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.     

Demonstration by freeze-etching of a single cleavage plane in the cell wall of a gram-negative bacterium

In the examination of protoplasts of a gram-negative bacterium classified as a Pseudomonas sp. by freeze-etching, we found a smooth external surface which is not seen if the preparations are not "etched." This external structure is seen as a sleeve surrounding and connecting the cells in unetched preparations, and we present evidence that it is a eutectic formed during the freezing of the specimen. In the system used in this study, the four layers of the cell wall of a gram-negative bacterium can be removed from the cell. The single cell wall cleavage plane is not affected by the removal of the loosely bound outer layer or of the peptidoglycan layer, but it is lost when the outer double track layer and the underlying soluble layer are simultaneously removed. Thus, we conclude that it is one of these two layers which is responsible for the cleavage plane which exposes variable areas of a smooth surface in the cell wall. This cell wall cleavage plane is more likely to deflect the actual cleavage of the frozen cell when cells are relatively old or when they are suspended in sucrose.     

Lipid-cell interactions. Induction of microvilli on the cell surface by liposomes

Treatment of cells with sonicated egg lecithin (EL) liposomes induced microvilli on the surface of mouse embryo fibroblasts and cells of an epithelioid MPTR strain. Unsonicated liposomes and sonicated dipalmitoyllecithin (DPL) liposomes do not induce microvilli on the surface of these cells. It is supposed that microvilli induction is caused by modification of the cell surface composition by small liquid-crystalline liposomes (e.g. by fusion with plasma membrane or transfer of some components from the cell surface to liposomes and vice versa). Microvilli induction by lipsomes provides a system for investigation of their role in cell life.     

Simple and complex synapses shown by freeze-etching of rat cortical synaptosomes

The shape of the synaptic sites (specialized contact areas) was examined on the synaptic membrane fracture faces of freeze-etched rat cortical synaptosomes. Simple and complex synapses were differentiated on the basis of the absence or presence of unspecialized areas in the synaptic sites. The particle-free nature of these areas is discussed.     

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.     

Trypanostatic activity of rat IgG purified from the surface coat of Trypanosoma lewisi

Host IgG is a component of the surface coat of Trypanosoma lewisi; it is specifically acquired during infection in the rat, concomitant with a rise in titer of trypanostatic (ablastic) activity of host serum. Host IgG was eluted from trypomastigotes at 7 to 9 days postinfection with a high salt-low pH buffer. Surface coats and trypanosome ultrastructure were not notably altered by the elution procedure, as determined by electron microscopy. Rat IgG was removed and purified from the trypanosome eluates on an immunoadsorbent column made with the IgG fraction of anti-rat IgG serum coupled to Sepharose beads. Concentrated column eluates, by comparison with a standard, were shown to be rat IgG by immunoelectrophoresis and SDS polyacrylamide gel electrophoresis. As a control, IgG from normal rat serum was purified by the same technique. IgG-negative trypanosomes harvested from immunosuppressed rats bound IgG purified from surface coats of trypanosomes, but not IgG purified from normal rat serum, as demonstrated by subsequent labelling with FITC-conjugated, rabbit anti-rat IgG. The IgG purified from surface coats inhibited the reproduction of T. lewisi in an in vitro assay, but purified, normal IgG did not. These data show that antigen-specific host IgG, adsorbed to the surface of T. lewisi, is ablastic antibody.     

Electron staining of the cell surface coat by osmium-low ferrocyanide

In aldehyde-fixed liver and renal cortex of rat and mouse several variations of postfixation with osmium tetroxide plus potassium ferrocyanide ( FeII ) were tried. Depending on the ferrocyanide concentration different staining patterns were observed in TEM. -Osmium-High Ferrocyanide [40 mM (approximately 1%) OsO4 + 36 mM (approximately 1.5%) FeII , pH 10.4], stains membranes and glycogen. Cytoplasmic ground substance, mitochondrial matrices and chromatin are partially extracted, cell surface coats remain unstained. Membrane contrast, but extraction too, are higher with solutions containing cacodylate- than phosphate-buffer. -Osmium-Low Ferrocyanide [40 mM (approximately 1%) OsO4 + 2 mM (approximately 0.08%) FeII , pH 7.4], stains cell surface coats and basal laminae, but not glycogen, except for special cases. The trilaminar structure of membranes is poorly delineated. Signs of cytoplasmic extraction are not visible. The surface coat staining is stronger and more widespread with solutions containing phosphate- instead of cacodylate-buffer; it is enhanced by section staining with lead citrate. The cell surface coat stain does not traverse tight junctions nor permeate membranes.     

