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. 2004 Aug 30;166(5):629-35.
doi: 10.1083/jcb.200404159.

Yeast actin patches are networks of branched actin filaments

Michael E Young  1 , John A Cooper, Paul C Bridgman
Affiliations

Yeast actin patches are networks of branched actin filaments

Michael E Young et al. J Cell Biol. .

Abstract

Cortical actin patches are the most prominent actin structure in budding and fission yeast. Patches assemble, move, and disassemble rapidly. We investigated the mechanisms underlying patch actin assembly and motility by studying actin filament ultrastructure within a patch. Actin patches were partially purified from Saccharomyces cerevisiae and examined by negative-stain electron microscopy (EM). To identify patches in the EM, we correlated fluorescence and EM images of GFP-labeled patches. Patches contained a network of actin filaments with branches characteristic of Arp2/3 complex. An average patch contained 85 filaments. The average filament was only 50-nm (20 actin subunits) long, and the filament to branch ratio was 3:1. Patches lacking Sac6/fimbrin were unstable, and patches lacking capping protein were relatively normal. Our results are consistent with Arp2/3 complex-mediated actin polymerization driving yeast actin patch assembly and motility, as described by a variation of the dendritic nucleation model.

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Figures

Figure 1.
Figure 1.
Comparison of isolated actin patches with patches in cells. (A) A lysed Cap1-GFP spheroplast, strain YJC1453, shows actin patches expelled with the cytoplasm (cyt), not retained on the plasma membrane ghost (pm). (B) Cap1-GFP actin patches in spheroplasts (left) are similar in size and intensity to those in a cell lysate (right). Fluorescence images were collected and displayed with the same settings, allowing one to compare the intensities. (C) Quantitation of the fluorescence intensity of Cap1-GFP labeled patches in B; n = 100. (D) Quantitation of filamentous actin in patches, by rhodamine-phalloidin, n = 100. (E) A Coomassie-stained SDS gel of fractions from an actin patch preparation. The cell lysate and fractions up to I3 were indistinguishable. P5 represents the entire contents of one preparation; other lanes are portions of another preparation.
Figure 2.
Figure 2.
Correlation of light and electron microscope images. The top portion shows GFP fluorescence (green) and bright field (blue) light microscope images of an EM grid coated with an actin patch preparation, overlaid on a colorized (red) low magnification EM image after processing the grid by negative staining. The large red area outlined by the blue diffraction pattern is the grid's copper lattice. The bottom portion shows EMs of structures that do or do not contain GFP. A meshwork of branched filaments is seen in GFP-containing structures. Some filaments are indicated by arrowheads. Structures lacking GFP always contained proteinaceous aggregates, sometimes membranes, but never filaments. Strain YJC1453.
Figure 3.
Figure 3.
Actin patches from preparations lacking cross-linker, with S1 decoration. Actin patches prepared without cross-linking were treated briefly with latrunculin A, exposing filamentous networks. Most patches were associated with membranes (A); others were not (B). Arrowheads mark some end to side branches. (C) Myosin S1 decoration. Strain YJC1453.
Figure 4.
Figure 4.
Actin patches from cross-linked preparations. (A) The branched filament network of a typical actin patch. Arrowheads mark some branches. (B) A graphical representation (bottom) of the actin filaments in another typical patch (top). Filament length and branching were quantitated for this patch, as indicated by the green lines and red dots, respectively. Strain YJC1453.
Figure 5.
Figure 5.
Cross-linked actin patches lacking Sac6/fimbrin. One of the larger patches from a sac6Δ strain, YJC3580. Most patches were far smaller (inset). Arrowheads mark some branches.
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