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Figure 3. Localization of Arp2/3 complex in lamellipodia. (aâc and eâg) Fluorescence microscopy of Xenopus keratocyte (aâc) or fibroblast (eâg). Staining with p21 antibody (green) and TRITC-phalloidin (red) shows ARP2/3 complex highly enriched in lamellipodia. Boxed region in g is enlarged in insets; it shows several filopodia lacking and only one filopodium containing Arp2/3 complex. (d and h) Immuno-EM of lamellipodia of Xenopus keratocyte (d) or fibroblast (h) stained with p21 primary antibody and 10-nm gold-conjugated secondary antibody after glutaraldehyde fixation and SDS treatment of detergent-extracted cells. Gold particles are highlighted in yellow. Bars: (a and e) 10 μm; (d and h) 0.1 μm.
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Figure 8. Localization of XAC in Xenopus keratocytes. (aâe) Fluorescence microscopy of a whole cell (aâc) and the intensity profile (d) of the enlarged lamellipodium (e) from the boxed region in c, double stained with XAC antibody (green) and TRITC-phalloidin (red). XAC in lamellipodium is excluded from the narrow zone at the extreme leading edge. (f) Phase-contrast sequence of a locomoting cell; time is shown in min. (gâi) Immuno-EM with XAC antibody of the cell shown in f, which was lysed and processed at 8 min time point. (g) Cell overview. (h) Intermediate magnification of the boxed region from g showing distribution of gold particles (yellow) in lamellipodia. (i) High magnification of the boxed region from d showing leading edge. XAC is excluded from the extreme front of the lamellipodium (h and i). Bars: (a and f) 10 μm; (h) 10 μm.
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Figure 2. Improved visualization of actin filament branching in lamellipodia. EM of keratocyte or fibroblast lamellipodial actin network after CD treatment (a and b, 0.2 μM for 30 min or 0.5 μM for 10 min), 1 min recovery from serum starvation of a mouse fibroblast (c), LA treatment (0.2 μM for 10 min), or unprotected extraction (d). All examples demonstrate frequent branching of actin filaments. Bars, 0.1 μm; b and d are shown at the same magnification as c.
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Figure 4. Localization of Arp2/3 complex at actin filament branching points. Xenopus keratocytes and fibroblasts were treated with CD (0.2 μM for 30 min or 0.5 μM for 10 min), extracted in the presence of phalloidin, fixed with glutaraldehyde, treated with 33% methanol, and immunostained with p21 antibody followed by 10-nm gold-conjugated secondary antibody. Gold particles are highlighted in yellow. Bar, 50 nm.
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Figure 5. Localization of cross-linking proteins in fibroblast cytoskeleton. (aâc) Fluorescence microscopy and corresponding intensity profiles (aâ²âcâ²) of Xenopus (a and c) or human 356 (b) fibroblast lamellipodia double stained with TRITC-phalloidin (red) and either p21 (a and aâ²), ABP-280 (b and bâ²), or α-actinin (c and câ²) antibodies (green). The protein/actin ratio at the leading edge of the lamellipodium is high for Arp2/3 complex (a and aâ²), medium for ABP-280 (b and bâ²), and low for α-actinin (c and câ²) compared with internal actin structures. (dâi) Immuno-EM of the cell edge (dâf) or interior (gâi) of CD-treated Xenopus (d, f, g, and i) or human 356 (e and h) fibroblasts stained with p21 (d and g), ABP-280 (e and h), or α-actinin (f and i) primary antibody and 10-nm (d, e, g, and h) or 18-nm (f and i) gold-conjugated secondary antibody. Gold particles (yellow) reveal Arp2/3 complex at Y-junctions at cell edge and ABP-280 and α-actinin at filament crossovers in the cell interior. Bars: (aâc) 1 μm; (dâi) 0.1 μm.
