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

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

On the dynamics of the microfilament system in HeLa cells

We measured the pools of unpolymerized and filamentous actin in homogenates of HeLa cells made in several different lysis buffers, as well as after treatment of cells with a variety of chemicals or trypsin, and after adenovirus (type 2) infection. This was possible when a series of factors concerning the basic culture conditions were kept constant: e.g., serum type used, serum batch, cell density, time after subcultivation of cells, and buffering substance in the medium. Homogenates from untreated cells usually contain 35-45 percent of the total actin in an unpolymerized form. With some batches of cells this number can be as high as 50 percent. In sparse cultures (3 x 10(4) cell/cm(2)), HeLa cells contain approximately 10 pg actin/cell, while the corresponding number is only 5 pg in dense cultures (3 x 10(5) cells/cm(2)). Treatment of cells with cytochalasin B increases the pool of unpolymerized actin by approximately 30-40 percent, while colchicine decreases the fraction of unpolymerized actin by 20 percent. The oxidant diamide increases the filamentous actin pool 25-50 percent. Glucose, sodium azide, dinitrophenol, serum starvation, or thymidine treatment does not affect the distribution between unpolymerized and filamentous actin to any significant extent. Trypsin and EDTA induced rounding up of cells but did not change the actin distribution. The distribution of actin between G- and F-forms was unchanged after adenovirus infection. These results show that significant changes in the actin pools can be induced in nucleated cells. However, several treatments which alter the morphology and motility of cells are not accompanied by an alteration in the G-/F-actin ratio

C-CAM (cell-CAM 105) is an adhesive cell surface glycoprotein with homophilic binding properties

C-CAM (Cell-CAM 105) is a cell surface glycoprotein that is involved in cell-cell adhesion of rat hepatocytes in vitro. To elucidate the adhesion mechanism the binding properties of purified C-CAM were investigated. Using proteins immobilized on nitrocellulose it was found that radiolabeled C-CAM bound to C-CAM but not to a variety of other proteins. Partitioning in Triton X-114 showed that C-CAM has hydrophobic properties. In accordance with this, C-CAM was effectively incorporated into phosphatidylcholine liposomes by dialysis from octylglucoside-containing solutions. The C-CAM-containing liposomes bound specifically to isolated hepatocytes. This binding was blocked by Fab fragments of anti-C-CAM antibodies. Furthermore, preincubation of hepatocytes with anti-C-CAM antibodies followed by washing of the cells blocked binding of C-CAM-containing liposomes. At increasing C-CAM contents in the reconstituted liposomes a marked self-aggregation of the liposomes occurred. This aggregation was blocked by Fab fragments of anti-C-CAM antibodies and by alkaline pH. After neutralization a rapid reaggregation occurred. Neither C-CAM binding to C-CAM immobilized on nitrocellulose nor C-CAM-liposome aggregation required calcium ions. Liposomes reconstituted with C-CAM-depleted membrane glycoproteins did not self-aggregate or bind to hepatocytes. Thus, it is concluded that C-CAM can bind specifically to C-CAM in a homophilic binding reaction that does not require calcium. Accordingly, C-CAM has the potential of directly mediating cell-cell adhesion via C-CAM-C-CAM binding between adjacent cells