New single-molecule speckle microscopy reveals modification of the retrograde actin flow by focal adhesions at nanometer scales

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 algori...

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Veröffentlicht in:	Molecular biology of the cell 2014-04, Vol.25 (7), p.1010-1024
Hauptverfasser:	Yamashiro, Sawako, Mizuno, Hiroaki, Smith, Matthew B, Ryan, Gillian L, Kiuchi, Tai, Vavylonis, Dimitrios, Watanabe, Naoki
Format:	Artikel
Sprache:	eng
Schlagworte:	Actin Cytoskeleton - drug effects Actin Cytoskeleton - metabolism Actins - metabolism Animals Cytoplasm - drug effects Cytoplasm - metabolism Electroporation Focal Adhesions - metabolism Heterocyclic Compounds, 4 or More Rings - pharmacology Humans Intracellular Signaling Peptides and Proteins - metabolism Light Lysine - metabolism Microscopy - methods Microtubule-Associated Proteins - metabolism Myosin Type II - antagonists & inhibitors Myosin Type II - metabolism Nanoparticles - chemistry Particle Size Pseudopodia - drug effects Pseudopodia - metabolism Rabbits Regression Analysis Staining and Labeling Stress Fibers - drug effects Stress Fibers - metabolism Time Factors
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Zusammenfassung:	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
ISSN:	1059-1524 1939-4586
DOI:	10.1091/mbc.e13-03-0162