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A Bretscher

Publications and source records attributed to A Bretscher.

88 records · Page 5Linked to original sources
PubMed1983

Induced morphological changes in isolated microvilli: regulation of membrane topology in vitro by submembranous microfilaments.

We have investigated the effects of treating isolated microvilli with 1 mM ATP, 5 mM CaCl2, and 25 mM MgCl2 in terms of the morphological changes induced and the solubilization of cytoskeletal proteins. Neither ATP nor MgCl2 treatment alone induced significant morphological changes, despite some solubilization of cytoskeletal proteins; yet combined ATP and MgCl2 treatment resulted in "beading" of the membrane and some loss of membrane from the basal end of the structures. CaCl2 treatment resulted in a very regular beading of the microvillus membrane together with partial disassembly and solubilization of the core. These changes could be induced by free Ca++ in the micromolar range, which therefore suggests that they may be of physiological importance. Induced morphological changes are discussed in terms of the effects of these treatments on the microvillus cytoskeleton.

Adenosine Triphosphate↗
PubMed1982

Immunohistochemical localization of several cytoskeletal proteins in inner ear sensory and supporting cells.

Several structural and contractile proteins have been searched for with immunohistochemical methods using antibodies directed against these proteins. Three types of preparations from the guinea pig have been used: isolated stereocilia from the utricle, organ of Corti fragments obtained by cellular dissociation and 0.2-1 micrometer sections obtained by cryoultramicrotomy. The main finding is that different sets of proteins compose the cytoskeleton in supporting cells and the mechanoreceptor structures of the sensory cells. Thus, actin was found in association with fimbrin in the mechanoreceptive region of hair cells, whereas supporting cells, although rich in actin, did not reveal fimbrin. Instead tubulin was seen together with actin in supporting cells which also exhibited prekeratin. Fimbrin appears to function as a protein capable of making bundles and networks from actin filaments. Its exclusive presence in the mechanosensitive region of the sensory cells is possibly related to the function of these cells as mechanoreceptors.

Actins↗
PubMed1981

Fimbrin is a cytoskeletal protein that crosslinks F-actin in vitro.

Fimbrin is a cytoskeletal protein associated with microfilaments in microvilli, microspikes, stereocilia, membrane ruffles, and cell--substratum attachment sites. Fimbrin purified from intestinal epithelial cell brush borders was found to be a monomeric protein of molecular weight 68,000. In a sedimentation assay, fimbrin bound to F-actin in a salt-dependent manner, with binding being optimal in 30 mM KCl and inhibited in greater than 100 mM KCl. In 50 mM KCl, which allows efficient polymerization of actin, the interaction was stabilized by the presence of polyethylene glycol. Under these conditions, binding was unaffected by the inclusion of up to 5 mM Ca2+ but was inhibited by greater than 0.5 mM Mg2+. Electron microscopy revealed that fimbrin crosslinked F-actin into relatively straight bundles with shorter bundles being formed at high fimbrin-to-actin ratios. The results suggest that fimbrin crosslinks F-actin in such a way as to confer some rigidity on the bundle formed. This proposed function for fimbrin is consistent with its in vivo localization in straight, highly organized, microfilament bundles such as microvilli, microspikes, and stereocilia.

Actins↗
PubMed1980

Plasma membranes from intestinal microvilli and erythrocytes contain cytochromes b5 and P-420.

The presence of cytochromes b5, P-450 and P-420 and activities of NADH- and NADPH-cytochrome c redutases were determined in plasma membranes isolated from microvilli of the chick and rat intestinal epithelium and erythrocyte membranes from chick, rat and man. The results are compared with the amounts of these components found in microsomal fractions from intestinal epithelium and in nuclear membranes from chick erythrocytes. Plasma membranes from intestinal microvilli and from erythrocytes contained significant amounts of NADH-cytochrome c reductase activity and of a pigment spectrophotometrically indistinguishable from rat liver microsomal cytochrome b5. In addition, cytochrome b5 fragments were prepared from the membranes by limited trypsin digestion and consisted of two to four components with Mr values in the range 10 000-13 500. In low-temperature difference spectra, the presence of a second cytochrome was noted which was similar to cytochrome P-420. Cytochrome P-450 and NADPH-cytochrome c reductase activities were not detected in plasma membrane fractions in significant concentrations but were present in the corresponding endomembrane fractions. These findings in highly purified, well defined plasma membrane fractions, in which contamination by endomembranes is minimal, strengthen the evidence for the existence of cytochrome-containing redox systems in plasma membranes of various cells and suggest that such redox components are general components of the cell surface. Possible functions and origins of these redox components in plasma membranes are discussed.

