Common and reversible regulation of wild-type p53 function and of ribosomal biogenesis by protein kinases in human cells.

Two specific inhibitors of cyclin-dependent kinase 2 (Cdk2), roscovitine and olomoucine, have been shown recently to induce nuclear accumulation of wt p53 and nucleolar unravelling in interphase human untransformed IMR-90 and breast tumor-derived MCF-7 cells. Here, we show that the early response of MCF-7 cells to roscovitine is fully reversible since a rapid restoration of nucleolar organization followed by an induction of p21(WAF1/CIP1), a downregulation of nuclear wt p53 and normal cell cycle resumption occurs if the compound is removed after 4 h. Interestingly, similar reversible effects are also induced by the casein kinase II (CKII) inhibitor, 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole. Upon short-term treatment also, both compounds significantly, but reversibly, reduce the level of 45S precursor ribosomal RNA. Cells exposed to the two types of protein kinase inhibitors for longer times keep exhibiting altered nucleolar and wt p53 features, yet they strikingly differentiate in that most roscovitine-treated cells fail to ever accumulate high levels of p21(WAF1/CIP1) in contrast with DRB-treated ones. In both cases, however, the cells eventually fall into an irreversible state and die. Moreover, we found that constitutive overexpression of p21(WAF1/CIP1) alters the nucleolar unravelling process in the presence of DRB, but not of roscovitine, suggesting a role for this physiological Cdk inhibitor in the regulation of nucleolar function. Our data also support the notion that both roscovitine- and DRB-sensitive protein kinases, probably including Cdk2 and CKII, via their dual implication in the p53-Rb pathway and in ribosomal biogenesis, would participate in coupling cell growth with cell division.

Potent inhibitors of cyclin-dependent kinase 2 induce nuclear accumulation of wild-type p53 and nucleolar fragmentation in human untransformed and tumor-derived cells.

The cdk2 gene has been identified as a human cdc2/CDC28-related gene that encodes a protein kinase essential for the G1/S transition in mammalian cells, but not for the G2/M transition, which requires Cdk1, another p34cdc2/CDC28 homolog. Novel potential functions of Cdk2 have been uncovered by using two potent and specific inhibitors of its kinase activity, roscovitine and olomoucine, on human wt p53-expresser untransformed and tumor-derived cells. At concentrations equal or superior to respectively 30- and 20-fold their in vitro IC50 values for cyclin B/Cdk1, cyclin A/Cdk2 and cyclin E/Cdk2, the Cdk inhibitors precipitately induce a dramatic nuclear accumulation of wt p53 and a delocalization of nucleolin from the nucleolus in all interphase cells, whatever their cell cycle status, acting in this way like the DNA-damaging drug, mitomycin C (7 microg/ml). These early events are soon followed by a nucleolar fragmentation in both normal and tumor cells in the presence of the Cdk inhibitors but not in the presence of the DNA-damaging drug. Yet, treatment with either type of compounds eventually triggers rapidly the death of the tumor cells and, much more slowly, that of the normal cells. The Cdk inhibitors, however, stimulate cell death from any stage of the cell cycle, whereas the DNA-damaging drug kills more efficaciously S phase cells. These observations provide a hint that the Cdk2 kinase might be involved in controlling the nuclear levels of the tumor suppressor wt p53 protein and in maintaining the nucleolar integrity and function, linking in this way the cell division cycle machinery to survival functions and overall cell metabolism via the control of nucleocytoplasmic transport and of ribosome production.

Sustained accumulation of the mitotic cyclins and tyrosine-phosphorylated p34cdc2 in human G1-S-arrested cancer cells but not untransformed cells.

