FRET-FLIM Shows Different DNM2 Interactions for HIVVSV-G and HIVJRFL
(A) Representative time correlated single photon counting intensity and FLIM micrographs for six different conditions are shown. FLIM images are pseudocolored and blue-cold pixels represent low lifetimes (FRET +), while red-warm pixels represent high lifetimes (FRET −). The negative controls (Dyn-GFP + mCherry with and without virions and Dyn-GFP + Dyn-mCherry) present high average lifetime values (FRET −), while the TZM-bl expressing Dyn-GFP + Dyn-mCherry and exposed to either HIVJRFL or HIVVSVG present blue-colder colors designating FRET+ detection and therefore DNM2 increased interactions upon virus addition. The scale bar represents 15 μm.
(B) Boxchart representing the average mean lifetime (in nanoseconds, ns) recovered from individual cells from at least three different FRET-FLIM experiments (n = 3) is shown for different conditions. The conditions are as follows: TZM-bl cells expressing Dyn-GFP + mCherry diffusing alone (n = 16), TZM-bl cells expressing Dyn-GFP + mCherry diffusing alone in the presence of HIVJRFL (n = 14), TZM-bl cells expressing Dyn-GFP + mCherry diffusing alone in the presence of HIVVSVG (n = 14), TZM-bl cells expressing Dyn-GFP + Dyn-mCherry in the presence of HIVJRFL (n = 14), TZM-bl cells expressing Dyn-GFP + Dyn-mCherry in the presence of HIVVSVG (n = 10), and TZM-bl cells expressing Dyn-GFP + Dyn-mCherry (n = 18).