Organization of actin, myosin, and intermediate filaments in the brush border of intestinal epithelial cells

Terminal webs prepared from mouse intestinal epithelial cells were examined by the quick-freeze, deep-etch, and rotary-replication method. The microvilli of these cells contain actin filaments that extend into the terminal web in compact bundles. Within the terminal web these bundles remain compact; few filaments are separated from the bundles and fewer still bend towards the lateral margins of the cell. Decoration with subfragment 1 (S1) of myosin confirmed that relatively few actin filaments travel horizontally in the web. Instead, between actin bundles there are complicated networks of the fibrils. Here we present two lines of evidence which suggest that myosin is one of the major cross-linkers in the terminal web. First, when brush borders are exposed to 1 mM ATP in 0.3 M KCl, they lose their normal ability to bind antimyosin antibodies as judged by immunofluorescence, and they lose the thin fibrils normally found in deep-etch replicas. Correspondingly, myosin is released into the supernatant as judged by SDS gel electrophoresis. Second, electron microscope immunocytochemistry with antimyosin antibodies followed by ferritin- conjugated second antibodies leads to ferritin deposition mainly on the fibrils at the basal part of rootlets. Deep-etching also reveals that the actin filament bundles are connected to intermediate filaments by another population of cross-linkers that are not extracted by ATP in 0.3 M KCl. From these results we conclude that myosin in the intestinal cell may not only be involved in a short range sliding-filament type of motility, but may also play a purely structural role as a long range cross-linker between microvillar rootlets.     

Reevaluation of brush border motility: calcium induces core filament solation and microvillar vesiculation

The report that microvillar cores of isolated, demembranated brush borders retract into the terminal web in the presence of Ca(++) and ATP has been widely cited as an example of Ca(++)-regulated nonmuscle cell motility. Because of recent findings that microvillar core actin filaments are cross-linked by villin which, in the presence of micromolar Ca(++), fragments actin filaments, we used the techniques of video enhanced differential interference contrast, immunofluorescence, and phase contrast microscopy and thin-section electron microscopy (EM) to reexamine the question of contraction of isolated intestinal cell brush borders. Analysis of video enhanced light microscopic images of Triton- demembranated brush borders treated with a buffered Ca(++) solution shows the cores disintegrating with the terminal web remaining intact; membranated brush borders show the microvilli to vesiculate with Ca(++). Using Ca(++)/EGTA buffers, it is found that micromolar free Ca(++) causes core filament dissolution in membranated or demembranated brush borders, Ca(++) causes microvillar core solation followed by complete vesiculation of the microvillar membrane. The lengths of microvilli cores and rootlets were measured in thin sections of membranated and demembranated controls, in Ca(++)-, Ca(++) + ATP-, and in ATP-treated brush borders. Results of these measurements show that Ca(++) alone causes the complete solation of the microvillar cores, yet the rootlets in the terminal web region remain of normal length. These results show that microvilli do not retract into the terminal web in response to Ca(++) and ATP but rather that the microvillar cores disintegrate. NBD-phallicidin localization of actin and fluorescent antibodies to myosin reveal a circumferential band of actin and myosin in mildly permeabilized cells in the region of the junctional complex. The presence of these contractile proteins in this region, where other studies have shown a circumferential band of thin filaments, is consistent with the hypothesis that brush borders may be motile through the circumferential constriction of this “contractile ring,” and is also consistent with the observations that ATP-treated brush borders become cup shaped as if there had been a circumferential constriction.     

Plastin 1 Binds to Keratin and Is Required for Terminal Web Assembly in the Intestinal Epithelium

Plastin 1 (I-plastin, fimbrin) along with villin and espin is a prominent actin-bundling protein of the intestinal brush border microvilli. We demonstrate here that plastin 1 accumulates in the terminal web and interacts with keratin 19, possibly contributing to anchoring the rootlets to the keratin network. This prompted us to investigate the importance of plastin 1 in brush border assembly. Although in vivo neither villin nor espin is required for brush border structure, plastin 1-deficient mice have conspicuous ultrastructural alterations: microvilli are shorter and constricted at their base, and, strikingly, their core actin bundles lack true rootlets. The composition of the microvilli themselves is apparently normal, whereas that of the terminal web is profoundly altered. Although the plastin 1 knockout mice do not show any overt gross phenotype and present a normal intestinal microanatomy, the alterations result in increased fragility of the epithelium. This is seen as an increased sensitivity of the brush border to biochemical manipulations, decreased transepithelial resistance, and increased sensitivity to dextran sodium sulfate-induced colitis. Plastin 1 thus emerges as an important regulator of brush border morphology and stability through a novel role in the organization of the terminal web, possibly by connecting actin filaments to the underlying intermediate filament network.     

