scholarly journals Ultrastructural localization of alpha-actinin and filamin in cultured cells with the immunogold staining (IGS) method.

1984 ◽  
Vol 99 (4) ◽  
pp. 1324-1334 ◽  
Author(s):  
G Langanger ◽  
J de Mey ◽  
M Moeremans ◽  
G Daneels ◽  
M de Brabander ◽  
...  
Keyword(s):  
Lung Epithelial Cells ◽  
Stress Fibers ◽  
Structural Basis ◽  
Cortical Networks ◽  
Specific Staining ◽  
Immunogold Staining ◽  
Chicken Heart ◽  
Dense Bodies ◽  
Microfilament System ◽  
Triton X

Monospecific antibodies to chicken gizzard actin, alpha-actinin, and filamin have been used to localize these proteins at the ultrastructural level: secondary cultures of 14-d-old chicken embryo lung epithelial cells and chicken heart fibroblasts were briefly lysed with either a 0.5% Triton X-100/0.25% glutaraldehyde mixture, or 0.1% Triton X-100, fixed with 0.5% glutaraldehyde, and further permeabilized with 0.5% Triton X-100, to allow penetration of the gold-conjugated antibodies. After immunogold staining (De Mey, J., M. Moeremans, G. Geuens, R. Nuydens, and M. De Brabander, 1981, Cell Biol. Int. Rep. 5:889-899), the cells were postfixed in glutaraldehyde-tannic acid and further processed for embedding and thin sectioning. This approach enabled us to document the distribution of alpha-actinin and filamin either on the delicate cortical networks of the cell periphery or in the densely bundled stress fibers and polygonal nets. By using antiactin immunogold staining as a control, we were able to demonstrate the applicability of the method to the microfilament system: the label was distributed homogeneously over all areas containing recognizable microfilaments, except within very thick stress fibers, where the marker did not penetrate completely. Although alpha-actinin specific staining was homogeneously localized along loosely-organized microfilaments, it was concentrated in the dense bodies of stress fibers. The antifilamin-specific staining showed a typically spotty or patchy pattern associated with the fine cortical networks and stress fibers. This pattern occurred along all actin filaments, including the dense bodies also marked by anti-alpha-actinin antibodies. The results confirm and extend the data from light microscopic investigations and provide more information on the structural basis of the microfilament system.

scholarly journals Myofibroblast contraction activates latent TGF-β1 from the extracellular matrix

2007 ◽  
Vol 179 (6) ◽  
pp. 1311-1323 ◽  
Author(s):  
Pierre-Jean Wipff ◽  
Daniel B. Rifkin ◽  
Jean-Jacques Meister ◽  
Boris Hinz
Keyword(s):  
Extracellular Matrix ◽  
Growth Factor ◽  
Mechanical Stress ◽  
Protease Activity ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β1 ◽  
Stress Fibers ◽  
Fibrotic Diseases ◽  
Triton X ◽  
Tgf Β1

The conjunctive presence of mechanical stress and active transforming growth factor β1 (TGF-β1) is essential to convert fibroblasts into contractile myofibroblasts, which cause tissue contractures in fibrotic diseases. Using cultured myofibroblasts and conditions that permit tension modulation on the extracellular matrix (ECM), we establish that myofibroblast contraction functions as a mechanism to directly activate TGF-β1 from self-generated stores in the ECM. Contraction of myofibroblasts and myofibroblast cytoskeletons prepared with Triton X-100 releases active TGF-β1 from the ECM. This process is inhibited either by antagonizing integrins or reducing ECM compliance and is independent from protease activity. Stretching myofibroblast-derived ECM in the presence of mechanically apposing stress fibers immediately activates latent TGF-β1. In myofibroblast-populated wounds, activation of the downstream targets of TGF-β1 signaling Smad2/3 is higher in stressed compared to relaxed tissues despite similar levels of total TGF-β1 and its receptor. We propose activation of TGF-β1 via integrin-mediated myofibroblast contraction as a potential checkpoint in the progression of fibrosis, restricting autocrine generation of myofibroblasts to a stiffened ECM.