Coupling mitosis to the completion of DNA replication in cycling embryonic extracts from Xenopus eggs appears to rely on blocking the activation of the tyrosine-phosphorylated p34cdc2/cyclin B, which continues to build up when S phase is inhibited by adding unreplicated DNA (Smythe, C., and Newport, J. W., Cell, 68: 787-797, 1992). We show here that a similar mechanism might be operative in human tumor-derived cells, which, during a thymidine-aphidicolin block, stop progressing through S phase and thereby fail to undergo mitosis. Under such conditions, indeed, cancer cells do continue to accumulate cyclin A, cyclin B1, and tyrosine-phosphorylated p34cdc2 to supranormal levels, a phenomenon that does not occur in untransformed, nonimmortalized human fibroblasts. Thus, in human cancer cells, the onset of active accumulation of cyclin A and cyclin B1 can be uncoupled from transit through the G1-S and S-G2 borders, respectively, and, as in simple embryonic cell cycles, the coupling of mitosis to the completion of S phase presumably relies, at least in part, on the prevention of premature activation of the tyrosine-phosphorylated p34cdc2/cyclin B1 complex.

Delaying the onset of M phase in NIH 3T3 cells blocked in early S phase occurs via accumulating cyclin B1 and tyrosine-phosphorylated p34cdc2 in the nucleus.

An affinity-purified antibody (anti-Cdc2C) raised against the carboxy terminal sequence LDNQIKKM of p34cdc2 uncovered in NIH 3T3 cells a protein subpopulation, the location and the level of accumulation of which evolve during progression through the cell cycle: it first emerges inside the nucleus in late G1/early S phase and continues to build up principally in this location throughout S phase; a cytoplasmic expression then becomes apparent near the end of S phase, develops during G2 and sometimes prevails over the nuclear expression; it finally relocates to the nucleus in early prophase. We propose that a major part of this subpopulation would represent p34cdc2 molecules existing inside a complex with cyclin B1. NIH 3T3 cells arrested in early S phase with aphidicolin do not commit prematurely to mitosis which indicates that the regulatory pathway involved in preserving the temporal order of S and M phases is functioning in these conditions. Conjugated Western blot analysis and immunofluorescence microscopy showed that cyclin A, cyclin B1 and tyrosine-phosphorylated p34cdc2 continue to build up predominantly in the nucleus of the arrested cells. After release from the block, the cells rapidly reenter S and G2 phases and, concomitantly, cyclin B1 and tyrosine-phosphorylated p34cdc2 relocate to the cytoplasm before redistributing again in the nucleus in early prophase. These data would suggest that delaying the onset of M phase in NIH 3T3 cells in which the rate of DNA replication is reduced, is first ensured by a mechanism that prevents the cytoplasmic relocation of inactive p34cdc2/cyclin B1 complexes continually forming in the nucleus once the G1 period of mitotic cyclin instability is over.

Phenotypic changes induced by wild type and variant c-src genes carrying C-terminal sequence alterations.

We previously reported the isolation of PR2257, a novel avian sarcoma retrovirus which transduced the c-src protooncogene. The v-src gene of PR2257 differs from the c-src gene by a sequence change after amino acid 525, resulting in the replacement of tyrosine 527 by a valine, and an extension of the open reading frame into the non coding region of c-src. We investigated the respective roles of Tyr527 mutation and of the C-terminal extension in activating the oncogenic properties of c-src. Therefore we overexpressed the wild type c-src gene and c-src variants, carrying either a substitution of tyrosine 527 or an extension of the C-terminus or both modifications in combination, in chicken embryo fibroblasts and post mitotic neuroretina (NR) cells, using replication defective retroviruses. We also used in vivo inoculation of plasmid DNA to assess the tumorigenicity of the various c-src genes. We report that, in contrast to previous results, overexpression of c-src is sufficient to induce NR cell division. While mutation of tyrosine 527 alone significantly activates c-src transforming and tumorigenic properties, its combination with the C-terminal extension of PR2257 confers to this gene full oncogenic properties and increased metastatic potential as compared to the v-src of Rous sarcoma virus strains.

Human cyclin B1 is targeted to the nucleus in G1 phase prior to its accumulation in the cytoplasm.