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

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.     

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

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.     

Developmental organization of the intestinal brush-border cytoskeleton

At the terminal web of chicken intestinal epithelial cell, the actin bundles are cross-linked by a fine filamentous network of actin-associated cross-linkers. Myosin, fodrin, and TW 260/240 have been identified as major components of the cross-linkers. We studied the development of the cross-linkers by quick-freeze, deep-etch electron microscopy, and the expression of cross-linker proteins (myosin, fodrin 240, and TW 260) by immunofluorescence and immunoblotting analysis during the embryogenesis. Microvilli start to form at 5-7 days, and the rootlets begin to elongate at 10 days. At an early stage of the development of the terminal web (13 days), fodrin 240 and a small amount of myosin are expressed, and a few actin-associated cross-linkers are present between the rootlets. However, TW 260 is not expressed at this stage. At an intermediate stage (19 days), the amount of myosin increases, and TW 260 begins to be expressed. The number of cross-linkers associated with the unit length of the rootlets is 24/microns. At the final stage of the terminal web formation (2 days after hatching), the amount of fodrin 240, myosin, and TW 260 is similar to the adult level, and the number of the actin-associated cross-linkers per unit length of the rootlet is 27/microns (approximately 85% of the adult). These results suggest that the synthesis of cross-linker proteins may be intricately regulated to achieve the desired density of cross-linkages at each developmental stage: at early and intermediate stages, sufficient and not an excess of cross-linkages are formed; and at a final stage, a higher complexity of cross-linkages is achieved. In addition, there is a differential expression of the components of the actin-associated cross-linkers: myosin and fodrin could be early components of the cross-linkers involved in the basic stabilization of the terminal web structure, whereas TW 260/240 becomes incorporated later, possibly involved in the stabilization preparatory to the rapid elongation of microvilli, which occurs after the formation of the terminal web.     

Identification of brush cells in the alimentary and respiratory system by antibodies to villin and fimbrin

Summary Brush cells represent a population of epithelial cells with unknown function, which are scattered throughout the epithelial lining of both the respiratory system and the alimentary system. These cells are reliably distinguished from other epithelial cells only at the ultrastructural level by the presence of an apical tuft of stiff microvilli and extremely long microvillar rootlets that may project down to the perinuclear space. In the present study we show that brush cells can be identified in tissue sections even at the light microscopic level by immunostaining with antibodies against villin and fimbrin, two proteins that crosslink actin filaments to form bundles. In brush cells, villin and fimbrin are not only present in the actin filament core bundles of apical microvilli and their long rootlets but, in addition, both proteins are also associated with microvilli extending from the basolateral cell surface of the brush cells. Basolateral immunostaining specific for villin and fimbrin does not occur in any other epithelial cell type of the respiratory and alimentary tract. Thus immunostaining with antibodies against both proteins allows unequivocal identification of individual brush cells even in sectional planes that do not contain the brightly stained apical tuft of microvilli and their long rootlets.     

The apical disposition of the Caenorhabditis elegans intestinal terminal web is maintained by LET-413

