1. Force Generation by Cytoskeletal Filament End-Tracking Proteins
Richard B. Dickinson, Luzelena Caro, Daniel L. Purich 
Biophysical Journal 
Volume 87, Issue 4, Pages (October 2004)
DOI: /biophysj
Copyright © 2004 The Biophysical Society Terms and Conditions

2. Figure 1
Bioenergetics of actin filament dynamics and treadmilling. (A) The key reactions in actin filament dynamics. Actin·ATP binds to actin filament (+)-ends (critical concentration [MT](+)-crit). Key reactions are enclosed in the dotted box: Filament-bound ATP hydrolyzes to form ADP·Pi. Phosphate dissociates reversibly (KP) from the filament, and Actin·ADP dissociates from the (−)-end (critical concentration, [MD](−)-crit). Also shown are the roles of profilin in catalyzing monomer addition and ATP/ADP exchange, and of ADF/cofilin in catalyzing depolymerization at (−)-ends. Consistent with the principle of detailed balance, the known catalyzing properties of these actin-binding proteins do not alter the net free energy change in the cycle going from (−)-end bound actin·ADP to (+)-end bound actin·ATP. (B) The free energy changes in the treadmilling steps, with one ATP molecule consumed per monomer (ΔGhydrolysis ∼ −2 kT). The largest energy decrease occurs at ATP hydrolysis to form ADP·Pi on the filament end, a reaction releasing ∼14 kT, but that plays no role in treadmilling of filaments in solution. This available energy is proposed to facilitate monomer addition and force generation when (+)-end is coupled to a filament end-tracking motor.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2004 The Biophysical Society Terms and Conditions

3. Figure 2
Hypothetical reaction schemes for cytoskeletal filament end-tracking motors. Each simple scheme shown requires two end-tracking units operating on an actin filament end. Actin·ATP subunits are shown in dark shading and actin·ADP (or actin·ADP·Pi) subunits are shown in light shading. The motor end-tracking units may be coupled together in a multimer or bound separately to the motile surface, as shown. Mechanism-A: end-tracking stepping motor (moving clockwise from the upper left). Step 1: The actin monomer binds to the filament end from solution. Step 2: The end-tracking unit advances to the new terminal subunit, in a manner that can be facilitated by hydrolysis. Mechanism-B: direct-transfer end-tracking motor (moving clockwise from the upper left). Step 1′: The actin monomer binds to the end-tracking unit. Step 2′: The end-tracking unit transfers the monomer to the filament end. Step 3′: Another end-tracking unit releases, in a manner that can be facilitated by ATP hydrolysis, which returns the system to the original state. In both Mechanisms, if hydrolysis were without effect, the principle of detailed balance would require that the net reaction would exhibit the same free energy change monomer addition to free filament ends, with critical concentration [MT](+)-crit and net free energy change, ΔG(+),add=−kT ln([MT]/[MT](+)-crit). With hydrolysis energy of the amount ɛ going to attenuate the affinity by the factor e−ɛ/kT, the net free energy change per assembled monomer is ΔG(+),add −ɛ.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2004 The Biophysical Society Terms and Conditions

4. Figure 3
Dimensionless elongation rate for the end-tracking stepping motor (Mechanism-A) versus dimensionless force. The elongation rate R, relative to the maximum rate k1[MT], is plotted as a function of dimensionless force, Fd/2kT, where k1 is the forward association rate constant in Step 1 of Mechanism-A (Fig. 2), [MT] is the actin monomer concentration, F is the force opposing the elongation, kT is the thermal energy, and d is the subunit length (5.4nm for actin). For each plot, [MT]/[MT](+)-crit is set equal to 3, and 9.2 kT of the hydrolysis energy is captured to provide a 10−4 reduction in affinity for Step 2 in the Mechanism-A reaction cycle. The solid lines that intersect the zero line at Fd/2kT=ln(3)=1.1 correspond to Mechanism-A without the benefit of hydrolysis (i.e., where K2,0=[MT](+)-crit/K1), and those that intersect at =10.3 are for Mechanism-A with hydrolysis modulating the affinity (i.e., where K2,0=10−4[MT](+)crit/K1). (A) Curves are shown for various values of the equilibrium dissociation constant K1 at a fixed value of α=k−2/k−1=10. (B) Curves are shown for the various values of α at a fixed value of K1=1.0.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2004 The Biophysical Society Terms and Conditions

5. Figure 4
Dimensionless elongation rate for the direct-transfer end-tracking motor (Mechanism-B) versus dimensionless force. The elongation rate R, relative to the maximum rate k'[MT], is plotted as a function of dimensionless force, Fd/2kT, where k′1 is the forward association rate constant of Step 1′ in Mechanism-B (Fig. 2). (Other parameters are defined in the Fig. 3 caption.) Step 2′ is assumed the only force-dependent step (K′2=K′2,0e−Fd/2kT). As in Fig. 3, [MT]/[MT](+)-crit is set equal to 3, and the 9.2 kT of the hydrolysis energy is captured to provide a 10−4 reduction in affinity for Step 3 of Mechanism-B. The solid lines that intersect the zero line at Fd/2kT=ln(3)=1.1 are for Mechanism-B without the benefit of hydrolysis (i.e., where K′3=K′1K′2,0/[MT](+)-crit), and those that intersect at =10.3 are for Mechanism-A with hydrolysis modulating the affinity (i.e., where K′3=104K′1K′2,0/[MT](+)crit). (A) Curves are for various shown values of the equilibrium constant K′20 with a fixed value of β=k′−2/k′−1 =10 and K′1=1.0. (B) Curves are for the various shown values of β with a fixed values of K′2,0=10−2 and K′1=1.0.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2004 The Biophysical Society Terms and Conditions

6. Figure 5
Mechanism-C: cofactor assisted end-tracking motor filament. Cofactor assisted pathways are shown for (A) actin polymerization, and (B) tubulin polymerization. Step 1: The soluble cofactor and monomer bind together to the end-tracking unit. Step 2: The cofactor and monomer add to the filament end. Step 3: Another tracking unit and cofactor dissociate from the adjacent protofilament, in a step that can be facilitated by ATP hydrolysis energy to modulate the affinity of the cofactor and/or the tracking unit for the filament. The cofactor may return immediately to solution, or remain transiently bound to the tracking unit or to the filament.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2004 The Biophysical Society Terms and Conditions

7. Figure 6
Schematic of filament with a free end flexed against a surface. The relevant parameters and variables for a flexed filament analyzed in Appendix A are shown, including the gap distance D, filament angle θ(s), arc length s, fixed initial angle of incidence, θ0, final angle of incidence θ1, and total length, L.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2004 The Biophysical Society Terms and Conditions

8. Figure 7
Maximum force versus initial angle of incidence for tethered and untethered filaments. The dimensionless buckling force for both cases is shown in the main plot, and the corresponding dimensional forces are shown for the case of a persistence length λ=10μm and a gap distance, D=70nm. The thermodynamic stall forces (dotted lines) for the Brownian ratchet model (BR Model) and the end-tracking motor (E-T motor) are also shown, assuming a factor of six greater monomer concentration above the critical concentration and 14 kT additional energy from ATP hydrolysis captured by the end-tracking motor.
Biophysical Journal  , DOI: ( /biophysj )
Copyright © 2004 The Biophysical Society Terms and Conditions