Organization of the cross-filaments in intestinal microvilli

We studied the arrangement of the cross-filaments in intestinal microvilli to understand how microfilaments interact with the membrane. Observations on thin-sectioned or negatively stained microvilli with the electron microscope demonstrate that the cross-filaments on the core bundle lie opposite to one another and are spaced 32.5 nm apart. In sections grazing through the membranes, the cross-filaments appear as transverse stripes in a barber-polelike arrangement. The cross- filaments point away from the microvillus tip. This subfragments S1 or HMM. The cross filaments are associated not only with the microfilaments but also with electron-dense patches on the inside surface of the membrane. These results suggest the cross-filaments are arranged as a double helix around the core bundle. Furthermore, the cross-filaments can serve as in situ markers for microvillar polarity. Lastly, the cross-filaments interact not only with specific portions on the actin filaments but also with dense patches on the membrane. These observations are summarized in a model of the microvillus cytoskeleton.     

The organization of actin filaments in human placental villi

The surface of the syncytial trophoblast of the human placenta is covered by a microvillous (brush) border that is in direct contact with maternal blood. Because of this location, it is the site of a variety of transport, enzymatic and receptor activities vital to many placental functions. The organization of the brush border as well as other features of placental villus organization may well be influenced by the distribution of cytoplasmic actin filaments. In order to determine the distribution of actin filaments in human placenta, small pieces of villi were briefly fixed in glutaraldehyde, permeabilized with saponin, and incubated in solutions containing subfragment 1 of myosin (S1). After S1 decoration of actin filaments, tissue was fixed in glutaraldehyde containing tannic acid in order to better visualize the polarity of the filaments, and prepared for electron microscopic examination. The microvilli each contained a core of actin filaments running from the tip of the microvillus to the apical cytoplasm. Most of the actin filaments displayed a distinct polarity, with the S1 arrowheads pointing away from the microvillar tips. These filaments extended only a short distance into the apical cytoplasm. There appeared to be another group of actin filaments in a matlike arrangement in the apical cytoplasm. Coated pits and vesicles were often observed between the microvilli. There appeared to be no clear association between the coated pits and decorated actin filaments, but this was difficult to establish with certainty because of the close proximity of the microvilli. Bundles of actin filaments were sometimes observed near the basal cell surface of the syncytial trophoblast, and in pericytes and capillary endothelial cells in the cores of the villi.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relation of cytoplasmic alkalinization to microvillar elongation and microfilament formation in the sea urchin egg

Experiments have been carried out to test the proposal that the pH increase at fertilization in sea urchin eggs promotes microvillar elongation. Results presented herein show that microvillar elongation and microfilament formation occurred when sea urchin eggs were incubated in sodium-free seawater containing the calcium ionophore A23187, a treatment which initiates activation, i.e., induces a transient increase in intracellular free calcium, but prevents subsequent cytoplasmic alkalinization. Within elongated microvilli and cortices of these eggs, microfilaments were arranged in a loose meshwork. However, if the pH of the egg cytoplasm was increased experimentally, microfilament bundles appeared within individual microvilli. These findings suggest that: (1) microvillar elongation and microfilament formation in the sea urchin egg at fertilization may occur when cytoplasmic alkalinization is inhibited, and (2) formation of the microvillus bundle of microfilaments at egg activation is pH sensitive. Additionally, if the cytoplasmic pH of unfertilized eggs was experimentally elevated by NH₄Cl, microvilli failed to elongate. These data indicate that elevation of intracellular pH by this method is not sufficient to induce microvillar elongation.     

Light-induced structural changes of cytoskeleton in squid photoreceptor microvilli detected by rapid-freeze method

The cytoskeleton in squid photoreceptor microvilli was studied by freeze-substitution electron microscopy combined with rapid freezing using liquid helium, under dark-adapted and light-illuminated conditions. In the dark-adapted microvilli, actin filaments were regularly associated with granular structures on their surface; these granular structures were cross-linked to the rhodopsin-bearing plasma membranes through slender strands. Upon exposure to light, the granular components detached from the actin filaments, which then appeared to be fragmented and/or depolymerized. These observations have led us to conclude that light stimulation triggers the breakdown of the microvillar actin filament complex in squid photoreceptor cells. The results are discussed with special reference to the physiological role of actin filaments in photoreception.     