We wish to understand how organ-specific structures assemble during embryonic development. In the present paper, we consider what determines the subapical position of the terminal web in the intestinal cells of the nematode Caenorhabditis elegans. The terminal web refers to the organelle-depleted, intermediate filament-rich layer of cytoplasm that underlies the apical microvilli of polarized epithelial cells. It is generally regarded as the anchor for actin rootlets protruding from the microvillar cores. We demonstrate that: (i) the widely used monoclonal antibody MH33 reacts (only) with the gut-specific intermediate filament protein encoded by the ifb-2 gene; (ii) IFB-2 protein accumulates near the gut lumen beginning at the lima bean stage of embryogenesis and remains associated with the gut lumen into adulthood; and (iii) as revealed by immunoelectron microscopy, IFB-2 protein is confined to a discrete circumferential subapical layer within the intestinal terminal web (known in nematodes as the "endotube"); this layer joins directly to the apical junction complexes that connect adjacent gut cells. To investigate what determines the disposition of the IFB-2-containing structure as the terminal web assembles during development, RNAi was used to remove the functions of gene products previously shown to be involved in the overall apicobasal polarity of the developing gut cell. Removal of dlg-1, ajm-1, or hmp-1 function has little effect on the overall position or continuity of the terminal web IFB-2-containing layer. In contrast, removal of the function of the let-413 gene leads to a basolateral expansion of the terminal web, to the point where it can now extend around the entire circumference of the gut cell. The same treatment also leads to concordant basolateral expansion of both gut cell cortical actin and the actin-associated protein ERM-1. LET-413 has previously been shown to be basolaterally located and to prevent the basolateral expansion of several individual apical proteins. In the present context, we conclude that LET-413 is also necessary to maintain the entire terminal web or brush border assembly at the apical surface of C. elegans gut cells, a dramatic example of the so-called "fence" function ascribed to epithelial cell junctions. On the other hand, LET-413 is not necessary to establish this apical location during early development. Finally, the distance at which the terminal web intermediate filament layer lies beneath the gut cell surface (both apical and basolateral) must be determined independently of apical junction position.     

A heterologous in-cell assay for investigating intermicrovillar adhesion complex interactions reveals a novel protrusion length-matching mechanism

Solute transporting epithelial cells build arrays of microvilli on their apical surface to increase membrane scaffolding capacity and enhance function potential. In epithelial tissues such as the kidney and gut, microvilli are length-matched and assembled into tightly packed “brush borders,” which are organized by ∼50-nm thread-like links that form between the distal tips of adjacent protrusions. Composed of protocadherins CDHR2 and CDHR5, adhesion links are stabilized at the tips by a cytoplasmic tripartite module containing the scaffolds USH1C and ANKS4B and the actin-based motor MYO7B. Because several questions about the formation and function of this “intermicrovillar adhesion complex” remain open, we devised a system that allows one to study individual binary interactions between specific complex components and MYO7B. Our approach employs a chimeric myosin consisting of the MYO10 motor domain fused to the MYO7B cargo-binding tail domain. When expressed in HeLa cells, which do not normally produce adhesion complex proteins, this chimera trafficked to the tips of filopodia and was also able to transport individual complex components to these sites. Unexpectedly, the MYO10–MYO7B chimera was able to deliver CDHR2 and CDHR5 to distal tips in the absence of USH1C or ANKS4B. Cells engineered to localize high levels of CDHR2 at filopodial tips acquired interfilopodial adhesion and exhibited a striking dynamic length-matching activity that aligned distal tips over time. These findings deepen our understanding of mechanisms that promote the distal tip accumulation of intermicrovillar adhesion complex components and also offer insight on how epithelial cells minimize microvillar length variability.     

Mechanism of brush border contractility studied by the quick-freeze, deep-etch method

We have analyzed terminal web contraction in sheets of glycerinated chicken small intestine epithelium and in isolated intestinal brush borders using a quick-freeze, deep-etch, rotary shadow replication technique. In the presence of Mg-ATP at 37 degrees C, the terminal web region of each cell in the glycerinated sheet and of each isolated brush border became severely constricted at the level of its zonula adherens (ZA). Consequently, the individual brush borders rounded up, splaying out their microvilli in fanlike patterns. The most prominent ultrastructural changes that occurred during terminal web contraction were a dramatic decrease in the diameter of the circumferential ring composed of a bundle of 8-9-nm filaments adjacent to the zonula adherens and a decrease in the number of cross-linkers between the microvillus rootlets. Microvilli were not retracted into the terminal web. We have used myosin S1 decoration to demonstrate that most of the circumferential bundle filaments are actin and that the actin filaments are arranged in the bundle with mixed polarity. Some filaments within the bundle did not decorate with myosin S1 and had tiny projections that appeared to be attached to adjacent actin filaments. Because of their morphology and immunofluorescent localization of myosin within this region of the terminal web, we propose that these undecorated filaments are myosin. From these results, we conclude that brush border contraction is caused primarily by an active sliding of actin and myosin filaments within the circumferential bundle of filaments associated with the ZA.     

