Fig. 40.1
Replica electron micrographs of intestinal smooth muscle cell s in living mice prepared by the IVCT followed by replica preparation method. (a) Freeze-fractured cytoplasm of three parallel-lining smooth muscle cells (M1 ~ M3). Many cytoskeletons and caveolar structures (small arrows) along sarcolemma (large arrows) are three-dimensionally seen. Inset, higher magnification. Bars, 0.5 μm. (b) Various types of caveolar neck parts (arrows) are seen on the freeze-fractured P-face of their sarcolemma (asterisks). Right upper inset, higher magnification. Bars, 0.2 μm. (c) and (d) Immunostaining for caveolin with the immunogold method. Bars, 0.1 μm. Replica immunoelectron microscopy for caveolin in smooth muscle cells of living scn (c) or mdx (d) mice. Each right-half panel shows a list of various caveolar structures labeled with the immunogold particles. In the mdx mice (d), some immunogold particles for caveolin are localized not only on caveolae (arrows) but also on flat cell membranes (arrowheads). Asterisks: freeze-fractured P-face
40.4 Morphofunctional Significance of Caveolae
In this way, our IVCT with the replica immuno-EM could show native intracellular localization of caveolin in functioning smooth muscle cell s . It could provide higher resolution images in the replica membrane and allowed us to examine membrane localizations of caveolin around caveolae . The freeze-fracture replica electron microscopy has some advantages to examine subcellular distribution of antigen proteins [8], but it has not been performed under physiological conditions of animal organs. Moreover, we have recently revealed that caveolae in smooth muscle cells are dynamic structures which could often detach from the cell membrane, as shown in another chapter of this review book. The caveolae contain various receptors and signaling molecules [3, 8, 9] and play some important roles as microdomains of the cell membrane.
40.5 Concluding Remarks
The in vivo ultrastructural examinations are needed to reveal dynamic processes of protein–protein interactions on caveolae in living smooth muscle cell s , which were hardly examined by the conventional electron microscopic or immuno-EM methods [3, 4]. Our IVCT with the replica immuno-EM can be directly used for living animal organ s under various experimental conditions, which will be effective not only for morphofunctional investigations of the caveolae [10] but also extracellular matrices in functioning organs [11].
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