A helical,polymeric extracellular protein associated with the luminal surface of Haemonchus contortus intestinal cells

The structure of the intestinal cells of the parasitic nematode Haemonchus contortus is described. The cells have numerous microvilli about 0.09 μ in diameter; most being 5.5–7.5 μ in length. The microvillar (plasma) membrane is coated with a layer of amorphous material (glycocalyx) about 60 Å thick which is electron dense in sectioned preparations. Associated with the surface of this material, and filling the spaces between the microvilli, are filaments in the form of helices about 400 Å in diameter and of variable pitch. The helices appear to be flexible but they are aligned approximately with the long axes of the microvilli. There are up to ten helices per microvillus; they extend beyond the tips of the microvilli and are up to 10 μ long. The material has been obtained nearly pure in small amounts. It is primarily protein and it is proposed that it should be called contortin. The monomeric form (of molecular weight about 60,000) has been identified with a Y-shaped structure with arms about 45 Å long and 25 Å wide seen in negatively stained preparations. The helical filament appears to be formed by lateral polymerization of pairs of these units.     

Characterization of intestinal microvillar membrane disks: detergent- resistant membrane sheets enriched in associated brush border myosin I (110K-calmodulin)

The actin bundle within each microvillus of the intestinal brush border (BB) is tethered laterally to the membrane by bridges composed of BB myosin I. Avian BB myosin I, formerly termed 110K-calmodulin, consists of a heavy chain with an apparent Mr of 110 kD and three to four molecules of calmodulin "light chains." Recent studies have shown that this complex shares many properties with myosin including mechanochemical activity. In this report, the isolation and characterization of a membrane fraction enriched in bound BB myosin I is described. This membrane fraction, termed microvillar membrane disks, was purified from ATP extracts of nonionic detergent-treated microvilli prepared from avian intestinal BBs. Ultrastructural analysis revealed that these membranes are flat, disk-shaped sheets with protrusions which are identical in morphology to purified BB myosin I. The disks exhibit actin-activated Mg-ATPase activity and bind and cross-link actin filaments in an ATP-dependent fashion. The mechanochemical activity of the membrane disks was assessed using the Nitella bead movement assay (Sheetz, M. P., and J. A. Spudich. 1983. Nature [Lond.]. 303:31-35). These preparations were shown to be free of significant contamination by conventional BB myosin. Latex beads coated with microvillar membrane disks move in a myosin-like fashion along Nitella actin cables at rates of 12-60 nm/s (average rate of 33 nm/s); unlike purified BB myosin I, the movement of membrane disk-coated beads was most reproducibly observed in buffers containing low Ca2+.     

Tetraspanins regulate the protrusive activities of cell membrane

Tetraspanins have gained increased attention due to their functional versatility. But the universal cellular mechanism that governs such versatility remains unknown. Herein we present the evidence that tetraspanins CD81 and CD82 regulate the formation and/or development of cell membrane protrusions. We analyzed the ultrastructure of the cells in which a tetraspanin is either overexpressed or ablated using transmission electron microscopy. The numbers of microvilli on the cell surface were counted, and the radii of microvillar tips and the lengths of microvilli were measured. We found that tetraspanin CD81 promotes the microvillus formation and/or extension while tetraspanin CD82 inhibits these events. In addition, CD81 enhances the outward bending of the plasma membrane while CD82 inhibits it. We also found that CD81 and CD82 proteins are localized at microvilli using immunofluorescence. CD82 regulates microvillus morphogenesis likely by altering the plasma membrane curvature and/or the cortical actin cytoskeletal organization. We predict that membrane protrusions embody a common morphological phenotype and cellular mechanism for, at least some if not all, tetraspanins. The differential effects of tetraspanins on microvilli likely lead to the functional diversification of tetraspanins and appear to correlate with their functional propensity.     

