Decoding information in cell shape.

Shape is an indicator of cell health. But how is the information in shape decoded? We hypothesize that decoding occurs by modulation of signaling through changes in plasma membrane curvature. Using analytical approaches and numerical simulations, we studied how elongation of cell shape affects plasma membrane signaling. Mathematical analyses reveal transient accumulation of activated receptors at regions of higher curvature with increasing cell eccentricity. This distribution of activated receptors is periodic, following the Mathieu function, and it arises from local imbalance between reaction and diffusion of soluble ligands and receptors in the plane of the membrane. Numerical simulations show that transient microdomains of activated receptors amplify signals to downstream protein kinases. For growth factor receptor pathways, increasing cell eccentricity elevates the levels of activated cytoplasmic Src and nuclear MAPK1,2. These predictions were experimentally validated by changing cellular eccentricity, showing that shape is a locus of retrievable information storage in cells.

Discrepancy between fluorescence correlation spectroscopy and fluorescence recovery after photobleaching diffusion measurements of G-protein-coupled receptors.

Fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) are the two most direct methods to measure the diffusion of molecules in intact living cells. Ideally, these methods should produce similar results for an identical system. We have used these methods to monitor the diffusion of two G-protein-coupled receptors and their associated proteins in the plasma membranes of cells that do not or do contain invaginated protein domains called caveolae. FRAP studies show that caveolae domains increase the immobile fraction of receptors without significantly changing their mobility. On the other hand, FCS studies show an unexpected increase the mobility of caveolae-associated proteins. Our data suggest that the geometry of caveolae domains gives rise to a confined diffusion of its attached proteins, resulting in an apparent increase in mobility.

A role for G-proteins in directing G-protein-coupled receptor-caveolae localization.

Caveolae are membrane domains that may influence cell signaling by sequestering specific proteins such as G-protein-coupled receptors (GPCRs). While previous reports largely show that Gα(q) subunits, but not other G-proteins, interact strongly with the caveolae protein, Caveolin-1 (Cav1), the inclusion of GPCRs in caveolae is controversial. Here, we have used fluorescence methods to determine the effect of caveolae on the physical and functional properties of two GPCRs that have been reported to reside in caveolae, bradykinin receptor type 2 (B(2)R), which is coupled to Gα(q), and the µ-opioid receptor (µOR), which is coupled to Gα(i). While caveolae do not affect cAMP signals mediated by µOR, they prolong Ca(2+) signals mediated by B(2)R. In A10 cells that endogenously express B(2)R and Cav1, downregulation of Cav1 ablates the prolonged recovery seen upon bradykinin stimulation in accord with the idea that the presence of caveolae prolongs Gα(q) activation. Immunofluorescence and Förster resonance energy transfer (FRET) studies show that a significant fraction of B(2)R resides at or close to caveolae domains while none or very little µOR resides in caveolae domains. The level of FRET between B(2)R and caveolae is reduced by downregulation of Gα(q) or by addition of a peptide that interferes with Gα(q)-Caveolin-1 interactions, suggesting that Gα(q) promotes localization of B(2)R to caveolae domains. Our results lead to the suggestion that Gα(q) can localize its associated receptors to caveolae domains to enhance their signals.