James R. Feramisco

Microinjection of the ras oncogene protein into PC12 cells induces morphological differentiation

To investigate the possible role of ras proteins in the differentiation process signaled by nerve growth factor, we have microinjected the proto-oncogenic and oncogenic (T24) forms of the human H-ras protein into living rat pheochromocytoma cells (PC12). PC12 cells, which have the phenotype of replicating chromaffin-like cells under normal growth conditions, respond to nerve growth factor by differentiating into nonreplicating sympathetic neuron-like cells. Microinjection of the ras oncogene protein promoted the morphological differentiation of PC12 cells into neuron-like cells. In contrast, microinjection of similar amounts of the proto-oncogene form of the ras protein had no apparent effect on PC12 cells. The induction of morphological differentiation by the ras oncogene protein occurred in the absence of nerve growth factor, was dependent on protein synthesis, and was accompanied by cessation of cell division. Treatment of PC12 cells with nerve growth factor or cAMP analogue prior to injection did not alter the phenotypic changes induced by the ras oncogene protein.

Transcriptional attenuation following cAMP induction requires PP-1-mediated dephosphorylation of CREB

We have examined the mechanism by which the transcriptional activity of the cAMP-responsive factor CREB is attenuated following induction with forskolin. Metabolic labeling studies reveal that, after an initial burst of phosphorylation in response to cAMP, CREB is dephosphorylated and transcription of the cAMP-responsive somatostatin gene is correspondingly reduced. The phosphatase inhibitor 1 protein and okadaic acid both prevented the dephosphorylation of CREB at Ser-133 in PC12 cells and also augmented the transcriptional response to cAMP. Of the four Ser/Thr phosphatases described to date, only PP-1 appears to be similarly inhibited by these agents. As PP-1 specifically dephosphorylates CREB at Ser-133 and inhibits cAMP-dependent transcription, we propose that this phosphatase is the major regulator of CREB activity in cAMP-responsive cells.

Microinjection and localization of a 130K protein in living fibroblasts: a relationship to actin and fibronectin

Summary A 130,000 dalton protein (130K protein), isolated from smooth muscle and having an as yet unknown function, has been studied through the use of microinjection of the protein covalently labeled with the fluorescent dye fluorescein isothiocyanate (FITC) into living cultured mammalian cells. The distribution of the 130K protein has also been investigated by several different methods including double label fluorescence microscopy of cells microinjected with FITC-130K protein and subsequently stained for 130K protein, actin or fibronectin by indirect immunofluorescence with rhodamine isothiocyanate (RITC)-labeled antibodies. Visualization of fluorescence in living fibroblasts 2–4 hr after microinjection of FITC-130K protein showed two major distributions of the fluorescent protein. One of these appeared as bright focal patches on the lower surface of the cells, and was similar to the distribution recently found by Geiger (1979) using immunofluorescent localization. Double label fluorescent microscopy of cells injected with FITC-130K protein and stained for actin 2 hr later by indirect immunofluorescence (RITC-antibodies) demonstrated that this focal distribution was at the ends of actin microfilament bundles. A second major distribution observed for the injected protein had a streaky or fibrillar appearance more commonly (but not always) located beneath the upper surface of the cell. Using antibodies against fibronectin and double label fluorescence microscopy, this second distribution of the injected protein was shown to correlate closely with the distribution of surface fibronectin on these cells. Where the fibronectin extended beyond a cell, the 130K protein remained strictly cellular and often appeared concentrated as if in an anchorage point. These findings suggest a possible role for the 130K protein in the organization of actin filaments at membrane attachment sites, and demonstrate that proteins of unknown function can be studied by the microinjection of their fluorescently labeled conjugates into living cells.

