3.1. Fluorescence Recovery After Photobleaching (FRAP) assay

Transfection. Plasmid transfection in N2a cells is carried out using lipofectamine 2000. N2a cells are plated at 90-95% confluent at the time of transfection on the 35mm poly-lysine coated glass-bottom dish (BD bioscience) with 2ml of the growth media. Plasmids (4μg of each plasmid expressing GFP-rab5a WT, dominant active GFP-rab5a Q79L, or dominant negative GFP-rab5a S34N) are diluted in 250 μl of Opti-MEM medium (Invitrogen), and 4 μl lipofectamine 2000 (Invitrogen) is mixed in 250 μl of Opti-Mem medium, incubated for 5 minutes and combined with diluted plasmids (total 500 μl) for 20 minutes at room temperature (RT). Mixture is added into the culture, and cells are incubated overnight at 37 °C.

Photobleaching. In a FRAP experiment (Fig. 2), an area of the cell containing a fluorescently tagged protein is photobleached and the recovery of the fluorescence in the bleached region is monitored as the fluorescent protein replaces the bleached protein [22] (Fig. 2a).The GTP-loaded active form of rab5 associates with endosomal membranes while inactive rab5 (GDP-bound) is cytosolic [1]. Because the rate of dissociation of rab5 from endosomal membranes is determined by the conversion of the GTP-bound active to the GDP-bound inactive forms, determination of the rate of rab5 exchange from early endosomes indicates rab5 activation state (Fig. 2a). Dissociation rates can be calculated from the rate of fluorescence recovery after early endosomes expressing GFP-tagged rab5 have been photobleached [23]. Thus, FRAP is a useful tool to evaluate rab5 activity when GFP-tagged rab5 is introduced in cells. Here, FRAP analysis is carried out on a Zeiss LSM510 confocal microscope (Fig. 2b). GFP-rab5a is used for visualizing early endosomes. 24 hours post-transfection cells are imaged live using a 40X oil immersion objective and a zoom of 6 using a single line excitation at 488nm and emission BP 520-550 nm filter sets. A ROI (region of interest) is drawn around the GFP-rab5 positive puncta and laser transmission increased to 100%. Photobleaching results in roughly 70-80% loss of fluorescence in the bleached area

a, The principle of FRAP is represented by a schematic diagram of the cell containing GFP-rab5a-positive endosomes. The graph of FRAP analysis shows a schematic recovery curve that corresponds to the target endosome (1), photobleached endosome (2), and recovered endosome (3). The recovery curve provides an estimate for the overall fraction of the GFP-rab5a molecules that exhibit activation. b, Representative images and graphs of GFP-rab5a WT and Q79L FRAP analysis in N2a cells. WT rab5 shows marked increased recovery, while dominant active mutant of rab5a (Q79L) significantly reduces GFP-rab5a recovery after photobleaching, indicating persistent activation of rab5 GTPase (n=20 endosomes in each cell).

The recovery. The recovery of the GFP fluorescence in each condition is recorded with 20 time-lapse image series by scanning the whole cell at 15 second intervals. The cells are maintained at 37°C in the growth media on a heating stage (Zeiss Pecon) throughout the experiments.

Calculation. To calculate percent of recovery after photobleaching, intensity profiles for the bleach spot are calculated by Zeiss LSM510 image analysis software.