Theoretical Model for the Formation of Caveolae and SimilarMembrane InvaginationsPierre Sens* and Matthew S. Turnery*Institut Charles Sadron, Strasbourg, France, and Centre National de la Recherche Scientifique/UMR 168, Institut Curie,Paris, France; andyDepartment of Physics, University of Warwick, Coventry, United KingdomABSTRACT We study a physical model for the formation of bud-like invaginations on fluid lipid membranes under tension, andapply this model to caveolae formation. We demonstrate that budding can be driven by membrane-bound proteins, providedthat they exert asymmetric forces on the membrane that give rise to bending moments. In particular, caveolae formation doesnot necessarily require forces to be applied by the cytoskeleton. Our theoretical model is able to explain several featuresobserved experimentally in caveolae, where proteins in the caveolin family are known to play a crucial role in the formation ofcaveolae buds. These include 1), the formation of caveolae buds with sizes in the 100-nm range and 2), that certain N- andC-termini deletion mutants result in vesicles that are an order-of-magnitude larger. Finally, we discuss the possible origin ofthe morphological striations that are observed on the surfaces of the caveolae.INTRODUCTIONIt has long been understood that invaginations formspontaneously on cell membranes (Alberts et al., 1994).These invaginations, which eventually separate from themembrane as mature, membrane-bound vesicles, play anessential role in cellular traf ficking and signaling (Stahlhutet al., 2000; Lisanti et al., 1994). The mechanism by whichsuch invagination is controlled is still far from fully un-derstood, although it is now widely accepted that certainmembrane-bound proteins, including clathrin and caveolin,play an important role. The formation of clathrin-coated pitsis thought to be driven by the controlled geometricaggregation of clathrin into rigid scaffolding, which forcesthe membrane to curve (Takei and Hauc ke, 2001; Mashl andBruinsma, 1998). The mechanism for formation of thesecond most common class of membrane invaginations,known as caveolae, is less well understood. Caveolae, whichare less morphologically distinct than clathrin-coated pits,resemble V-shaped invaginations with a typical size of ;100nm (Rothberg et al., 1992; Schlegel et al., 1998; Westermannet al., 1999). They are present at high concentrations onprimary adipocytes, fibroblasts, muscle cells, and pulmonarytype 1 cells as well as endothelial cells, and perform a varietyof functions ranging from signal transduction to intracellulartransport (Gilbert et al., 1999; Schlegel and Lisanti, 2001). A‘‘striated coat’’ can be seen on the cytoplasmic side of thecaveolae membrane. It is believed to reflect the organizationof a recently discovered class of membrane-bound proteins,called caveolins, which are crucial to the formation ofcaveolae (Lisanti e t al., 1994).The protein caveolin has a hairpin structure, with a shortmembrane-spanning sequence, flanked by two hydrophilictermini, both found on the cytoplasmic side of the cellmembrane: a 101-amino-acid polypeptide N-terminus tail(polymer), and a shorter (44 a-a) C-terminal, which is stro nglyattached to the membrane (Schlegel and Lisanti, 2001). Thesecaveolin molecules are typically found in small aggregates of15–17 molecules (Schlegel et al., 1998; Sargiacomo et al.,1995), the aggregation being driven by residues of theN-terminal located close to the membrane. Furthermore, itis believed (Schlegel and Lisanti, 2001) that there existsome specific C-terminal to C-terminal attractions, which areresponsible for the organization of the protein aggregates atthe surface of the caveolae membrane. Mutational analysis ofcaveolin-induced vesicle formation have been recentlyperformed (Li et al., 1998) and is discussed in relation withour theory in the Conclusions section.Caveolae are now thought to influence cell physiology inmany ways, including growth and cell division, adhesion, andhormonal response (Fielding and Fielding, 2000). Theseinvaginations have been associated with the formation of lipidrafts (Kurzchalia and Parton, 1999)—glycosphingolipid- andcholesterol-enriched microdomains within the plasma mem-brane of eukaryotic cells. Their ability to perform manydifferent tasks might be achieved by their involvement inreporting change in membrane composition by signa l trans-duction to the nucleus. It may also be connected to theirregulation of signal traffic in response to extracellular stimuli,including mechanical stress (Park et al., 2000).From a physical point of view, spontaneous vesicleformation has been observed in vitro by adding amphiphilicpolymers to various lipid systems (Lasic et al., 2001). It can beviewed as an example of the so-called curvature instability offluid membranes containing inclusions, predicted to occur forinclusions that locally influence the membrane curvature(Leibler, 1986; Leibler and Andelman, 1987). There havebeen physical studies of the inclusion-induced budding ofvesicles (Kim and Sung, 1999; Seifert, 1993; Ju¨licher andSubmitted September 24, 2003, and accepted for publication December 29,2003.Address reprint requests to Pierre Sens, Tel.: 33-142-34-6474; Fax: 33-140-51-0636; E-mail: [email protected].Ó 2004 by the Biophysical Society0006-3495/04/04/2049/09 $2.00Biophysical Journal Volume 86 April 2004 2049–2057 2049Lipowsky, 1996) and works on the effect of single (Lipowsky,1997; Hiergeist et al., 1996) and distributed (Nicolas, 2002)polymers grafted on membranes.Many theoretical studies have also been devoted tounderstanding physical coupling between integral membraneproteins and biological membranes. Such couplings includelocal disruption of the bilayer molecular structure in thevicinity of the protein (hydrophobic mismatch, local mem-brane tilt), and the behavior of foreign bodies in a fluctuatingenvironment (for reviews, see Goulian, 1996 and Mouritsenand Andersen, 1998).Our aim is to study the effect of small inclusions, such asproteins, that affect the shape of the cell membrane. Weassume that this ‘‘foreign’’ object exerts a force on themembrane, which may be due either to entropic effects,similar in origin to the pressure exerted by a gas onto thewalls of its container, or to specific mechanochemical forces.Throughout we will attempt to compare our rather generaltheory with the specific phenomenon of caveolin-mediatedformation of caveolae. The fact
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