Microinjection of the oncogene form of the human H-ras (t-24) protein results in rapid proliferation of quiescent cells

Using an E. coli expression-vector system we have efficiently produced, purified, and characterized the full-length, nonfused, protooncogenic and oncogenic (T-24) forms of the human H-ras gene product. These purified ras proteins have been introduced by microinjection into a variety of somatic cells in an effort to examine their function. Within several hours after injection of the oncogenic form of the human H-ras protein into quiescent cells, we observe dramatic morphological changes followed by transient proliferation of the cells. In contrast, microinjection of the normal, protooncogenic form of the ras protein at the same level appears to have only little effect on the cells. Additional experiments indicate that the effect of the ras protein requires entry into the cells, is temporary, is inhibited by cycloheximide or actinomycin D, and is seen only in established cell lines. This experimental approach demonstrates that the bacterially derived and purified human H-ras proteins retain their ability to function when put back into mammalian cells and furthermore, provides a novel assay for transformation induced in established cells by the human H-ras oncogene protein.

Disruption of the in vivo distribution of the intermediate filaments in fibroblasts through the microinjection of a specific monoclonal antibody

Monoclonal antibodies (JLB1 and JLB7) that recognize minor components of the intermediate filament system of cultured cells were introduced into living fibroblasts by microinjection. Several minutes after injection of the JLB7 antibody virtually all of the intermediate filaments of the cells were found to be aggregated into tight bundles near or around the nucleus. In contrast, injection of the JLB1 antibody caused little or no aggregation of the intermediate filaments. Electron microscopy showed that the perinuclear bundles that formed after injection of the JLB7 antibody each consisted of ten or more filaments apparently crosslinked together. Double-label immunofluorescence microscopy showed that virtually all of the vimentin-containing intermediate filaments in the JLB7 antibody-injected cells were redistributed to the perinuclear region and remained there for at least 24 hr. The distributions of actin microfilaments and microtubules were seemingly undisturbed following microinjection. No obvious changes in cell morphology or behavior were apparent in the cells injected with JLB7 antibody; the cells displayed a flat appearance, showed a polarity, were able to ruffle and bleb and even appeared to show the normal saltatory movements of intracellular vesicles, granules and mitochondria, suggesting that intermediate filaments are not involved in these activities. The microinjection of highly specific monoclonal antibodies that recognize and alter components of the cell provides an additional approach to determine the in vivo functions of intracellular elements.

Monoclonal Antibodies to Cytoskeletal Proteins

Cytoskeletal proteins are found, as a general rule, in highly organized arrays within the cytoplasm of higher eukaryotic cells. The three filamentous networks that comprise the cytoskeleton are the microfilaments, composed of actin and many accessory proteins; the microtubules, composed of tubulin and several accessory proteins; and the intermediate filaments, composed of vimentin (or one of four other related proteins) and at least two accessory proteins. These networks have been the objects of intense study over the past 10 years in terms of cell motility, structure, and adhesion, and have proven to be most amenable to immunofluorescence and immunoelectron microscopic examination [e.g., R. Goldman et al., 1976)]. Because of the numerous applications of immunological techniques to investigate the structure and function of the cellular cytoskeleton systems, it is easily understood why many researchers in the field have begun utilizing the lymphocyte hybridoma method (Kohler and Milstein, 1975) as a source of antibodies. Studying something as complex as the cytoskeleton requires the use of the purest immunological reagents available, i.e., monoclonal antibodies.

Regulation of membrane turnover by ras proteins.

Becauseras oncogenes mediate abnormal cellular growth,ras proteins have been presumed to play a role primarily in growth control. The biological function ofras proteins may, however, prove to be much more diverse:ras proteins may be involved in cellular functions that control endocytosis and/or exocytosis.

Identification and Isolation of Clones by Immunological Screening of cDNA Expression Libraries

Various methods are available for the construction of essentially complete cDNA or genomic libraries from many sources. Since such libraries contain thousands of members, a major difficulty is the identification of clones of interest. Final identifications are usually made by sequencing the DNA, by using the DNA to select a specific mRNA for in vitro translation, or both. Such assays are not usually suited for screening large libraries for individual clones and steps are taken to enrich for the clones of interest. The most commonly used procedures rely on cDNA hybridization probes made from an mRNA preparation that contains high levels of the sequence of interest. Variations of this general approach include the physical fractionation of the mRNA by sedimentation, gel electrophoresis, or immunoprecipitation (or absorption) of polysomes. Alternatively, if part or all of the amino acid sequence of a protein is known, it is possible to synthesize specific oligonucleotides for use either as hybridization probes themselves or as primers for the enzymatic synthesis of cDNA.