Localisation of actin, villin, fimbrin, ezrin and ankyrin in rat taste receptor cells

Mammalian taste buds consist of 50–150 pear- or spindle-shaped taste receptor cells which contain, at their apical cell surface, a bundle of microvillar projections. The microvilli probably serve to increase the receptive membrane surface of the chemosensory receptor cells. The molecular basis controlling the ultrastructure of taste receptor microvilli is present unknown. In the present study we analysed, by immunostaining at the light and electron microscopic levels and by immunoblotting, components of the cytoskeleton of these microvilli. We show here that taste cell microvilli contain the major cytoskeletal proteins of intestinal microvilli, actin, fimbrin and villin. Another actin-binding, peripheral membrane protein of intestinal microvilli, ezrin, was also localised to taste cell microvilli, where ezrin might play a role, for example, in placement of specific membrane proteins to the microvillus membrane. In search of further linkage proteins, we found ankyrin localised along the basolateral cell surface of taste receptor cells, where ankyrin might be involved in the immobilisation of the Na⁺, K⁺-ATPase or other ion-translocating proteins of taste cells to the membrane cytoskeleton. Accepted: 26 April 1999     

Measuring orientation of actin filaments within a cell: orientation of actin in intestinal microvilli.

Orientational distribution of actin filaments within a cell is an important determinant of cellular shape and motility. To map this distribution we developed a method of measuring local orientation of actin filaments. In this method actin filaments within cells are labeled with fluorescent phalloidin and are viewed at high magnification in a fluorescent microscope. Emitted fluorescence is split by a birefringent crystal giving rise to two images created by light rays polarized orthogonally with respect to each other. The two images are recorded by a high-sensitivity video camera, and polarization of fluorescence at any point is calculated from the relative intensity of both images at this point. From the value of polarization, the orientation of the absorption dipole of the dye, and thus orientation of F-actin, can be calculated. To illustrate the utility of the method, we measured orientation of actin cores in microvilli of chicken intestinal epithelial cells. F-actin in microvillar cores was labeled with rhodamine-phalloidin; measurements showed that the orientation was the same when microvillus formed a part of a brush border and when it was separated from it suggesting that "shaving" of brush borders did not distort microvillar structure. In the absence of nucleotide, polarization of fluorescence of actin cores in isolated microvilli was best fitted by assuming that a majority of fluorophores were arranged with a perfect helical symmetry along the axis of microvillus and that the absorption dipoles of fluorophores were inclined at 52 degrees with respect to the axis. When ATP was added, the shape of isolated microvilli did not change but polarization of fluorescence decreased, indicating statistically significant increase in disorder and a change of average angle to 54 degrees. We argue that these changes were due to mechanochemical interactions between actin and myosin-I.     

Parathyroid hormone-induced changes of the brush border topography and cytoskeleton in cultured renal proximal tubular cells

Summary In order to examine the possibility of parathyroid hormone-mediated ultrastructural rearrangements in target epithelium, isolated canine renal proximal tubular cells were grown on a collagen-coated semipermeable membrane in a defined medium. Scanning and transmission electron microscopy of these monolayers revealed abundant microvilli. Exposure of the proximal tubular cells to parathyroid hormone resulted in a biphasic changes involving: (1) dramatic shortening and rarefaction of microvilli within 1 min; and (2) recovery of microvillar topography after 5 min. A similar shortening of microvilli was observed following exposure to ionomycin, whereas incubation with cyclic AMP resulted in an elongation of microvilli. Parathyroid hormone stimulated cyclic AMP production and increased cytoplasmic free calcium concentration in cultured proximal tubular cells. Pretreatment of cells with a calmodulin inhibitor abolished the effect of parathyroid hormone on brush border topography. Shortening of microvilli was associated with a disappearance of microvillar core filaments. Staining of F-actin with fluoresceinphalloidin showed that parathyroid hormone resulted in fragmentation of stress fibers. It is concluded that parathyroid hormoneinduced cell activation involves cytoplasmic-free calcium, calmodulin, and the cytoskeleton.     

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).     

The paracrystalline structure of an insect rhabdomere (Calliphora erythrocephala)

Summary By use of a modified fixation technique, the receptor cells of the compound eye of the blowfly Calliphora erythrocephala were found to contain a regular, paracrystalline array of alternating rows of hexagonally shaped microvilli. The receptor cells R1 to R6 have a cell-specific number of microvilli per row in a cross section. Every microvillus has a filament cluster connecting the axial skeleton with the microvillar membrane. This cluster is preferentially right-left oriented relative to the longitudinal axis of the microvillar array. Three adjacent microvilli are interconnected by an electron-dense substance. A mirroring technique indicated that this intermicrovillar structure consists of three subunits, although these subunits could not be conclusively demonstrated by classical densitometry or image subtraction techniques. The electron-dense substance can be seen in all cross sections of the proximal and distal parts of the microvilli. They are cylindrical structures separating the microvilli along their entire length. It is suggested that these cylindrical aggregates contain an enzymatic complex separating the rhodopsin-containing microvillar membrane into six compartments.     

