title: New single-molecule speckle microscopy reveals modification of the retrograde actin flow by focal adhesions at nanometer scales.
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.
authors: Laboratory of Single-Molecule Cell Biology, Tohoku University Graduate School of Life Sciences, Sendai, Miyagi 980-8578, Japan Department of Physics, Lehigh University, Bethlehem, PA 18015.
authors: Sawako
authors: S
authors: Yamashiro
authors: Hiroaki
authors: H
authors: Mizuno
authors: Matthew B
authors: MB
authors: Smith
authors: Gillian L
authors: GL
authors: Ryan
authors: Tai
authors: T
authors: Kiuchi
authors: Dimitrios
authors: D
authors: Vavylonis
authors: Naoki
authors: N
authors: Watanabe
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D000199:Actins / Q000378:metabolism*; D000818:Animals; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D018274:Electroporation; D022001:Focal Adhesions / Q000378:metabolism*; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008027:Light; D008239:Lysine / Q000378:metabolism; D008853:Microscopy / Q000379:methods*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D024462:Myosin Type II / Q000037:antagonists & inhibitors / Q000378:metabolism; D053758:Nanoparticles / Q000737:chemistry*; D010316:Particle Size; D011554:Pseudopodia / Q000187:drug effects / Q000378:metabolism; D011817:Rabbits; D012044:Regression Analysis; D013194:Staining and Labeling; D022502:Stress Fibers / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors
chemical_list: D000199:Actins; D006576:Heterocyclic Compounds, 4 or More Rings; D047908:Intracellular Signaling Peptides and Proteins; D008869:Microtubule-Associated Proteins; C472645:blebbistatin; D024462:Myosin Type II; D008239:Lysine