Use of fluorescently labelled deoxyribonuclease I to spatially measure G-actin levels in migrating and non-migrating cells.
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.
Cramer
L P
LP
MRC, Laboratory for Molecular Cell Biology and Department of Biology, University College London, London, United Kingdom. l.cramer@ucl.ac.uk
Briggs
L J
LJ
Dawe
H R
HR
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D002642:Chick Embryo; D003599:Cytoskeleton / Q000737:chemistry; D003850:Deoxyribonuclease I / Q000378:metabolism*; D004173:Dipodomys; D005347:Fibroblasts / Q000201:enzymology; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D016707:Tissue Fixation
D000199:Actins; D005456:Fluorescent Dyes; D003850:Deoxyribonuclease I