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.     

Actin filaments and photoreceptor membrane turnover

The shape and turnover of photoreceptor membranes appears to depend on associated actin filaments. In dipterans, the photoreceptor membrane is microvillar. It is turned over by the addition of new membrane at the bases of the microvilli and by subsequent shedding, mostly from the distal ends. Each microvillus contains actin filaments as a component of its cytoskeletal core. Two myosin I-like proteins co-localize with the actin filaments. It is suggested that one of the myosin I-like proteins might be linked to the microvillar membrane. By interacting with the actin filaments, this motor should move the membrane of a microvillus in a distal direction, thus providing a possible mechanism for the turnover of the membrane. A vertebrate photoreceptor cell contains a small cluster of actin filaments in its connecting cilium at the site where new transductive disk membranes are formed. Disruption of the actin filaments perturbs disk morphogenesis. The most likely explanation for this perturbation is that the process of initiating a new disk is inhibited. Conventional myosin (myosin II) is found in the connecting cilium with the same distribution as actin. A simple model is proposed to illustrate how the actin-myosin system of the connecting cilium might function to initiate the morphogenesis of a disk membrane.     

Characterization of actin microfilaments at the apical pole of thyroid cells

In thyroid cells (rat or hog), actin has been detected by immunofluorescence with an antiactin antibody and, in electron microscopy by decoration “in situ” with heavy meromyosin. The antibody as the heavy meromyosin method have shown that actin microfilaments are especially localized at the apical pole of the cells, in a region where thin filaments are usually observed by conventional methods of electron microscopy. These microfilaments are attached to the apical membrane at the ends of the microvilli and form dense bundles at their cores. They are polarized towards the interior of the cell. Decorated filaments are also organized in a clear network, parallel to the apical membrane; they are associated with microvillar bundles, but also with small apical vesicles and lateral membranes, in tight or gap junctions.     

Ultrastructural study of sensory cells of the proboscidial glandular epithelium of Riseriellus occultus (Nemertea,Heteronemertea)

Only one sensory cell type has been observed within the glandular epithelium of the proboscis in the heteronemertine Riseriellus occultus. These bipolar cells are abundant and scattered singly throughout the proboscis length. The apical surface of each dendrite bears a single cilium enclosed by a ring of six to eight prominent microvilli. The cilium has the typical 9×2 + 2 axoneme arrangement and is equipped with a cross-striated vertical rootlet extending from the basal body. No accessory centriole or horizontal rootlet was observed. Large, modified microvilli (stereovilli) surrounding the cilium are joined together by a system of fine filaments derived from the glycocalyx. Each microvillus contains a bundle of actin-like filaments which anchor on the indented inner surface of a dense, apical ring situated beneath the level of the ciliary basal body. The tip of the cilium is expanded and modified to form a bulb-like structure which lies above the level where the surrounding microvilli terminate. In the region where the cilium emerges from the microvillar cone, the membrane of the microvillar apices makes contact with a corresponding portion of the ciliary membrane. At this level microvilli and cilium are apparently firmly linked by junctional systems resembling adherens junctions. The results suggest that these sensory cells may be mechanoreceptors. © 1996 Wiley-Liss, Inc.     

Ezrin regulates microvillus morphogenesis by promoting distinct activities of Eps8 proteins

The mechanisms that regulate actin filament polymerization resulting in the morphogenesis of the brush border microvilli in epithelial cells remain unknown. Eps8, the prototype of a family of proteins capable of capping and bundling actin filaments, has been shown to bundle the microvillar actin filaments. We report that Eps8L1a, a member of the Eps8 family and a novel ezrin-interacting partner, controls microvillus length through its capping activity. Depletion of Eps8L1a leads to the formation of long microvilli, whereas its overexpression has the opposite effect. We demonstrate that ezrin differentially modulates the actin-capping and -bundling activities of Eps8 and Eps8L1a during microvillus assembly. Coexpression of ezrin with Eps8 promotes the formation of membrane ruffles and tufts of microvilli, whereas expression of ezrin and Eps8L1a induces the clustering of actin-containing structures at the cell surface. These distinct morphological changes are neither observed when a mutant of ezrin defective in its binding to Eps8/Eps8L1a is coexpressed with Eps8 or Eps8L1a nor observed when ezrin is expressed with mutants of Eps8 or Eps8L1a defective in the actin-bundling or -capping activities, respectively. Our data show a synergistic effect of ezrin and Eps8 proteins in the assembly and organization of actin microvillar filaments.     

