We used useful wild-type and mutant artificial nuclear localization sign peptides of SV-40 T antigen cross-linked to human serum albumin (peptide conjugates) to assay their binding to proteins of rat liver nuclei on Western blots. Proteins of 140 and 55 kD (p140 and p55) had been solely acknowledged by wild-type peptide conjugates. Free wild-type peptides competed for the wild-type peptide conjugate binding to p140 and p55 whereas free mutant peptides, which differed by a single amino acid from the wild sort, competed much less effectively. The 2 proteins had been extractable from nuclei by both low or excessive ionic energy buffers. We purified p140 and raised polyclonal antibodies in hen in opposition to the protein excised from polyacrylamide gels.
The anti-p140 antibodies had been monospecific as judged by their reactivity with a single nuclear protein band of 140 kD on Western blots of subcellular fractions of complete cells. Oblique immunofluorescence microscopy on mounted and permeabilized Buffalo rat liver (BRL) cells with anti-p140 antibodies exhibited a definite punctate nucleolar staining. Rhodamine-labeled wild-type peptide conjugates additionally sure to nucleoli in an analogous sample on mounted and permeabilized BRL cells. Primarily based on biochemical characterization, p140 is a novel nucleolar protein. It’s attainable that p140 shuttles between the nucleolus and the cytoplasm and capabilities as a nuclear import service.
Caldesmon is an F-actin cross-linking protein of hen gizzard clean muscle whose F-actin binding exercise will be regulated in vitro by Ca2+-calmodulin (Sobue, Okay., Y. Muramoto, M. Fujita, and S. Kakiuchi, 1981, Proc. Natl. Acad. Sci. USA, 78:5652-5655). It’s a rod-shaped, heat-stable, F-actin bundling protein and is essentially the most ample F-actin cross-linking protein of hen gizzard clean muscle presently recognized (Bretscher, A., 1984, J. Biol. Chem., 259:12873-12880). We report using polyclonal antibodies to caldesmon to research its distribution and localization in different cells.
Utilizing immune blotting procedures, we’ve got detected immunoreactive, heat-stable types of caldesmon in cultured cells having both roughly the identical obvious polypeptide molecular weight as gizzard caldesmon (120,000-140,000) or a considerably decrease molecular weight (71,000-77,000). By use of affinity-purified antibodies in oblique immunofluorescence microscopy, we’ve got localized the immunoreactive varieties to the terminal internet of the comb border of intestinal epithelial cells and to the stress fibers and ruffling membranes of cultured cells. On the gentle microscope stage caldesmon is distributed in a periodic style alongside stress fibers that’s coincident with the distribution of tropomyosin and complementary to the distribution of alpha-actinin.
Filaments and fibrils that exhibit a 100-nm axial periodicity and happen within the medium and within the deposited extracellular matrix of hen embryo and human fibroblast cultures have been tentatively recognized with sort VI collagen on the idea of their related structural traits (Bruns, R. R., 1984, J. Ultrastruct. Res., 89:136-145). Utilizing oblique immunoelectron microscopy and particular monoclonal and polyclonal antibodies, we now report their optimistic identification with collagen VI and their distribution in fibroblast cultures and in tendon.
Major human foreskin fibroblast cultures, labeled with anti-type VI antibody and studied by fluorescence microscopy, confirmed a progressive enhance in labeling and modifications in distribution with time as much as eight d in tradition. With immunoelectron microscopy and monoclonal antibodies to human sort VI collagen adopted by goat anti-mouse IgG coupled to colloidal gold, they confirmed in skinny sections particular 100-nm periodic labeling on extracellular filaments and fibrils: one monoclonal antibody (3C4) connected to the band area and one other (4B10) to the interband area of the filaments and fibrils. Rabbit antiserum to sort VI collagen additionally localized on the band area, however the staining was much less properly outlined.
Management experiments with antibodies to fibronectin and to procollagen sorts I and III labeled different filaments and fibrils, however not these with a 100-nm interval. Heavy metal-stained fibrils with the identical periodic and structural traits even have been present in each grownup rat tail tendon and embryonic hen tendon subjected to extended incubation in tradition medium or therapy with adenosine 5′-triphosphate at pH 4.6. We conclude that the 100-nm periodic filaments and fibrils symbolize the native mixture type of sort VI collagen. It’s probably that banded fibrils of the identical periodicity and look, reported by many observers through the years in a variety of regular and pathological tissues, are at the very least partly, sort VI collagen.
We’ve examined the extent of tyrosine phosphorylation of talin, a element of the cytoskeleton localized within the focal adhesions and, subsequently, a possible substrate of p60v-src, the reworking protein of Rous sarcoma virus. p60v-src is a tyrosine kinase that induces excessive ranges of phosphotyrosine and the disorganization of the cytoskeleton in reworked cells. With a polyclonal antibody utilized in a earlier examine [Maher, P. A., Pasquale, E. B., Wang, J. Y. J. & Singer, S. J. (1985) Proc. Natl. Acad. Sci. USA 82, 6576-6580] for the detection of tyrosine-phosphorylated proteins, we’ve got detected phosphotyrosine residues in talin molecules immunoprecipitated from Rous sarcoma virus-transformed, however not regular, hen embryo fibroblasts.
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Phospho amino acid evaluation of talin from the contaminated cells confirmed the presence of phosphotyrosine, along with phosphoserine and phosphothreonine. The extent of tyrosine modification in talin was in comparison with that in vinculin, the opposite focal adhesion element beforehand discovered to include enhanced ranges of phosphotyrosine in numerous retrovirus-transformed cells. A significantly (three occasions) bigger fraction of the talin than of the vinculin molecules was discovered to be phosphorylated on tyrosine. The phosphorylation of talin on tyrosine could also be essential for the expression of the irregular morphology attribute of cells reworked by Rous sarcoma virus.
