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.     

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.     

The terminal web. A reevaluation of its structure and function

The apical cytoplasm of epithelial cells of the small and large intestines has been examined by freeze-etch techniques as well as conventional and high voltage electron microscopy of sectioned material to gain a better understanding of the fine structural organization of the terminal web region. In the small intestine the terminal web exhibits a distinct stratification caused by the association of different sets of filaments with the three members of the junctional complex. Individual filaments of this network are closely associated with the sealing elements of the tight junctions, the surface of the core microfilament bundles, and the intermicrovillar plasma membrane. This region of the terminal web is the apical zone. The adherens zone appears as a band of interwoven filaments of two different diameters extending across the cytoplasm at the level of the intermediate junction. Within this region of the terminal web, individual 60-70 A actin-like filaments separate from the bundles of core microfilaments to interact with one another and with filaments of similar diameter from the zonula adherens. 100 A tonofilaments also contribute to the adherens zone, presumably stabilizing the orientation of the actin-like filaments. The basal zone which underlies the adherens zone consists of closely interwoven bundles of tonofilaments that are anchored to and interconnect the spot desmosomes. Within the large intestine the cytoplasmic microfilaments form a looser and less clearly stratified network which nevertheless retains the same basic organization found in the small intestine. Transmembrane linkers appear to originate within the cytoplasmic plaques of the spot desmosomes, pass through the plasma membranes, and meet in a staggered configuration in the intercellular space; these linkers may thus mediate the actual mechanical coupling between the cytoskeletal networks of tonofilament bundles of adjacent cells. This integrated system of cytoplasmic filaments and intercellular junctions endows the apical cytoplasm with both the flexibility and the stability necessary for the normal functioning of the epithelium.     

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

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.     

Filament-containing chromophobe in anterior pituitary of the guinea-pig

Numerous 95 ± 5 Å filaments have been observed in a chromophobe within the anterior pituitary gland of the guinea-pig. The cytoplasm of the chromophobe is somewhat electron lucid and long processes of the cell extend between granulated parenchymal cells. A basement membrane borders the plasma membrane of chromophobes facing the pericapillary space. Adjacent chromophobes are connected by maculae adherentes (desmosomes) and zonula adherens and often form a lumen; microvilli project into the lumen from the chromophobes.     

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.     

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

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.     

Three-dimensional visualization of basal body structures and some cytoskeletal components in the apical zone of tracheal ciliated cells

The ciliated cells of tracheal epithelium were mechanically fragmented to remove the cytoplasmic soluble contents, and the apical zone was examined to clarify the three-dimensional structures of basal body and cytoskeletal filaments using freeze-fracture-etch approaches. The basal body was connected to the apical plasma membrane by definite laminae, formerly called alar sheets. The distal one-half of the basal foot was composed of several smooth-surfaced 12-nm fibrils. Intermediate filament networks extended to the lower half plane of the basal body, and enmeshed the basal body tightly by tiny 5- to 8-nm fibrils. Actin core bundles of microvilli also had tiny crosslinking fibrils. Some actin filaments were seen to run horizontally at the upper half plane of the basal body. Tracheal cilated cells also had circular actin filament bundles just inside the zonula adherens as many other epithelial cells. These cytoskeletal networks which enmeshed both basal bodies and core filaments of microvilli may function as a coordinator of ciliary beating.     

Nucleated polymerization of actin from the membrane-associated ends of microvillar filaments in the intestinal brush border

We examined the nucleated polymerization of actin from the two ends of filaments that comprise the microvillus (MV) core in intestinal epithelial cells by electron microscopy. Three different in vitro preparations were used to nucleate the polymerization of muscle G- actin: (a) MV core fragments containing "barbed" and "pointed" filament ends exposed by shear during isolation, (b) isolated, membrane-intact brush borders, and (c) brush borders demembranated with Triton-X 100. It has been demonstrated that MV core fragments nucleate filament growth from both ends with a strong bias for one end. Here we identify the barbed end of the core fragment as the fast growing end by decoration with myosin subfragment one. Both cytochalasin B (CB) and Acanthamoeba capping protein block filament growth from the barbed but not the pointed end of MV core fragments. To examine actin assembly from the naturally occurring, membrane-associated ends of MV core filaments, isolated membrane-intact brush borders were used to nucleate the polymerization of G-actin. Addition of salt (75 mM KCl, 1 mM MgSO4) to brush borders preincubated briefly at low ionic strength with G- actin induced the formation of 0.2-0.4 micron "growth zones" at the tips of microvilli. The dense plaque at the tip of the MV core remains associated with the membrane and the presumed growing ends of the filaments. We also observed filament growth from the pointed ends of core filaments in the terminal web. We did not observe filament growth at the membrane-associated ends of core filaments when the latter were in the presence of 2 microM CB or if the low ionic strength incubation step was omitted. Addition of G-actin to demembranated brush borders, which retain the dense plaque on their MV tips, resulted in filament growth from both ends of the MV core. Again, 2 microM CB blocked filament growth from only the barbed (tip) end of the core. The dense plaque remained associated with the tip-end of the core in the presence of CB but usually was dislodged in control preparations where nucleated polymerization from the tip-end of the core occurred. Our results support the notion that microvillar assembly and changes in microvillar length could occur by actin monomer addition/loss at the barbed, membrane-associated ends of MV core filaments.     