The immunolocalisation patterns of cyclin B1 have been investigated in various tumour-derived and untransformed human cells, using one polyclonal (B7/B8) and two different monoclonal anti-human cyclin B1 antibodies, GNS1 and GNS11. In actively dividing cell populations, GNS11 reveals uniquely a cytoplasmic pool of cyclin B1 that rapidly increases after the onset of S phase; yet, B7/B8 and GNS1 detect, besides this cytoplasmic cyclin B1 population, a moderate but clear nuclear concentration of the protein in cells that have not yet entered S phase. As for confluent populations of untransformed and tumour cells, they become enriched in G1- and G2-arrested cells that characteristically display a discrete nuclear or an intense cytoplasmic GNS1 immunostaining respectively. Altogether, our immunofluorescence data conjugated with the results of a detailed biochemical analysis suggest that human cyclin B1 would exist in situ as two distinguishable molecular entities differentially susceptible to in vitro degradation and exhibiting different timing, kinetics and site of accumulation during the cell cycle: one form (recognized by the polyclonal and GNS1 antibodies but apparently not by GNS 11) starts to build up inside the nucleus prior to entry in S phase and the other (in which both the GNS11 and GNS1 epitopes are readily accessible) emerges and accumulates in the cytoplasm beyond the G1/S boundary.

Cell cycle-dependent regulation of nuclear p53 traffic occurs in one subclass of human tumor cells and in untransformed cells.

We have analyzed the regulation of subcellular compartmentation of mutant and wild-type (WT) p53 proteins as a function of the cell cycle using immunofluorescence microscopy and referring to different markers of position in the cell cycle in different human cells expressing either mutated (KHOS-240, A 431, and T47-D cells) or WT (WI 38 and MCF-7 cells) p53. The mutant p53 proteins present in the KHOS-240, A 431, and T47-D tumor-derived cell lines enter very rapidly in the nucleus in early postmitotic cells before the chromosomes have fully decondensed; they continue accumulating in this location without any obvious cytoplasmic retention throughout the cell cycle until prophase. Such behavior is similar to that observed for the WT p53 associating with SV40 large T antigen in human WI 38 cells transformed by SV40, but it is in contrast to the behavior of the WT p53 protein present in both the untransformed WI 38 and the tumor-derived MCF-7 cells. In these latter systems, the highest nuclear concentrations of the WT protein are always found in G1 cells that still fail to exhibit a high rate of nuclear cyclin A; past the G1-S transition, the nuclear level of WT p53 tends to decrease, possibly to the benefit of cytoplasmic expression, whereas that of cyclin A concomitantly increases, suggesting that the nuclear accumulation of WT p53 becomes restricted during the phase of DNA replication. As for Saos-2 cells stably transfected with the temperature-sensitive p53Ala-143 mutant, they become arrested before the G1-S transition with a heavy pool of nuclear p53 at 32.5 degrees C, the temperature at which the transcriptional activity of p53Ala-143 is restored. All these data are compatible with the presently acknowledged primary role for WT p53, which would be to brake transit through the G1-S border possibly by directly transactivating the p21cip1 protein.

Highly specific antibody to Rous sarcoma virus src gene product recognizes nuclear and nucleolar antigens in human cells.

An antiserum to the Rous sarcoma virus-transforming protein pp60v-src, raised in rabbits immunized with the bacterially produced protein alpha p60 serum (M. D. Resh and R. L. Erikson, J. Cell Biol. 100:409-417, 1985) previously reported to detect very specifically a novel population of pp60v-src and pp60c-src molecules associated with juxtareticular nuclear membranes in normal and Rous sarcoma virus-infected cells of avian and mammalian origin, was used here to investigate by immunofluorescence microscopy localization patterns of Src molecules in human cell lines, either normal or derived from spontaneous tumors. We found that the alpha p60 serum reveals nuclear and nucleolar concentrations of antigens in all the human cell lines tested and in two rat and mouse hepatoma cell lines derived from adult tumorous tissues but not in any established rat and mouse cell lines either untransformed or transformed by the src and ras oncogenes. Both the nuclear and nucleolar stainings can be totally extinguished by preincubation of the serum with highly purified chicken c-Src. We show also that the partitioning of the alpha p60-reactive proteins among the whole nucleus and the nucleolus depends mostly on two different parameters: the position in the cell cycle and the degree of cell confluency. Our observations raise the attractive possibility that, in differentiated cells, pp60c-src and related proteins might be involved not only in mediating the transduction of mitogenic signals at the plasma membrane level but also in controlling progression through the cell cycle and entry in mitosis by interacting with cell division cycle regulatory components at the nuclear level.

