Use of fluorescently labelled deoxyribonuclease I to spatially measure G‐actin levels in migrating and non‐migrating cells

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Abstract
Lamellipodium protrusion is linked to actin filament disassembly in migrating fibroblasts [Cramer, 1999: Curr. Biol. 9:1095–1105]. To further study this relationship, we have identified a method to specifically and sensitively detect G‐actin in distinct spatial locations in motile cells using deoxyribonuclease I (DNase I). Although DNase I can bind both G‐ and F‐actin in vitro [Mannherz et al., 1980: Eur. J. Biochem. 95:377–385], when cells were fixed in formaldehyde and permeabilized in detergent, fluorescently‐labelled DNase I specifically stained G‐actin and not F‐actin. 92–98% of actin molecules were stably retained in cells during fixation and permeabilization. Further, increasing or decreasing cellular G‐actin concentration by treating live cells with latrunculin‐A or jasplakinolide, respectively, caused a respective increase and decrease in DNase I cell‐staining intensity as expected. These changes in DNase I fluorescence intensity accurately reflected increases and decreases in cellular G‐actin concentration independently measured in lysates prepared from drug‐treated live cells (regression coefficient = 0.98). This shows that DNase I cell‐staining is very sensitive using this method. Applying this method, we found that the ratio of G‐/F‐actin is lower in both the lamellipodium and in a broad band immediately behind the lamellipodium in migrating compared to non‐migrating fibroblasts. Thus, we predict that protrusion of the lamellipodium in migrating fibroblasts requires tight coupling to filament disassembly at least in part because G‐actin is relatively limited within and behind the lamellipodium. This is the first report to directly demonstrate high sensitivity of cell‐staining for any G‐actin probe and this, together with the ready commercial accessibility of fluorescently‐labelled DNase I, make it a simple, convenient, and sensitive tool for cell‐staining of G‐actin. Cell Motil. Cytoskeleton 51:27–38, 2002.