scholarly journals Increase in actin contents and elongation of apical projections in retinal pigmented epithelial cells during development of the chicken eye.

1985 ◽  
Vol 101 (2) ◽  
pp. 590-596 ◽  
Author(s):  
K Owaribe ◽  
G Eguchi
Keyword(s):  
Epithelial Cells ◽  
Actin Filaments ◽  
Immunofluorescence Microscopy ◽  
Photoreceptor Cells ◽  
Optical Diffraction ◽  
Heavy Meromyosin ◽  
Actin Bundles ◽  
The Core ◽  
Sds Page ◽  
Retinal Pigmented Epithelial Cells

The structural and biochemical changes of cytoskeletal components of retinal pigmented epithelial cells were studied during the development of chicken eyes. When the cytoskeletal components of the pigmented epithelial cells from various stages of development were examined by SDS PAGE, actin contents in the cells markedly increased between the 15-d-old and hatching stages. Immunofluorescence microscopy showed that chicken pigmented epithelial cells have two types of actin bundles. One is the circumferential bundle associated with the zonula adherens region as previously reported (Owaribe, K., and H. Masuda, 1982, J. Cell Biol., 95:310-315). The other is the paracrystalline bundle forming the core of the apical projections. The increase in actin contents after the 15-d-old stage is accompanied by the formation and elongation of core filaments of apical projections in the cells. During this period the apical projections extend into extracellular space among outer and inner segments of photoreceptor cells. Accompanying this change is an elongation of the paracrystalline bundles of actin filaments in the core of the projection. By electron microscopy, the bundles decorated with muscle heavy meromyosin showed unidirectional polarity, and had transverse striations with approximately 12-nm intervals, as determined by optical diffraction of electron micrographs. Since the shape of these bundles was not altered in the presence or absence of Ca2+, they seemed not to have villin-like proteins. Unlike the circumferential bundles, the paracrystalline bundles did not contract when exposed to Mg-ATP. These observations indicate that the paracrystalline bundles are structurally and functionally different from the circumferential actin bundles.

scholarly journals FORMATION OF ARROWHEAD COMPLEXES WITH HEAVY MEROMYOSIN IN A VARIETY OF CELL TYPES

1969 ◽  
Vol 43 (2) ◽  
pp. 312-328 ◽  
Author(s):  
Harunori Ishikawa ◽  
Richard Bischoff ◽  
Howard Holtzer
Keyword(s):  
Epithelial Cells ◽  
Actin Filaments ◽  
Cell Types ◽  
Nerve Cells ◽  
Collagen Fibrils ◽  
Heavy Meromyosin ◽  
Thin Filaments ◽  
Cellular Components

Heavy meromyosin (HMM) forms characteristic arrowhead complexes with actin filaments in situ. These complexes are readily visualized in sectioned muscle. Following HMM treatment similar complexes appear in sectioned fibroblasts, chondrogenic cells, nerve cells, and several types of epithelial cells. Thin filaments freshly isolated from chondrogenic cells also bind HMM and form arrowhead structures in negatively stained preparations. HMM-filament complexes are prominent in the cortex of a variety of normal metaphase and Colcemid-arrested metaphase cells. There is no detectable binding of HMM with other cellular components such as microtubules, 100-A filaments, tonofilaments, membranes, nuclei, or collagen fibrils. The significance of HMM-filament binding is discussed in view of the finding that arrowhead complexes form in types of cells not usually thought to contain actin filaments.

scholarly journals Isolation and characterization of circumferential microfilament bundles from retinal pigmented epithelial cells.

