Tropomyosin antibody: the specific localization of tropomyosin in nonmuscle cells

An antibody against purified chicken skeletal muscle tropomyosin is used in indirect immunofluorescence to visualize the localization of tropomyosin in a variety of nonmuscle cells. The antibody produces a fluorescent pattern which is very similar to that obtained with an actin-specific antibody. This pattern is composed of fluorescent fibers which are shown to be coincident with the fibers seen with phase-contrast optics. High resolution epifluorescent microscopy reveals that fibers stained with the actin antibody show a continuous fluorescence, while fibers reacted with the tropomyosin antibody show a periodic fluorescence. Measurements indicate that the lengths of the fluorescent segments are variable with an average of 1.2 μm while the spacing between segments is approximately 0.4 μm

Actin, α-actinin, and tropomyosin interaction in the structural organization of actin filaments in nonmuscle cells

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, α-actinin, and tropomyosin indicate that this network is composed of foci containing actin and α-actinin, connected by tropomyosin-associated actin filaments. Actin filaments, having both tropomyosin and α-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 α-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

Synthesis of spectrin in avian erythroid cells: association of nascent polypeptide chains with the cytoskeleton

The site of synthesis of spectrin was investigated in erythroid cells from 10-day chicken embryos. After various periods of [35S]methionine incorporation the cells were lysed in a Triton X-100 (TX-100)-containing buffer and were separated into a TX-100-soluble and -insoluble (cytoskeletal) fraction. Analysis of these two fractions by two-dimensional gel electrophoresis after a short pulse-labeling period reveals that alpha-spectrin nascent polypeptides are present predominantly in the TX-100-insoluble fraction. These polypeptides can be immunoprecipitated with alpha-spectrin antisera and the [35S]methionine incorporated into them during a short pulse can be chased into mature alpha-spectrin molecules. The alpha-spectrin nascent polypeptide chains are released quantitatively from the TX-100 cytoskeleton by treatment of lysed cells with puromycin, suggesting that they themselves are not associated with the cytoskeleton. A small fraction of the newly synthesized mature alpha-spectrin molecules is rapidly incorporated into the cytoskeleton, as shown by the fact that they are not released by the puromycin treatment; the rest are recovered in the soluble fraction. These results suggest that alpha-spectrin is synthesized in association with the cytoskeleton during chicken erythropoiesis and assembles onto the membrane-cytoskeleton posttranslationally

Assembly of Protein 4.1 during Chicken Erythroid Differentiation

Protein 4.1 is a peripheral membrane protein that strengthens the actin-spectrin based membrane skeleton of the red blood cell and also serves to attach this structure to the plasma membrane. In avian erythrocytes it exists as a family of closely related polypeptides that are differentially expressed during erythropoiesis. We have analyzed the synthesis and assembly onto the membrane skeleton of protein 4.1 and in this paper we show that its assembly is extremely rapid and highly efficient since greater than 95% of the molecules synthesized are assembled in less than 1 min. The remaining minor fraction of unassembled protein 4.1 differs kinetically and is either degraded or assembled with slower kinetics. All protein 4.1 variants exhibit a similar kinetic behavior irrespective of the stage of erythroid differentiation. Thus, the amount and the variants ratio of protein 4.1 assembled are determined largely at the transcriptional or at the translational level and not posttranslationally. During erythroid terminal differentiation the molar amounts of protein 4.1 and spectrin assembled change. In postmitotic cells, as compared with proliferative cells, far more protein 4.1 than spectrin is assembled onto the membrane-skeleton. This modulation may permit the assembly of an initially flexible membrane skeleton in mitotic erythroid cells. As cells become postmitotic and undergo the final steps of maturation the membrane skeleton may be gradually stabilized by the assembly of protein 4.1

Isolation of a new high molecular weight protein associated with desmin and vimentin filaments from avian embryonic skeletal muscle

Filaments with a diameter of 80-120 Å have been prepared from 14-d-old chick embryonic skeletal muscle, using a physiological salt solution and gel filtration chromatography. The filaments obtained are composed of the two known muscle intermediate-filament proteins, vimentin and desmin, as well as the vimentin- and desmin-associated high molecular weight protein, synemin (230,000 mol. wt). In addition, they contain a previously unidentified high molecular weight protein (280,000 mol wt) which differs from synemin by isoelectric point, molecular weight, and immunological reactivity. Immunofluorescence on cultured myogenic cells,using antisera to the 280,000-dalton polypeptide, has revealed that this protein has the same spatial distribution as desmin, vimentin, and synemin in both early myotubes, where it associates with cytoplasmic filaments, and late in myotubes, where it is associated with myofibril Z lines. Examination by immunofluorescence of frozen sections of developing embryonic skeletal muscle reveals a gradual diminution in the presence of the 280,000-dalton protein. The 280,000-dalton protein is undetectable in adult skeletal and smooth muscle, as shown by immunofluorescence and immunoautoradiography. In chick embryonic fibroblasts grown in tissue culture, only a subpopulation of the cells is reactive with antibodies to the 280,000-dalton protein even though all these cells contain vimentin. In the reactive cells, vimentin and the 280,000-dalton polypeptide exhibit an indistinguishable cytoplasmic filamentous network, which aggregates into filamentious bundles when the cells are exposed to colcemid. These results suggest that this newly identified high molecular weight protein is closely associated with intermediate filaments containing either vimentin alone or vimentin, desmin and synemin. The expression of this protein appears to be developmentally regulated and does not appear to parallel the expression of any of the other three intermediate-filament proteins. The absence of the 280,000-dalton polypeptide in adult muscle cells and its gradual reduction during development implies that is probably not required for the maintenance of Z-disk structure after the assembly of the sarcomere

