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. 2011 Dec 1;124(23):4106–4114. doi: 10.1242/jcs.091538

Fig. 5.

Fig. 5.

Diffusion dynamics within membrane cups indicate that the barrier localizes to the cup walls. (A–C) Fluorescence redistribution following asymmetric activation of PAGFP-MEM in macropinocytic cups. (A) Representative images. From left to right: mCherry–Mem image 1 second prior to activation, PAGFP:mCherry ratio images 1, 10 and 20 seconds after activation. Yellow lines indicate position of linescans. Green boxes indicate the perimeter of the activation region. Scale bar: 1.0 μm. Color bars indicate relative fluorescence intensities of ratio images. (B) Linescan measurements of mCherry–MEM pixel intensities. (C) Linescan measurements of PAGFP:mCherry ratio values at 1, 10, and 20 seconds after photoactivation. Similar fluorescence patterns were seen in five macropinocytic cups. Paired vertical lines indicate the location of the cup wall. Green lines indicate the edge of the activation region. (D) Fluorescence measurements of activated and non-activated regions in the base of the cup over time, n=5. (E–J) Modeling of diffusion within cups. (E,F) Diagrams of activation patterns used to model diffusion inside the cup, showing views from above (C) and in sagital section (F). (G,H) Distributions of molecules along the diameter of the base of the cup normal to the activation boundary, measured at 1, 10 and 20 seconds after activation. (G) When the diffusion coefficient in the walls is 10−11 cm2/second (i.e., a barrier) and in the base of the cup is 10−9 cm2/second, fluorophore redistribution resembles the experimental data in C. (H) When the diffusion coefficients of the walls and base are both set to 10−11 cm2/second, fluorophore redistribution does not resemble the experimental data. (I,J) Modeling of the time course of fluorophore decrease from the activated region (triangles) and increase in the non-activated region (circles) when diffusion coefficients are set as in G (I) and H (J). The model resembles the experimental observations in D when the base of the cup has the same diffusion coefficient as membrane outside the cup (I).