Characterization and localization of myosin in the brush border of intestinal epithelial cells

Mooseker, M S; Pollard, T.D.; Fujiwara, Keigi

doi:10.1083/jcb.79.2.444

Cited by 129 publications

(87 citation statements)

References 30 publications

(30 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Brush Border Isolation : Brush borders were isolated from intestinal epithelium of chickens according to the method of Mooseker et al (31) using modifications described in Keller and Mooseker (37). In this method, brush borders, once isolated by homogenization of epithelial cells in low ionic strength medium containing divalent cation chelators, are transferred to a buffer termed brush border stabilization solution (BBSB) containing 75 mM KCI, 5 .0 mM MgSO,, 1 .0 mM EGTA, 0 .2 mM dithiothreitrol .…”

Section: Methodsmentioning

confidence: 99%

“…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 .…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Pearl¹,

Fishkind²,

Mooseker³

et al. 1984

The Journal of Cell Biology

View full text Add to dashboard Cite

The terminal web of the intestinal brush border contains a spectrin-like protein, TW 260/240 (Glenney, J. R ., Jr., P. Glenney, M. Osborne, and K. Weber, 1982, Cell, 28:843-854 .) that interconnects the "rootlet" ends of microvillar filament bundles in the terminal web (Hirokawa, N ., R. E. Cheng, and M . Willard, 1983, Cell, 32 :953-965; Glenney J . R ., P. Glenney, and K. , /. Cell Biol., 96:1491-1496 . We have investigated further the structural properties of TW 260/240 and the interaction of this protein with actin . Salt extraction of TW 260/240 from isolated brush borders results in a loss of terminal web cross-linkers primarily from the apical zone directly beneath the plasma membrane. Morphological studies on purified TW 260/240 using the rotary shadowing technique confirm earlier results that this protein is spectrin-like and is in the tetrameric state in buffers of low ionic strength . However, examination of TW 260/240 tetramers by negative staining revealed a molecule much straighter and more uniform in diameter than rotary-shadowed molecules . At salt concentrations at (150 mM KCI) and above (300 mM KCI) the physiological range, we observed a partial dissociation of tetramers into dimers that occurred at both 0°and 37°C. We also observed (in the presence of 75 mM KCI) a concentration-dependent self-association of TW 260/240 into sedimentable aggregates .We have studied the interaction of TW 260/240 with actin using techniques of cosedimentation, viscometry, and both light and electron microscopy. We observed that TW 260/240 can bind and cross-link actin filaments and that this interaction is salt-and pHdependent . Under optimum conditions (25-75 mM KCI, at pH 7 .0) TW 260/240 cross-linked F-actin into long, large-diameter bundles. The filaments within these bundles were tightly packed but loosely ordered . At higher pH (7.5) such bundles were not observed, although binding and cross-linking were detectable by co-sedimentation and viscometry. At higher salt (>150 mM KCI), the binding of TW 260/240 to actin was inhibited . The presence of skeletal muscle tropomyosin had no significant effect on the salt-dependent binding of TW 260/240 to F-actin .The ubiquitous presence of spectrin-like proteins in the "cortical" cytoplasm of cells has been recently established by numerous biochemical and immunological studies on a wide variety of cell types and tissues (reviewed in references 1 and 2). By analogy with erythrocyte spectrin

show abstract

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Pearl¹,

Fishkind²,

Mooseker³

et al. 1984

The Journal of Cell Biology

View full text Add to dashboard Cite

show abstract

“…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 .…”

Section: The Cytoskeleton Of Intestinal Cellsmentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Hirokawa¹,

Heuser²

1981

The Journal of Cell Biology

262

173

View full text Add to dashboard Cite

The cytoskeleton that supports microvilli in intestinal epithelial cells was visualized by the quick-freeze, deep-etch, rotary-replication technique (Heuser and Salpeter. 1979 . J. Cell Biol. 82 :150) . Before quick freezing, cells were exposed to detergents or broken open physically to clear away the granular material in their cytoplasm that would otherwise obscure the view . After such extraction, cells still displayed a characteristic organization of cytoskeletal filaments in their interiors. Platinum replicas of these cytoskeletons had sufficient resolution to allow us to identify the filament types present, and to determine their characteristic patterns of interaction . The most important new finding was that the apical "terminal web" in these cells, which supports the microvilli via their core bundles of actin filaments, does not itself contain very much actin but instead is comprised largely of narrow strands that interconnect adjacent actin bundles with one another and with the underlying base of intermediate filaments . These strands are slightly thinner than actin, do not display actin's 53 A periodicity, and do not decorate with myosin subfragment S1 . On the contrary, two lines of evidence suggested that these strands could include myosin molecules. First, other investigators have shown that myosin is present in the terminal web (Mooseker et al . 1978 . J. Cell Biol . 79 : 444-453), yet we could find no thick filaments in this area . Second, we found that the strands were removed completely in the process of decorating the core filament bundles with the myosin subfragment S1 , suggesting that they had been competitively displaced by exogenous myosin . We conclude that myosin may play a structural role in these cells, via its cross-linking distribution, in addition to whatever role it plays in microvillar motility .In an earlier study, we used the quick-freeze, deep-etch, rotaryreplication technique to visualize the cytoskeletons of detergent-extracted fibroblasts (10) . This study illustrated the feasibility of using such a preparative procedure to determine the overall organization of cytoskeletal filament arrays, and to identify the filaments that comprise them . However, this study did not contribute toward understanding how cytoskeletal filaments could be linked together to form such arrays.In the present report, we use this same technique to visualize the cytoskeleton inside differentiated epithelial cells of the vertebrate intestine . These cells are particularly interesting because they possess apical microvilli that have long been known to be supported by a distinct cytoplasmic specialization called the "terminal web" (5,12,20,22) . 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 demon...

show abstract

Structure of Human Placental Microvilli

Booth

Vanderpuye

1983

Ciba Foundation Symposium 95 ‐ Brush Border Membranes

View full text Add to dashboard Cite

Characterization and localization of myosin in the brush border of intestinal epithelial cells

Cited by 129 publications

References 30 publications

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

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

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

Structure of Human Placental Microvilli

Contact Info

Product

Resources

About