The morphology of caveolae and caveolar membrane organization by caveolin 1. (from Parton & Simons, ...
Protein complexes interacting with specific lipids mediate the modulation of membrane curvature, thus enabling basic cellular processes like endocytosis and exocytosis. Compared to the well-characterized clathrin-based machinery, the caveolae based one has remained enigmatic. Caveolae are highly abundant small (diameter 60–80 nm) membrane micro-domains forming omega-shaped invaginations at the plasma membrane of many mammalian cell types. They are involved in various cellular processes including endocytosis, transcytosis, cell motility, lipid homeostasis, signaling and cancer. Caveolar transport does not involve cycles of coat assembly and disassembly. Instead, once assembled, caveolar coats remain stable throughout transport. The group of Ari Helenius (ETH Zurich) – with whom we collaborate – has recently been able to purify two major building blocks of caveolae. Caveolin 1 (CAV1), an integral membrane protein is the main component of the major caveolar coat large complex that most likely corresponds to mature caveolae. Moreover, a smaller building block of the caveolar coat was found. Further biochemical purification and characterization is ongoing.
We will study caveolar assembly structurally by cryo-electron-microscopy and complementary methods. The aim is a mechanistic understanding of how the caveolar coat is assembled. We will analyze the structures at different levels of complexity by ultrastructural studies of the isolated small and large caveolae complexes as well as by in situ imaging of fluorescently labeled caveolar components inside cells. Ultimately, in vitro reconstitution assays will be established to identify the minimal building blocks and their stoichiometry in caveolar assembly. In collaboration with the group in Zurich analysis by mass spectrometry approaches and state-of-the-art live-cell imaging of caveolae at the plasma membrane of living cells will be applied to reveal the assembly process spatio-temporally.
The multi-disciplinary project will provide training in a variety of methods. This will encompass biochemical and biophysical analyses, correlation microscopy spanning numerous imaging means and ranges of resolution (from advanced life cell light and fluorescent microscopy to cryo electron microscopy and tomography) and the use of state of the art image reconstruction and processing techniques for structure determination and analysis. There will also be the opportunity to perform molecular biology experiments, cell culture techniques and to be involved in the analysis of the mass spectrometry based proteomic analyses.
Protein Science & Structural Biology and Physiology, Cellular & Molecular Biology
Project reference number: 239
| Name | Department | Institution | Country | |
|---|---|---|---|---|
| Dr Kay Grunewald | Structural Biology | Oxford University | UK | kay@strubi.ox.ac.uk |
2007. The multiple faces of caveolae. Nat. Rev. Mol. Cell Biol., 8 (3), pp. 185-94. Read abstract | Read more
Caveolae are a highly abundant but enigmatic feature of mammalian cells. They form remarkably stable membrane domains at the plasma membrane but can also function as carriers in the exocytic and endocytic pathways. The apparently diverse functions of caveolae, including mechanosensing and lipid regulation, might be linked to their ability to respond to plasma membrane changes, a property that is dependent on their specialized lipid composition and biophysical properties. Hide abstract
2005. Assembly and trafficking of caveolar domains in the cell: caveolae as stable, cargo-triggered, vesicular transporters. J. Cell Biol., 170 (5), pp. 769-79. Read abstract | Read more
Using total internal reflection fluorescence microscopy (TIR-FM), fluorescence recovery after photobleaching (FRAP), and other light microscopy techniques, we analyzed the dynamics, the activation, and the assembly of caveolae labeled with fluorescently tagged caveolin-1 (Cav1). We found that when activated by simian virus 40 (SV40), a non-enveloped DNA virus that uses caveolae for cell entry, the fraction of mobile caveolae was dramatically enhanced both in the plasma membrane (PM) and in the caveosome, an intracellular organelle that functions as an intermediate station in caveolar endocytosis. Activation also resulted in increased microtubule (MT)-dependent, long-range movement of caveolar vesicles. We generated heterokaryons that contained GFP- and RFP-tagged caveolae by fusing cells expressing Cav1-GFP and -RFP, respectively, and showed that even when activated, individual caveolar domains underwent little exchange of Cav1. Only when the cells were subjected to transient cholesterol depletion, did the caveolae domain exchange Cav1. Thus, in contrast to clathrin-, or other types of coated transport vesicles, caveolae constitute stable, cholesterol-dependent membrane domains that can serve as fixed containers through vesicle traffic. Finally, we identified the Golgi complex as the site where newly assembled caveolar domains appeared first. Hide abstract
2004. Caveolin-stabilized membrane domains as multifunctional transport and sorting devices in endocytic membrane traffic. Cell, 118 (6), pp. 767-80. Read abstract | Read more
Endocytosis comprises several routes of internalization. An outstanding question is whether the caveolar and endosomal pathways intersect. Following transport of the caveolar protein Caveolin-1 and two cargo complexes, Simian Virus 40 and Cholera toxin, in live cells, we uncovered a Rab5-dependent pathway in which caveolar vesicles are targeted to early endosomes and form distinct and stable membrane domains. In endosomes, the low pH selectively allowed the toxin to diffuse out of the caveolar domains into the surrounding membrane, while the virus remained trapped. Thus, we conclude that, unlike cyclic assembly and disassembly of coat proteins in vesicular transport, oligomeric complexes of caveolin-1 confer permanent structural stability to caveolar vesicles that transiently interact with endosomes to form subdomains and release cargo selectively by compartment-specific cues. Hide abstract