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Figure 6. Structural differentiation of actin network in lamellipodium. EM of Xenopus keratocytes (aâc) or Xenopus fibroblasts (dâf) after regular extraction in the presence of PEG and phalloidin (a and d) or after unprotected extraction without PEG and phalloidin (b, c, e, and f). Boxed areas from b and e are enlarged in c and f, respectively. Actin network at lamellipodial rear disassembled in the course of unprotected extraction, whereas front zone remained as dense as in control cells. Bar, 1 μm.
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Figure 7. Differential response of lamellipodial actin network to LA. (A) Phase-contrast sequence of a locomoting Xenopus keratocyte. After addition of 0.1 μm LA at 9 min time point, the cell continued to translocate, retaining the crescent-like shape. (B) Plot showing rate of front edge protrusion versus time of the cell shown in A. LA addition (arrow) decreased rate of protrusion from â¼4 μm/min before LA application to 1 μm/min at the end of the sequence. (C) Fluorescence microscopy of the boxed region of the cell shown in A, which was lysed at 34 min time point, fixed, and stained with TRITC-phalloidin. Actin-staining reveals narrow bright lamellipodium at the leading edge, separated by the wide actin-depleted zone from the internal actin structures. (D and E) EM of a Xenopus keratocyte (D) or Xenopus fibroblast (E) lamellipodium treated with LA (D, 0.1 μM for 30 min; E, 0.25 μM for 10 min) reveals actin depletion from the lamellipodial rear. Bars: (A) 10 μm; (D and E) 1 μm.
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Figure 9. Localization of XAC to posterior regions of depolymerization-resistant actin brush. Electron (a and c) and fluorescence (b) microscopy of lamellipodia of Xenopus keratocytes after unprotected extraction (a) or LA treatment (b, 0.15 μM for 30 min; c, 0.25 μM for 10 min) and subsequent staining with XAC antibody (a and c) or double staining with TRITC-phalloidin and XAC antibody (b).
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Figure 10. Localization of XAC in Xenopus fibroblasts. (aâe) Fluorescence microscopy of a cell fragment (aâc) and the intensity profile (d) of the enlarged lamellipodium (e) from the boxed region in c, double stained with XAC antibody (green) and TRITC-phalloidin (red). XAC is distributed throughout the entire lamellipodium. (f and g) Phase-contrast sequence showing protruding lamellipodium of a motile cell (f) and the overview of the same cell (g) at 80 s time point. (h and i) Immuno-EM with XAC antibody of the cell shown in g. (h) Cell overview. Nucleus and surrounding regions look bright because of high content of cellular proteins, which creates high electron density (brightness in inverted contrast). Immunogold-labeling in these central regions is very low. (i) High magnification of the protruding region pointed to by arrow in h. Gold particles (yellow) revealing XAC localization are found at the extreme leading edge, as well as throughout the lamellipodia.
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Figure 11. Two treadmilling models for actin turnover in lamellipodia. Left, treadmilling of individual filaments suggests that each actin filament in the actin network simultaneously assembles subunits at its barbed end and releases subunits from the pointed end, thus continuously reproducing itself by treadmilling mechanism. Treadmilling of individual filaments collectively results in the treadmilling of the lamellipodial network. Right, treadmilling of the dendritic array suggests frequent formation of new filaments by de novo nucleation, which occurs within the narrow zone at the leading edge, the actin brush. Newborn filaments become immediately incorporated into the actin array as branches of pre-existing filaments. Within the actin brush, filaments are protected from depolymerization at pointed ends. Nucleation, cross-linking, and pointed end capping are proposed to be mediated by the Arp2/3 complex. Many barbed ends are predicted to be capped to prevent exponential increase in filament mass. Release of the Arp2/3 complex from Y-junctions behind the actin brush, followed by ADF/cofilin-mediated dissociation of actin subunits from pointed ends, may be a major pathway for actin array disassembly. By this model, an individual filament in the dendritic array does not treadmill, but rather first grows at the barbed end and later shrinks at the pointed end. However, the actin filament array as a whole treadmills, reproducing itself at the cell front and dismantling itself at the lamellipodial rear. Growing barbed ends are shaded in gray.
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