Sorting of actin isoforms in chicken auditory hair cells

1997 ◽  
Vol 110 (6) ◽  
pp. 765-770 ◽  
Author(s):  
D. Hofer ◽  
W. Ness ◽  
D. Drenckhahn
Keyword(s):  
Actin Filament ◽  
Hair Cells ◽  
Auditory Hair Cells ◽  
Actin Isoforms ◽  
Cytoplasmic Actin ◽  
Beta Actin ◽  
Nonmuscle Cells ◽  
Filament Bundles ◽  
Actin Isoform ◽  
Cellular Compartments

Most nonmuscle cells of higher vertebrates contain two different actin isoforms, beta- and gamma-cytoplasmic actin. The beta-isoform is with few exceptions the predominant isoform in nonmuscle cells and tissues. Perturbation of the beta:gamma ratio has been shown to affect the organization of bundled actin filaments indicating that the beta- and gamma-genes encode functionally distinct cytoarchitectural information. In the present study we localized by immunostaining beta- and gamma-actin in chicken auditory hair cells. These highly specialized cells serve as model system for studying certain developmental and structural aspects of a complex actin filament system with high architectural precision. We show that gamma-actin is the predominant actin isoform in auditory hair cells with an apparent beta:gamma ratio of approximately 1:2. gamma-Actin is not sorted and occurs in all three actin assemblies of the hair border, i.e. the cores of sensory hairs (stereocilia), the subjacent gel-like actin filament meshwork (cuticular plate) and the zonula adherens ring. In contrast to gamma-actin, the beta-isoform is specifically sorted to the actin filament core bundle of stereocilia that is extensively crosslinked by fimbrin. In view of recent studies showing that L-plastin, the leukocyte homolog of fimbrin, has a higher binding affinity for beta-actin than for gamma-actin, a mechanism is proposed for how hair cells might restrict formation of actin filament bundles to a single cellular site (i.e. the stereocilia). The limited level of expression of beta-actin in hair cells may help to prevent ectopic bundle formation in other cellular compartments.

scholarly journals Polarized distribution of actin isoforms in gastric parietal cells.

1995 ◽  
Vol 6 (5) ◽  
pp. 541-557 ◽  
Author(s):  
X Yao ◽  
C Chaponnier ◽  
G Gabbiani ◽  
J G Forte
Keyword(s):  
Epithelial Cells ◽  
Basolateral Membrane ◽  
Parietal Cells ◽  
Gastric Epithelial Cells ◽  
Intense Staining ◽  
Actin Isoforms ◽  
Specific Antibodies ◽  
Beta Actin ◽  
Actin Isoform ◽  
Gastric Parietal Cells

The actin genes encode several structurally similar, but perhaps functionally different, protein isoforms that mediate contractile function in muscle cells and determine the morphology and motility in nonmuscle cells. To reveal the isoform profile in the gastric monomeric actin pool, we purified actin from the cytosol of gastric epithelial cells by DNase I affinity chromatography followed by two-dimensional gel electrophoresis. Actin isoforms were identified by Western blotting with a monoclonal antibody against all actin isoforms and two isoform-specific antibodies against cytoplasmic beta-actin and gamma-actin. Densitometry revealed a ratio for beta-actin/gamma-actin that equaled 0.73 +/- 0.09 in the cytosol. To assess the distribution of actin isoforms in gastric glandular cells in relation to ezrin, a putative membrane-cytoskeleton linker, we carried out double immunofluorescence using actin-isoform-specific antibodies and ezrin antibody. Immunostaining confirmed that ezrin resides mainly in canaliculi and apical plasma membrane of parietal cells. Staining for the beta-actin isoform was intense along the entire gland lumen and within the canaliculi of parietal cells, thus predominantly near the apical membrane of all gastric epithelial cells, although lower levels of beta-actin were also identified near the basolateral membrane. The gamma-actin isoform was distributed heavily near the basolateral membrane of parietal cells, with much less intense staining of parietal cell canaliculi and no staining of apical membranes. Within parietal cells, the cellular localization of beta-actin, but not gamma-actin, isoform superimposed onto that of ezrin. In a search for a possible selective interaction between actin isoforms and ezrin, we carried out immunoprecipitation experiments on gastric membrane extracts in which substantial amounts of actin were co-eluted with ezrin from an anti-ezrin affinity column. The ratio of beta-actin/gamma-actin in the immunoprecipitate (beta/gamma = 2.14 +/- 0.32) was significantly greater than that found in the cytosolic fraction. In summary, we have shown that beta- and gamma-actin isoforms are differentially distributed in gastric parietal cells. Furthermore, our data suggest a preferential, but not exclusive, interaction between beta-actin and ezrin in gastric parietal cells. Finally, our results suggest that the beta- and gamma-actin-based cytoskeleton networks might function separately in response to the stimulation of acid secretion.

scholarly journals Drosophila singed, a fascin homolog, is required for actin bundle formation during oogenesis and bristle extension.

1994 ◽  
Vol 125 (2) ◽  
pp. 369-380 ◽  
Author(s):  
K Cant ◽  
B A Knowles ◽  
M S Mooseker ◽  
L Cooley
Keyword(s):  
Sea Urchin ◽  
Actin Filament ◽  
Nurse Cell ◽  
Structural Integrity ◽  
Molar Ratio ◽  
Cell Nuclei ◽  
Cytoplasmic Actin ◽  
Filament Bundles ◽  
Filament Bundle

Drosophila singed mutants were named for their gnarled bristle phenotype but severe alleles are also female sterile. Recently, singed protein was shown to have 35% peptide identity with echinoderm fascin. Fascin is found in actin filament bundles in microvilli of sea urchin eggs and in filopodial extensions in coelomocytes. We show that Drosophila singed is required for actin filament bundle formation in the cytoplasm of nurse cells during oogenesis; in severe mutants, the absence of cytoplasmic actin filament bundles allows nurse cell nuclei to lodge in ring canals and block nurse cell cytoplasm transport. Singed is also required for organized actin filament bundle formation in the cellular extension that forms a bristle; in severe mutants, the small disorganized actin filament bundles lack structural integrity and allow bristles to bend and branch during extension. Singed protein is also expressed in migratory cells of the developing egg chamber and in the socket cell of the developing bristle, but no defect is observed in these cells in singed mutants. Purified, bacterially expressed singed protein bundles actin filaments in vitro with the same stoichiometry reported for purified sea urchin fascin. Singed-saturated actin bundles have a molar ratio of singed/actin of approximately 1:4.3 and a transverse cross-banding pattern of 12 nm seen using electron microscopy. Our results suggest that singed protein is required for actin filament bundle formation and is a Drosophila homolog of echinoderm fascin.

scholarly journals Immunolocalization of the gamma isoform of nonmuscle actin in cultured cells.

1986 ◽  
Vol 102 (5) ◽  
pp. 1726-1737 ◽  
Author(s):  
C A Otey ◽  
M H Kalnoski ◽  
J L Lessard ◽  
J C Bulinski
Keyword(s):  
Cultured Cells ◽  
Contractile Ring ◽  
Western Blots ◽  
Actin Isoforms ◽  
Amino Terminal ◽  
Microfilament Bundles ◽  
Peptide Antibody ◽  
Nonmuscle Cells ◽  
Actin Isoform ◽  
Amino Terminal Sequence

In many vertebrate nonmuscle cells, the microfilament subunit protein, actin, exists as two isoforms, called beta and gamma, whose sequences differ only in their amino-terminal regions. We have prepared a peptide antibody specifically reactive with the amino-terminal sequence of gamma actin. This antibody reacted with nonmuscle actin as determined by Western blots of SDS gels, and reacted with the gamma, but not the beta, nonmuscle actin isoform as shown by Western blots of isoelectric focusing gels. In immunofluorescence experiments, the gamma peptide antibody stained microfilament bundles, ruffled edges, and the contractile ring of a variety of cultured cells, including mouse L cells, which have previously been reported to contain only the beta actin isoform (Sakiyama, S., S. Fujimura, and H. Sakiyama, 1981, J. Biol. Chem., 256:31-33). Double immunofluorescence experiments using the gamma peptide antibody and an antibody reactive with all actin isoforms revealed no differences in isoform localization. Thus, at the level of resolution of light microscopy, we have detected the gamma actin isoform in all microfilament-containing structures in cultured cells, and have observed no subcellular sorting of the nonmuscle actin isoforms.

scholarly journals Actin, alpha-actinin, and tropomyosin interaction in the structural organization of actin filaments in nonmuscle cells.

1976 ◽  
Vol 68 (2) ◽  
pp. 202-219 ◽  
Author(s):  
E Lazarides
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Specific Interaction ◽  
Three Dimensional ◽  
Culture Cells ◽  
Dimensional Network ◽  
Nonmuscle Cells ◽  
Embryo Cells ◽  
Filament Bundles ◽  
Regular Network

During the spreading of a population of rat embryo cells, approximately 40% of the cells develop a strikingly regular network which precedes the formation of the straight actin filament bundles seen in the fully spread out cells. Immunofluorescence studies with antibodies specific for the skeletal muscle structural proteins actin, alpha-actinin, and tropomyosin indicate that this network is composed of foci containing actin and alpha-actinin, connected by tropomyosin-associated actin filaments. Actin filaments, having both tropomyosin and alpha-actinin associated with them, are also seen to extend from the vertices of this network to the edges of the cell. These results demonstrate a specific interaction of alpha-actinin and tropomyosin with actin filaments during the assembly and organization of the actin filament bundles of tissue culture cells. The three-dimensional network they form may be regarded as the structural precursor and the vertices of this network as the organization centers of the ultimately formed actin filament bundles of the fully spread out cells.

scholarly journals Identification of actin from hepatoma Morris 5123 cells.

