1. Model of Formin-Associated Actin Filament Elongation
Dimitrios Vavylonis, David R. Kovar, Ben O'Shaughnessy, Thomas D. Pollard 
Molecular Cell 
Volume 21, Issue 4, Pages (February 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1
Model of Actin Filament Growth Mediated by a Bni1(FH1FH2)p Formin Dimer
We assume that all formins obey the same mechanism. FH2 domains (red) are shown as a dimer encircling the barbed end of the filament (blue) (Otomo et al., 2005b). The FH1 domain (black, green) is shown unstructured with three profilin binding sites (green). In reality, FH1 may be more compact than depicted. We used the PDB entries 1Y64 (FH2-actin), 2BTF (profilin-actin), and 1CJF (profilin-poly-proline) and incorporated the proposed FH2 lasso domain swap (Otomo et al., 2005b). The filament model (Holmes et al., 1990) was constructed with FilaSitus. The strained conformation of the tip subunits (Otomo et al., 2005b) is not shown in this coarse-grained picture.
(A) FH1-mediated polymerization: (1) assembly of profilin-actin to FH1; (2) transfer of actin from FH1 to the barbed end; (3) detachment of profilin.
(B) Mechanism of FH2 processivity based on Figure 5 of Otomo et al. (2005b) showing FH2 in states preventing or allowing subunit incorporation and the association or dissociation of an actin subunit. Our model is consistent with this picture, but other mechanisms of FH2 processivity are also conceivable. We assume that FH2 is in rapid equilibrium among its accessible states. FH2 steps are assumed to be uncoupled from hydrolysis, but we also discuss the effects of a coupled hydrolysis mechanism.
(C) FH1 domain sequences of four formins and putative profilin binding sites (highlighted). Most mDia1 experiments of Kovar et al. (2006) involved a deletion of the underlined part of FH1.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
Scheme of Formin-Mediated Actin Assembly with mDia1(FH1FH2) with 11 Profilin Binding Sites Shown as an Example
(A) Polymerization paths and associated rates connecting states I and VII, which has an identical FH1 configuration as state I but one extra polymerized actin monomer. For fast growing filaments, the nucleotide bound at the monomers near the tip is ATP; the filament interior consists of ADP-Pi-actin and ADP-actin. n indicates the nth profilin binding site.
(B) Legend of protein shapes and arrow colors. Rate constants of green arrows derived from detailed balance.
(C) Bulk profilin-actin association.
(D) Polymerization paths of scheme (A). Path 1 (I→II, direct assembly of profilin-actin onto FH1) and path 2 (II→III, assembly of free actin onto profilin-FH1) assembly is catalyzed by FH1. Paths 3 (I→VI) and 4 (I→VII) represent direct addition of profilin-actin or actin to the barbed end, respectively. The net elongation rate is the sum of the four paths.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
Fit of Formin-Associated Elongation Rates to Previous Experiment
Experimental data is from Kovar et al. (2006). Parameters are from Table 1, but for mDia1 kB−P = 3000 s−1 and for Cdc12p kB−P = 5000 s−1.
(A) Barbed-end elongation rate versus total profilin concentration, [ATP-actin] = 1μM and KdPA = 6 μM.
(B) As in (A), but the total actin concentration is varied and [profilin] = 2.5 μM.
(C) Elongation rate (color scale, sub/s) versus [actin] and [profilin] for Bni1p. Gray indicates depolymerization.
(D) Elongation rate as a function of [profilin] and contribution of each of the four paths of Figure 2D to the theoretical curves of (A) for Bni1p. Curve labeled “Total” is the sum of all paths. Inset: Average number of poly-proline sites out of the total six of the formin dimer that are free, bound to profilin, or bound to profilin-actin. As a consequence of detailed balance, the rates of all four paths vanish at the same profilin concentration (experimentally, it is unclear if elongation vanishes at high [profilin], see Discussion).
(E) Same as (D) but showing the four paths as a function of [actin].
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Filament Fluoresence Intensity, Φ, as a Function of [Profilin] Obtained from Previously Published Movies
Movies are from Kovar et al. (2006).The intensity of formin-associated filaments is normalized with respect to formin-free filaments whose intensity did not change with [profilin], within experimental error. Inset: 1/Φ versus formin-associated elongation rate from Figure 3A and Figure 6B; the relationship is approximately linear: straight lines are linear regression fits for mDia1, Bni1p, and mDia2.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
Elongation Rate versus [Profilin] and Its Dependence on Parameter Values for Bni1p
The varied parameters values are indicated in each plot. The nonvaried parameters are the same as in Figure 3 and Table 1. In (B), all p-dependent values of Table 1 were varied accordingly. In (C), the ring opening rate r−PF is equal to kB−P.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6
Elongation Rate as a Function of Profilin Concentration for ADP-Actin and for mDia1(FH2)
(A) Elongation rate of ADP-actin versus [profilin], tentative theoretical fit to experiment (Kovar et al., 2006). [ADP-actin] = 5 μM, parameters from Table 1. KdPA = 4.5 μM, chosen such that elongation rate vanishes between 6 and 12 μM profilin (Kovar et al., 2006). For [profilin] > 10 μM, formins are predicted to catalyze ADP-actin disassembly.
(B) Elongation rate versus [profilin] for mDia1(FH2) lacking the FH1 domain. Dashed line indicates parameters from Table 1, KdPA = 6 μM. Continuous line is the same as the dashed but kB+PA = 3 μM−1s−1, kB−P = 80 s−1. Inset: elongation rate versus [profilin] for mDia1(FH1FH2). Dashed line: same as Figure 3A. Continuous line: same as dashed but kB+PA = 3 μM−1s−1, kB−P = 80 s−1, r−PF = 3000 s−1.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions