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

Abstract

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

Contributions of the β‐subunit to spectrin structure and function

“…TW 260/240 was purified by a modification of the method of Glenney et al [ 1982bl as described by Pearl et al [1984]. Chickens erythrocytes were harvested according to established procedures [Granger and Lazarides, 19841.…”

Functional diversity among spectrin isoforms

“…The direct visualization of ankyrin [Davis and Bennett, 1984b], calmodulin [Tsukita et al, 19831, and antigenic sites [Glenney et al, 1983al on mammalian brain spectrin and the mapping of human brain spectrin functional domains [Harris and Morrow, 1988b;Harris et al, 19881 indicate that these tetramers are arranged in an erythroid-like head-to-head manner. Given the similar composition and morphology of other nonerythroid spectrins, it is likely that all spectrin tetramers are arranged in a similar Bennett [ 19851;3, Bloch and Morrow [1989]; 4, Branton et al [1981]; 5, Byers et al [ 19871;6 , Coleman et al [1989]; 7, Dubreuil et al [1987]; 8, Fishkind et al [1987]; 9, Giebelhaus et al 119871;10, Glenney and Glenney [1983a]; 11, ; 12, Glenney et al [1982b]; 13, ; 14, Goodman and Zagon [1986]; 15, Kuramochi et al [1986]; 16, Lazarides and Nelson [1985]; 17, Marchesi [1985]; 18, ; 19, Pearl et al [1984];20, Pollard 119841;21, Shatten et al [1986]; 22, Shile et al [1985]; 23, Repasky et al [1982]; 24, Riederer et al [1986].…”

“…In any event, the fact that TW 260/240 stimulates actomyosin ATPase activity and cross-links actin filaments in a Ca2+-independent manner [Coleman and Mooseker, 19851 suggests that this spectrin's actin binding is very different. Unlike other nonerythroid spectrins, TW 260/240 does not bind to or cross-link actin under physiological conditions [Pearl et al, 1984;Fishkind et al, 19851, even in the presence of erythroid protein 4.1 [Coleman et al, 1987,19891.…”

“…role of the spectrins is that of regulating receptorfilament interactions. As such, it is conceivable that the spectrins may be involved with such processes as membrane trafficking in the intestinal brush border and sea urchin egg/embryo [Hirokawa et al, 1982[Hirokawa et al, , 1983Pearl et al, 1984;Bonder et al, 1989;Fishkind et al, 19881, secretion in chromaffin cells [Perrin and Aunis, 19851, topographic membrane assembly in epithelial cells Veshnock, 1986, 1987b;Morrow et al, 19891, receptor regulation and presentation in the nervous system [Lynch and Baudry, 19841, lymphocyte capping [reviewed in Bourguignon and Bourguignon, 19841, or substrate-mediated endothelial cell differentiation [Pratt et al, 19841 (Fig. 4).…”

Apico-basal elongation requires a drebrin-E–EB3 complex in columnar human epithelial cells

“…Actin rearrangements must occur in order to generate the typical narrow, elongated shape of the columnar epithelial cells and the dense meshwork consisting of the apical terminal web and bundles of microvilli (Bretscher and Weber, 1980;Ezzell et al, 1989;Fath et al, 1990;Lin et al, 1994). The actin-binding proteins required to obtain this highly specialized apical morphology and a properly organized terminal web include villin, fimbrin, spectrin, myosin II and ankyrin (Heintzelman et al, 1994;Pearl et al, 1984;. Proteins belonging to the ezrin-radixin-moesin (ERM) family are also involved in regulating the formation of the subapical cytoskeleton (Berryman et al, 1993;Saotome et al, 2004), and it seems very likely that apico-basal elongation and compaction processes might require many other actin-interacting proteins.…”