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.     

Apical spectrin is essential for epithelial morphogenesis but not apicobasal polarity in Drosophila.

Changes in cell shape and position drive morphogenesis in epithelia and depend on the polarized nature of its constituent cells. The spectrin-based membrane skeleton is thought to be a key player in the establishment and/or maintenance of cell shape and polarity. We report that apical beta(Heavy)-spectrin (beta(H)), a terminal web protein that is also associated with the zonula adherens, is essential for normal epithelial morphogenesis of the Drosophila follicle cell epithelium during oogenesis. Elimination of beta(H) by the karst mutation prevents apical constriction of the follicle cells during mid-oogenesis, and is accompanied by a gross breakup of the zonula adherens. We also report that the integrity of the migratory border cell cluster, a group of anterior follicle cells that delaminates from the follicle epithelium, is disrupted. Elimination of beta(H) prevents the stable recruitment of alpha-spectrin to the apical domain, but does not result in a loss of apicobasal polarity, as would be predicted from current models describing the role of spectrin in the establishment of cell polarity. These results demonstrate a direct role for apical (alphabeta(H))(2)-spectrin in epithelial morphogenesis driven by apical contraction, and suggest that apical and basolateral spectrin do not play identical roles in the generation of apicobasal polarity.     

Localization of capping protein in chicken epithelial cells by immunofluorescence and biochemical fractionation

We have localized capping protein in epithelial cells of several chicken tissues using affinity-purified polyclonal antibodies and immunofluorescence. Capping protein has a distribution in each tissue coincident with proteins of the cell-cell junctional complex, which includes the zonula adherens, zonula occludens, and desmosome. "En face" views of the epithelial cells showed capping protein distributed in a polygonal pattern coincident with cell boundaries in intestinal epithelium, sensory epithelium of the cochlea, and the pigmented epithelium of the retina and at regions of cell-cell contact between chick embryo kidney cells in culture. "Edge-on" views obtained by confocal microscopy of intact single intestinal epithelial cells and of retinal pigmented epithelium showed that capping protein is located in the apical region of the epithelial cells coincident with the junctional complexes. These images do not resolve the individual types of junctions of the junctional complex. Immunolabeling of microvilli or stereocilia was faint or not detectable. Capping protein was also detected in the cytoplasm of intact intestinal epithelial cells and in nuclei of cells in the pigmented retina and in the kidney cell cultures, but not in nuclei of cells of the intestinal epithelium or sensory epithelium. Biochemical fractionation of isolated intestinal epithelial cells shows capping protein in the brush border fraction, which contains the junctional complexes, and in the soluble fraction. These results are consistent with the results of the immunolabeling experiments. Highly purified microvilli of the brush borders also contained capping protein; this result was unexpected based on the low intensity of immunofluorescence staining of microvilli and stereocilia. The microvilli were not contaminated with junctional complexes, as defined by the absence of several markers for cell junctions. The cause and significance of this discrepancy is not certain at this time. Since capping protein binds the barbed end of actin filaments in vitro, we hypothesize that capping protein is bound to the barbed ends of actin filaments associated with one or more of the junctions of the junctional complex.     

Immunolocalization of the 110,000 molecular weight cytoskeletal protein of intestinal microvilli