1982 ◽  
Vol 95 (1) ◽  
pp. 310-315 ◽  
Author(s):  
K Owaribe ◽  
H Masuda
Keyword(s):  
Epithelial Cells ◽  
Electrophoretic Pattern ◽  
Isolation And Characterization ◽  
Microfilament Bundles ◽  
Myosin Subfragment ◽  
Sds Page ◽  
Myosin Subfragment 1 ◽  
Retinal Pigmented Epithelial Cells ◽  
Large Contraction

Chicken retinal pigmented epithelial cells have circumferential microfilament bundles (CMBs) at the zonula adherens region. We have isolated these CMBs in intact form and characterized them structurally and biochemically. Pigmented epithelia obtained from 11-d-old chick embryos were treated with glycerol and Triton. Then, the epithelia were homogenized by passing them through syringe needles. Many isolated CMBs were found in the homogenate by phase-contrast microscopy. They formed polygons, mostly pentagons and hexagons, or fragments of polygons. Polygons were filled with meshwork structures, i.e. they were polygonal plates. Upon exposure to Mg-ATP, isolated CMBs showed clear and large contraction. The contraction was inhibited by treatment with N-ethylmaleimide-modified myosin subfragment-1. After purification by centrifugation with the density gradient of Percoll, CMBs were analyzed by SDS PAGE. The electrophoretic pattern gave three major components of 200, 55, and 42 kdaltons and several minor components. Electron microscopy showed that the polygons were composed of thick bundles of actin-containing microfilaments, and the meshworks were composed primarily of intermediate filaments.

Concentration-dependent effects of cytochalasin D on tight junctions and actin filaments in MDCK epithelial cells

1994 ◽  
Vol 107 (3) ◽  
pp. 367-375 ◽  
Author(s):  
B.R. Stevenson ◽  
D.A. Begg
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Actin Filaments ◽  
Mdck Cells ◽  
Microscopic Analysis ◽  
Cytochalasin D ◽  
Transepithelial Resistance ◽  
Actin Bundles ◽  
Filament Bundles ◽  
Tight Junction Permeability

The effects of different concentrations of the actin-disrupting drug cytochalasin D on tight junction permeability and distribution of actin filaments in MDCK epithelial cells were examined. Consistent with previous studies, 2 micrograms/ml cytochalasin D caused a significant decrease in transepithelial resistance, indicative of an increase in tight junction permeability. Surprisingly, increasing concentrations of cytochalasin D caused progressively smaller decreases in transepithelial resistance. The effects of cytochalasin D were reversible. Light microscopic analysis utilizing rhodamine-conjugated phalloidin demonstrated two distinct populations of actin filaments in MDCK cells: an apical peripheral ring of actin, presumably associated with the zonula adherens, and larger actin bundles more basally situated. When treated with 2 micrograms/ml cytochalasin D, both actin populations were severely disrupted and cells became flattened. Actin in the apical ring aggregated along cell boundaries, and these aggregates co-localized with similarly disrupted focal accumulations of the tight junction-associated protein ZO-1. The basal actin filament bundles also reorganized into focal aggregates. Increasing concentrations of cytochalasin D caused gradually less perturbation of the apical actin ring, consistent with the transepithelial resistance observations. However, the basal actin bundles were disrupted at all concentrations of cytochalasin D tested, demonstrating that the two actin populations are differentially sensitive to cytochalasin D and that apical actin filaments are more important in the regulation of tight junction permeability. Finally, treatment of cells with cytochalasin D inhibited the decrease in transepithelial resistance induced by the chelation of extracellular Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals The spectrin-related molecule, TW-260/240, cross-links the actin bundles of the microvillus rootlets in the brush borders of intestinal epithelial cells.

1983 ◽  
Vol 96 (5) ◽  
pp. 1491-1496 ◽  
Author(s):  
J R Glenney ◽  
P Glenney ◽  
K Weber
Keyword(s):  
Epithelial Cells ◽  
Actin Filaments ◽  
Intestinal Epithelial Cells ◽  
Intestinal Epithelial ◽  
Cross Link ◽  
Actin Bundles ◽  
Terminal Web ◽  
Brush Borders ◽  
Cross Links

