Polymerase I and transcript release factor (PTRF)/Cavin is a cytoplasmic protein whose expression is obligatory for caveola formation. Using biochemistry and fluorescence resonance energy transfer–based approaches, we now show that a family of related proteins, PTRF/Cavin-1, serum deprivation response (SDR)/Cavin-2, SDR-related gene product that binds to C kinase (SRBC)/Cavin-3, and muscle-restricted coiled-coil protein (MURC)/Cavin-4, forms a multiprotein complex that associates with caveolae. This complex can constitutively assemble in the cytosol and associate with caveolin at plasma membrane caveolae. Cavin-1, but not other cavins, can induce caveola formation in a heterologous system and is required for the recruitment of the cavin complex to caveolae. The tissue-restricted expression of cavins suggests that caveolae may perform tissue-specific functions regulated by the composition of the cavin complex. Cavin-4 is expressed predominantly in muscle, and its distribution is perturbed in human muscle disease associated with Caveolin-3 dysfunction, identifying Cavin-4 as a novel muscle disease candidate caveolar protein.
The caveolar membrane microdomain plays an integral role in stabilizing the muscle fiber surface in mice and zebrafish.
Caveolar proteins and caveolae negatively regulate a second clathrin-independent endocytic CLIC/GEEC pathway; caveolin-1 affects membrane diffusion properties of raft-associated CLIC cargo, and the scaffolding domain of caveolin-1 is required and sufficient for endocytic inhibition.
†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 ...
Muscle contraction depends on tightly regulated Ca 2+ release. Aberrant Ca 2+ leak through ryanodine receptor 1 (RyR1) on the sarcoplasmic reticulum (SR) membrane can lead to heatstroke and malignant hyperthermia (MH) susceptibility, as well as severe myopathy. However, the mechanism by which Ca 2+ leak drives these pathologies is unknown. Here, we investigate the effects of four mouse genotypes with increasingly severe RyR1 leak in skeletal muscle fibers. We find that RyR1 Ca 2+ leak initiates a cascade of events that cause precise redistribution of Ca 2+ among the SR, cytoplasm, and mitochondria through altering the Ca 2+ permeability of the transverse tubular system membrane. This redistribution of Ca 2+ allows mice with moderate RyR1 leak to maintain normal function; however, severe RyR1 leak with RYR1 mutations reduces the capacity to generate force. Our results reveal the mechanism underlying force preservation, increased ATP metabolism, and susceptibility to MH in individuals with gain-of-function RYR1 mutations.
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