In this work, we report the development of a computational model which can address how changes in caveolin-1 oligomerization and phosphorylation impact upon membrane invagination, budding, and internalization by (i) controlling the stability of caveolin polymers in the plasma membrane and (ii) their ability to facilitate changes in membrane curvature. These studies will assess the role of Src-mediated phosphorylation of caveolin-1 N-terminal tyrosine residue Y14 in controlling the stability of caveolin-1 oligomers in endothelial cells and thereby in the mechanism of caveolae-mediated endocytosis. Central to this mechanism is the interaction between (i) Brownian motion of the individual caveolin chains in the heptameric structures, and (ii) repulsion between the charged groups resultant from phosphorylation – to produce asymmetric forces leading to deformation and invagination.
Biophysical background of the modeling hypothesis: Caveolae, small, flask-shaped invaginations of mammalian plasma membranes, are ubiquitous features of endothelial cells. They comprise about 15% of the cell volume1 and account for >95% of the plasmalemmal vesicles within the cells2. Assessment of caveolar internalization using cholera toxin subunit B3, a caveolae-specific tracer4, showed that plasma membrane-bound caveolae are released into the cytosol2, 5. Using methyl-β-cyclodextrin (MβC), a specific cholesterol-binding agent that flattens caveolae6, we observed a reduction in [125I]-albumin endocytosis dependent on the concentration of MβC5. Thus, caveolae are implicated as essential vesicle carriers mediating endocytosis in endothelial cells7-11.
Caveolin-1, the 22 kDa protein that coats the cytoplasmic surface of caveolae, is the defining protein constituent of caveolae6, 12. The characteristic invaginations were absent in endothelial cells from caveolin-1 knockout mice7, 8, 10, 11 – indicating the importance of caveolin-1 in determining the caveolar structure. An important property of caveolin-1 is its ability to form oligomers in the cytoplasmic domain of the caveolar membrane12. Caveolin-1 oligomerization is crucial in regulating the invagination of the membrane6, 13, 14. Moreover, the stability of caveolin oligomers may be an important determinant of the release of caveolae from the plasma membrane15, 16; that is, destabilization of caveolin-1 oligomers may facilitate the release of caveolae.
Caveolin-1 is a well-described substrate of Src17-20. In vitro phosphorylation of caveolin-1-derived synthetic peptides and site-directed mutations showed that Tyr14 is the primary Src phosphorylation site17. Aoki and coworkers20 demonstrated that phosphorylation of caveolin-1 Tyr14 in rat tissue occurred primarily in endothelial cells of capillaries and venules. Importantly, this study suggested that phosphorylation of caveolin-1 at Tyr14 induced caveolae endocytosis; these investigators observed fewer plasma membrane-attached and invaginated caveolae and more free cytoplasmic vesicles in endothelial cells treated with the tyrosine phosphatase inhibitor pervanadate. Although studies from the Fujimoto lab19, 20 have implicated an important role of caveolin-1 phosphorylation in regulating caveolae internalization, the mechanism by which phosphorylation mediates this process has not been described.
Features of the model: A coarse-grained atomistic (CGA) approach is developed to simulate key mechanistic features of the budding of vesicles, thereby illuminating specific biochemical hypotheses. It is postulated that Coulombic inter-chain repulsion resulting from phosphorylation-induced charge creates asymmetric forces that cause the formation of curvature on the cell membrane. A Brownian dynamics simulation adapted from polymer kinetic theory represents the protein chains as bead-spring assemblies, and tracks their stochastic motion as forces are transmitted to the caveola. A novel coarse-grained, particulate model is developed for the lipid bilayer, in which local directionality (surface normal vector) is extracted from the relative arrangement of isotropic particles rather than being embedded in the more complex (i.e., rod-like or multi-bead) internal structure of lipid particles used thus far in CGA simulations. An anisotropic inter-particle force law results in effective bending elasticity of the sheet-like aggregate of particles, and preserves two-dimensional Lennard-Jones liquid behavior in the tangential direction. By combining Brownian dynamics of heptamers anchored to the membrane with the elastic membrane response, a model of caveolin-mediated curvature is obtained.
Future extensions of the model are planned to incorporate the stabilizing influence of the adjoining cytoskeleton (using a bead-spring girder model) and also viscous damping by the surrounding cytoplasm and extracellular fluid (by superposing Stokes hydrodynamic kernels).
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