Previous studies have shown that molecules related to erythrocyte spectrin are present in the cortical cytoplasm of nonerythroid cells. We report here the localization by immunoelectron microscopy of one such molecule, TW-260/240, in the brush border of intestinal epithelial cells. Using highly specific antibodies against TW-260 and TW-240 as well as antibodies against fodrin, another spectrinlike molecule, we have found that the TW-260/240 molecules are displayed between rootlets at all levels of the terminal web. Occasionally, extended structures appear labeled suggestive of the fine filaments known to cross-link actin bundles. These results are in line with previous in vitro studies showing that TW-260/240 binds to, and cross-links, actin filaments. The results are discussed in terms of a model in which rootlets are immobilized in the terminal web in a matrix of TW-260/240.

scholarly journals Calcium-regulated cooperative binding of the microvillar 110K-calmodulin complex to F-actin: formation of decorated filaments.

1987 ◽  
Vol 105 (1) ◽  
pp. 325-333 ◽  
Author(s):  
L M Coluccio ◽  
A Bretscher
Keyword(s):  
Electron Microscopy ◽  
Actin Filaments ◽  
Protein Concentration ◽  
High Yield ◽  
Cooperative Binding ◽  
Compelling Evidence ◽  
Calmodulin Antagonists ◽  
Heavy Meromyosin ◽  
The Core ◽  
Purification Scheme

The 110K-calmodulin complex of intestinal microvilli is believed to be the link between the actin filaments comprising the core bundle and the surrounding cell membrane. Although not the first study describing a purification scheme for the 110K-calmodulin complex, a procedure for the isolation of stable 110K-calmodulin complex both pure and in high yield is presented; moreover, isolation is without loss of the associated calmodulin molecules since a previously determined ratio in isolated microvillar cytoskeletons of calmodulin to 110-kD polypeptide of 3.3:1 is preserved. We have found that removal of calmodulin from the complex by the calmodulin antagonists W7 or W13 results in precipitation of the 110-kD polypeptide with calmodulin remaining in solution. The interaction of 110K-calmodulin with beef skeletal muscle F-actin has been examined. Cosedimentation assays of 110K-calmodulin samples incubated with F-actin show the amount of 110K-calmodulin associating with F-actin to be ATP, calcium, and protein concentration dependent; however, relatively salt independent. In calcium, approximately 30% of the calmodulin remains in the supernatant rather than cosedimenting with the 110-kD polypeptide and actin. Electron microscopy of actin filaments after incubation with 110K-calmodulin in either calcium- or EGTA-containing buffers show polarized filaments often laterally associated. Each individual actin filament is seen to exhibit an arrowhead appearance characteristic of actin filaments after their incubation with myosin fragments, heavy meromyosin and subfragment 1. In some cases projections having a 33-nm periodicity are observed. This formation of periodically spaced projections on actin filaments provides further compelling evidence that the 110K-calmodulin complex is the bridge between actin and the microvillar membrane.

The Organization of Actin Filaments in the Stereocilia of the Bird Cochlea

1980 ◽  
Vol 38 ◽  
pp. 554-555
Author(s):  
E.J. Battles ◽  
D. DeRosier ◽  
J.C. Saunders ◽  
L.G. Tilney
Keyword(s):  
Hair Cell ◽  
Diffraction Pattern ◽  
Sea Urchin ◽  
Actin Filaments ◽  
Optical Diffraction ◽  
Dense Material ◽  
Apical Surface ◽  
Structural Rigidity ◽  
High Degree ◽  
Degree Of Order

