Table 1.
Average particle velocities in 8.5 µM profilin-actin, 100 nM Arp2/3.
Figure 1.
Actin filament and branch geometry in comet tails under TIRF microscopy.
Conditions: A–B, 8.5 µM (8% labeled) actin, 9 µM profilin, 100 nM Arp2/3, CP as indicated; C–E, 2 µM (10% labeled) actin, 3 µM profilin, 20 nM Arp2/3, 40 nM CP; F–H, 8.5 µM (8% labeled) actin, 9 µM profilin, 100 nM Arp2/3, CP as indicated; nanofibers coated with 10 µM GST-WCA from N-WASP, motility buffer, 0.38 mM total ATP. (A–B) Actin architecture in comet tails (T) of moving nanofibers (dashed outline) was visible under TIRF microscopy. In 100 nM CP, comet tails consisted primarily of long filament bundles. Increasing CP to 200 nM generated a branched actin networks with short bundles (Black arrowhead). (C) Lowering profilin-actin, Arp2/3, and CP concentrations showed individual filaments and branches (white arrowheads) in the comet tail (T). Some filaments (white arrows) crossed the nanofiber boundary, while others terminated at the nanofiber (black arrows). Brighter filament bundles (black arrowhead) terminated at the nanofiber. (D) Epi-fluorescence image of panel C. (E) Magnified image of box in B showing bundle (black arrowhead) dissociation. The bundle was formed from daughter filaments from the same mother filament (white arrowheads). (F) In high CP, nanofibers sometimes formed two comet tails. (G) Kymograph of line in F showing tail expansion at the nanofiber surface (dashed outline) under TIRF (left) and DIC (right) microscopy. (H) Nanofibers sometimes formed two comet tails in low CP. Long actin bundles (black arrowheads) appeared within and beyond the comet tails. Scale bars are 1 µm for E and 5 µm for all others. Times are shown in min∶sec.
Figure 2.
Filament bundles processively attach to the nanofiber.
Conditions: 8.5 µM (8 to 20% labeled) Mg-ATP actin, 9 µM profilin, 100 nM Arp2/3, and indicated CP. Nanofiber coating and buffers as in Figure 1. (A–B) Bright filament spots grew against the nanofiber surface. Spots either remained attached to the same location on the moving nanofiber (A, black arrowhead) or oscillated back and forth along the moving nanofiber (B, blacks arrowhead). Bright spots grew faster than the surrounding comet tail network (T) and often buckled from compression between the nanofiber and tail network (arrow). White arrowhead in B indicates bundle starting position. (C) CP was lowered from 200 to 50 nM after the establishment of two comet tails (T) by motility buffer exchange. Short, bright bundles (top panels, black arrowheads) became rapidly polymerizing bundled loops (black arrowheads, bottom panels) that remained attached to the nanofiber at their growing ends (white arrowhead) and to the tail at their pointed ends (white arrows). Growing bundles splayed into filaments of different loop lengths. (D) Traces of bundles lengths over time before CP reduction. Zero seconds represents the point of buffer exchange, 163 min after the experiment start. (E–H) Traces of individual filament lengths over time after CP reduction showed that filament within each bundle grew at different rates. Plots represent filaments from the same bundle. Some filaments continuously elongated while others show pulsed growth. Scale bars are 1 µm for A–B and 5 µm for C. Times are shown in min∶sec for A–B and hr∶min∶sec for C.
Table 2.
Frequency of bundle formation on nanofibers.
Figure 3.
Cellular Mg2+ concentrations bundle actin filaments at high densities.
