Membrane Diffusion Barriers Localize Signal Amplification during Macropinocytosis.

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 plasma membrane, thereby organizing the contractile activities that close macropinosomes. Using quantitative fluorescence microscopy, we demonstrate that macropinosome formation is directed by a sequence of chemical changes within the cups of plasma membrane circular ruffles. Stages of receptor-dependent signaling were organized into distinct transient waves of phosphoinositides, diacylglycerol, PKCα, Rac and Ras activities, which preceded cup closure and peak recruitment of Rab5 to macropinosomes. Thus, circular ruffles enclose plasma membrane subdomains that focus receptor signal amplification and the signal transitions that coordinate cell movements.