Electron staining of the cell surface coat by osmium-low ferrocyanide

Summary In aldehyde-fixed liver and renal cortex of rat and mouse several variations of postfixation with osmium tetroxide plus potassium ferrocyanide (Fe^II) were tried. Depending on the ferrocyanide concentration different staining patterns were observed in TEM.-Osmium-High Ferrocyanide [40 mM (1%) OsO₄+36 mM (1.5%) Fe^II, pH 10.4], stains membranes and glycogen. Cytoplasmic ground substance, mitochondrial matrices and chromatin are partially extracted, cell surface coats remain unstained. Membrane contrast, but extraction too, are higher with solutions containing cacodylate- than phosphate-buffer.-Osmium-Low Ferrocyanide [40 mM (1%) OsO₄+2 mM (0.08%) Fe^II, pH 7.4], stains cell surface coats and basal laminae, but not glycogen, except for special cases. The trilaminar structure of membranes is poorly delineated. Signs of cytoplasmic extraction are not visible. The surface coat staining is stronger and more widespread with solutions containing phosphate- instead of cacodylate-buffer; it is enhanced by section staining with lead citrate. The cell surface coat stain does not traverse tight junctions nor permeate membranes.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Identification of soybean cultivars by the surface relief of the seed coat

Summary The relief of the outer surface of the seed coat of 16 diverse cultivars of soybeans, Glycine max (L.) Merrill, was compared on the basis of pattern, mound number and mound height in both air and water media. Significant differences among cultivars were found indicating that surface relief characteristics were cultivar-specific and could be used to describe and distinguish cultivars.     

A coat of glycoconjugates on the inner surface of the lysosomal membrane in the rat kidney

The immunogold-silver staining technique is shown to be of great value in the detection of regulatory peptide-containing nerves and endocrine cells in routinely fixed, paraffin-wax-embedded tissues. The method appears to be better for this system than peroxidase anti-peroxidase (PAP) which can yield poor or variable results. Antibodies to regulatory peptides, including calcitonin gene-related peptide (CGRP), substance P, neuropeptide tyrosine (NPY), glucagon, pancratic polypeptide, and somatostatin 14 and 28, as well as to neurofilaments, neuron-specific enolase (NSE) and S-100, were used on sections of a variety of tissues from rat and pig including respiratory tract, skin, gut, pancreas, vagina, uterus, fallopian tube and kidney. In all cases, stronger immunostaining of nerves was obtained with the immunogold-silver technique than with PAP. The inherent density of the staining was also found to improve the visibility of endocrine cells in the section, and to permit the use of routine histological stains for counterstaining. As immunogold-silver staining is sensitive, rapid, cheap and avoids hazardous reagents, we feel it has great potential for the immunostaining of nerves and endocrine cells that contain regulatory peptides in routinely fixed and embedded tissues and may prove useful in pathology.     

Ultrastructure of cell surface coat (glycocalyx) in rat urinary bladder epithelium

Earlier statements to the contrary, the present study demonstrates the presence of a cell surface coat (glycocalyx) on the luminal plasma membrane of the superficial transitional epithelial cells lining the urinary bladder of male Buffalo rats. This coat was demonstrated with ruthenium red, an electron dense stain, which revealed a surface layer, 60-80 A thick, separated from the outer leaflet of the plasma membrane by an electron lucent layer, approximately 30 A thick. The structure of the glycocalyx was not affected by 12 weeks of treatment with dibutylnitrosamine, a known bladder carcinogen.     

