(571m) A Computational and Experimental Investigation of Mechanisms for Caveolae-Mediated Endocytosis

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 cytosol^{2, 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 cells^7-11. 

Caveolin-1, the 22 kDa protein
that coats the cytoplasmic surface of caveolae, is the defining protein
constituent of caveolae^{6, 12}.  The characteristic invaginations were absent in endothelial cells from caveolin-1 knockout mice^{7, 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 membrane^{6, 13, 14}.   Moreover, the stability of caveolin oligomers may be an important determinant of the release of caveolae from the plasma membrane^{15, 16}; that is, destabilization of caveolin-1 oligomers may facilitate the release of caveolae.

Caveolin-1 is a well-described
substrate of Src^17-20.  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 lab^{19, 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).

References:

1. D. Predescu and G. E. Palad. Am. J. Physiol. 265, H725-H733 (1993)

2. D. N.
Predescu, S. A. Predescu, et al. Mol. Biol. Cell. 14, 4997-5010 (2003)

3. A. Gilbert, J.
P. Paccaud, et al. J. Cell Sci. 112, 1101-1110 (1999)

4. R. G. Parton,
B. Joggerst, et al. J. Cell Biol. 127, 1199-1215 (1994)

5. T. A. John, S.
M. Vogel, et al. Am. J. Physiol. Lung Cell. Mol. Physiol. 284, L187-L196
(2003)

6. K. G.
Rothberg, J. E. Heuser, et al. Cell. 68, 673-682 (1992)

7. M. P. Drab, P.
Verkade, et al. Science. 293, 2449-2452 (2001)

8. B. Razani, J.
A. Engelman, et al. J. Biol. Chem. 276, 38121-38138 (2001)

9. W. Schubert,
P. G. Frank, et al. J. Biol. Chem. 276, 48619-48622 (2001)

10. Y. Y. Zhao,
Y. Liu, et al. Proc. Natl. Acad. Sci. USA. 99, 11375-11380 (2002)

11. D. Predescu,
S. M. Vogel, et al. Am. J. Physiol. Lung Cell. Mol. Physiol. L895-901
(2004)

12. T.
Kurzchalia, P. Dupree, et al. J. Cell Biol. 118, 1003-1014 (1992)

13. S. Monier, R.
G. Parton, et al. Mol. Biol. Cell. 6, 911-927 (1995)

14. R. G.
Anderson. Annu. Rev. Biochem. 67, 199-225 (1998)

15. B. van Deurs,
K. Roepstorff, et al. Trends Cell Biol. 13, 92-100 (2003)

16. I. R. Nabi
and P. U. Le. J. Cell Biol. 161, 673-677 (2003)

17. S. Li, J.
Couet, et al. J. Biol. Chem. 271, 29182-29190 (1996)

18. C.
Tiruppathi, W. Song, et al. J. Biol. Chem. 272, 25968-25975 (1997)

19. R. Nomura and
T. Fujimoto. Mol. Biol. Cell. 10, 975-986 (1999)

20. T. Aoki, R.
Nomura, et al. Exp. Cell Res. 253, 629-636 (1999)