Differential localization patterns of myristoylated and nonmyristoylated c-Src proteins in interphase and mitotic c-Src overexpresser cells.

Myristoylation of pp60src is required for its membrane attachment and transforming activity. The mouse monoclonal antibody, mAb327, which recognizes both normal, myristoylated pp60c-src and a nonmyristoylated mutant, pp60c-src/myr-, has been used to compare the effects of preventing myristoylation on the localization of c-Src in NIH 3T3-derived overexpresser cells using immunofluorescence microscopy. During interphase, pp60c-src partitions between the plasma membrane and the centrosome, while pp60c-src/myr- is predominantly cytoplasmic but also partly nuclear. The cytoplasmic, but not the nuclear, staining can be readily washed out by brief pretritonization of the cells before fixation, indicating that the cytoplasmic pool of pp60c-src/myr-, in contrast with the nuclear one, does not associate tightly with structures that are insoluble in the presence of nonionic detergents. We have previously shown that during G2 phase, pp60c-src leaves the plasma membrane and is redistributed diffusely throughout the cytoplasm and to two clusters of patches surrounding the two separating centriole pairs. In contrast, we now find that pp60c-src/myr- translocates to the nucleus in late G2 or early prophase prior to there being any clear evidence of nuclear membrane breakdown or nuclear lamina disassembly. Similar nuclear translocation of pp60c-src/myr-, but not of pp60c-src, is also observed when cells are arrested in G0 or at the G1/S transition. Furthermore, during mitosis, pp60c-src is found primarily in diffuse and patchy structures dispersed throughout the cytoplasm while pp60c-src/myr- more specifically associates with the main components of the spindle apparatus (poles and fibers) and inside the interchromosomal space. These results suggest that a possible role for myristoylation might be to prevent unregulated nuclear transport of proteins whose nonmyristoylated counterparts are readily moved into the nucleus. They also raise the possibility that a subfraction of wild-type pp60c-src may behave, at specific times, like its nonmyristoylated counterpart, and may translocate to the nucleus and exert specific functions in that location.

Slow time-dependent cellular transformation induced at restrictive temperature by ts-src mutants.

Cell lines infected with a temperature-sensitive Rous sarcoma virus have been widely used to study the temporal dynamics of various transformation parameters following downshift from the non-permissive temperature to the permissive temperature as it is considered that, at the non-permissive temperature, the infected cells exhibit the morphological and growth characteristics of normal cell whereas, at the permissive temperature, they exhibit characteristics of the transformed state. We show here that the apparently normal state in which tsPA1-infected FR3T3 cells are directed at the restrictive temperature is not a stationary stable state, but rather a transient one which continuously drifts as the cells are grown and passaged at this temperature and which eventually ends up as a new transformed state (T2) with morphological and growth properties definitely different from those belonging to the transformed state (T1) at the permissive temperature. The establishment of the transformed T2 state at the restrictive temperature occurs concomitantly with a steady accumulation of an intracellular pool of pp60v-src in the vicinity of the nucleus, whose traffic towards the plasma membrane is released following downshift to the permissive temperature, leading to the reappearance of transformation parameters characteristic of the transformed T1 state. Our finding raises the possibility that the v-src protein encoded by the tsPA1 mutant of Rous sarcoma virus may induce cellular transformation via two different pathways, leading to two different transformation states, depending on at which temperature the infected cells are grown. Various possible mechanisms that could be involved in the time-dependent establishment of a transformed state by ts-src mutants at the restrictive temperature are discussed.

