Figure 4.

NED-19 compromises plasma membrane repair by preventing lysosomal fusion with the plasma membrane. (A) Sample transmission (top) and fluorescent (bottom) images of pyramidal cells in hippocampal slice cultures incubated with fluoroscein dextran during biolistic wounding. Only cells that have successfully re-sealed cells become fluorescently labelled. Pre-treatment with Ned-19, GPN, or Mon (monensin) abolished fluorescent labelling seen in vehicle control (Con), suggesting that membrane repair was compromised. Scale bar = 10 µm. (B) Quantification of fluoroscein dextran fluorescent labelling (n > 5 slices/condition). (C) Sample image of dissociated neurons transfected with LAMP2-pHlourin prior to and following wounding with a sharp microelectrode (red). Local fluorescence increase was triggered by wounding in control conditions, and reduced with NED-19 treatment. Scale bar = 10μm (D). Quantification of LAMP2-pHlourin fluorescence over time (n = 3 cells/condition). Error bars represent S.E.M. Non-significant group comparisons are denoted by n.s.