Visualization of Actin Polymerization in Invasive Structures of Macrophages and Carcinoma Cells Using Photoconvertible β-Actin – Dendra2 Fusion Proteins (original) (raw)
Figure 3
The orientation of the Dendra2 fluorescent protein relative to β-actin affects the localization of the fusion protein in carcinoma, but not macrophage, cells.
(A, B) MTLn3 cells (A) or RAW macrophages (B) were transfected with either D2BA (upper panels), BAD2 (middle panels) or fixed and stained with an antibody against endogenous β-actin (lower panels) and stained for F-actin using phalloidin-Alexa 647. In MTLn3 cells, the D2BA construct localizes to stress fibers, lamellipodia and invadopodia, whereas the BAD2 construct localizes to cytoplasm, lamellipodia and invadopodia, similar to endogenous β-actin, without affecting endogenous stress fibers. In macrophages, both constructs show indistinguishable localization with endogenous β-actin and F-actin. (C) Incorporation of photoconverted BAD2 in macrophage podosomes is similar to that observed with D2BA. i. Still images of a cell expressing BAD2 photoconverted in the encircled region in widefield at 0 and 30 seconds after photoconversion. ii. Blow-up of the podosome indicated in i during the entire post-conversion time-lapse showing rapid incorporation of photoconverted BAD2 monomers into the podosome. Scale bars, 5 µm (A) and10 µm (B, C).