The core structures of microvilli from absorptive cells of the intestinal epithelium are primarily composed of calmodulin (M_r 16,000), actin (M_r 43,000), villin (M_r 95,000) and a protein of M_r 110,000. We have isolated this protein and raised antibodies against it. The antibodies interact specifically with villin and M_r 110,000 polypeptides present in isolated microvilli or brush borders. However, after absorption on an immobilized villin preparation, these antibodies still immunoprecipitate the M_r 110,000 protein but not villin. Thus, these two proteins appear to share some antigenic determinants but also contain other determinants specific for each protein. Immunolocalization studies have been performed using specific antibodies against the M_r 110,000 protein. Immunofluorescent studies on thin frozen sections of intestinal cells show that this protein is located in the brush border and at the basolateral faces of these polarized cells. Immunoferritin studies on rat brush borders demembranated with the detergent Triton X-100 show the association of the M_r 110,000 protein with core filaments of microvilli, as well as with some filaments localized in the terminal web network.Using sealed, right-side-out vesicles prepared from pig intestinal mucosa in the presence of Ca²⁺ and Mg²⁺, a polypeptide of M_r 140,000 was found to be a major component of the Triton X-100 insoluble pellet. This protein is a minor component of an equivalent pellet obtained from isolated microvilli prepared in the presence of EDTA. The significance of this M_r 140,000 polypeptide associated with the core residue of intestinal microvilli is discussed.     

FACTORS CONTROLLING THE REASSEMBLY OF THE MICROVILLOUS BORDER OF THE SMALL INTESTINE OF THE SALAMANDER

Hydrostatic pressure, when applied to segments of the small intestine of the salamander, causes a tremendous reduction in number of microvilli and a loss of the terminal web. The intestinal epithelium strips off from its deeper layers at the level of the basement membrane. When the pressure is released and this epithelial sheet is allowed to recover, the microvilli and its terminal web reappear. Stages in the reformation of microvilli are described. In the earliest stages, foci of dense material seem to associate with the cytoplasmic surface of the apical plasma membrane. From this material, filaments appear and their regrowth is correlated with the extension of the microvilli. We suggest that the dense material nucleates the assembly of the filaments which, in turn, appear instrumental in the redevelopment of microvilli. This concept is supported by the existing literature. Further, since neither the microvilli nor the terminal web reappear on any surface but the apical surface, even though the apical and basal surfaces are bathed with the same medium, we suggest that information in the membrane itself or directly associated with the membrane dictates the distribution of the dense material which leads to the formation of the microvilli and ultimately to the polarity of the cell.     

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.     

N-WASP regulates the epithelial junctional actin cytoskeleton through a non-canonical post-nucleation pathway

N-WASP is a major cytoskeletal regulator that stimulates Arp2/3-mediated actin nucleation. Here, we identify a nucleation-independent pathway by which N-WASP regulates the cytoskeleton and junctional integrity at the epithelial zonula adherens. N-WASP is a junctional protein whose depletion decreased junctional F-actin content and organization. However, N-WASP (also known as WASL) RNAi did not affect junctional actin nucleation, dominantly mediated by Arp2/3. Furthermore, the junctional effect of N-WASP RNAi was rescued by an N-WASP mutant that cannot directly activate Arp2/3. Instead, N-WASP stabilized newly formed actin filaments and facilitated their incorporation into apical rings at the zonula adherens. A major physiological effect of N-WASP at the zonula adherens thus occurs through a non-canonical pathway that is distinct from its capacity to activate Arp2/3. Indeed, the junctional impact of N-WASP was mediated by the WIP-family protein, WIRE, which binds to the N-WASP WH1 domain. We conclude that N-WASP-WIRE serves as an integrator that couples actin nucleation with the subsequent steps of filament stabilization and organization necessary for zonula adherens integrity.     

Role of myosin in terminal web contraction in isolated intestinal epithelial 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.     

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

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.     

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

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).     

Cytoskeletal proteins of the rat kidney proximal tubule brush border

Cytoskeletal components backing the brush border of the rat kidney proximal tubule cell were identified and compared with those of the well characterized intestinal brush border by immuneoverlay and immunocytochemistry. Antibodies reactive against the intestinal microvillus core components, villin and fimbrin, as well as against the terminal web components, spectrin (fodrin) and myosin, were used. Proteins of similar molecular weight to these intestinal brush border cytoskeletal components were identified in isolated kidney brush borders by immuneoverlay. Spectrin, a major component of the terminal web region of both cell types, was more concentrated in the kidney brush border relative to both actin and myosin. By immunofluorescence, villin and fimbrin were localized in the microvilli, and spectrin and myosin were localized to the terminal web region of the brush border. In addition, spectrin was found along the basolateral membranes of the proximal tubule cell, and myosin was detected in a punctate staining pattern throughout its cytoplasm. By immunoelectron microscopy using immunogold labeling procedures, fimbrin and villin were localized in the terminal web as well as in microvilli, and spectrin and myosin were localized to fibrils in the terminal web. A key difference between the epithelia of the two organs is the extensive network of clathrin coated pits found in the terminal web region of the kidney but not the intestinal brush border. The clathrin-rich terminal web region of the kidney, like the intestinal brush border, proved to be quite stable and resistant to disruption by non-ionic detergents and harsh mechanical treatment.