Simultaneous mapping of filamentous actin flow and turnover in migrating cells by quantitative fluorescent speckle microscopy.

Abstract

We report advances in quantitative fluorescent speckle microscopy to generate simultaneous maps of cytoskeleton flow and rates of net assembly and disassembly in living cells. We apply this tool to analyze the filamentous actin (F-actin) dynamics at the front of migrating cells. F-actin turnover and flow are both known to be factors of cell locomotion. However, how they are orchestrated to produce directed cell movements is poorly understood. Our approach to data analysis allows us to examine their interdependence. Our maps confirm the previously described organization of flow into a lamellipodium and a lamellum, both exhibiting retrograde flow; and a convergence zone, where lamellum retrograde flow meets with slow anterograde flow of cortical F-actin at the ventral side of the cell body. The turnover maps show the well known actin polymerization at the leading edge, but also indicate that approximately 90% of the polymer disassembles at the lamellipodium-lamellum junction. Strong depolymerization is also found in the convergence zone, where meshwork contraction is prominent. To determine whether contraction and depolymerization are coupled events, we have treated cells with calyculin A, which is known to promote myosin activity. Stimulated contraction was accompanied by accelerated retrograde flow and increased depolymerization throughout the lamellum, whereas disassembly at the lamellipodium-lamellum junction remained unaffected. There appear to be two distinct depolymerization mechanisms, of which one depends directly on meshwork contraction.