1999 ◽  
Vol 46 (4) ◽  
pp. 949-959 ◽  
Author(s):  
D Nowak ◽  
A Kochman ◽  
M Malicka-Błaszkiewicz
Keyword(s):  
Gel Electrophoresis ◽  
Dnase I ◽  
2D Gel Electrophoresis ◽  
Muscle Actin ◽  
Actin Isoforms ◽  
Beta Actin ◽  
Sds Page ◽  
Actin Isoform ◽  
2D Gel ◽  
Differential Affinity

The hepatoma Morris 5123 tumor growth is accompanied by changes in actin content and polymerization (Malicka-Błaszkiewicz et al. (1995) Mat. Med. Pol., 27, 115-118; Nowak et al. (1995) J. Exp. Cancer Res. 14, 37-40). Presently actin isoforms from cytosol and cytoskeleton fractions were separated by SDS/PAGE and identified with antibodies directed against different actin isoforms. Actin isolated from the cytosol by affinity chromatography on DNase I bound to agarose shows the presence of only one protein spot on 2D gel electrophoresis corresponding to the mobility of the rabbit a skeletal muscle actin (Mr 43,000) and isoelectric point equal to 5.3. It interacts only with monoclonal anti beta actin isoform antibodies, posing the question of differential affinity of actin isoforms to DNase I.

Actin mRNA isoforms are differentially sorted in normal osteoblasts and sorting is altered in osteoblasts from a skeletal mutation in the rat

1998 ◽  
Vol 111 (9) ◽  
pp. 1287-1292 ◽  
Author(s):  
H. Watanabe ◽  
E.H. Kislauskis ◽  
C.A. Mackay ◽  
A. Mason-Savas ◽  
S.C. Marks
Keyword(s):  
In Situ Hybridization ◽  
Bone Resorption ◽  
Cell Types ◽  
Mrna Isoforms ◽  
Actin Isoforms ◽  
Beta Actin ◽  
Cell Processes ◽  
Actin Mrna ◽  
Actin Isoform

Actin isoform sorting has been shown to occur in a variety of cell types in culture. To this list we add osteoblasts, in which we show by in situ hybridization that beta-actin is distributed primarily in cell processes and on one side of the nucleus and gamma-actin has a perinuclear distribution. Osteoblasts from the skeletal mutation toothless (tl), evaluated under identical conditions, fail to sort these actin isoforms differentially and exhibit diffuse labeling as their major manifestation. Northern analyses of actin mRNAs showed no differences between normal and mutant cultures. Shortened osteoblast life span and an inability to direct osteoclast-mediated bone resorption have recently been demonstrated in tl mutants. The present results suggest that a failure of osteoblasts to sort actin mRNAs may be related to one or both of these pathological manifestations in this mutation and represent, to our knowledge, the first correlation of an actin mRNA-sorting abnormality with a mammalian disease.

scholarly journals Impaired Expression of Cytoplasmic Actins Leads to Chromosomal Instability of MDA-MB-231 Basal-Like Mammary Gland Cancer Cell Line

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2151
Author(s):  
Vera Dugina ◽  
Galina Shagieva ◽  
Mariya Novikova ◽  
Svetlana Lavrushkina ◽  
Olga Sokova ◽  
...  
Keyword(s):  
Cell Line ◽  
Chromosomal Instability ◽  
Cell Transformation ◽  
Cancer Cell Line ◽  
Neoplastic Cell ◽  
Chromosome Morphology ◽  
Human Carcinoma ◽  
Actin Isoforms ◽  
Cytoplasmic Actin ◽  
Neoplastic Cell Transformation

We have shown previously that two cytoplasmic actin isoforms play different roles in neoplastic cell transformation. Namely, β-cytoplasmic actin acts as a tumor suppressor, whereas γ-cytoplasmic actin enhances malignant features of tumor cells. The distinct participation of each cytoplasmic actin in the cell cycle driving was also observed. The goal of this study was to describe the diverse roles of cytoplasmic actins in the progression of chromosomal instability of MDA-MB-231 basal-like human carcinoma cell line. We performed traditional methods of chromosome visualization, as well as 3D-IF microscopy and western blotting for CENP-A detection/quantification, to investigate chromosome morphology. Downregulation of cytoplasmic actin isoforms alters the phenotype and karyotype of MDA-MB-231 breast cancer cells. Moreover, β-actin depletion leads to the progression of chromosomal instability with endoreduplication and aneuploidy increase. On the contrary, γ-actin downregulation results not only in reduced percentage of mitotic carcinoma cells, but leads to chromosome stability, reduced polyploidy, and aneuploidy.

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