Monoclonal antibody P-31 recognizes a novel intermediate filament-associated protein (p250) in rat podocytes

1998 ◽  
Vol 274 (5) ◽  
pp. F986-F997 ◽  
Author(s):  
Hidetake Kurihara ◽  
Norio Sunagawa ◽  
Tatsuo Kobayashi ◽  
Kazuhiro Kimura ◽  
Nobuo Takasu ◽  
...  
Keyword(s):  
Intermediate Filament ◽  
Immunoblot Analysis ◽  
Puromycin Aminonucleoside ◽  
Immunogold Staining ◽  
Glomerular Epithelial Cells ◽  
Developing Kidney ◽  
Triton X ◽  
Filament Network ◽  
Aminonucleoside Nephrosis ◽  
Puromycin Aminonucleoside Nephrosis

The visceral glomerular epithelial cells (GECs) or podocytes of the renal glomerulus constitute a highly specialized epithelium. To study the nature of podocytes, we established mouse monoclonal antibodies against GEC. Clone P-31 reacted exclusively with the cytoplasm of GEC by immunofluorescence. Immunoblot analysis with P-31 showed that a single band of 250 kDa was detectable in a glomerular lysate. The 250-kDa polypeptide (p250) was recovered from Triton X-100-insoluble fractions of isolated glomeruli, suggesting that this molecule is associated with the cytoskeleton. Immunogold staining with P-31 demonstrated that the gold particles were located at the intersections of vimentin-type intermediate filaments of podocytes. In developing kidney, this protein first appeared in immature GECs during the S-shaped body stage. In puromycin aminonucleoside nephrosis, p250 was dramatically increased in glomeruli where enhanced desmin expression was observed in GECs. These results indicate that p250 is a novel intermediate filament-associated protein and plays a role in the organization of the intermediate filament network in both normal and diseased conditions.

scholarly journals Synaptopodin is required for stress fiber and contractomere assembly at the epithelial junction

2021 ◽  
Author(s):  
Timothy Morris ◽  
Eva Sue ◽  
Caleb Geniesse ◽  
William M Brieher ◽  
Vivian W Tang
Keyword(s):  
Control Cell ◽  
Stress Fiber ◽  
Force Generation ◽  
Stress Fibers ◽  
Structural Basis ◽  
Macromolecular Structure ◽  
Cell Geometry ◽  
Summary Statement ◽  
Myosin Iia ◽  
Epithelial Junction

AbstractThe apical junction of epithelial cells can generate force to control cell geometry and perform contractile processes while maintaining barrier function and cell-cell adhesion. Yet, the structural basis of force generation at the apical junction is not completely understood. Here, we describe 2 actomyosin structures at the apical junction containing synaptopodin, myosin IIB, and alpha-actinin-4. We showed that synaptopodin is required for the assembly of E-cadherin-associated apical stress fibers and a novel macromolecular structure, which we named contractomere. Knockdown of synaptopodin abolished both apical stress fiber and contractomere formation. Moreover, depletion of synaptopodin abolished basal stress fibers, converting myosin IIA sarcomere-like arrangement into a meshwork-type actomyosin organization. We propose a new model of junction dynamics that is dependent on contractomere movement to control epithelial cell boundary and geometry. Our findings reveal 2 actomyosin structures at the epithelial junction and underscore synaptopodin in the assembly of stress fibers and contractomeres.Summary StatementSynaptopodin assembles 2 actomyosin structures at the epithelial junction: apical stress fiber and contractomere. Synaptopodin selectively regulates myosin IIB without altering the level of myosin IIA and is responsible for converting evolutionary-conserved actomyosin meshwork into vertebrate-specific stress fibers.Graphic Abstract

Mechanical anisotropy of adherent cells probed by a three-dimensional magnetic twisting device

2004 ◽  
Vol 287 (5) ◽  
pp. C1184-C1191 ◽  
Author(s):  
Shaohua Hu ◽  
Luc Eberhard ◽  
Jianxin Chen ◽  
J. Christopher Love ◽  
James P. Butler ◽  
...  
Keyword(s):  
Magnetic Beads ◽  
Three Dimensional ◽  
Magnetic Bead ◽  
Mechanical Anisotropy ◽  
Stress Fibers ◽  
Structural Basis ◽  
Mechanical Stiffness ◽  
Cytoskeletal Tension ◽  
Coated Surface ◽  
Deformation Patterns