Conditions: 2.5 µM (20% labeled) Mg-ATP actin, 50 mM KCl, 1.05 mM EGTA, 10 mM imidazole, pH 7.0, 100 mM DTT, 0.2 mM ATP, 15 mM glucose, 20 µg/ml catalase, 100 ug/ml glucose oxidase 0.25% 1500 cP methylcellulose. (A) Time-lapse TIRF microscopy images of de novo nucleated actin filaments. Images in each column were taken at the same time (min∶sec) after addition of salts. MgCl2 was added to low Mg-EGTA polymerization buffer to set the total Mg2+ as indicated. Free [Mg2+] was calculated from pH, ionic strength, and total Ca2+, Mg2+, EGTA, and ATP. Actin bundles readily formed at 1 mM total Mg2+ once filament densities increased. Increasing total Mg2+ to 5 or 10 mM increased the speed and extent of bundle formation. (B) Fraction of filaments forming bundles over time. Free Mg2+ concentration of at least 0.7 mM significantly increased bundle formations. (C) Time at which the first bundle was observed as a function of Mg2+ concentration. Scale bar is 2 µm.
Figure 4.
Polycations or fascin are required for bead motility.
Conditions: 8.5 µM (20% labeled) Mg-ATP actin, 9 µM profilin, 100 nM Arp2/3, 200 nM CP, 4.5 µm diameter bead coated with 8.5 µM GST-WCA, low Mg2+ buffer (50 mM KCl, 0.105 mM MgCl2, 1.05 mM EGTA, 10 mM imidazole pH 7.0, 100 mM DTT, 0.2 mM ATP, 15 mM glucose, 0.25% methylcellulose, 20 µg/ml catalase, 100 ug/ml glucose oxidase) supplemented with MgCl2, Lys-Lys·2HCl, KCl, or fascin as indicated. (A) TIRF and Epi-fluorescence microscopy images show representative actin comet tails (T) grown from GST-WCA coated beads (B, dashed circle). All images were recorded 40 minutes after the reaction start. In low Mg2+, beads formed a shell (S) that broke symmetry but rarely a comet tail. Tails that did form remained short and detached from the bead. Restoration of cellular, 1 mM Mg2+ restored comet tail growth. Additional Mg2+ accelerated comet tail growth. (B) TIRF microscopy images of actin comet tails grown in 0.1 mM total Mg2+ with added Lys-Lys2+ as indicated. All images were recorded 40 minutes after the reaction start. Lys-Lys2+ substituted for Mg2+ to restore motility. (C–D) Comet tail growth over time after the reactions start. The lengths of actin comet tails from A–B were recorded in each frame. Line segments represent growth of individual comet tails. Comet tail growth increased with the concentration of divalent cation, either in the form of (C) Mg2+ or (D) Lys-Lys2+. (E) Comet tail growth rates from C–D as a function of free cation. Both MgCl2 and Lys-Lys-2HCl restored motility in a concentration dependent manner. Removal of methylcellulose did not influence the trend of comet tail growth rates as a function of Mg2+. Addition of 5, 10, or 15 mM KCl did not restore motility in low Mg2+ buffers. (F–G) Comet tail growth over time in low Mg2+ with added fascin. Line segments represent individual comet tails. (F) In low, 0.03 mM free Mg2+, 80 nM fascin optimally restored motility while (G) 500 nM fascin optimally restored motility in 0.3 mM free Mg2+. Line breaks (arrows) in no fascin represent growth of an actin shell followed by shell detachment during an observation. (H) Comet tail growth rates from F–G as a function of fascin concentration. Errors bars in E and H show S.D. of tail growth rates. Scale bars in A–B are 5 µm.
Figure 5.
Polycation-dependent motility does not require cofilin.
Conditions: 8.5 µM (20% labeled) Mg-ATP actin, 9 µM profilin, 100 nM Arp2/3, 200 nM CP, 2 µM cofilin, 4.5 µm diameter bead coated with 8.5 µM GST-WCA, low Mg2+ buffer as in Figure 4 supplemented with MgCl2 as indicated. (A–B) Comet tail growth over time after the reactions start in the presence (A) and absence (B) of cofilin. The lengths of actin comet tails were recorded in each frame. Line segments represent growth of individual comet tails. Comet tail growth increased with the concentration of Mg2+, either in the presence or absence of cofilin. (C) Comet tail growth rates from A–B as a function of free Mg2+. MgCl2 restored motility in a concentration-dependent manner in the presence or absence of cofilin. Errors bars show S.D. of tail growth rates.
