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

1978 ◽  
Vol 79 (2) ◽  
pp. 444-453 ◽  
Author(s):  
MS Mooseker ◽  
TD Pollard
Keyword(s):  
Ionic Strength ◽  
Epithelial Cells ◽  
Atpase Activity ◽  
Brush Border ◽  
Intestinal Epithelial Cells ◽  
Gel Filtration ◽  
Intestinal Epithelial ◽  
Terminal Web ◽  
Brush Borders ◽  
Low Ionic Strength

The brush border of intestinal epithelial cells consists of a tightly packed array of microvilli, each of which contains a core of actin filaments. It has been postulated that microvillar movements are mediated by myosin interactions in the terminal web with the basal ends of these actin cores (Mooseker, M.S. 1976. J. Cell. Biol. 71:417-433). We report here that two predictions of this model are correct: (a) The brush border contains myosin, and (b) myosin is located in the terminal web. Myosin is isolated in 70 percent purity by solubilization of Triton-treated brush borders in 0.6 M KI, and separation of the components by gel filtration. Most of the remaining contaminants can be removed by precipitation of the myosin at low ionic strength. This yield is approximately 1 mg of myosin/30 mg of solubilized brush border protein. The molecule consists of three subunits with molecular weights of 200,000, 19,000, and 17,000 daltons in a 1:1:1 M ratio. At low ionic strength, the myosin forms small, bipolar filaments with dimensions of 300 X 11nm, that are similar to filaments seen previously in the terminal web of isolated brush borders. Like that of other vertebrate, nonmuscle myosins, the ATPase activity of isolated brush border myosin in 0.6 M KCI is highest with EDTA (1 μmol P(i)/mg-min; 37 degrees C), intermediate with Ca++ (0.4 μmol P(i)/mg-min), and low with Mg++ (0.01 μmol P(i)/mg-min). Actin does not stimulate the Mg-ATPase activity of the isolated enzyme. Antibodies against the rod fragment of human platelet myosin cross-react by immunodiffusion with brush border myosin. Staining of isolated mouse or chicken brush borders with rhodamine-antimyosin demonstrates that myosin is localized exclusively in the terminal web.

The Fine-Structural Organization of the Brush Border of Intestinal Epithelial Cells

1971 ◽  
Vol 8 (3) ◽  
pp. 573-599
Author(s):  
T. M. MUKHERJEE ◽  
L. A. STAEHELIN
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Brush Border ◽  
Intestinal Epithelial Cells ◽  
Membrane Surface ◽  
Intestinal Epithelial ◽  
The Core ◽  
Unit Structure ◽  
Terminal Web ◽  
Freeze Etching

The fine structure of the brush border of intestinal epithelial cells of the mouse has been studied with both normal sectioning and freeze-etching techniques. Freeze-etching reveals the plasma membrane of the microvilli as consisting of a continuous layer, that is split during the cleaving process, in which numerous particles, 5-9 nm in diameter, are embedded, while other particle-like structures, with diameters of 7-10 nm, appear attached to the true outer membrane surface. The mucopolysaccharide surface coats of the microvilli show up more clearly in sectioned material than in freeze-etched specimens. Inside each microvillus 2 different filament systems can be demonstrated: (1) bundles of fairly closely packed and straight core microfilaments, which lead into the tip of the microvillus, and (2) short cross-filaments. Under suitable conditions the core microfilaments display a sub-unit structure with a repeating distance of approximately 6 nm. The diameter of a microfilament can vary along its length from 6 to 11 nm. Two strands of globular particles wound helically around each other seem to make up each microfilament. These and other data support the idea that the core microfilaments are actin-like. No substructure has been found on the cross-filaments, which have an orientation approximately radial to the axis of the microvilli and seem to be attached at one end to the core microfilaments and at the other to the inner surface of the microvillous membrane. The interwoven terminal web filaments also show no substructure. They form a continuous flexible platform-like structure into which the bundles of core microfilaments extend. Some terminal web filaments appear attached to the plasma membrane between the microvilli. It is suggested that the core microfilaments represent mechanical supporting elements and that the terminal web and cross-filaments are tensile elements of the brush border. In addition all 3 filament systems may also be involved in possible contractile movements of the microvilli.

scholarly journals STUDIES ON THE ORGANIZATION OF THE BRUSH BORDER IN INTESTINAL EPITHELIAL CELLS

1965 ◽  
Vol 26 (3) ◽  
pp. 687-691 ◽  
Author(s):  
Alexander Eichholz ◽  
Robert K. Crane
Keyword(s):  
Epithelial Cells ◽  
Brush Border ◽  
Intestinal Epithelial Cells ◽  
Companion Paper ◽  
Density Gradients ◽  
Intestinal Epithelial ◽  
Fibrillar Material ◽  
Brush Borders ◽  
Individual Bands ◽  
Positively Charged

Brush borders isolated from the epithelial cells of hamster jejunum have been dissociated by treatment with 1 M Tris(hydroxymethyl)aminomethane into several subfractions which can be separated by means of centrifugation on glycerol density gradients. Investigation of the chemical specificity of disrupting agents suggests that the amino group of Tris, in its positively charged state, is involved. Five individual bands or fractions have been routinely recovered from density gradients. The distribution of alkaline phosphatase and maltase activities among these fractions has been studied and the results indicate that both enzymes are predominantly associated with one fraction which has been identified in a companion paper as being composed of the membranes of the brush border microvilli. A fibrillar material of unidentified origin has also been obtained from Tris-disrupted brush borders.

scholarly journals Role of myosin in terminal web contraction in isolated intestinal epithelial brush borders.