Immunolocalization of the cellular src protein in interphase and mitotic NIH c-src overexpresser cells.

The mouse mAb, mAb 327, that recognizes specifically both pp60v-src and pp60c-src in a wide variety of cells, has been used to determine precisely the various locations of pp60c-src in NIH c-src overexpresser cells, using the technique of immunofluorescence microscopy. In interphase cells, the protein exhibits two main distributions: one that appears uniform and in association with the cell surface and the other that is patchy and juxtanuclear and coincides with the centrosomes. The juxtanuclear aggregation of pp60c-src-containing patches depends on microtubules and does not seem to occur within the Golgi apparatus and the rough ER. At the G2-to-M-phase transition, a drastic change in the localization patterns of pp60c-src takes place. We also report experiments in which the NIH c-src overexpresser cells were exposed to Con A for various times to induce a redistribution of the cell surface Con A receptors. We show that, at each stage of the Con A-mediated endocytotic process, the Con A-receptor complexes redistribute into structures to which pp60c-src appears also to be associated: at first, into patches that form at the cell surface level and then, into a cap that stands at the cell center in a juxtanuclear position and that coincides with the Golgi apparatus. During this capping process, pp60c-src-containing vesicles continue to accumulate in a centriolar spot, as in interphase, Con A-untreated cells, from which Con A is excluded. The significance of the intracellular locations of pp60c-src to the possible functions of the protein is discussed. Also, the distribution patterns of the cellular protein in the NIH c-src overexpresser cells are compared with those of pp60v-src in RSV-transformed cells. The differences observed are discussed in relation with the differences in transforming capacities of the two proteins. Finally, the possible physiological significance of the association between pp60c-src and the structures generated after the binding of Con A to its surface receptors is addressed.

Serine/threonine-specific protein kinase activity associated with viral pp60src protein.

Three different types of experiments are presented in this paper, the results of which converge to indicate that the viral src protein associates with and modulates the activity and/or the specificity of a serine/threonine protein kinase. Firstly, a 60-kDa protein from extracts of FR3T3 rat fibroblasts transformed by wild-type Rous sarcoma virus (SRD-FR3T3) is shown to be immunoprecipitated with a monoclonal antibody (mAb) raised against bacterially produced pp60v-src, the mAb327 [Lipsich, L. A., Lewis, A. J. & Brugge, J. S. (1983) J. Virol. 48, 352-360] and to be phosphorylated in vitro at serine/threonine/tyrosine residues, in the ratio 25:53:22. Under the same experimental conditions, the pp60c-src protein immunoprecipitated with mAb327 from extracts of NIH c-src overexpresser cells is phosphorylated exclusively on tyrosine residues. Secondly, the results of immunoprecipitation experiments using a tumor-bearing rabbit (TBR) serum and reported in an earlier work [David-Pfeuty, T. & Hovanessian, A. (1984) Eur. J. Biochem. 140, 325-342], together with those reported here, suggest that the TBR-immunoprecipitated pp60v-src coprecipitates with a cellular protein related to the 60-kDa subunit of the Ca2+/calmodulin protein kinase II from brain. Finally, partially purified preparations of pp60v-src, but not of pp60c-src, are shown to contain a Ca2+/calmodulin-dependent protein kinase activity that phosphorylates a 52-kDa protein substrate.

Studies in pig heart tissue on various 60,000 Da phosphoproteins.

