Ruffles limit diffusion in the plasma membrane during macropinosome formation

Abstract

In murine macrophages stimulated with macrophage-colony-stimulating factor (M-CSF), signals essential to macropinosome formation are restricted to the domain of plasma membrane enclosed within cup-shaped, circular ruffles. Consistent with a role for these actin-rich structures in signal amplification, microscopic measures of Rac1 activity determined that disruption of actin polymerization by latrunculin B inhibited ruffling and the localized activation of Rac1 in response to M-CSF. To test the hypothesis that circular ruffles restrict the lateral diffusion of membrane proteins that are essential for signaling, we monitored diffusion of membrane-tethered, photoactivatable green fluorescent protein (PAGFP-MEM) in ruffling and non-ruffling regions of cells. Although diffusion within macropinocytic cups was not inhibited, circular ruffles retained photoactivated PAGFP-MEM inside cup domains. Confinement of membrane molecules by circular ruffles could explain how actin facilitates positive feedback amplification of Rac1 in these relatively large domains of the plasma membrane, thereby organizing the contractile activities that close macropinosomes.

Fig. 1.

Focal activation of Rac1 during macropinocytosis. (A–D) FRET interactions of Cerulean-PBD and Citrine–Rac1 in control (A,C) and latrunculin B-treated BMM (B,D) in response to M-CSF. (A,B) Left: Phase contrast; right: G*. Scale bars: 4 μm. (C,D) Time series for subregions of the cells shown in A and B, highlighting a forming macropinosome (C), and a comparable region of a latrunculin B-treated cell (D). Top row: Phase contrast; bottom row: G*. Scale bars: 1 μm. All color bars indicate G* values. (E) Quantification of total Citrine-Rac1 activity in control and latrunculin B- or wiskostatin-treated BMM (n=5 for all conditions). Dotted line indicates when M-CSF was added. (F) To quantify GTPase activation, the average G* in a forming macropinosome was divided by the average G* in the entire cell at each time point (n=10 for each). The resulting ratio indicates the relative change in GTPase activity in the forming macropinosomes, with numbers greater than 1.0 indicating localized increases in activity. Sequences were aligned by the timing of ruffle closure (t=60 seconds). Ratios for Rac1 were significantly higher than cytoplasm values (*P<0.001) from 80 to 100 seconds following the beginning of macropinosome formation. Ratios for Cdc42 did not significantly change during macropinosome formation. Error bars indicate s.d.

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.

Fig. 3.

4D reconstruction of activated PAGFP-MEM in an open macropinocytic cup. Through-focus z-stacks were collected of PAGFP-MEM and mCherry at regular intervals after photoactivation of PAGFP-MEM in a cup. (A,B) Projections of a macropinocytic cup. Top row: mCherry–MEM; bottom row: PAGFP:mCherry ratio. From left to right: 6.5, 13.0 and 19.5 seconds after activation of PAGFP-MEM. (A) XY projection of a macropinocytic cup and surrounding cellular region. Yellow boxes delineate the macropinocytic cup. (B) XZ projections of the macropinocytic cup (side view). Fields correspond to the regions marked by the yellow boxes in A. (C) Cross-sections of macropinocytic cups showing distribution of mCherry–MEM (red) and PAGFP:mCherry ratio (pseudocolor) at 13 seconds after photoactivation of PAGFP-MEM. Green line shows region of cross-section. Color bars indicate relative fluorescence intensities of ratio images. Scale bars: 1.0 μm.

Fig. 4.

Fluorescence intensity linescans of cupped and flat membrane. (A,D) Representative linescans (yellow lines) in cupped and flat membrane, respectively. From left to right: mCherry–MEM image 1 second prior to activation, PAGFP:mCherry ratio image 1 second after activation, ratio image 10 seconds after activation, ratio image 20 seconds after activation. Scale bars: 1.0 μm. Color bars indicate relative fluorescence intensities of ratio images. (B,E) Linescans of mCherry–MEM fluorescence intensities in cupped and flat membrane, respectively. (C,F) Linescans of PAGFP/mCherry fluorescence ratios in cupped and flat membrane, respectively, at 1, 10 and 20 seconds after photoactivation. Green lines indicate the perimeter of the activation region. Similar fluorescence patterns were seen for seven macropinocytic cups and for ten regions of flat membrane.

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).