11 research outputs found

    Mechanism of Filament Nucleation and Branch Stability Revealed by the Structure of the Arp2/3 Complex at Actin Branch Junctions

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    <div><p>Actin branch junctions are conserved cytoskeletal elements critical for the generation of protrusive force during actin polymerization-driven cellular motility. Assembly of actin branch junctions requires the Arp2/3 complex, upon activation, to initiate a new actin (daughter) filament branch from the side of an existing (mother) filament, leading to the formation of a dendritic actin network with the fast growing (barbed) ends facing the direction of movement. Using genetic labeling and electron microscopy, we have determined the structural organization of actin branch junctions assembled in vitro with 1-nm precision. We show here that the activators of the Arp2/3 complex, except cortactin, dissociate after branch formation. The Arp2/3 complex associates with the mother filament through a comprehensive network of interactions, with the long axis of the complex aligned nearly perpendicular to the mother filament. The actin-related proteins, Arp2 and Arp3, are positioned with their barbed ends facing the direction of daughter filament growth. This subunit map brings direct structural insights into the mechanism of assembly and mechanical stability of actin branch junctions.</p> </div

    Visualization of NPFs in the Actin Branch Junction by Difference Mapping

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    <div><p>(A-F) WASp protein dissociate from the actin branch. 2D average projection obtained (A) using the unlabeled yeast Arp2/3 complex and N-WASp WA, and (B) using the full-length GST-N-WASp complexed with its activator GST-Nck. (C) Difference map between (A) and (B). 2D average projection obtained using (D) the amoeba Arp2/3 complex and Scar WA, and (E) MBP-Scar1WA. (F) Difference map between (D) and (E).</p> <p>(G-I) Cortactin is present in the actin branch. (G) 2D average projection obtained using GST-cortactin and the amoeba Arp2/3 complex. (H) Difference map between (G) and (D). (I) The peak in the difference map shown in yellow superimposed with the projection map.</p> <p>Bar = 10 nm.</p></div

    Localization of the Labels Attached to Arp2, Arp3, Arc40/ARPC1, and Arc18/ARPC3 at the Actin Branch Junction

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    <div><p>Color codes used: Arp2 (pink), Arp3 (orange), Arc40/ARPC1 (green), and Arc18/ARPC3 (red).</p> <p>(A) 2D average projection maps of the branches obtained with Arp2-GFP (row 1), Arp3-GFP (row 2), Arc40/ARPC1-YFP (row 3), and Arc18/ARPC3-GFP (row 4).</p> <p>(B) Difference maps calculated between maps obtained with labeled and unlabeled complexes.</p> <p>(C) Difference maps superimposed with the projection maps. The position of the difference peaks was cross-validated (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030383#s3" target="_blank">Materials and Methods</a>).</p> <p>(D) The average projection map obtained with the unlabeled complex.</p> <p>(E) The main difference peaks are superimposed with the unlabeled projection map.</p> <p>(F and G) Circles of 3.9-nm radius centered on the difference peaks indicate the possible locations of the C-termini of each labeled subunit. The GFP/YFP label was attached to the C-terminus of the relevant subunit with an eight-amino-acid flexible linker that in fully extended conformation can reach a length of up to approximately 3.2 nm. The distance of the N-terminus of GFP or YFP from the center of mass of its beta-barrel (14 Ă— 8 Ă— 8 nm) is approximately 2.5 nm. The centers of the peaks determined from the difference maps probably coincide with the center of mass.</p> <p>Bar = 10 nm.</p></div

    Structure Models of the Arp2/3 Complex at Actin Branch Junction

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    <div><p>Color codes used: Arp2 (light pink), Arp3 (orange), Arc40/ARPC1 (green), Arc35/ARPC2 (cyan), Arc19/ARPC4 (blue), Arc18/ARPC3 (dark pink), and Arc15/ARPC5 (yellow). Gray arrows indicate the mother and daughter filaments.</p> <p>(A) Orientation of the Arp2/3 complex relative to the mother and daughter filaments as determined using the labeling constraints.</p> <p>(B) Model rotated vertically anticlockwise by 90° from view in (A) and tilted so that the mother filament coincides with the vertical axis. The gray arrow is positioned to pass through the center of the complex.</p> <p>(C) Model rotated vertically by 180° from the view in (A).</p> <p>(D) Label positions and their corresponding C-termini localization. GFP or YFP, shown as ribbon diagram with the same color coding as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030383#pbio-0030383-g003" target="_blank">Figure 3</a>, were superimposed on the respective difference peak (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030383#pbio-0030383-g003" target="_blank">Figure 3</a>) with their orientation matching the peak shape.</p> <p>(E) Model superimposed on the projection density map (white corresponds to high density).</p> <p>(F) Model and ribbon diagram of a daughter filament (white) as it would grow after small relative rotations of Arp2 and Arp3 (see text) superimposed on the projection density map.</p> <p>(G and H) Model proposed by Beltzner and Pollard [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030383#pbio-0030383-b06" target="_blank">6</a>] (G) and by Aguda et al. [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030383#pbio-0030383-b05" target="_blank">5</a>] (H) shown for comparison. Note that in (G), the daughter filament will be oriented out of the paper plane toward the reader.</p> <p>(I)Arp2/3 crystal structure in the same orientation as originally presented in Robinson et al. [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030383#pbio-0030383-b12" target="_blank">12</a>].</p></div

    FISH with two color probes: chromosome 7 centromere (green) and <i>MET</i> gene (red).

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    <p>A) True <i>MET</i> gene amplification in 10% of cells: 4–8 centromere signals and 16–20 <i>MET</i> signals, ratio >2.0. B) High polysomy: the same number of control and <i>MET</i> gene spots were seen in 15% of giant cells, ratio is 1.0. C) Chromosome 7 monosomy: only one control and one <i>MET</i> signal were detected for this case. In some cells there is only one signal (or no signal) due to a nuclei section. D) Normal hybridization pattern: two control spots and two <i>MET</i> gene spots. Again, some cells show only one of two signals due to a nuclei section.</p

    The location and predicted effect of each <i>MET</i> variation found, numbered from the reference sequence NM 000245.2.

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    <p>Mutation Chromatograms include reference sequences and variant description sequence variations described using IUPAC code (<a href="http://www.insdc.org/" target="_blank">http://www.insdc.org/</a>).</p><p>Align-Grantham Variation Grantham Deviation.</p><p>Sorts Intolerant From Tolerant.</p
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