Turnover Dynamics of Diffuse Actin and Regulators at the Leading Edge

The lamellipodium at the leading edge of motile cells is a dynamic structure consisting of a dense network of branched actin filaments. These actin filaments polymerize in a region close to the leading edge and undergo retrograde flow towards the main body of the cell. A large number of experimental techniques have been used to monitor the structural aspects of actin networks at the leading edge. However, the role of concentration gradients and local heterogeneity of soluble actin, actin oligomers, and actin regulators in the crowded filament network is little explored.

In collaboration with the lab of Naoki Watanabe (Tohoku University), we use modeling, image analysis, and single molecule fluorescence microscopy to quantify the role of diffuse actin species and their gradients in actin reorganization at the leading edge of motile cells. We use the numbers obtained from experiments in numerical simulations of turnover dynamics. The simulations are used to predict FRAP recovery curves for comparison to experiments. We further model how these mechanisms contribute to dynamical patterns of protrusion and retraction at the leading edge.

To aid our quantification of actin patterns at the leading edge, we developed Speckle TrackerJ, a particle tracking software for ImageJ to mark the appearance and disappearance of bright spots that correspond to proteins becoming associated to, or dissociated from, the actin network.  We also developed a set of ImageJ plugins (LEAP) to measure leading edge position and intensity.

References
  1. S. Yamashiro, H. Mizuno, M. B. Smith, G. L. Ryan, T. Kiuchi, D. Vavylonis, N. Watanabe, "New single-molecule speckle microscopy reveals modification of the retrograde actin flow by focal adhesions at nanometer scales," MOL. BIOL. CELL 25:1010-1024 (2014).
  2. N. Watanabe, Y. Sawako, D. Vavylonis and T. Kiuchi, "Molecular viewing of actin polymerizing actions and beyond: combination analysis of SiMS microscopy with modeling, FRAP and s-FDAP," DEVELOPMENT, GROWTH AND DIFFERENTIATION, 55:508-514 (2013).
  3. M. B. Smith, T. Kiuchi, N. Watanabe and D. Vavylonis, "Distributed Actin Turnover in the Lamellipodium and FRAP Kinetics," BIOPHYS. J. 104:247-257 (2013).  
  4. G. L. Ryan, N. Watanabe and D. Vavylonis, "Image Analysis Tools to Quantify Cell Shape and Protein Dynamics near the Leading Edge" (Cell Structure and Function, 2013)  
  5. G. L. Ryan, N. Watanabe and D. Vavylonis, "A review of models of fluctuating protrusion and retraction patterns at the leading edge of motile cells," CYTOSKELETON 69:195-206 (2012).   
  6. G. L. Ryan, H. Petroccia, N. Watanabe and D. Vavylonis, "Excitable actin dynamics in lamellipodial protrusion and retraction," BIOPHYS J., 102:1493-1502 (2012).  
  7. M. B. Smith, E. Karatekin, A. Gohlke, H. Mizuno, N. Watanabe and D. Vavylonis, "Interactive, computer-assisted tracking of speckle trajectories in fluorescence microscopy: application to actin polymerization and membrane fusion," BIOPHYS J., 101:1794-1804 (2011). (Evaluated in Faculty of 1000)