Pig heart tissue have been shown to contain 3 different 60,000 Da phosphoproteins. Different purification procedures were used in order to separate them, suggesting that the 3 phosphoproteins differ in their environmental parameters. The 2 major ones appear essentially as peripheral phosphoproteins that are associated with cellular membranes through ionic forces, whereas the third minor phosphoprotein behaves as an integral plasma membrane protein. The three phosphoproteins also differ in their relative amount of phosphorylated serine, threonine and tyrosine residues after in vitro protein kinase assay. Evidence that the 3 phosphoproteins are related arises from the similarity between their respective phosphopeptide maps after partial hydrolysis with proteases, an experiment that also points out relatedness in primary structure between them and the transforming protein of Rous sarcoma virus, pp60v-src. The 3 phosphoproteins, however, do not appear to be immunologically related to pp60v-src since none of them is immunoprecipitated by sera that precipitate pp60v-src. The possibility that the three 60,000 Da phosphoproteins under study represent 3 differentially localized and phosphorylated products of c-src and/or c-src related genes is discussed.

Transformed and tumorigenic phenotypes induced by avian retroviruses containing the v-mil oncogene.

Avian retrovirus MH2 contains two oncogenes, v-mil and v-myc. We have previously shown that a spontaneous mutant of MH2 (PA200-MH2), expressing only the v-mil oncogene, is able to induce proliferation of quiescent neuroretina cells. In this study, we investigated the transforming and tumorigenic properties of v-mil. PA200 induced fibrosarcomas in about 60% of the injected chickens, whereas inoculation of MH2 resulted mainly in the appearance of kidney carcinomas. Analysis of several parameters of transformation showed that PA200, in contrast to MH2, induced only limited in vitro transformation of fibroblasts and neuroretina cells. These results suggest that v-myc is the major transforming and tumorigenic gene in MH2-infected cells. This low in vitro transforming capacity differentiates v-mil not only from other avian oncogenes, but also from the homologous murine v-raf gene.

Rous sarcoma virus mutant dlPA105 induces different transformed phenotypes in quail embryonic fibroblasts and neuroretina cells.

dlPA105 is a spontaneous variant of Rous sarcoma virus, subgroup E, which carries a deletion in the N-terminal portion of the v-src gene coding sequence. This virus was isolated on the basis of its ability to induce proliferation of quiescent quail neuroretina cells. The altered v-src gene encodes a phosphoprotein of 45,000 daltons which possesses tyrosine kinase activity. DNA sequencing of the mutant v-src gene has shown that deletion extends from amino acid 33 to 126 of wild-type p60v-src. We investigated the tumorigenic and transforming properties of this mutant virus. dlPA105 induced fibrosarcomas in quails with an incidence identical to that induced by wild-type virus. Quail neuroretina cells infected with the mutant virus were morphologically transformed and formed colonies in soft agar. In contrast, dlPA105 induced only limited morphological alterations in quail fibroblasts and was defective in promoting anchorage-independent growth of these cells. Synthesis and tyrosine kinase activity of the mutant p45v-src were similar in both cell types. These data indicate that the portion of the v-src protein deleted in p45v-src is dispensable for the mitogenic and tumorigenic properties of wild-type p60v-src, whereas it is required for in vitro transformation of fibroblasts. The ability of dlPA105 to induce different transformation phenotypes in quail fibroblasts and quail neuroretina cells is a property unique to this Rous sarcoma virus mutant and provides evidence for the existence of cell-type-specific response to v-src proteins.

Epidermal growth factor stimulates serine and tyrosine phosphorylation in a 59-kD protein in purified plasma membranes from rat liver.

Preincubation of purified plasma membranes from rat liver with EGF stimulates the level of phosphorylation on serine and tyrosine residues in a 59-kD protein. Such an increased phosphoserine and phosphotyrosine content of the 59-kD protein occurs at the expense of the phosphorylation on threonine residues. The effect is observed under conditions where the plasma membranes have been extracted at pH 10. It is not observed when the membranes are simply washed at pH 7.5 before further purification. A number of experiments, including TBR-IgG phosphorylation in immunoprecipitates and partial hydrolysis with varying concentrations of the V8 protease, suggest that the 59-kD protein modified upon EGF treatment could be a representative of the c-src gene product from hepatocytes.

The coordinate organization of vinculin and of actin filaments during the early stages of fibroblast spreading on a substratum.

