Periodic patterns of actin turnover in lamellipodia and lamellae of migrating epithelial cells analyzed by Quantitative Fluorescent Speckle Microscopy

¹ The Scripps Research Institute

^* To whom correspondence should be addressed. E-mail: gdanuser{at}scripps.edu.

Submitted on December 23, 2004
Revised on March 17, 2005
Accepted on 14 July 2005

	Abstract

We measured actin turnover in lamellipodia and lamellae of migrating cells, using quantitative Fluorescent Speckle Microscopy (qFSM). Lamellae disassembled at low rates from the front to the back. However, the dominant feature in their turnover was a spatially random pattern of periodic polymerization and depolymerization moving with the retrograde flow. Power spectra contained frequencies between 0.5 and 1 cycle per minute. The spectra remained unchanged when applying Latrunculin A and Jasplakinolide in low doses, except that additional frequencies occurred beyond 1 cycle per minute. Whereas Latrunculin did not change the rate of mean disassembly, jasplakinolide halted it completely, indicating that the steady state and the dynamics of actin turnover are differentially affected by pharmacological agents. Lamellipodia assembled in recurring bursts at the leading edge and disassembled ~2.5m behind. Events of polymerization correlated spatially and temporally with transient formation of Arp2/3 clusters. In lamellae, Arp2/3 accumulation and polymerization correlated only spatially, suggesting an Arp2/3-independent mechanism for filament nucleation. To acquire these data we had to enhance the resolution of qFSM to the submicron level. Several algorithmic advances in speckle image processing are described enabling the analysis of kinetic and kinematic activities of polymer networks at the level of single speckles.

Key Words: actin, cytoskeleton, fluorescent speckle microscopy, particle tracking

This article has been cited by other articles:

				I. L. Novak, B. M. Slepchenko, and A. Mogilner Quantitative Analysis of G-Actin Transport in Motile Cells Biophys. J., August 15, 2008; 95(4): 1627 - 1638. [Abstract] [Full Text] [PDF]

				W. R. Hendee, K. Cleary, R. L. Ehman, G. D. Fullerton, W. S. Grundfest, J. Haller, C. A. Kelley, A. E. Meyer, R. F. Murphy, W. Phillips, et al. Bioengineering and Imaging Research Opportunities Workshop V: Summary of Findings on Imaging and Characterizing Structure and Function in Native and Engineered Tissues Radiology, August 1, 2008; 248(2): 342 - 347. [Full Text] [PDF]

				E. A. Cavalcanti-Adam, T. Volberg, A. Micoulet, H. Kessler, B. Geiger, and J. P. Spatz Cell Spreading and Focal Adhesion Dynamics Are Regulated by Spacing of Integrin Ligands Biophys. J., April 15, 2007; 92(8): 2964 - 2974. [Abstract] [Full Text] [PDF]

				S. T. Hess, T. P. K. Girirajan, and M. D. Mason Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy Biophys. J., December 1, 2006; 91(11): 4258 - 4272. [Abstract] [Full Text] [PDF]

				M. Machacek and G. Danuser Morphodynamic Profiling of Protrusion Phenotypes Biophys. J., February 15, 2006; 90(4): 1439 - 1452. [Abstract] [Full Text] [PDF]