Figure 4.
Figure 4. Processivity of barbed end–associated VASP tetramers. (A) Actin filament lengths before and after burst-phase imaging of 0.25 nM Cy3-VASP in the presence of 1 µM Mg-ATP-actin (30% Alexa Fluor 488). Maximum intensity projections of 0.25 nM Cy3-VASP (1,000 frames) superimposed on the Alexa Fluor 488 actin filament (t = 100 s). Bar, 5 µm. (B). Barbed end localization of Cy3-VASP (middle image; arrow) merged with maximum intensity projection of Cy3-VASP actin filament binding (frames 1–387; top image). Two molecules are also laterally bound to the actin filament (arrowheads). Bar, 5 µm. (C) Representative kymographs showing single Cy3-VASP tetramers processively tracking on actin filament barbed ends. Kymographs from left to right show molecules with increasing processivity. The dashed line connecting two processive VASP tetramers has a slope equivalent to an ∼10 sub/sec barbed end elongation rate in the absence of VASP. Arrowheads point to molecules transiently binding to ATP/ADP-Pi actin, near barbed ends (τ1 = 0.22 ± 0.01 s, n = 280). Vertical bar, 5 s. (D) Magnification of dashed line box in C showing: (1) side binding, (2) diffusion toward barbed end, and (3) barbed end attachment and surfing of Cy3-VASP. Horizontal bar, 2 µm; vertical bar, 2.5 s. (E) Histogram of barbed end dwell times measured in the presence of 0.25 nM Cy3-VASP and 1 µM actin (30% Alexa Fluor 488). Inset plot is an exponential fit of 1-cumulative frequency (τ1 = 1.45 ± 0.02 s, n = 1,166). (F) Time series for a highly processive Cy3-VASP tetramer tracking an actin filament barbed end. (G) Barbed end growth rates measured in the presence of 1 µM Mg-ATP-actin (30% Alexa Fluor 488), plus varying concentrations of VASP. Data fit to Michaelis-Menten equation with a y-axis offset of 10 (Kd = 9.2 ± 1.4 nM). From the Kd and Koff (0.69 s−1) we calculated a barbed end association rate constant of 75 µM−1 sec−1 for VASP. (H) Barbed end polymerization rates in the presence of 0–50 nM VASP, plus 0.25–2 µM Mg-ATP-actin (30% Alexa Fluor 488). (I) Barbed end dwell times for 0.25 nM Cy3-VASP in the presence of 0.5–2 µM Mg-ATP-actin. The number of actin monomers delivered to the barbed end by single VASP tetramers is the product of the barbed end dwell time and the polymerization rate from H.

Processivity of barbed end–associated VASP tetramers. (A) Actin filament lengths before and after burst-phase imaging of 0.25 nM Cy3-VASP in the presence of 1 µM Mg-ATP-actin (30% Alexa Fluor 488). Maximum intensity projections of 0.25 nM Cy3-VASP (1,000 frames) superimposed on the Alexa Fluor 488 actin filament (t = 100 s). Bar, 5 µm. (B). Barbed end localization of Cy3-VASP (middle image; arrow) merged with maximum intensity projection of Cy3-VASP actin filament binding (frames 1–387; top image). Two molecules are also laterally bound to the actin filament (arrowheads). Bar, 5 µm. (C) Representative kymographs showing single Cy3-VASP tetramers processively tracking on actin filament barbed ends. Kymographs from left to right show molecules with increasing processivity. The dashed line connecting two processive VASP tetramers has a slope equivalent to an ∼10 sub/sec barbed end elongation rate in the absence of VASP. Arrowheads point to molecules transiently binding to ATP/ADP-Pi actin, near barbed ends (τ1 = 0.22 ± 0.01 s, n = 280). Vertical bar, 5 s. (D) Magnification of dashed line box in C showing: (1) side binding, (2) diffusion toward barbed end, and (3) barbed end attachment and surfing of Cy3-VASP. Horizontal bar, 2 µm; vertical bar, 2.5 s. (E) Histogram of barbed end dwell times measured in the presence of 0.25 nM Cy3-VASP and 1 µM actin (30% Alexa Fluor 488). Inset plot is an exponential fit of 1-cumulative frequency (τ1 = 1.45 ± 0.02 s, n = 1,166). (F) Time series for a highly processive Cy3-VASP tetramer tracking an actin filament barbed end. (G) Barbed end growth rates measured in the presence of 1 µM Mg-ATP-actin (30% Alexa Fluor 488), plus varying concentrations of VASP. Data fit to Michaelis-Menten equation with a y-axis offset of 10 (Kd = 9.2 ± 1.4 nM). From the Kd and Koff (0.69 s−1) we calculated a barbed end association rate constant of 75 µM−1 sec−1 for VASP. (H) Barbed end polymerization rates in the presence of 0–50 nM VASP, plus 0.25–2 µM Mg-ATP-actin (30% Alexa Fluor 488). (I) Barbed end dwell times for 0.25 nM Cy3-VASP in the presence of 0.5–2 µM Mg-ATP-actin. The number of actin monomers delivered to the barbed end by single VASP tetramers is the product of the barbed end dwell time and the polymerization rate from H.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal