Document Type

Article

Publication Date

2-2014

Publication Title

Molecular Biology of the Cell

Abstract

Speckle microscopy directly visualizes the retrograde actin flow, which is believed to promote cell-edge protrusion when linked to focal adhesions (FAs). However, it has been argued that, due to rapid actin turnover, the use of green fluorescent protein–actin, the lack of appropriate analysis algorithms, and technical difficulties, speckle microscopy does not necessarily report the flow velocities of entire actin populations. In this study, we developed a new, user-friendly single-molecule speckle (SiMS) microscopy using DyLight dye-labeled actin. Our new SiMS method enables in vivo nanometer-scale displacement analysis with a low localization error of ±8–8.5 nm, allowing accurate flow-velocity measurement for actin speckles with lifetime <5 s. In lamellipodia, both short- and long-lived F-actin molecules flow with the same speed, indicating they are part of a single actin network. These results do not support coexistence of F-actin populations with different flow speeds, which is referred to as the lamella hypothesis. Mature FAs, but not nascent adhesions, locally obstruct the retrograde flow. Interestingly, the actin flow in front of mature FAs is fast and biased toward FAs, suggesting that mature FAs attract the flow in front and actively remodel the local actin network.

Volume

25

Issue

7

First Page

1010

Last Page

1024

DOI

10.1091/mbc.E13-03-0162

ISSN

1059-1524

Comments

This work was supported by Human Frontiers Science program grant RGP0061∕2009-C (N.W. and D.V.); NEXT program grant LS013 from the Cabinet Office, Government of Japan (N.W.); a grant from the Takeda Science Foundation (N.W.); a Grant-in-Aid for Young Scientists (S.Y.); and a Grant-in-Aid for Scientific Research on Innovative Areas (S.Y.).

Supplemental Material can be found at: http://www.molbiolcell.org/content/suppl/2014/02/03/mbc.E13-03-0162v1.DC1

Rights Statement

© 2014 Yamashiro et al., posted with permission.

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