FIGURE 5:

Dyn2 colocalizes with α-actinin at invadopodia, and the interaction between Dyn2 and α-actinin 4 is required for ECM remodeling. (A, B) Immunofluorescence staining of Dyn2 and α-actinin 1/4 on fluorescent gelatin-coated coverslips shows colocalization of these proteins at invadopodia actively degrading matrix. The region highlighted in the Merge image is shown in the individual channel insets. Images were collected from n = 3 experiments. (C–E) Gelatin degradation assays were used to measure invadopodia activity in DanG cells expressing the WT or deletion mutant forms of each protein after knockdown of the endogenous protein. DanG cells were plated on Cy3-fluorescent gelatin-coated coverslips for 7 h to allow for gelatin degradation, and then fixed. (F–H) The area of matrix degradation was quantified by dividing the total area of degraded gelatin by the cell area. Graphed data represent the mean ± SEM, and data points represent average values for independent experiments relative to the nontargeting control. α-Actinin 4: n = 5 biological replicates, 9–42 cells per condition in each experiment. α-Actinin 1: n = 3 biological replicates, 20–34 cells per condition in each experiment. Dyn2: n = 3 biological replicates, 26–37 cells per condition in each experiment. Scale bars: 5 μm (A, B) and 10 μm (C–E). Student’s t test was used to measure statistical significance. * indicates p < 0.05; ** indicates p < 0.01.