Figure 7. Bis-T-23 induces formation of Dyn^OLIGO in podocytes at distinct sites.

a) Color-coded FLIM images showing the fluorescence lifetime (τ1) of the donor fluorophore (Alexa Fluor 488). Lifetimes are shown as continuous colors and as discrete colors: fast lifetimes between 0–2000 ps are shown in red (positive FRET), and slow lifetimes above 2001 ps are shown in blue (no FRET). When indicated, cells were treated with 30 µM of Bis-T-23 for 20 min prior to fixation. Cells were stained using monoclonal anti-GTPase antibody conjugated with Alexa Fluor 488 (donor fluorophore) and monoclonal anti-PRD antibody conjugated with Alexa Fluor 594 (acceptor fluorophore). The data display FRET for a narrow optical section at the bottom of the cell.

b) Histogram of fluorescence lifetimes (τ1) of the donor fluorophore (Alexa Fluor 488) in the whole image after the lifetimes were curve fitted to a two exponential decay curves with one exponential fixed at the average lifetime for donor only fluorophore (τ2=2500 ps). Cells expressing Dyn1, when indicated were treated with 30 µM Bis-T-23. Note similar distribution of the lifetimes in the presence of Bis-T-23 and cells expressing Dyn1^E/K mutant. Fast lifetimes between 0–2000 ps correspond to FRET efficiency of ≥20% FRET efficiency.

c) Dyn^OLIGO localize at the cell membrane and transition zone where cortical actin transitions to stress fibers. Positive FRET signal (in red) was overlaid on the actin cytoskeleton (light blue). Inset (A) shows higher magnification of the section of the cell.