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FIGURE 1:. Arp2/3 complex inhibition radically transforms the coelomocyte LP actin cytoskeleton. Live-cell phase contrast images from two digital movies shows that 100 μM CK666 treatment results in two basic phenotypes—a more common, transverse arc form (A, B) and a less common, filopodial form (C). In each case, the dense dendritic array of the control cells is replaced with less dense assemblages of elongate filaments. The effects of CK666 are readily reversible, as illustrated in the sequential wash out (+WO) images in B, in which the actin arcs are completely replaced by the control actin organization. The box in the 30-s post-CK666 treatment image in A denotes a region of the cell that generates a response analogous to that seen in the TEM image in Figure 3D. Bar, 10 μm; magnifications of A–C are equivalent.
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FIGURE 2:. Fluorescence staining of actin in Arp2/3 complex–inhibited coelomocytes. Fluorescent phalloidin (A, E–G) and actin antibody staining (B–D) of cells treated with 100 μM CK666 corroborates the live-cell imaging in depicting the transition of the control dendritic network (A) into the actin arcs (B, C, E) and actin filopodia (D, F) present in CK666-affected cells. Washing the cells out of CK666 with control buffer (+WO; G) rapidly reestablishes the control dendritic array at the cell edge, which then moves centripetally. Bar, 10 μm; magnifications of A, B, and E–G are equivalent.
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FIGURE 3:. Ultrastructure of the alteration of actin organization during Arp2/3 complex inhibition. Control cells (A, C) contain a dense dendritic network of actin filaments in their LP region, which, upon treatment with 100 μM CK666, rapidly transitions to an array of less dense elongate filaments oriented diagonally and parallel to the membrane (D, E). The pyramidal projections seen in D are analogous to the similar structures apparent in the live-cell imaging in the boxed region of the 30-s image in Figure 1A. Over time, the transverse actin arcs become apparent, decrease in density, and tend to occupy more of the cell diameter (B, F) and consist of loosely affiliated and highly elongate actin filaments. Washout of CK666 (G, H) results in the immediate restart of the dense dendritic actin network at the cell edge, and this pushes centripetally, and elongate actin filaments oriented at low angles to the membrane are apparent within this network. Bar, 5 μm (A), 1 μm (C); magnifications of A, B, and C–H are equivalent.
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FIGURE 4:. CK666-induced actin arcs delaminate from the cell edge, undergo decreased centripetal flow, and are arrested by CytoD treatment. Phase contrast digital movies reveal the delamination-like generation of actin arcs in a cell treated with 100 μM CK666 (A–E; 50 s between images; 1–3 mark three consecutive arcs) and that arc production can be arrested by treatment with 500 nM CytoD (F–J; time between images is 60 s for F–I and 180 s for I to J). Quantification of average (K) and percentage previous (L) centripetal flow rates shows that CK666 treatment significantly slows centripetal flow. ***Statistically significant differences at the p < 0.001 level. Bar, 10 μm; magnifications of A–J are equivalent.
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FIGURE 5:. The distributions of Arp2/3 complex and myosin II are altered by CK666 treatment. Immunofluorescence labeling of actin (A, D, green in C and F) and Arp3 (B, E, magenta in C and F) in control (A–C) and 100 μM CK666–treated (D–F) cells shows that the dense Arp3 labeling apparent in control cells is substantially decreased in cells containing the CK666-induced actin arc phenotype (right-hand cell in D–F). Staining for actin (G, J, green in I and J) and myosin II (H, K, magenta in I and L) in control (G–I) and 100 μM CK666–treated (J–L) cells demonstrates that the tightly compacted perinuclear myosin II ring apparent in control cells becomes more spread in cells exhibiting a CK666-induced actin arc phenotype. Nuclei in C, F, I, and L are labeled blue. Bar, 10 μm; magnifications of A–F and G–L are equivalent.
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FIGURE 6:. Arp2/3 complex inhibition retards the process of coelomocyte spreading and induces a novel actin organization. Cells treated in suspension with 100 μM CK666 undergo a lamellipodial-to-filopodial shape change, as seen when allowed to settle for 5 min and stained with phalloidin (D). In contrast, cells washed out of the drug just after substrate attachment and imaged 5 min later (A) show numerous LP-like spreading regions. With increasing time post settling (B and E, 15 min; C and F, 20 min; G–J, 45 min), control cells (B, C, G, H) spread into the typical discoidal morphology, with prominent LP dendritic arrays, whereas the spreading CK666-treated cells (E, F, I, J) have processes containing noticeable actin arcs and numerous filopodia. In these cells, the processes produced in regions of spreading from filopodia making focal substrate contacts can contain novel concentric circle patterns of actin arcs (arrow in F). Quantification shows that CK666 treatment of coelomocytes in suspension induces a significantly larger number of filopodial cells than control treatment with CK689 (K; p < 0.001) and that control/washout cells spread faster than cells kept in the presence of the drug (L). Bars, 10 μm; magnifications of A–F and G–J are equivalent.
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FIGURE 7:. Ultrastructural organization of actin in control and Arp2/3 complex–inhibited spreading cells. TEM imaging of control/washout cells settled for 15 min (A–C, G) shows that the spreading peripheral regions contain the expected LP-like dendritic actin network (A, B). In contrast, cells spreading in the presence of 100 μM CK666 for 15 min (D–F, H) possess more- limited spread regions containing actin arcs of elongated filaments (D, E). In spreading filopodial regions, control cells show evidence of small regions of dendritic actin arrays (C, G), whereas CK666-affected cells display actin arcs arrayed in unusual concentric circle patterns (F, H). Bars, 2 μm (A, D), 0.5 μm (B, C, E–H).
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FIGURE 8:. The generation of actin arcs in Arp2/3 complex–inhibited cells is arrested by treatment with the formin inhibitor SMIFH2. Phase contrast digital movie of a cell treated with 100 μM CK666 followed by treatment with CK666 plus 5 μM SMIFH2 (A; time between the first and second images is 10 s and between subsequent images is 60 s). Arc generation and centripetal movement continued briefly after SMIFH2 application (note movement of arcs 1 and 2 and production of arc 3) and then was clearly arrested. Treating control cells with 2.5–5 μM SMIFH2 (B–E) for 10 min resulted in the patchy, discontinuous distribution of actin networks as seen in live-cell phase contrast (B), fluorescent phalloidin staining of actin (C), and TEM imaging (D, E). A control cell (F) treated with 2.5 μM SMIFH2 for 10 min (G) shows disruption in the actin cytoskeleton. Washing out the drug for 2 (H) or 15 min (I) restores the control actin structural organization (I). Bars,10 μm (A, F), 5 μm (E); magnifications of A–C and F–I are equivalent.
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FIGURE 9:. Inhibition of the Arp2/3 complex in fish keratocytes and S2 cells induces actin arc formation. Phase contrast (A, C, E, G) and fluorescent phalloidin (B, D, F, H) imaging of control (A–D) and 100 μM CK666–treated (E–H) fish keratocytes demonstrates that the drug transforms the LP region from a dendritic network into a collection of transverse, elongate filaments. A similar response is seen when control S2 cells (I–L) are treated with 100 μM CK666 (M–P), suggesting that actin arcs are a common feature of Arp2/3 complex inhibition in cells with broad LP regions. Bar, 10 μm; magnifications of A–P are equivalent.
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