of each FH2 subunit (Bni1p residues 1401-1417) are depicted as either stretched or relaxed springs (see [B]). The FH2 subunits of the transient translocation intermediate between states 3 and 4 are partially transparent. (B) Space-filling models of the proposed closed state with a 180° filament conformation and the open state with a 167° filament conformation. The yellow lines show the distances that the FH2 flexible linker must span between the leading subunit's knob and the trailing subunit's post (from the carboxyl-terminus of residue 1400 to the amino-terminus of 1418 of the leading subunit) in the two states. Illustration of the linkers as stretched or relaxed springs emphasizes the differences in the extension of the linkers in the two states. The 180° filament conformation with Bni1(FH2)p is from PDB file 1Y64. The 167° conformation was modeled by overlaying the Bni1(FH2)p-actin structure with the atomic model of the actin filament (from K.C. Holmes, ftp://149.217.48.3/pub/holmes/pdb/actin_helix_93.pdb); the FH2 subunits were rotated about the filament axis while the knob-site attachments of the FH2 subunits to the actin subunits were maintained as in the structure [18]. Images were rendered with PYMOL (Delano Scientific, Palo Alto, California).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Sagot I, Klee SK, Pellman D. Yeast formins regulate cell polarity by controlling the assembly of actin cables. Nat Cell Biol. 2002;4:42–50. - PubMed
    1. Chang F, Drubin D, Nurse P. cdc12p, a protein required for cytokinesis in fission yeast, is a component of the cell division ring and interacts with profilin. J Cell Biol. 1997;137:169–182. - PMC - PubMed
    1. Feierbach B, Chang F. Roles of the fission yeast formin for3p in cell polarity, actin cable formation and symmetric cell division. Curr Biol. 2001;11:1656–1665. - PubMed
    1. Watanabe N, Kato T, Fujita A, Ishizaki T, Narumiya S. Cooperation between mDia1 and ROCK in Rho-induced actin reorganization. Nat Cell Biol. 1999;1:136–143. - PubMed
    1. Pellegrin S, Mellor H. The Rho family GTPase Rif induces filopodia through mDia2. Curr Biol. 2005;15:129–133. - PubMed

Publication types

MeSH terms

LinkOut - more resources