We describe a three-dimensional magnetic twisting device that is useful in characterizing the mechanical properties of cells. With the use of three pairs of orthogonally aligned coils, oscillatory mechanical torque was applied to magnetic beads about any chosen axis. Frequencies up to 1 kHz could be attained. Cell deformation was measured in response to torque applied via an RGD-coated, surface-bound magnetic bead. In both unpatterned and micropatterned elongated cells on extracellular matrix, the mechanical stiffness transverse to the long axis of the cell was less than half that parallel to the long axis. Elongated cells on poly-l-lysine lost stress fibers and exhibited little mechanical anisotropy; disrupting the actin cytoskeleton or decreasing cytoskeletal tension substantially decreased the anisotropy. These results suggest that mechanical anisotropy originates from intrinsic cytoskeletal tension within the stress fibers. Deformation patterns of the cytoskeleton and the nucleolus were sensitive to loading direction, suggesting anisotropic mechanical signaling. This technology may be useful for elucidating the structural basis of mechanotransduction.

scholarly journals An enhanced method for post-embedding immunocytochemical staining which preserves cell membranes.

1990 ◽  
Vol 38 (2) ◽  
pp. 159-170 ◽  
Author(s):  
M A Berryman ◽  
R D Rodewald
Keyword(s):  
Osmium Tetroxide ◽  
Cell Membranes ◽  
Uranyl Acetate ◽  
Cell Ultrastructure ◽  
Neonatal Rat ◽  
Immunocytochemical Staining ◽  
Membrane Phospholipids ◽  
Specific Staining ◽  
Immunogold Staining ◽  
Thin Sections

We have devised a method for immunogold staining of unosmicated, plastic-embedded tissue which gives high levels of specific staining without scrificing cell ultrastructure. The key to this method is a combination of several standard techniques optimized to preserve cell membranes as well as antigen. Important conditions include (a) a combination primary fixative, (b) post-fixation with uranyl acetate to preserve membrane phospholipids, (c) dehydration with acetone to minimize extraction of phospholipids, (d) low-temperature embedding in LR Gold resin, and (e) use of osmium tetroxide to stain thin sections after immunogold labeling. We have developed this method specifically to localize the membrane receptor for immunoglobulin G in the jejunal epithelium of the neonatal rat. Ultra-thin sections of embedded tissue were stained with a monoclonal primary antibody and colloidal gold-labeled secondary antibody, followed by 2% osmium tetroxide and lead citrate. The receptor was resolved in the well-preserved network of tubules, endosomes, and other membrane compartments involved in immunoglobulin transport. In several other tissues processed by this method, cell ultrastructure resembled that seen after conventional osmium post-fixation and epoxy embedding. In addition to its usefulness in these studies, this general method should be applicable to many other immunocytochemical problems.

scholarly journals The molecular organization of myosin in stress fibers of cultured cells.

1986 ◽  
Vol 102 (1) ◽  
pp. 200-209 ◽  
Author(s):  
G Langanger ◽  
M Moeremans ◽  
G Daneels ◽  
A Sobieszek ◽  
M De Brabander ◽  
...  
Keyword(s):  
Light Chain ◽  
Cultured Cells ◽  
Staining Pattern ◽  
Central Zone ◽  
Molecular Organization ◽  
Stress Fibers ◽  
General Distribution ◽  
Immunogold Staining ◽  
Myosin Subfragment ◽  
Myosin Subfragment 1

Antibodies to chicken gizzard myosin, subfragment 1, light chain 20, and light meromyosin were used to visualize myosin in stress fibers of cultured chicken cells. The antibody specificity was tested on purified gizzard proteins and total cell lysates using immunogold silver staining on protein blots. Immunofluorescence on cultured chicken fibroblasts and epithelial cells exhibited a similar staining pattern of antibodies to total myosin, subfragment 1, and light chain 20, whereas the antibodies to light meromyosin showed a substantially different reaction. The electron microscopic distribution of these antibodies was investigated using the indirect and direct immunogold staining method on permeabilized and fixed cells. The indirect approach enabled us to describe the general distribution of myosin in stress fibers. Direct double immunogold labeling, however, provided more detailed information on the orientation of myosin molecules and their localization relative to alpha-actinin: alpha-actinin, identified with antibodies coupled to 10-nm gold, was concentrated in the dense bodies or electron-dense bands of stress fibers, whereas myosin was confined to the intervening electron-lucid regions. Depending on the antibodies used in combination with alpha-actinin, the intervening regions revealed a different staining pattern: antibodies to myosin (reactive with the head portion of nonmuscle myosin) and to light chain 20 (both coupled to 5-nm gold) labeled two opposite bands adjacent to alpha-actinin, and antibodies to light meromyosin (coupled to 5-nm gold) labeled a single central zone. Based on these results, we conclude that myosin in stress fibers is organized into bipolar filaments.