1985 ◽  
Vol 100 (5) ◽  
pp. 1647-1655 ◽  
Author(s):  
T C Keller ◽  
K A Conzelman ◽  
R Chasan ◽  
M S Mooseker
Keyword(s):  
Atpase Activity ◽  
Intestinal Epithelium ◽  
Brush Border ◽  
Time Course ◽  
Intestinal Epithelial ◽  
Terminal Web ◽  
Brush Borders

We have investigated the role of myosin in contraction of the terminal web in brush borders isolated from intestinal epithelium. At 37 degrees C under conditions that stimulate terminal web contraction (1 microM Ca++ and ATP), most (60-70%) of the myosin is released from the brush border. Approximately 80% of the myosin is also released by ATP at 0 degree C, in the absence of contraction. Preextraction of this 80% of the myosin from brush borders with ATP has no effect on either the time course or extent of subsequently stimulated contraction. However, contraction is inhibited by removal of all of the myosin with 0.6 M KCl and ATP. Contraction is also inhibited by an antibody to brush border myosin, which inhibits both the ATPase activity of brush border myosin and its ability to form stable bipolar polymers. These results indicate that although functional myosin is absolutely required for terminal web contraction only approximately 20% of the brush border myosin is actually necessary. This raises the possibility that there are at least two different subsets of myosin in the terminal web.

scholarly journals Localization of actin and microfilament-associated proteins in the microvilli and terminal web of the intestinal brush border by immunofluorescence microscopy.

1978 ◽  
Vol 79 (3) ◽  
pp. 839-845 ◽  
Author(s):  
A Bretscher ◽  
K Weber
Keyword(s):  
Epithelial Cells ◽  
Indirect Immunofluorescence ◽  
Brush Border ◽  
Immunofluorescence Microscopy ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
Indirect Immunofluorescence Microscopy ◽  
Terminal Web ◽  
Intestinal Brush Border ◽  
Associated Proteins

Indirect immunofluorescence microscopy was used to localize microfilament-associated proteins in the brush border of mouse intestinal epithelial cells. As expected, antibodies to actin decorated the microfilaments of the microvilli, giving rise to a very intense fluorescence. By contrast, antibodies to myosin, tropomyosin, filamin, and alpha-actinin did not decorate the microvilli. All these antibodies, however, decorated the terminal web region of the brush border. Myosin, tropomyosin, and alpha-actinin, although present throughout the terminal web, were found to be preferentially located around the periphery of the organelle. Therefore, two classes of microfilamentous structures can be documented in the brush border. First, the highly ordered microfilaments which make up the cores of the microvilli apparently lack the associated proteins. Second, seemingly less-ordered microfilaments are found in the terminal web, in which region the myosin, tropomyosin, filamin and alpha-actinin are located.

scholarly journals The spectrin-related molecule, TW-260/240, cross-links the actin bundles of the microvillus rootlets in the brush borders of intestinal epithelial cells.

1983 ◽  
Vol 96 (5) ◽  
pp. 1491-1496 ◽  
Author(s):  
J R Glenney ◽  
P Glenney ◽  
K Weber
Keyword(s):  
Epithelial Cells ◽  
Actin Filaments ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
Cross Link ◽  
Actin Bundles ◽  
Terminal Web ◽  
Brush Borders ◽  
Cross Links

Previous studies have shown that molecules related to erythrocyte spectrin are present in the cortical cytoplasm of nonerythroid cells. We report here the localization by immunoelectron microscopy of one such molecule, TW-260/240, in the brush border of intestinal epithelial cells. Using highly specific antibodies against TW-260 and TW-240 as well as antibodies against fodrin, another spectrinlike molecule, we have found that the TW-260/240 molecules are displayed between rootlets at all levels of the terminal web. Occasionally, extended structures appear labeled suggestive of the fine filaments known to cross-link actin bundles. These results are in line with previous in vitro studies showing that TW-260/240 binds to, and cross-links, actin filaments. The results are discussed in terms of a model in which rootlets are immobilized in the terminal web in a matrix of TW-260/240.

scholarly journals Organization of an actin filament-membrane complex. Filament polarity and membrane attachment in the microvilli of intestinal epithelial cells.

