Probing f-actin flow by tracking shape fluctuations of radial bundles in lamellipodia of motile cells

We examined the dynamics of radial actin bundles based on time-lapse movies of polarized light images of living neuronal growth cones. Using a highly sensitive computer vision algorithm for tracking, we analyzed the small sha pe fluctuations of radial actin bundles that otherwise remained stationary in their positions in the growth cone lamellipodium. Using the tracking sof tware, we selected target points on radial bundles and measured both the lo cal bundle orientations and the lateral displacements between consecutive m ovie frames. We found that the local orientation and the lateral displaceme nt of a target point are correlated. The correlation can be explained using a simple geometric relationship between the lateral travel of tilted actin bundles and the retrograde flow of f-actin structures. Once this relations hip has been established, we have turned the table and used the radial bund les as probes to measure the velocity field of f-actin flow. We have genera ted a detailed map of the complex retrograde flow pattern throughout the la mellipodium. Such two-dimensional flow maps will give new insights into the mechanisms responsible for f-actin-mediated cell motility and growth.