Figure 6.
Divalent cations or fascin rescues comet tail attachment.
Conditions as in Figure 4. (A) Time-lapse epi-fluorescence and TIRF microscopy sequence showing detachment of primary actin shell (1°S) from GST-WCA coated bead in 0.03 free Mg2+. Filament density between the shell and the bead surface (dotted circle) is gradually lost. (B) Epi-fluorescence fluorescence microscopy showing formation of secondary actin shell (2°S) after detachment of primary actin shell (1°S) in low Mg2+. Times are shown as min∶sec. Scale bars are 5 µm. (C) Kymograph of line in B showing the detachment of primary shell (1) and establishment of a secondary shell (2). (D–O) Percentage of GST-WCA coated beads with either an actin shell or comet tail over time. At the reaction start, all beads developed a thin actin shell. In low 0.03 mM free Mg2+ buffer, actin shells detached over time (D) and many beads formed a secondary actin shell (E). Addition of 0.3 mM (F), 0.7 mM (G), 4.5 mM (H), or 9.4 mM (I) free Mg2+ restored shell or comet tail attachment. Addition of either 1 mM (J), 5 mM (K), or 10 mM (L) Lys-Lys2+ restored actin shell or tail attachment in 0.03 mM free Mg2+ buffer. Addition of 80 nM (M), 0.5 µM (N), or 1 µM (O) fascin restored actin shell or tail attachment in 0.03 mM free Mg2+ buffer. Means and S.D. were calculated from three independent experiments. At least 50 beads were counted in each experiment.
Figure 7.
Bundling promotes barbed end attachment to WCA domains in the absence of Arp2/3 complex.
Conditions: 8 µM (30% labeled) actin was polymerized in low Mg2+ F buffer. Filaments were incubated with GST-WCA coated microspheres with 1 µM ATP-actin monomers and the indicated final concentration of Mg2+, Lys-Lys2+, fascin, or K+ for 10 minutes. Beads were centrifuged, resuspended in low Mg2+ buffer in the absence of methylcellulose, and imaged on poly-lysine coated coverslips. (A) TIRF microscopy images of actin filaments and bundles attached to the bottom of coated microspheres in the indicated concentration of Mg2+, Lys-Lys2+, fascin, or K+. The number of actin filaments and bundles crossing the bead boundary (dashed circle) were counted for each bead. Scale bar is 2 µm. An example measurement is shown in Figure S6. (B) Count of average number of captured filaments per bead as a function of Mg2+ (○), Lys-Lys2+ (•), fascin (□), or K+ (⋄) concentration. Bundles were counted as two filaments. Error bars show S.D. from at least 60 beads for each condition from three independent experiments. Coincidental filament overlap with control, BSA coated microsphere (⧫) was negligible. (C) Stacked bar chart showing average number of filaments (light gray) or bundles (dark gray) captured by WCA-coated microspheres, with indicated Mg2+, Lys-Lys2+, fascin concentrations. The proportion of captured bundles increased with increasing polycation or fascin.
Figure 8.
Model of bundle and branch cooperativity.
(A) Bundles cooperate efficiently to maintain barbed end orientation. (1) WASP binding to membrane and Rho family GTPase frees active WCA domains that can either bind to a free profilin-actin or the barbed end of a nearby filament bundle. (2) One filament in a bundle is attached to WCA (red), while sister filaments are free to either polymerize by subunit addition (green), bind to a nearby free WCA, or (3) bind to a profilin-actin bound WCA. The force of polymerization is efficiently transmitted through the stiff bundle to tethered barbed ends and promotes tether dissociation. (4) Dissociation of bound WCA frees a sister barbed end for polymerization. (B) Branches cooperative inefficiently to maintain barbed end orientation. (1) While one barbed end is tethered to the membrane through WCA, a nearby barbed end polymerizes against the membrane (green). The force of polymerization is transmitted through branches and flexible filaments to tethered barbed ends to promote tether dissociation. (2) WCA dissociation frees a barbed end for polymerization while other filaments within the branched network become tethered.