Localization of myosin, actin, and tropomyosin in rat intestinal epithelium: immunohistochemical studies at the light and electron microscope levels.

Abstract

Myosin, tropomyosin, and actin were localized in the epithelial cells of rat intestine by means of specific antibodies to chicken gizzard smooth muscle myosin, tropomyosin, and actin by immunohistochemical studies at both the light and electron microscope levels (unlabeled antibody enzyme technique) . The pattern of antibody staining was the following : (a) Anti-actin was associated with the microfilament bundles of the microvilli in their entire length, as well as with the microfilament network in the terminal web . (b) Anti-myosin was concentrated along the rootlets of the microvillar microfilament bundles and within the filamentous feltwork forming the terminal web. (c) Anti-tropomyosin showed a distribution similar to that of anti-myosin . In addition, the three antibodies also labeled the subplasmalemmal web underneath the cell membrane bordering on the basal lamina . Utilizing the above ultrastructural findings, we wish to propose a functional model of microvillar contraction .In recent years much interest has been focused on the forcegenerating apparatus responsible for the motility of intestinal epithelial brush border . This highly ordered structure proved to be an ideal model for studying the structural basis of nonmuscle cell motility .By phase-contrast microscopy, epithelia of rat intestine and of kidney tubules were shown to perform fast microvillar movements (31, 35) . Studies on isolated apical segments of intestinal epithelium demonstrated rapid microvillar retraction or contraction of the whole apical segments in response to ATP, Ca z+, and Mg t + (26, 30) . Biochemical analysis indicated the presence of myosin, tropomyosin, actin, and other associated proteins in the apical cytoplasm (3,26,28) . Recently, these biochemical findings have been confirmed by the immunofluorescent localization of myosin, tropomyosin, and actin in the apical cytoplasm of mouse, rat, and chicken intestinal epithelial cells (4,13,14,28). Ultrastructurally, the microvilli of rat intestinal epithelium contain an orderly axial bundle of actin filaments in parallel alignment, which penetrates -0 .5-1 pin into the apical cytoplasm to form a rootlet, which terminates abruptly (5) . As judged by heavy meromyosin binding, the actin filaments are Tne JOURNAL Of CELL Bioro(,y " Voruti+e

Studies on the spectrin-like protein from the intestinal brush border, TW 260/240, and characterization of its interaction with the cytoskeleton and actin.

“…The rationale for these experiments was that tropomyosin is present on the actin filaments of the microvillus core rootlets (33,20) to which TW 260/ 240 molecules are presumably bound. Results of these experiments (Fig.…”

“…Electron microscopy-immunolocalization studies using antibodies to either TW 260/240 (29) or brain fodrin (30) indicate that TW 260/240 (or its equivalent in mammalian brush borders) comprises at least one type of "interdigitating" filaments that cross-link core rootlets. A second class of cross-linking filaments that appear to be greater in diameter than the TW 260/240 filaments when visualized by the quick-freeze, deep-etch rotary replication (QFDERR)' technique (27) may be comprised ofbrush border myosin that, like TW 260/240, is localized exclusively in the terminal web (31)(32)(33)(34). The studies of Hirokawa et al .…”

Quick-freeze, deep-etch visualization of the cytoskeleton beneath surface differentiations of intestinal epithelial cells.

“…Light and EM immunocytochemical studies to date (2,7,9,18) have shown that the apices of intestinal cells contain the essential contractile proteins, including actin, myosin, tropomyosin, and a-actinin . EM has not revealed how all these proteins could fit together to provide rigid support and flexible contractility in an appropriate manner .…”

“…A large number of biochemical and immunocytochemical studies have focused on this region of the intestinal cell . These studies have defined the proteins that are present in the terminal web, but they have THE JOURNAL OF CELL BIOLOGY " VOLUME 91 NOVEMBER 1981 399-409 © The Rockefeller University Press -0021-9525/81/11/399/11 $1 .00not been able to demonstrate exactly how these proteins are put together (2,3,4,(6)(7)(8)(9)18) . The micrographs presented here display certain advantages over traditional transmission electron micrographs of plastic-embedded and sectioned material, and thus help to reveal how the terminal web is organized.…”

“…not been able to demonstrate exactly how these proteins are put together (2,3,4,(6)(7)(8)(9)18) . The micrographs presented here display certain advantages over traditional transmission electron micrographs of plastic-embedded and sectioned material, and thus help to reveal how the terminal web is organized.…”