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. 2016 Jan 15;27(2):247–259. doi: 10.1091/mbc.E14-11-1555

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© 2016 Borinskaya et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 3: — Allowing VCA turnover in membrane clusters does not result in formation of comet tails. (A) Schematic of CD16/7-mCherry-Nck aggregation and binding by endogenous N-WASP protein. (B) Schematic of VCA clusters with turnover through SynZip-mediated interaction. CD16/7-eYFP-SynZip1 aggregation results in recruitment of cytosolic SynZip2-mCherry-VCA fusion proteins. (C) Actin recruitment and shape of actin structures induced by clustering of SynZip2-mCherry-VCA through interaction with CD16/7-eYFP-SynZip1. Left, merged staining of SynZip2-mCherry-VCA (red) and CD16/7-eYFP-SynZip1 (green). Insets, magnified views of CD16/7-eYFP-SynZip1 (left inset), SynZip2-mCherry-VCA (middle inset), and a composite of the two (right inset). Middle, actin staining alone. Right, merged staining of SynZip2-mCherry-VCA (red) and mTFP1-actin (green). Scale bar, 10μm. (D) Morphology analysis of actin structures induced by VCA clustered through SynZip binding. Nck SH3 clustering: three cells, 124 aggregates; VCA clustering though SynZip interaction: eight cells, 152 aggregates; VCA clustering: three cells, 233 aggregates. Error bars are mean ± SEM. *p < 0.001, **p < 0.01. (E) Velocity of actin structures induced by VCA clustered through SynZip binding. Nck SH3 clustering: three cells, 121 aggregates; VCA clustering though SynZip interaction: four cells, 174 aggregates; VCA clustering: three cells, 246 aggregates.