Animals↗
PubMed1980

Calcium control of the intestinal microvillus cytoskeleton: its implications for the regulation of microfilament organizations.

The microvillus core-filament bundle from intestinal epithelial cells is a highly ordered structure containing actin and four major associated proteins. Two of these, villin and calmodulin, bind calcium ions (Kd approximately 10(-6) M) in the physiologically important range. Because ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid is present throughout the purification and the isolated cores contain levels of calcium substoichiometric to calmodulin, the protein is bound in the structure without calcium saturation. 10-[3-(4-Methyl-1-piperazinyl)propyl]-2-trifluoromethylphenothiazine, a calmodulin-specific drug, removes the protein from the cores without visibly affecting their ultrastructure. Calmodulin-depleted cores rebind exogenously supplied brain calmodulin. Although the core filaments are stable when the calcium level is less than 10(-7) M, they dissassemble when it is greater than 10(-6) M. This appears to be due to the calcium-sensitive allosteric transition of villin from an F-actin bundling protein to an F-actin severing protein. The actions of the two calcium-binding proteins, villin and calmodulin, are discussed in terms of the calcium sensitivity of the filament bundle. We suggest that villin may act as a calcium-sensitive factor regulating microfilament assembly and disassembly and that calmodulin serves as a buffer modulating the free calcium concentration. This hypothesis may explain some aspects of the physiological process of calcium uptake in the intestine and of the effects of calcium fluxes on the submembranous organization of microfilaments in other cells and tissues.

Actins↗
PubMed1980

Fimbrin, a new microfilament-associated protein present in microvilli and other cell surface structures.

A 68,000 mol wt polypeptide has been identified as one of the few major proteins in the microfilament bundles of the microvilli present on intestinal epithelial cells. Antibodies against the purified protein have been used in indirect immunofluorescence microscopy on several cultured cells. The protein have been used in indirect immunofluorescence microscopy on several cultured cells. The protein is found particularly prominent in membrane ruffles, microspikes, and microvilli.

Actins↗
PubMed1979

Villin: the major microfilament-associated protein of the intestinal microvillus.

The major protein associated with actin in the microfilament core of intestinal microvilli has been purified. This protein, for which we propose the name villin, has a polypeptide molecular weight of approximately 95,000. Two arguments suggest that villin may be the microvillus crossfilament protein that links the microfilament core laterally down its length to the cytoplasmic side of the plasma membrane. First, electron microscopy shows that crossfilaments stay attached to isolated membrane-free microvillus cores. Calculation of the expected abundance of the crossfilament protein shows that only villin is present in sufficient quantity to account for these structures. Second, decoration of microvillus cores by antibodies to either actin or villin, followed by ferritin-labeled second antibody in a sandwich procedure, results in specific labeling of the cores in both cases. The antivillin decoration, however, gives rise to a greater increase in diameter, in agreement with a model in which villin projects from the F-actin microfilament core. Villin is distinct from alpha-actinin, a protein suggested to be involved in membrane anchorage of microfilaments in nonmuscle cells. The two proteins differ in molecular weight. Specific antibodies against villin and alpha-actinin show no immunological crossreactivity. Immunofluorescence microscopy reveals that villin is located in the microvilli of the brush border whereas alpha-actinin is absent from the microvilli but is found in the terminal web. In addition, villin is not found in microfilament bundles of tissue culture cells, which are rich in alpha-actinin. Thus, villin and alpha-actinin appear to be immunologically and functionally different proteins.

Actinin↗
PubMed1978

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.

Actinin↗