Cultured normal fibroblasts adhere to their support essentially through the focal adhesion plaques which are greatly enriched with the 130 000 dalton protein, vinculin, along with the newly described 215 000 dalton protein, talin, and at which actin bundles terminate. In order to explore a role for vinculin in the formation of the adhesion plaques and of the actin bundles, we have studied and compared the development of these two cellular structures during the spreading of trypsinized and replated chicken embryonic fibroblasts. The techniques used were double indirect immunofluorescence and interference reflection microscopy. At the earliest stage of cell spreading observed, vinculin distributes into small patches that are located along actin filaments and at the basis of the ruffling membrane. At later spreading stage, vinculin markedly redistributes into larger striations which coincide with focal contacts. Some of these vinculin striations are associated with the ends of microfilaments while the others are not. These observations would suggest that two types of focal contacts can form simultaneously in early cell spreading. Hypotheses are made concerning the role of vinculin in the formation of the adhesive cell structures in the light of these new data and of previous reports on the subject.

Partial purification and characterization of a 60 000-dalton phosphoprotein from pig heart tissue.

A 60 000-dalton phosphoprotein (pp60) was purified up to 10(4)-fold by a combination of low-ionic-strength extraction, ammonium sulfate fractionation, on-exchange and affinity chromatography, all in detergent-free buffer. Fractionation on omega-aminohexylagarose column shows that pp60 actually consists of two different polypeptides of similar molecular mass (pp60 omega 1 and pp60 omega 2). Partial hydrolysis with proteases of the proteins 32P-labeled in vitro indicates that pp60 omega 1 and pp60 omega 2 are similar but not identical. On the other hand, individual phosphoamino acid analysis reveals that pp60 omega 1 is phosphorylated primarily at serine residues while pp60 omega 1 is phosphorylated almost equally at serine and threonine residues. Partial hydrolysis with proteases has been also used to explore a possible relationship between the pp60's and the transforming protein of Rous sarcoma virus (pp60v-src). Our data suggest that pp60v-src also consists of two different polypeptides chemically homologous to the presently purified pp60's.

The effect of microtubule disruption on the distribution of the cytoskeletal proteins, vinculin, alpha-actinin, and actin in normal and RSV-transformed fibroblasts.

By double indirect immunofluorescence and interference electron microscopy, we have observed the effect of microtubule disruption by antimitotic drugs and coldness treatment on the distribution of adhesion sites and of the three cytoskeletal proteins, vinculin, alpha-actinin, and actin in normal rat cells and in rat cells transformed by Rous sarcoma virus. This study shows that the state of organization of the microtubule--intermediate filament complex modulates the location and the arrangement of intracellular structures containing vinculin, alpha-actinin, and actin in normal as well as in transformed cells. The most important alterations are observed in transformed cells on the distribution of the rosette clusters that have been shown to characterize the transformation by Rous sarcoma virus [8]. These results suggest the microtubule-intermediate filament complex is directly or indirectly connected with the microfilament network.

Altered distributions of the cytoskeletal proteins vinculin and alpha-actinin in cultured fibroblasts transformed by Rous sarcoma virus.

It was recently shown by combined immunofluorescence and interference reflection microscopy that a protein named vinculin, along with alpha-actinin, is concentrated at focal adhesion plaques inside cultured normal fibroblasts [Geiger, B. (1979) Cell 18, 193-205]. These plaques are the discrete, isolated sites of strong adhesions formed between the ventral surfaces of the cells and the substrata on which they are grown. We show that after transformation of fibroblasts by Rous sarcoma virus a majority of the cells have many fewer focal adhesion plaques and now exhibit a cluster of small patches that are immunolabelled for both vinculin and alpha-actinin. Such a cluster (rosette) is located near the ventral surface of the cell, usually partly under the nucleus. The significance that these altered distributions of vinculin and alpha-actinin may have for the rounding up and loss of adherence of transformed cells is discussed.