Direct real-time observation of actin filament branching mediated by Arp2/3 complex using total internal reflection fluorescence microscopy

Abstract

Existing methods for studying actin filament dynamics have allowed analysis only of bulk samples or individual filaments after treatment with the drug phalloidin, which perturbs filament dynamics. Total internal reflection fluorescence microscopy with rhodamine-labeled actin allowed us to observe polymerization in real time, without phalloidin. Direct measurements of filament growth confirmed the rate constants measured by electron microscopy and established that rhodamine actin is a kinetically inactive tracer for imaging. In the presence of activated Arp2/3 complex, growing actin filaments form branches at random sites along their sides, rather than preferentially from their barbed ends.

Figure 1

Elongation of actin filaments visualized by TIRFM. Actin monomers (1 μM, 40% rhodamine-labeled) were polymerized in fluorescence buffer (50 mM KCl/1 mM MgCl₂/10 mM imidazole, pH 7.0/1 mM EGTA/100 μM CaCl₂/200 μM ATP/3 mM NaN₃/3 mg/ml glucose/100 μg/ml glucose oxidase/20 μg/ml catalase/10 mM DTT) in a flow cell with the coverslip coated with 100 μg/ml NEM-myosin for 1 min, and blocked with 1% BSA for 1 min. Exposures of 300 ms were collected every 3 s for 3 min. Shown are frames representing the time course of elongation. (Scale bar = 14 μm.)

Figure 2

Dependence of barbed-end elongation rates on actin monomer concentration. Actin filament elongation was observed as described in Fig. 1. (A) Dependence on total actin concentration with constant 40% rhodamine-actin. (B) Dependence on fraction of rhodamine-labeled actin with 1 μM total actin. Values shown are the mean ± SD of at least six randomly chosen growing filaments per condition. Lines represent linear least-squares fits to the mean values.

Figure 3

Actin filament branch formation. Filament elongation of 20% rhodamine actin was visualized by TIRFM as described in Fig. 1. (A and B) Actin monomers (1 μM) were visualized polymerizing for 2 min before addition of 100 nM Arp2/3 complex, 300 nM Scar WA, and 2 μM actin monomers. Frames shown were subsequently captured at 100, 130, 170, and 210 (A) or 105, 135, 165, 270, and 305 (B) s after addition. (C) Actin monomers (2 μM) were polymerized for 3 min before growing branches for 3 min in the presence of 60 nM Arp2/3 complex, 500 nM Scar WA, 4 μM profilin, and 2 μM actin. Frames shown were collected at 0, 36, 75, 132, and 180 s after addition of branching mixture. (D) Unbound components were washed from the branched structures in C and 2 μM monomeric actin was added to the cell. Frames shown were collected 0, 32, 72, and 108 s after monomer addition. (E and F) Histograms of the distances from branch points to mother filament barbed ends for branches growing from the sides of filaments that had grown before addition of Arp2/3 complex (E) or from portions of filaments that had grown in the presence of Arp2/3 complex (F). (Scale bar = 4 μm.)