1975 ◽  
Vol 67 (3) ◽  
pp. 725-743 ◽  
Author(s):  
M S Mooseker ◽  
L G Tilney
Keyword(s):  
Epithelial Cells ◽  
Actin Filament ◽  
Brush Border ◽  
Actin Filaments ◽  
Functional Organization ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
Membrane Complex ◽  
Brush Borders ◽  
Filament Bundles

The association of actin filaments with membranes is now recognized as an important parameter in the motility of nonmuscle cells. We have investigated the organization of one of the most extensive and highly ordered actin filament-membrane complexes in nature, the brush border of intestinal epithelial cells. Through the analysis of isolated, demembranated brush borders decorated with the myosin subfragment, S1, we have determined that all the microvillar actin filaments have the same polarity. The S1 arrowhead complexes point away from the site of attachment of actin filaments at the apical tip of the microvillar membrane. In addition to the end-on attachment of actin filaments at the tip of the microvillus, these filaments are also connected to the plasma membrane all along their lengths by periodic (33 nm) cross bridges. These bridges were best observed in isolated brush borders incubated in high concentrations of Mg++. Their visibility is attributed to the induction of actin paracrystals in the filament bundles of the microvilli. Finally, we present evidence for the presence of myosinlike filaments in the terminal web region of the brush border. A model for the functional organization of actin and myosin in the brush border is presented.

scholarly journals Brush border motility. Microvillar contraction in triton-treated brush borders isolated from intestinal epithelium.

1976 ◽  
Vol 71 (2) ◽  
pp. 417-433 ◽  
Author(s):  
M S Mooseker
Keyword(s):  
Molecular Weight ◽  
Brush Border ◽  
Intestinal Epithelial Cells ◽  
Major Protein ◽  
Intestinal Epithelial ◽  
Terminal Web ◽  
Brush Borders ◽  
Filament Bundles ◽  
Triton X

The brush border of intestinal epithelial cells consists of an array of tightly packed microvilli. Within each microvillus is a bundle of 20-30 actin filaments. The basal ends of the filament bundles are embedded in and interconected by a filamentous meshwork, the terminal web, which lies directly beneath the microvilli. When calcium and ATP are added to isolated brush borders that have been treated with the detergent, Triton X-100, the microvillar filament bundles rapidly retract into and through the terminal web region. Biochemical studies of brush border contractile proteins suggest that the observed microvillar contraction is actomyosin mediated. We have shown previously that the major protein of the brush border's actin (Tilney, L. G., and M. S. Mooseker. 1971. Proc. Natl. Acad. Sci. U. S. A. 68:2611-2615). The brush border also contains a protein with the same molecular weight as the heavy chain subunit of myosin (200, 000 daltons). In addition, preparations of demembranated brush borders exhibit potassium-EDTA ATPase activity of 0.02 mumol phosphate/mg-min (22 degrees C); this assay is diagnostic for myosin-like ATPase isolated from vertebrate sources. Other proteins of the brush border include a 30,000 dalton protein with properties similar to those of tropomyosin, and a protein with the same molecular weight as the Z band protein, alpha-actinin (95,000 daltons). How these observations bear on the basis for microvillar movements in vivo is discussed within the framework of our recent model for the organization of actin and myosin in the brush border (Mooseker, M. S., and L. G. Tilney. 1975. J. Cell Biol. 67:725-743).

scholarly journals Ca++-calmodulin-dependent phosphorylation of myosin, and its role in brush border contraction in vitro.

1982 ◽  
Vol 95 (3) ◽  
pp. 943-959 ◽  
Author(s):  
T C Keller ◽  
M S Mooseker
Keyword(s):  
Light Chain ◽  
Brush Border ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
Anti Psychotic ◽  
Terminal Web ◽  
Brush Borders

We have reinvestigated the effects of Ca++ and ATP on brush borders isolated from intestinal epithelial cells. At 37 degrees C, Ca++ (1 microM) and ATP cause a dramatic contraction of brush border terminal webs, not a retraction of microvilli as previously reported (M. S. Mooseker, 1976, J. Cell Biol. 71:417-433). Terminal web contraction, which occurs over the course of 1-5 min at 37 degrees C, actively constricts brush borders at the level of their zonula adherens. Contraction requires ATP, is stimulated by Ca++ (1 microM), and occurs in both membrane-intact and demembranated brush borders. Ca++ -dependent-solation of microvillus cores requires a concentration of Ca++ slightly greater (10 microM) than that required for contraction. Under conditions in which brush borders contract, many proteins in the isolated brush borders become phosphorylated. However, the phosphorylation of only one of the brush border proteins, the 20,000 dalton (20-kdalton) light chain of brush border myosin (BBMLC20), is stimulated by Ca++. At 37 degrees C, BBMLC20 phosphorylation correlates directly with brush border contraction. Furthermore, both BBMLC20 phosphorylation and brush border contraction are inhibited by trifluoperazine, an anti-psychotic phenothiazine that inhibits calmodulin activity. These results indicate that Ca++ regulates brush border contractility in vitro by stimulating cytoskeleton-associated, Ca++- and calmodulin-dependent brush border myosin light chain kinase.

Sign in / Sign up

Export Citation Format

Share Document