Vimentin filaments are assembled from a soluble precursor in avian erythroid cells

The synthesis and assembly of vimentin was studied in erythroid cells from 10-d-old chicken embryos. After various periods of [35S]methionine incorporation, cells were lysed in a Triton X-100-containing buffer and separated into a soluble and an insoluble (cytoskeletal) fraction. Analysis of these two fractions by two-dimensional gel electrophoresis shows that vimentin is almost exclusively present in the cytoskeletal fraction and that newly synthesized vimentin is rapidly incorporated into this fraction. However, after a short pulse-labeling period, a prominent labeled protein at the position of vimentin is present in the soluble fraction. By immunoautoradiography and immunoprecipitations with vimentin antibodies, this protein was identified as vimentin. The vimentin in the soluble fraction is not sedimented by high speed centrifugation, suggesting that it does not consist of short filaments. After different pulse-labeling periods, assembly of newly synthesized vimentin in the cytoskeletal fraction increases linearly, while the radioactivity in the soluble vimentin remains constant. During a 2-h pulse-chase period, the vimentin in the soluble fraction is chased into the cytoskeletal fraction, with a half-life of 7 min. These results suggest that in chicken embryo erythroid cells newly synthesized vimentin is rapidly assembled into filaments from a soluble precursor

Highly Homologous Filamin Polypeptides Have Different Distributions in Avian Slow and Fast Muscle Fibers

The high molecular weight actin-binding protein filamin is located at the periphery of the Z disk in the fast adult chicken pectoral muscle (Gomer, R. H., and E. Lazarides, 1981, Cell, 23: 524-532). In contrast, we have found that in the slow anterior latissimus dorsi (ALD) muscle, filamin was additionally located throughout the l band as judged by immunofluorescence with affinity-purified antibodies on myofibrils and cryosections. The Z line proteins desmin and alpha-actinin, however, had the same distribution in ALD as they do in pectoral muscle. Quantitation of filamin and actin from the two muscle types showed that there was approximately 10 times as much filamin per actin in ALD myofibrils as in pectoral myofibrils. Filamin immunoprecipitated from ALD had an electrophoretic mobility in SDS polyacrylamide gels identical to that of pectoral myofibril filamin and slightly greater than that of chicken gizzard filamin. Two-dimensional peptide maps of filamin immunoprecipitated and labeled with ^(125)I showed that ALD myofibril filamin was virtually identical to pectoral myofibril filamin and was distinct from chicken gizzard filamin

Canavanine Inhibits Vimentin Assembly But Not Its Synthesis in Chicken Embryo Erythroid Cells

In chicken embryo erythroid cells, newly synthesized vimentin first enters a Triton X-100 (TX-100)-soluble pool and subsequently assembles posttranslationally into TX-100-insoluble vimentin filaments (Blikstad I., and E. Lazarides, J. Cell Biol., 96:1803-1808). Here we show that incubation of chicken embryo erythroid cells in a medium in which arginine has been substituted by its amino acid analogue, canavanine, results in the inhibition of the posttranslational assembly of vimentin into the TX-100-insoluble filaments. Immunoprecipitation and subsequent SDS gel electrophoresis showed that the synthesis of canavanine-vimentin is not inhibited and that it accumulates in the TX-100-soluble compartment. Pulse-chase experiments with [35S]methionine demonstrated that while arginine-vimentin can be rapidly chased from the soluble to the cytoskeletal fraction, canavanine-vimentin remains in the soluble fraction, where it turns over. The effect of canavanine on the assembly of vimentin did not prevent the assembly of arginine-vimentin, as cells labeled with [35S]methionine first in the presence of canavanine and then in the presence of arginine contained labeled canavanine-vimentin only in the soluble fraction, and arginine-vimentin in both the soluble and cytoskeletal fractions. These results suggest that arginine residues play an essential role in the assembly of vimentin in vivo

Continuous growth of vimentin filaments in mouse fibroblasts

We have investigated the dynamics of intermediate filament assembly in vivo by following the fate of heterologous chicken vimentin subunits expressed under the control of an inducible promoter in transfected mouse fibroblasts. Using RNase protection, metabolic protein pulse-chase and immunofluorescence microscopy, we have examined the fate of newly assembled subunits under physiological conditions in situ. Following induction and subsequent removal of inducer, chicken vimentin mRNA had a half-life of approximately 6 h while both chicken and mouse vimentin protein polymer had long half-lives--roughly equivalent to the cell generation time. Moreover, following deinduction, chicken vimentin immunolocalization progressed from a continuous (8-10 h chase) to a discontinuous (> or = 20 h chase) pattern. The continuous chicken vimentin staining reflects the uniform incorporation of chicken vimentin throughout the endogenous mouse vimentin network while the discontinuous or punctate chicken vimentin staining represents short interspersed segments of assembled chicken vimentin superimposed on the endogenous polymer. This punctate staining pattern of chicken vimentin was present throughout the entire array of intermediate filaments, with no bias toward the perinuclear region. These results are consistent with a continuous growth model of intermediate filament assembly, wherein subunit addition occurs at discrete sites located throughout the cytoskeleton

Localization of actin in Dictyostelium amebas by immunofluorescence

Antibody prepared against avian smooth muscle actin has been used to localize actin in the slime mold, Dictyostelium discoideum. The distribution of actin in migrating cells is different from that in feeding cells. Migrating amebas display fluorescence primarily in advancing regions whereas feeding amebas show uniform fluorescence throughout. The reaction is specific for actin since the fluorescence observed is blocked when the antibody is absorbed by actin purified from avian skeletal muscle, human platelets, and Dictyostelium. These results, in addition to describing the distribution of actin in D. discoideum, demonstrate that actins from these diverse sources share at least one common antigenic determinant