A two-photon FRAP analysis of the cytoskeleton dynamics in the microvilli of intestinal cells

The molecular structure of the brush-border of enterocytes has been investigated since the 1980s, but the dynamics of this highly specialized subcellular domain have been difficult to study due to its small size. To perform a detailed analysis of the dynamics of cytoskeleton proteins in this domain, we developed two-photon fluorescence recovery after photobleaching and a theoretical framework for data analysis. With this method, fast dynamics of proteins in the microvilli of the brush border of epithelial intestinal cells can be measured on the millisecond timescale in volumes smaller than 1 microm3. Two major proteins of the cytoskeleton of the microvilli, actin and myosin 1a (Myo1a; formerly named brush border myosin I), are mobile in the brush-border of Caco-2 cells, an enterocyte-like cellular model. However, the mobility of actin is very different from that of Myo1a and they appear to be unrelated (diffusion coefficient of 15 microm2 s(-1) with a mobile fraction of 60% for actin, and 4 microm2 s(-1) with a mobile fraction of 90% for Myo1a). Furthermore, we show for the first time, in vivo, that the dynamics of Myo1a in microvilli reflect its motor activity.     

Characterization of an integral membrane glycoprotein associated with the microfilaments of pig intestinal microvilli

An integral membrane glycoprotein of pig intestinal microvilli which exists in two polypeptide forms [mol. wt. 140 K and 200 K as measured by SDS-polyacrylamide gel electrophoresis (SDS-PAGE)] was purified to homogeneity and characterized. The 200-K form is probably a precursor of the 140-K species. We have localized the glycoprotein by electron microscope immunochemistry using specific antibodies and determined its topological organization with respect to the membrane bilayer. Triton X-100 treatments which solubilize most other microvillar membrane glycoproteins from purified, closed, right-side out vesicles do not efficiently extract this protein. The protein can be partially solubilized from the detergent-insoluble residue, either by treatment with proteases (trypsin or papain) or by exposure to low ionic strength buffer in the presence of chelating agents and detergents. Once solubilized by papain or trypsin, the protein co-migrates on SDS-PAGE with the protein obtained by low ionic strength extraction. However, the form of the protein released by papain does not bind detergents and exhibits hydrophilic properties. Our observations are consistent with the 140-K protein having a small hydrophobic domain that anchors it to the microvillar membrane. The 140-K glycoprotein binds in vitro to a 110-K protein of the core cytoskeleton residue. These observations suggest that the 140-K glycoprotein may be a transmembrane protein which may in vivo provide attachment sites for direct or indirect association with polypeptides of the microvillus cytoskeleton.     

Endothelial microvilli in the vessels of the rat gasserian ganglion and testis

Summary In the Gasserian ganglion and testis of the rat the endothelium of the small blood vessels has a singular appearance due to the presence of a high number of microvillous processes. These arise from the luminal surface, either individually, or in small groups. The significance of this structure is not known. It is pointed out that these vessels are particularly sensitive to cadmium intoxication.This investigation was performed during a tenure by Dr. Gabbiani of a fellowship of the Medical Research Council of Canada and was supported by the National Institutes of Health (Grant No. 5R01 HE-08794-04) and the Ministère de la Santé du Québec (projet 604-7-635).The authors wish to thank Dr. S. M. Shea for his advice in the measurement of the villi. The technical assistance of Miss Virginia Gilmore and of Mr. Eduardo Garriga is gratefully acknowledged.     

Electron microscope observations on the carbohydrate-rich cell coat present at the surface of cells in the rat

Periodic acid-silver methenamine, a fairly specific technique for glycoprotein detection, was used to stain a variety of rat tissues, in the hope of confirming the existence of a carbohydrate-rich "cell coat" at the surface of mammalian cells. It was found that nearly all cells are coated by a thin layer of stained material. Around fibrocytes and migrating blood cells, the layer is uniform and merges with the ground substance. In the nervous system, cells and processes are surrounded with a layer whose density increases in synaptic clefts. Around epithelial cells, the layer outlines apical microvilli, follows lateral interspaces, and extends between cells and basement membrane. The layer is continuous with the middle plate of desmosomes and can be followed within the wide portion of terminal bars. In contrast, staining usually vanishes when two adjacent plasma membranes fuse to form tight junctions. These findings indicate that the stained layer is a "cell coat" located outside the plasma membrane. Since the cell coat is also stained by colloidal thorium, a technique for detection of acidic carbohydrates, this structure presumably contains not only glycoprotein(s) but also acidic residues. The carbohydrates may play a role in holding cells together and in controlling the interactions between cells and environment.