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

Abstract

<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 × 8 × 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

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