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Fig. 4. In vivo analysis of Tfn plasma membrane flow and internalization. (A) For analysis of Tfn lateral diffusion on the plasma membrane, ice-chilled cells were incubated with Tfn-A488 for 5 minutes, washed and bleached at their rear halve membranes. Incubation at 37°C of the representative cell quickly restored the membrane diffusion, and fluorescence intensity recovered 
50% within 300 seconds at the uropod membrane (100% is the pre-bleaching intensity). Similar analyses were repeated in other five cells and the average recovery ±s.d. was 43±18%. The uptake curve is also shown as the mean intensity measured within the uropod over time (see D). (B) Recovery ratios of Tfn-A488 or anti-CD45-FITC (control). Recovery ratio at one bleached pole corresponds to the mean intensity of the bleached membrane, divided by the intensity of the opposite unbleached membrane. (C) Similar analyses to A were performed bleaching the front membrane. In this way, the membrane fluorescence recovery was minimal (5±2%, n=5), while Tfn uptake at the uropod was comparable with that of the neighbor cells during the 300 second assays (91±4% of control cells, n=5). (D) Tfn internalization in the rear-bleached cell was almost undetectable in the 300 seconds of the assay (arrow), while uptake in control neighbor unbleached cells was as usual (normalized to 100%). The average uptake in these conditions compared with control cells was 4±6% (n=6). Projection of a complete z scanning – taken at time 350 seconds – is shown to allow the identification of any out-of-focus intracellular Tfn. Simultaneous membrane recovery and uptake can be checked in Movie 3 (see supplementary material). Bar, 10 µm.
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