Organization of the actin filament cytoskeleton in the intestinal brush border: a quantitative and qualitative immunoelectron microscope study

In the present study we have used immunogold labeling of ultrathin sections of the intact chicken and human intestinal epithelium to obtain further insight into the molecular structure of the brush-border cytoskeleton. Actin, villin, and fimbrin were found within the entire microvillus filament bundle, from the tip to the basal end of the rootlets, but were virtually absent from the space between the rootlets. This suggests that the bulk of actin in the brush border is kept in a polymerized and cross-linked state and that horizontally deployed actin filaments are virtually absent. About 70% of the label specific for the 110-kD protein that links the microvillus core bundle to the lipid bilayer was found overlying the microvilli. The remaining label was associated with rootlets and the interrootlet space, where some label was regularly observed in association with vesicles. Since the terminal web did not contain any significant amounts of tubulin and microtubules, the present findings would support a recently proposed hypothesis that the 110-kD protein (which displays properties of an actin-activated, myosin-like ATPase) might also be involved in the transport of vesicles through the terminal web. Label specific for myosin and alpha-actinin was confined to the interrootlet space and was absent from the rootlets. About 10-15% of the myosin label and 70-80% of the alpha-actinin label was observed within the circumferential band of actin filaments at the zonula adherens, where myosin and alpha-actinin displayed a clustered, interrupted pattern that resembles the spacing of these proteins observed in other contractile systems. This circular filament ring did not contain villin, fimbrin, or the 110-kD protein. Finally, actin-specific label was observed in close association with the cytoplasmic aspect of the zonula occludens, suggesting that tight junctions are structurally connected to the microfilament system.     

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

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 53A 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.     

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

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.     

Purification of microvilli and an analysis of the protein components of the microfilament core bundle

A fast and convenient method for the purification of microvilli from chicken intestinal brush borders is described. The microvilli appear morphologically very similar to those found on intact brush borders. Removal of the microvillus membrane from the microvilli by Triton X-100 treatment reveals compact bundles of microfilaments with small regularly spaced projections along their length. SDS-polyacrylamide gel analysis of the protein components of the brush border, the microvilli and the microvillus core bundles shows that little or no tropomyosin, myosin or filamin is found in the microvillus, whereas polypeptide chains with mobilities characteristic for these proteins are present in the whole brush border. The majority of the microvillus core protein is actin, and the other major protein present has a polypeptide molecular weight of 95 000. Total actin from both brush borders and microvilli, characterized by isoelectric focussing analysis, contained about 40% β actin and 60% γ actin. The presence of both the β and γ cytoplasmic actins in the highly ordered parallel arrays of microfilaments of the microvilli is discussed in light of hypotheses for different functional roles of these two actin species.     

Reorganization of the apical cytoskeleton of uterine epithelial cells during early pregnancy in the rat: a study with myosin subfragment 1.

Actin filaments were identified in the epithelial cells of rat uterus following detergent extraction and decoration of microfilaments (MF) with myosin subfragment 1 (S1). MF connections with cytoplasmic organelles and the apical plasma membrane are also described. Transmission electron microscopy revealed that the regular microvilli of non-pregnant, oestrous animals contain several decorated MF with rootlets descending into a densely filamentous terminal web. Following mating, the actin cytoskeleton was examined on days 1, 3 and 6 of pregnancy. In this period, the irregular projections that replace MV assumed an underlying, dense network of decorated MF, whilst smoother surfaces displayed few cytoplasmic filaments. At the time of blastocyst implantation, a structured terminal web was no longer present. Structural details were revealed concerning the contents of large, bleb-like projections found on the apical surface.     