scholarly journals Expression in Escherichia Coli, Purification, and Functional Reconstitution of Human Steroid 5α-Reductases

Endocrinology ◽  
2020 ◽  
Vol 161 (8) ◽  
Author(s):  
Hwei-Ming Peng ◽  
Juan Valentín-Goyco ◽  
Sang-Choul Im ◽  
Bing Han ◽  
Jiayan Liu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Functional Characterization ◽  
Catalytic Efficiency ◽  
Biochemical Properties ◽  
Exchange Chromatography ◽  
Structural Basis ◽  
Cancer Drug ◽  
Ph Optimum ◽  
Hek 293 ◽  
Triton X

Abstract The potent androgen 5α-dihydrotestosterone irreversibly derives from testosterone via the activity of steroid 5α-reductases (5αRs). The major 5αR isoforms in most species, 5αR1 and 5αR2, have not been purified to homogeneity. We report here the heterologous expression of polyhistidine-tagged, codon-optimized human 5αR1 and 5αR2 cDNAs in Escherichia coli. A combination of the nonionic detergents Triton X-100 and Nonidet P-40 enabled solubilization of these extremely hydrophobic integral membrane proteins and facilitated purification with affinity and cation-exchange chromatography methods. For functional reconstitution, we incorporated the purified isoenzymes into Triton X-100-saturated dioleoylphosphatidylcholine liposomes and removed excess detergent with polystyrene beads. Kinetic studies indicated that the 2 isozymes differ in biochemical properties, with 5αR2 having a lower apparent Km for testosterone, androstenedione, progesterone, and 17-hydroxyprogesterone than 5αR1; however, 5αR1 had a greater capacity for steroid conversion, as reflected by a higher Vmax than 5αR2. Both enzymes preferred progesterone as substrate over other steroids, and the catalytic efficiency of purified reconstituted 5αR2 exhibited a sharp pH optimum at pH 5. Intriguingly, we found that the prostate-cancer drug-metabolite 3-keto-∆ 4-abiraterone is metabolized by 5αR1 but not 5αR2, which may serve as a structural basis for isoform selectivity and inhibitor design. The functional characterization results with the purified reconstituted isoenzymes paralleled trends obtained with HEK-293 cell lines stably expressing native 5αR1 and 5αR2. Access to purified human 5αR1 and 5αR2 will advance studies of these important enzymes and might help to clarify their contributions to steroid anabolism and catabolism.

scholarly journals A novel approach to study of the structural basis of enzyme polymorphism. Analysis of carboxylesterase B of Escherichia coli as model

1987 ◽  
Vol 241 (3) ◽  
pp. 877-881 ◽  
Author(s):  
B Picard ◽  
P Goullet ◽  
R Krishnamoorthy
Keyword(s):  
Escherichia Coli ◽  
Theoretical Data ◽  
Enzyme Polymorphism ◽  
Structural Basis ◽  
Staining Procedure ◽  
Polymorphism Analysis ◽  
Specific Staining ◽  
Titration Curves ◽  
Novel Approach ◽  
Original Approach

In order to understand the structural basis of charge differences among enzyme variants without undertaking purification and sequencing of the protein, an original approach was developed. The approach is applicable to any enzyme or protein provided that there is a specific staining procedure. This consists, as a first step, in the projection of electrophoretically obtained mobility values versus pI of all variants into a two-dimensional profile. In a second step, starting from the most common variant, various theoretical possibilities of substitutions are envisaged, taking into consideration the pH of the electrophoretic conditions, pI of the variants and range of variations of the pK values of several amino acid side chains. In a third step, verification of the theoretical data is obtained through comparative protein titration curves by combined isoelectrofocusing-electrophoresis of several pairs of relevant variants. The validity of this approach is tested on the highly polymorphic carboxylesterase B enzyme of Escherichia coli and is found to provide valuable information.

