No abstract

Caveolin plays an essential role in the formation of characteristic surface pits, caveolae, which cover the surface of many animal cells. The fundamental principles of caveola formation are only slowly emerging. Here we show that caveolin expression in a prokaryotic host lacking any intracellular membrane system drives the formation of cytoplasmic vesicles containing polymeric caveolin. Vesicle formation is induced by expression of wild-type caveolins, but not caveolin mutants defective in caveola formation in mammalian systems. In addition, cryoelectron tomography shows that the induced membrane domains are equivalent in size and caveolin density to native caveolae and reveals a possible polyhedral arrangement of caveolin oligomers. The caveolin-induced vesicles or heterologous caveolae (h-caveolae) form by budding in from the cytoplasmic membrane, generating a membrane domain with distinct lipid composition. Periplasmic solutes are encapsulated in the budding h-caveola, and purified h-caveolae can be tailored to be targeted to specific cells of interest.

The caveolar membrane microdomain plays an integral role in stabilizing the muscle fiber surface in mice and zebrafish.

Caveolae are characteristic invaginations of the mammalian plasma membrane (PM) implicated in lipid regulation, signal transduction and endocytosis. We have employed electron microscope tomography (ET) to quantify caveolae structure-function relationships in threedimension (3D) at high resolution both in conventionally fixed and in fast-frozen/freeze-substituted (intact) cells as well as immunolabelled PM lawns. Our findings provide a detailed quantitative comparison of the average caveola dimensions for different cell types including tissue endothelial cells and cultured 3T3-L1 adipocytes. These studies revealed the presence of a spiked caveolar coat and a wide caveolar neck open to the extracellular milieu that is sensitive to conventional fixation; the neck region appeared to form a specialized microdomain with associated cytoplasmic material. In endothelial cells in situ in pancreatic islets of Langerhans, the diaphragm spanning the caveolar opening was clearly resolved by ET, and the involuted 3D topology of the cell surface mapped to measure the contribution of caveolar membranes to local increases in the surface area of the PM. The complexity of connections among caveolae and to the actin cytoskeleton and microtubules suggests that individual caveolae may be interconnected through a complex filamentous network to form a single functional unit.

†These authors contributed equally to this work. The zebrafish is a powerful vertebrate system for cell and developmental studies. In this study, we have optimized methods for fast freezing and processing of zebrafish embryos for electron microscopy (EM). We show that in the absence of primary chemical fixation, excellent ultrastructure, preservation of green fluorescent protein (GFP) fluorescence, immunogold labelling and electron tomography can be obtained using a single technique involving high-pressure freezing and embedding in Lowicryl resins at low temperature. As well as being an important new tool for zebrafish research, the maintenance of GFP fluorescence after fast freezing, freeze substitution and resin embedding will be of general use for correlative light and EM of biological samples. Zebrafish are an excellent model system for cell and developmental biology. In addition to the power of this vertebrate system for genetic screening (1,2), the transparency of the zebrafish embryo has made it an ideal system for light microscopy that has been used with full advantage to study morphogenetic processes during development. Genetic screens are now being increasingly complemented by reverse genetic approaches, particularly involving mor-pholino-based approaches (3) but more recently using a powerful zinc finger nuclease-based approach (4,5). Zebrafish embryos are also ideal for electron microscopic (EM) studies; one transverse section of a 72 h embryo is easily contained in a single EM section, yet can contain numerous different cell types and tissues. However, conventional methods of EM do not provide optimal preservation of all tissues and are usually incompatible with immunolabeling and visualization of expressed fluorescently tagged proteins. An exception to this is the Tokuyasu frozen sectioning method, but the absence of a resin (6), while providing excellent antigen detection, is less suitable for preservation of large fluid-filled structures, such as the notochord of the zebrafish embryo, a crucial structural and signalling structure. Until recently, most EM analysis of the zebrafish has been performed using standard techniques that involve chemical fixation. In this study, we aimed to develop a technique that avoided chemical fixation, which allowed us to use the power of green fluorescent protein (GFP) as a fluorescent marker for light microscopy, while also allowing us to correlate light microscopic observations with immunogold EM on the same sections. Ideally, this technique would also allow us to perform three-dimensional (3D) electron tomography on the same sections. We now describe a method for high-pressure freezing of the zebrafish embryo that fulfils these criteria. The described method provides improved ultrastructure over conventional methods. Most importantly, with this method, we show that GFP fluorescence is still retained after freeze substitution (FS) and Lowicryl embedding of high-pressure frozen (HPF) embryos. As well as the detection of GFP-labelled proteins , endogenous antigens ...