Constitutively active ARF1 induces F-actin–rich puncta (labeled by mCherry-UtrCH) in mouse fibroblasts. (A) Transfection with wild-type CFP-ARF1 did not change actin cytoskeleton of fibroblast. (B) Numerous F-actin–rich puncta in the fibroblast transfected with constitutively active ARF1 mutant, CFP–ARF1 Q71L. (B, right) Images of the boxed area (6 × 4.5 µm2) in the left panel showing F-actin (red, top row), CFP–ARF1 Q71L (green, middle row), and their superimposition (bottom row) at three time points taken with a 3-s time interval. Transient contacts (yellow) of CFP–ARF1 Q71L–containing puncta with the F-actin–rich puncta are seen. (C) Cell transfected with dominant-negative RhoA (GFP–RhoA T19N) did not contain stress fibers. (D) Cell cotransfected with dominant-negative RhoA (GFP–RhoA T19N) and constitutively active ARF1 (CFP–ARF1 Q71L) formed numerous F-actin–rich puncta. (E) Control fibroblast forming stress fibers (left) did not demonstrate localization of mCherry-WIP (right). (F) F-actin–rich puncta in fibroblast expressing CFP–ARF1 Q71L are enriched with mCherry-WIP. (G and H) Fibroblasts transfected with constitutively active ARF1 (CFP–ARF1 Q71L) were plated on fluorescent gelatin-coated coverslips in control medium containing 0.2% DMSO (G) or in medium containing 25 µM MMP inhibitor GM6001 (H) and incubated for 4 h. The matrix degradation sites in the boxed areas are seen at high magnification on the left in the bottom panels of G but not H. Actin puncta were visualized by phalloidin staining. High magnifications of boxed areas in G and H as well as merged images of actin and fluorescent gelatin are shown at left, middle, and right in the bottom panels of G and H, respectively. Note that actin puncta colocalize with dark areas corresponding to degraded fluorescent gelatin in control fibroblasts (G, bottom, white arrowheads), whereas gelatin degradation is completely prevented in cells treated with GM6001 (H, bottom). Bars: (A and C–H) 5 µm; (B) 1 µm.
