scholarly journals Non-muscle myosin-2 contractility-dependent actin turnover limits the length of epithelial microvilli

2020 ◽  
Author(s):  
Colbie R. Chinowsky ◽  
Julia A. Pinette ◽  
Leslie M. Meenderink ◽  
Matthew J. Tyska
Keyword(s):  
Brush Border ◽  
Isotropic Layer ◽  
Intestinal Epithelial ◽  
Actin Bundles ◽  
Terminal Web ◽  
Actin Turnover ◽  
Genetic Perturbations ◽  
And Function ◽  
Pointed End ◽  
Muscle Myosin

ABSTRACTEpithelial brush borders are large arrays of microvilli that enable efficient solute uptake from luminal spaces. In the context of the intestinal tract, brush border microvilli drive functions that are critical for physiological homeostasis, including nutrient uptake and host defense. However, cytoskeletal mechanisms that regulate the assembly and morphology of these protrusions are poorly understood. The parallel actin bundles that support microvilli have their pointed-end rootlets anchored in a highly crosslinked filamentous meshwork referred to as the “terminal web”. Although classic EM studies revealed complex ultrastructure, the composition, organization, and function of the terminal web remains unclear. Here, we identify non-muscle myosin-2C (NM2C) as a major component of the brush border terminal web. NM2C is found in a dense, isotropic layer of puncta across the sub-apical domain, which transects the rootlets of microvillar actin bundles. Puncta in this network are separated by ∼210 nm, dimensions that are comparable to the expected size of filaments formed by NM2C. In primary intestinal organoid cultures, the terminal web NM2C network is highly dynamic and exhibits continuous remodeling. Using pharmacological and genetic perturbations to disrupt NM2C activity in cultured intestinal epithelial cells, we found that this motor controls the length of growing microvilli by regulating actin turnover in a manner that requires a fully active motor domain. Our findings answer a decades old question on the function of terminal web myosin and hold broad implications for understanding apical morphogenesis in diverse epithelial systems.

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 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 Reactivation of intestinal epithelial cell brush border motility: ATP-dependent contraction via a terminal web contractile ring.

1982 ◽  
Vol 95 (3) ◽  
pp. 853-863 ◽  
Author(s):  
D R Burgess
Keyword(s):  
Electron Microscopy ◽  
Epithelial Cell ◽  
Brush Border ◽  
Intestinal Epithelial Cell ◽  
Intercellular Junctions ◽  
Intestinal Epithelial ◽  
Differential Interference Contrast Microscopy ◽  
Contractile Ring ◽  
Terminal Web ◽  
Brush Borders

Various models have been put forward suggesting ways in which brush borders from intestinal epithelial cells may be motile. Experiments documenting putative brush border motility have been performed on isolated brush borders and have generated models suggesting microvillar retraction or microvillar rootlet interactions. The reported Ca++ ATP-induced retraction of microvilli has been shown, instead, to be microvillar dissolution in response to Ca++ and not active brush border motility. I report here studies on the reactivation of motility in intact sheets of isolated intestinal epithelium. Whole epithelial sheets were glycerinated, which leaves the brush border and intercellular junctions intact, and then treated with ATP, PPi, ITP, ADP, GTP, or delta S-ATP. Analysis by video enhanced differential interference-contrast microscopy and thin-section transmission electron microscopy reveals contractions in the terminal web region causing microvilli to be fanned apart in response to ATP and delta S-ATP but not in response to ADP, PPi, ITP, or GTP. Electron microscopy reveals that the contractions occur at the level of the intermediate junction in a circumferential constriction which can pull cells completely apart. This constriction occurs in a location occupied by an actin-containing circumferential band of filaments, as demonstrated by S-1 binding, which completely encircles the terminal web at the level of the intermediate junction. Upon contraction, this band becomes denser and thicker. Since myosin, alpha-actinin and tropomyosin, in addition to actin, have been localized to this region of the terminal web, it is proposed that the intestinal epithelial cell can be motile via a circumferential terminal web contractile ring analogous to the contractile ring of dividing cells.

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 Proteomic analysis of the enterocyte brush border

2011 ◽  
Vol 300 (5) ◽  
pp. G914-G926 ◽  
Author(s):  
Russell E. McConnell ◽  
Andrew E. Benesh ◽  
Suli Mao ◽  
David L. Tabb ◽  
Matthew J. Tyska
Keyword(s):  
Brush Border ◽  
Intestinal Tract ◽  
Molecular Mechanisms ◽  
Actin Dynamics ◽  
Intestinal Epithelial ◽  
Future Studies ◽  
Nutrient Processing ◽  
Shotgun Mass Spectrometry ◽  
Membrane Bending ◽  
And Function

The brush border domain at the apex of intestinal epithelial cells is the primary site of nutrient absorption in the intestinal tract and the primary surface of interaction with microbes that reside in the lumen. Because the brush border is positioned at such a critical physiological interface, we set out to create a comprehensive list of the proteins that reside in this domain using shotgun mass spectrometry. The resulting proteome contains 646 proteins with diverse functions. In addition to the expected collection of nutrient processing and transport components, we also identified molecules expected to function in the regulation of actin dynamics, membrane bending, and extracellular adhesion. These results provide a foundation for future studies aimed at defining the molecular mechanisms underpinning brush border assembly and function.

scholarly journals Cellular titin localization in stress fibers and interaction with myosin II filaments in vitro.

1994 ◽  
Vol 126 (5) ◽  
pp. 1201-1210 ◽  
Author(s):  
K J Eilertsen ◽  
S T Kazmierski ◽  
T C Keller
Keyword(s):  
Epithelial Cell ◽  
Brush Border ◽  
Intestinal Epithelial Cell ◽  
Myosin Ii ◽  
Gel Filtration ◽  
Stress Fibers ◽  
Myosin Isoforms ◽  
Intestinal Epithelial ◽  
Muscle Myosin

We previously discovered a cellular isoform of titin (originally named T-protein) colocalized with myosin II in the terminal web domain of the chicken intestinal epithelial cell brush border cytoskeleton (Eilertsen, K.J., and T.C.S. Keller. 1992. J. Cell Biol. 119:549-557). Here, we demonstrate that cellular titin also colocalizes with myosin II filaments in stress fibers and organizes a similar array of myosin II filaments in vitro. To investigate interactions between cellular titin and myosin in vitro, we purified both proteins from isolated intestinal epithelial cell brush borders by a combination of gel filtration and hydroxyapatite column chromatography. Electron microscopy of brush border myosin bipolar filaments assembled in the presence and absence of cellular titin revealed a cellular titin-dependent side-by-side and end-to-end alignment of the filaments into highly ordered arrays. Immunogold labeling confirmed cellular titin association with the filament arrays. Under similar assembly conditions, purified chicken pectoralis muscle titin formed much less regular aggregates of muscle myosin bipolar filaments. Sucrose density gradient analyses of both cellular and muscle titin-myosin supramolecular arrays demonstrated that the cellular titin and myosin isoforms coassembled with a myosin/titin ratio of approximately 25:1, whereas the muscle isoforms coassembled with a myosin:titin ratio of approximately 38:1. No coassembly aggregates were found when cellular myosin was assembled in the presence of muscle titin or when muscle myosin was assembled in the presence of cellular titin. Our results demonstrate that cellular titin can organize an isoform-specific association of myosin II bipolar filaments and support the possibility that cellular titin is a key organizing component of the brush border and other myosin II-containing cytoskeletal structures including stress fibers.

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.

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).

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