Fig. 2.

Selective photoactivation of PAGFP-MEM in plasma membranes. (A) Schematic of experimental protocol for XYT experiments. PAGFP-MEM was photoactivated in regions of flat (a), ruffled (b) or cupped membrane (c). Fluorescence intensities were collected in these activation regions over time. Loss of fluorescence indicated diffusion of activated PAGFP-MEM out of the activation region; conversely, retention of activated PAGFP-MEM indicated restricted diffusion. (B–D) Images of different macropinocytic structures. Top row, mCherry–MEM; middle row: PAGFP-MEM; bottom row, PAGFP:mCherry ratio. From left to right: 1 second pre-activation, 1 second post-activation, 10 seconds post-activation, 20 seconds post-activation. Scale bars: 1 μm. Color bars indicate relative fluorescence intensities of ratio images. (B) Photoactivation in flat membrane. (C) Photoactivation in ruffle membrane. (D) Photoactivation in a macropinocytic cup. (E) Quantification of the fluorescence decrease in plasma membrane (n=5 for each condition). Membrane ruffles and cups demonstrate significant retention of photoactivated PAGFP-MEM. (F) Modeling of the effects of cup height on probe retention. Increasing cup height without adding a barrier or decreasing the diffusion coefficient did not affect molecule retention and could not account for the experimental values. Error bars indicate s.d.