De-coating Caveolae: Understanding the Caveolar Protein Machinery in Endocytosis
Caveolae are flask-shaped invaginations with a diameter of 50-80nm in the plasma membrane of many types of mammalian cells. They are particularly abundant in fat cells, muscle cells, and cells that line blood vessels and serve as a source for clathrin-dependent endocytosis. Although they are likely to be important for cellular responses to mechanical stress, intracellular trafficking, and signaling events, the precise molecular mechanisms that determine how they form and carry out these functions have yet to be understood.
The basis for more complete understanding of the network of protein interactions that produces caveolae has been provided by the work of a team comprised of researchers from Cambridge University, UCSD, and Nanyang Technical University (Singapore) that published their findings in the August 27, 2013, issue of PLoS Biology.
Recent years have witnessed the expansion of proteins known to be present in caveolae. They include caveolin, cavin, EHD2, and pacsin 2. This variety implies that caveolar biogenesis and function involve a complex set of proteins. But what’s not understood is how they assemble physically and spatially to generate caveolae.
This study showed that the main protein components, cavins and caveolins (but not EHD2 and pacsin 2), assemble into one specific complex. It also showed how different amounts of two caveolar proteins, cavin 2 and cavin 3, may be incorporated into this single type of complex, thereby potentially conferring different functional properties on caveolae.
Aided by NCMIR scientists, researchers from the Laboratory of Molecular Biology in Cambridge, England used immunofluorescence and electron microscopy aided by a recently developed protein tag for electron microscopy (MiniSOG) from Roger Tsien’s laboratory and NCMIR to demonstrate that the protein complex is distributed all around the membrane bulb of caveolae, which leads to its being named the caveolar coat. The caveolar coat excludes the protein EHD2, which defines a spatially and biochemically distinct domain at the caveolar neck and is not required for formation of the coat complex. (MiniSOG, which stands for mini Singlet Oxygen Generator, is a fluorescent flavoprotein engineered from Arabidopsis phototropin 2. It fusions to well-characterized proteins, enabling the proteins to be localized by electron microscopy at high spatial [low-nanometer] resolution.)
Furthermore, 3D electron tomograms of the caveolar coat show that the coat is composed of repeating units of a unitary caveolar coat complex. The 3D electron tomography was conducted at the National Center for Microscopy and Imaging Research (NCMIR) led by Mark H. Ellisman at UCSD on 250-300nm sections of tissue using an FEI Titan TEM system. Dual-tilt series were recorded at +/−60° with 1° intervals and a pixel size of 0.5 nm (at 18k). Images were captured using a 4k°—4k Gatan Ultrascan 4000 camera. Reconstruction was accomplished using two software packages developed at NCMIR: IMOD (for rough alignment of the two tilt series) and TxBR (fine alignment and reconstruction).
The identity, basic stoichiometry, and distribution of this unitary complex around the caveolar bulb obtained from this 3D data provides an excellent foundation for further structural characterization of the protein machinery for generating caveolae.
Citation: Alexander Ludwig, Gillian Howard, Carolina Mendoza-Topaz, Thomas Deerinck, Mason Mackey, Sara Sandin, Mark H. Ellisman, and Benjamin J. Nichols, Molecular Composition and Ultrastructure of the Caveolar Coat Complex, PLoS Biology 2014, 11:1-20, e1001640. doi: 10.1371/journal.pbio.1001640. Epub 2013 Aug 27. PMID: 24013648 PMCID: PMC3754886.
Also includes two animations showing (1) 3D electron tomography of the caveolar coat (a sequence of z slices through a 150-200 nm section) and (2) a maximum-intensity projection of a subregion from the first animation.