Dense-body aggregates as plastic structures supporting tension in smooth muscle cells

2010 ◽  
Vol 299 (5) ◽  
pp. L631-L638 ◽  
Author(s):  
Jie Zhang ◽  
Ana M. Herrera ◽  
Peter D. Paré ◽  
Chun Y. Seow
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Striated Muscle ◽  
Dense Body ◽  
Muscle Cells ◽  
Cell Length ◽  
Structural Basis ◽  
Airway Smooth Muscle Cells ◽  
Dense Bodies ◽  
Large Length

The wall of hollow organs of vertebrates is a unique structure able to generate active tension and maintain a nearly constant passive stiffness over a large volume range. These properties are predominantly attributable to the smooth muscle cells that line the organ wall. Although smooth muscle is known to possess plasticity (i.e., the ability to adapt to large changes in cell length through structural remodeling of contractile apparatus and cytoskeleton), the detailed structural basis for the plasticity is largely unknown. Dense bodies, one of the most prominent structures in smooth muscle cells, have been regarded as the anchoring sites for actin filaments, similar to the Z-disks in striated muscle. Here, we show that the dense bodies and intermediate filaments formed cable-like structures inside airway smooth muscle cells and were able to adjust the cable length according to cell length and tension. Stretching the muscle cell bundle in the relaxed state caused the cables to straighten, indicating that these intracellular structures were connected to the extracellular matrix and could support passive tension. These plastic structures may be responsible for the ability of smooth muscle to maintain a nearly constant tensile stiffness over a large length range. The finding suggests that the structural plasticity of hollow organs may originate from the dense-body cables within the smooth muscle cells.

scholarly journals Incorporation and turnover of biotin-labeled actin microinjected into fibroblastic cells: an immunoelectron microscopic study.

1989 ◽  
Vol 109 (4) ◽  
pp. 1581-1595 ◽  
Author(s):  
S Okabe ◽  
N Hirokawa
Keyword(s):  
Actin Filaments ◽  
Actin Polymerization ◽  
Stress Fibers ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Actin Bundles ◽  
Actin Networks ◽  
Focal Contacts ◽  
Microfilament System ◽  
Fibroblastic Cells

We investigated the mechanism of turnover of an actin microfilament system in fibroblastic cells on an electron microscopic level. A new derivative of actin was prepared by labeling muscle actin with biotin. Cultured fibroblastic cells were microinjected with biotinylated actin, and incorporated biotin-actin molecules were detected by immunoelectron microscopy using an anti-biotin antibody and a colloidal gold-labeled secondary antibody. We also analyzed the localization of injected biotin-actin molecules on a molecular level by freeze-drying techniques. Incorporation of biotin-actin was rapid in motile peripheral regions, such as lamellipodia and microspikes. At approximately 1 min after injection, biotin-actin molecules were mainly incorporated into the distal part of actin bundles in the microspikes. Heavily labeled actin filaments were also observed at the distal fringe of the densely packed actin networks in the lamellipodium. By 5 min after injection, most actin polymers in microspikes and lamellipodia were labeled uniformly. These findings suggest that actin subunits are added preferentially at the membrane-associated ends of preexisting actin filaments. At earlier times after injection, we often observed that the labeled segments were continuous with unlabeled segments, suggesting the incorporation of new subunits at the ends of preexisting filaments. Actin incorporation into stress fibers was a slower process. At 2-3 min after injection, microfilaments at the surface of stress fibers incorporated biotin-actin, but filaments in the core region of stress fibers did not. At 5-10 min after injection, increasing density of labeling along stress fibers toward their distal ends was observed. Stress fiber termini are generally associated with focal contacts. There was no rapid nucleation of actin filaments off the membrane of focal contacts and the pattern of actin incorporation at focal contacts was essentially identical to that into distal parts of stress fibers. By 60 min after injection, stress fibers were labeled uniformly. We also analyzed the actin incorporation into polygonal nets of actin bundles. Circular dense foci, where actin bundles radiate, were stable structures, and actin filaments around the foci incorporated biotin-actin the slowest among the actin-containing structures within the injected cells. These results indicate that the rate and pattern of actin subunit incorporation differ in different regions of the cytoplasm and suggest the possible role of rapid actin polymerization at the leading margin on the protrusive movement of fibroblastic cells.

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