Extending from the apical surface of each hair cell of the chick cochlea are from 75 to 200 microvilli or stereocllia and one true cllium, the kinocilium. The stereocllia are arranged in rows of progressively increasing length (Fig. 1). Within each tapering sterocilium is a bundle of actin filaments with over 900 filaments near the tip yet only approximately 25 at the base where filaments are enmeshed in a dense material (Fig. 1); from here some of the filaments enter the apical surface of the cell (cuticular plate) as a rootlet. Examination of longitudinal sections of the stereocilia (Fig. 2) show that the filaments are aligned parallel to each other and show considerable order. Examination of an optical diffraction pattern of this bundle (Fig. 4) reveal that the actin filaments are packed such that the crossover points of adjacent actin filaments are inregister. A prominent reflection at 125Å−1 demonstrates that the filaments are cjossbridged by a macromolecular bridge situated at an average of 125Å−1 intervals (Fig. 4) in transverse sections the filaments appear hexagonally packed although there are regions where the filaments are less ordered (Fig. 3). In images processed in the computer to remove, noise and enhance detail periodic nature of the bridge can be clearly seen (see arrows Fig. 5). This image resembles that of an actin paracrystal formed from sea urchin extract composed of bundles of actin filaments crossbridged by a second protein. Thus the actin filaments in the bird stereocilia by being cross-bridged and packed with a high degree of order and produces a structure with considerable structural rigidity. Embryos were studied at various stages in development in an attempt to determine how the stereocilia form and how does the actin packing develops. These stages will be discussed.

scholarly journals Actin-binding protein promotes the bipolar and perpendicular branching of actin filaments.

1980 ◽  
Vol 87 (3) ◽  
pp. 841-848 ◽  
Author(s):  
J H Hartwig ◽  
J Tyler ◽  
T P Stossel
Keyword(s):  
Actin Filaments ◽  
Actin Polymerization ◽  
Average Length ◽  
Binding Protein ◽  
Actin Binding ◽  
Branch Points ◽  
Heavy Meromyosin ◽  
Actin Binding Protein ◽  
Protein Dimers ◽  
Protein Molecules

Branching filaments with striking perpendicularity form when actin polymerizes in the presence of macrophage actin-binding protein. Actin-binding protein molecules are visible at the branch points. Compared with actin polymerized in the absence of actin-binding proteins, not only do the filaments branch but the average length of the actin filaments decreases from 3.2 to 0.63 micrometer. Arrowhead complexes formed by addition of heavy meromyosin molecules to the branching actin filaments point toward the branch points. Actin-binding protein also accelerates the onset of actin polymerization. All of these findings show that actin filaments assemble from nucleating sites on actin-binding protein dimers. A branching polymerization of actin filaments from a preexisting lattice of actin filaments joined by actin-binding protein molecules could generate expansion of cortical cytoplasm in amoeboid cells.

scholarly journals Cell-substratum interaction of cultured avian osteoclasts is mediated by specific adhesion structures.

1984 ◽  
Vol 99 (5) ◽  
pp. 1696-1705 ◽  
Author(s):  
P C Marchisio ◽  
D Cirillo ◽  
L Naldini ◽  
M V Primavera ◽  
A Teti ◽  
...  
Keyword(s):  
Electron Microscope ◽  
Intermediate Filaments ◽  
Immunofluorescence Microscopy ◽  
Laying Hens ◽  
Cell Types ◽  
Medullary Bone ◽  
Transformed Cell ◽  
The Core ◽  
Low Calcium Diet

The cell-substratum interaction was studied in cultures of osteoclasts isolated from the medullary bone of laying hens kept on low calcium diet. In fully spread osteoclasts, cell-substratum adhesion mostly occurred within a continuous paramarginal area that corresponded also to the location of a thick network of intermediate filaments of the vimentin type. In this area, regular rows of short protrusions contacting the substratum and often forming a cup-shaped adhesion area were observed in the electron microscope. These short protrusions showed a core of F-actin-containing material presumably organized as a network of microfilaments and surrounded by a rosette-like structure in which vinculin and alpha-actinin were found by immunofluorescence microscopy. Rosettes were superposable to dark circles in interference-reflection microscopy and thus represented circular forms of close cell-substratum contact. The core of ventral protrusions also contained, beside F-actin, fimbrin and alpha-actinin. Villin was absent. This form of cell-substratum contact occurring at the tip of a short ventral protrusion differed from other forms of cell-substratum contact and represented an osteoclast-specific adhesion device that might also be present in in vivo osteoclasts as well as in other normal and transformed cell types.

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