A new view of mechanotransduction and strain amplification in cells with microvilli and cell processes

In this paper we shall describe new mechanical models for the deformation of the actin filament bundles in kidney microvilli and osteocytic cell processes to see whether these cellular extensions, like the stereocilia on hair cells in the inner ear, can function as mechanotransducers when subject to physiological flow. In the case of kidney microvilli we show that the hydrodynamic drag forces at the microvilli tip are <0.01 pN, but there is a 38-fold force amplification on the actin filaments at the base of the microvilli due to the resisting moment in its terminal web. This leads to forces that are more than sufficient to deform the terminal web complex of the microvillus where ezrin has been shown to couple the actin cytoskeleton to the Na(+)/H(+) exchanger. In the case of bone cell processes we show that the actin filament bundles have an effective Young's modulus that is 200 times > the measured modulus for the actin gel in the cell body. It is, therefore, unlikely that bone cell processes respond in vivo to fluid shear stress, as proposed in [59]. However, we show that the fluid drag forces on the pericellular matrix which tethers the cell processes to the canalicular wall can produce a 20-100 fold amplification of bone tissue strains in the actin filament bundle of the cell process.     

Maintenance of the intestinal tube in Caenorhabditis elegans: the role of the intermediate filament protein IFC-2

The Caenorhabditis elegans intestinal lumen is surrounded by a dense cytoplasmic network that is laterally attached to the junctional complex and is referred to as the endotube. It localizes to the terminal web region which anchors the microvillar actin filament bundles and is particularly rich in intermediate filaments. To examine their role in intestinal morphogenesis and function, C. elegans reporter strains were generated expressing intestine-specific CFP-tagged intermediate filament polypeptide IFB-2. When these animals were treated with dsRNA against intestinal intermediate filament polypeptide IFC-2, the endotube developed multiple bubble-shaped invaginations that protruded into the enterocytic cytoplasm. The irregularly widened lumen remained surrounded by a continuous IFB-2::CFP-labeled layer. Comparable but somewhat mitigated phenotypic changes were also noted in wild-type N2 worms treated with ifc-2 (RNAi). Junctional complexes were ultrastructurally and functionally normal and the apical domain of intestinal cells was also not altered. These observations demonstrate that IFC-2 is important for structural maintenance of the intestinal tube but is not needed for establishment of the endotube and epithelial cell polarity.     

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

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.     

A CD317/tetherin–RICH2 complex plays a critical role in the organization of the subapical actin cytoskeleton in polarized epithelial cells

CD317/tetherin is a lipid raft–associated integral membrane protein with a novel topology. It has a short N-terminal cytosolic domain, a conventional transmembrane domain, and a C-terminal glycosyl-phosphatidylinositol anchor. We now show that CD317 is expressed at the apical surface of polarized epithelial cells, where it interacts indirectly with the underlying actin cytoskeleton. CD317 is linked to the apical actin network via the proteins RICH2, EBP50, and ezrin. Knocking down expression of either CD317 or RICH2 gives rise to the same phenotype: a loss of the apical actin network with concomitant loss of apical microvilli, an increase in actin bundles at the basal surface, and a reduction in cell height without any loss of tight junctions, transepithelial resistance, or the polarized targeting of apical and basolateral membrane proteins. Thus, CD317 provides a physical link between lipid rafts and the apical actin network in polarized epithelial cells and is crucial for the maintenance of microvilli in such cells.     

Sequence of human villin: a large duplicated domain homologous with other actin-severing proteins and a unique small carboxy-terminal domain related to villin specificity

Villin is a calcium-regulated actin-binding protein that caps, severs, and bundles actin filaments in vitro. This 92,500-D protein is a major constituent of the actin bundles within the microvilli of the brush border surface of intestinal and kidney proximal tubule cells. Villin is a very early marker of cells involved in absorption and its expression is highly increased during intestinal cell differentiation. The amino acid sequence deduced from the cDNA sequence revealed that human villin is composed of three domains. The first two domains appear as the result of a duplication: their structural organization is similar. We can then define a basic unit in which a slightly hydrophilic motif is followed by three hydrophobic motifs, similar between themselves and regularly spaced. The duplicated domain is highly homologous to three other actin-severing proteins and this basic structure represents the whole molecule in severin and fragmin, while two basic units compose gelsolin. The third domain which is carboxy terminal is villin specific: it is unique among actin modulating proteins so far known. It could account for its actin-binding properties (dual regulation by calcium of severing and bundling activities). We propose that it may also be related to the subcellular localization of villin in different epithelial cell types.