Visualization of Actin Polymerization in Invasive Structures of Macrophages and Carcinoma Cells Using Photoconvertible β-Actin – Dendra2 Fusion Proteins (original) (raw)
Figure 5
Photoconversion of β-actin-Dendra2 monomers in MTLn3 cells reveals extended actin polymerization at barbed ends in invadopodial precursors stimulated by EGF.
(A,B) Still images of cells that were either left untreated (A) or treated with cytochalasin D (B) taken before the photoconversion (0s), after photoconversion (20s) and after EGF stimulation (240s). Each cell is shown in green, 488nm channel (upper panels) and red, 561nm channel (lower panels). White circle marks the site of photoconversion. Scale bar, 2µm. (C) Representative plots of actin fluorescence of unconverted (488 nm) and photoconverted (561 nm) BAD2 species in an invadopodium of an untreated and a cytochalasin D (cytoD)-treated cell. Arrows point to the time of photoconversion (407 nm) and EGF addition. Cytochalasin D blocks EGF-induced BAD2 incorporation into the invadopodium. (D) Average fluorescence intensity of unconverted (488 nm) and photoconverted (561 nm) BAD2 in invadopodia of cells that were left untreated or treated with cytochalasin D (+cytoD). 0: pre-EGF addition; 1: 1 min after EGF addition; 2: 2 min after EGF addition; 3: 3 min after EGF addition. Values were normalized to pre-photoconversion values and average of intensity in invadopodial precursors from 5 different cells per condition are shown +/−s.e.m.