Isolation of microvillar microfilaments and associated transmembrane complex from ascites tumor cell microvilli

The association of microvillar microfilaments with the microvillar membrane actin-containing transmembrane complex of MAT-C1 13762 ascites tumor cell microvilli has been investigated by differential centrifugation, gel electrophoresis and electron microscopy of detergent extracts of the isolated microvilli. Several methods have been used to reduce breakdown and solubilization of the microfilament core actin during the detergent extractions for preparation of microvillar core microfilaments. Gel electrophoresis of differential centrifugation fractions demonstrated that over 70% of the total microvillus actin could be pelleted with microfilament cores at 10 000 g under extraction conditions which reduce filament breakdown. Transmission electron microscopy (TEM) of all of the core preparations showed arrays of microfilaments and small microfilament bundles. The major protein components of the microfilament cores, observed by sodium dodecyl sulfate (SDS) electrophoresis, were actin and alpha-actinin. Among the less prominent polypeptide components was a 58 000 Dalton polypeptide (58 K), previously identified as a member of the MAT-Cl transmembrane complex. This three-component complex contains, in addition to 58 K, actin associated directly and stably with a cell surface glycoprotein (Carraway, CAC, Jung, G & Carraway, K L, Proc. natl acad. sci. US 80 (1983) 430). Evidence that the apparent association of complex with the microfilament core was not due simply to co-sedimentation was provided by myosin affinity precipitation. These results provide further evidence that the transmembrane complex is a site for the interaction of microfilaments with the microvillar plasma membrane.     

Structural and biochemical differentiation of the guinea-pig colon during foetal development

Summary We have studied some aspects of the morphological and biochemical differentiation of the foetal guinea-pig colonic epithelium. At day 40 the epithelium was organised in ridges and appeared pseudo-stratified. Folding of the epithelium, followed by villus formation, occurred between days 45 and 55, and by day 50 mucus-secreting goblet cells appeared at the bases of the colonic villi. By day 55 most epithelial cells, including goblet cells, possessed numerous microvilli which, by day 65, had become organised into well developed brush-borders. Between day 55 and term (day 65–68) mucosal depth increased markedly and the colon attained its final glandular morphology.Biochemical studies showed the specific activities of the microvillar hydrolases to be much lower in the washed colon than in either foetal meconium or small intestine at all times during development. Furthermore, a membrane fraction highly enriched in microvillus hydrolase activities was prepared from foetal colonic meconium using techniques originally devised to isolate the foetal small intestinal microvillus membrane. This meconial subfraction was almost identical in polypeptide composition to the highly-purified foetal small intestinal microvillus membrane. Identification of the colonic microvillus membrane was hampered by the absence of reliable membrane markers. Nevertheless, a fraction 14-fold enriched in aminopeptidase activity was prepared from day 40 foetal colon and its polypeptide composition compared by SDS-PAGE to that of the small intestinal microvillus membrane at the same age.     

Effect of hyper- and hypoosmotic solutions on the structure of theAstacus retina

Summary Alterations of the retinula cells in the retina of the light-adapted crayfish in response to hyper- and hypoosmotic van Harreveld solutions was examined by transmission electron microscopy. Increased osmolarity of the bathing medium to twice that of the physiological value leads to shrinkage of the retinula-cell somata. Microvilli, on the other hand, do not undergo shrinkage. Some other characteristic irreversible changes do, however, take place, including detachment of microvilli from the soma, showing a two- or threefold increase in diameter, and a concomitant decrease in number, probably due to fusion of microvilli.Prolonged incubation or higher osmolarities (5 isosmol) cause alteration of the microvillar membranes to whorls. Structural changes are often restricted to microvillar stacks evaginating from certain individual retinular cells. The number of affected stacks increases depending on the duration of incubation or the osmotic pressure. Hypoosmotic solution (0.5 isosmol) also induces an increase of microvillar diameters and a concomitant reduction in number of microvilli per stack. Exposure to a 20% solution of glycerol causes destruction of the rhabdom structure and the formation of whorls from microvillar membranes. The present findings suggest that the structure of the microvilli is stabilized by an axial cytoskeleton.