Robert W. Rubin

Lack of tropomyosin correlates with the absence of stress fibers in transformed rat kidney cells

We have utilized epithelial rat kidney cells and their Kirsten viral transformant (442) to examine the role of actin-binding proteins in cellular morphogenesis. Normal rat kidney cells are well spread while the transformed cells are more spherical, poorly adherent, and lack actin stress fibers (Rubin, R.W., Warren, R.H., Lukeman, D.S. and Clements, E. (1978) J. Cell Biol. 78, 28-35). By immunofluorescence, antitropomyosin prominently stains normal rat kidney cell stress fibers while only a weak, nonspecific fluorescence is observed in 442 cells. Using two-dimensional gel electrophoresis, tropomyosin can be detected in normal rat kidney cells homogenates. The tropomyosin subunits are enriched in Triton-extracted filamentous normal rat kidney cell models, and in extracts of normal rat kidney cell homogenate produced by using a rapid myosin affinity technique to isolate actin and actin-associated proteins. The identity of the tropomyosin subunits has been confirmed by electrophoretic mobility, lack of proline, and the peptide map generated by limited proteolysis. None of these techniques have detected tropomyosin in the corresponding 442 preparations. Our results suggest that the transformation of normal rat kidney cells has led to an overall reduction in tropomyosin content. This may be related to the inability of 442 cells to organize filamentous actin stress fibers.

Quantitation of microgram amounts of protein in SDS-mercaptoethanol-tris electrophoresis sample buffer.

Abstract We have investigated the utilization of a previously published dye-binding protein quantitation method for the analysis of proteins solubilized in SDS, mercaptoethanol-tris electrophoresis sample buffer. Although there is a direct relationship between the amount of protein applied and the absorbance of Coomassie blue G for BSA standard curves, complicated protein mixtures such as whole cell homogenates of tissue culture cells show more complicated functions. Apparently, components present in whole cell homogenates altered the dye-binding phenomenon to the extent that absolute values of the total amount of protein of such a mixture cannot be obtained by comparison to standard curve. However, over a relatively wide range of protein concentrations, the dye-binding method can provide a relative value for the amount of total protein present in a given complex protein mixture solubilized in SDS-mercaptoethanol electrophoresis buffer.

Evidence for secretory granule membrane recycling in cultured adrenal chromaffin cells

Abstract The ability to initiate exocytosis upon carbachol addition to primary cultures of bovine adrenal chromaffin cells provides a system for a complimentary morphological and biochemical determination of the fate of secretory granule membrane. Concurrent with stimulation of exocytosis, cultures were either exposed to the electron dense tracer cationized ferritin (CF) or to conditions which label exposed proteins with 125I. Ultrastructural examination of CF-treated cells reveals that membrane labeled during exocytosis becomes incorporated into new granule membrane. Secretory granule membrane was isolated two hours after initiation of exocytosis and its polypeptides characterized by two-dimensional (2-D) gel electrophoresis. Autoradiography reveals that 125I is primarily found in dopamine-β-hydroxylase (DBH), a biochemical marker of the inner surface of chromaffin granule membranes. Thus, our results demonstrate that secretory granule membrane is removed from the cell surface by endocytosis and subsequently major components are incorporated into the membrane of intact intracellular chromaffin granules.

Subpicogram analysis of sweat proteins using two-dimensional polyacrylamide gel electrophoresis

Sweat collected from six normal volunteers was analyzed to determine if reproducible protein patterns could be obtained using two-dimensional polyacrylamide gel electrophoresis of 125I-labeled sweat proteins. This method has the capability of easily detecting picogram quantities of protein. Once the methods of collection of the sweat had been standardized, reproducible patterns were obtained from these volunteers. Over 100 discrete spots were revealed by a combination of fluorography and rare earth screen radioautography of dried two-dimensional gels. This method will allow analysis of sweat for qualitative and quantitative variations in protein content in pathologic conditions such as cystic fibrosis, renal failure, and diabetes.

Identification of a cytoskeleton-associated glycoprotein from isolated microvilli of a mammary ascites tumor

Microvilli isolated from MAT-C1 13762 ascites tumor cells were extracted with Triton X-100 in phosphate-buffered saline (PBS) to yield cytoskeletal residues. Analysis of the residues by two-dimensional isoelectric focusing-dodecyl sulfate electrophoresis and silver staining suggested that one of the major components is a glycoprotein (CAG). Neuraminidase treatments and glucosamine labeling demonstrated that CAG is a glycoprotein, and lactoperoxidase iodination showed its presence at the microvillar surface. DNase treatments and myosin affinity analysis suggested an association between CAG and the microvillar microfilaments. Thus, CAG has the properties expected of a transmembrane-linking molecule connecting the cell surface to the cytoskeleton.

Effects of colchicine on cardiac cell function indicate possible role for membrane surface tubulin.

The effects of the tubulin-binding drug colchicine on cultured neonate cardiac cell function were investigated. Application of low doses of colchicine (but not lumicolchicine) caused an early reversible increase in beating rate with a concomitant decrease in amplitude. Treatment of the cells with trypsin at a dose that removes surface tubulin but does not inhibit spontaneous beating, diminished the colchicine effect. Surface radio-iodination of the live cultures followed by two-dimensional gel electrophoresis and radioautography revealed that two spots were heavily labeled. These spots co-migrated with purified brain tubulin. Fibroblasts derived from the cardiac cultures did not label over the tubulin spots. Trypsin treatment removed the presumptive tubulin from the radioautographs but only removed the most basic portion of the alpha-tubulin spot from the stained gel. These results are consistent with a surface membrane role for an iso-form of tubulin in neonate cardiac cells.

Actin turnover during encystation in Acanthamoeba.

Abstract A thin-slab, SDS polyacrylamide gel electrophoresis system is described in which actin within whole cell homogenates can be quantitated within a wide range of protein values (0.05–1.4 μg/band). After demonstrating the absence of appreciable contaminants in the actin band, and the lack of appreciable reincorporation of label during pulse-chase experiments, the turnover of actin was examined in pre-labeled cells during normal log growth and during induced encystation in Acanthamoeba. During log growth, no actin degradation was detected. However, as the cells approached the end of log phase growth and entered stationary phase, a dramatic increase in the amount of actin/cell and the percentage of total protein represented by actin was recorded. The encystation process per se was accompanied by a rapid reduction in these values to preencystment levels.

Further studies on dividing sea urchin eggs exposed to the volatile anesthetic halothane

Abstract Mitotic apparatus (MA)-isolation techniques and SDS-gel quantitation show that halothane, a widely used volatile anesthetic, inhibits the growth of the mitotic apparatus of echinoderm eggs in vivo, but has no detectable effect on the amount of actin associated with the cell cortex. These studies confirm and extend long standing observations on anesthetic-treated echinoderm eggs and support the hypothesis that anesthetics indirectly prevent cleavage by inhibiting the growth of mitotic asters.

A biochemical and ultrastructural comparison of Triton X-100 models of normal and transformed cells

We have compared the two-dimensional gel profiles of Triton models of normal rat kidney (NRK) cells and their Kirsten viral transformant, 442. Several protein differences were detected. The models of the transformed line lacked five acidic polypeptides and possessed a much higher intermediate filament to actin ratio. Scanning microscopy reveals significant ultrastructural differences in these models, with the NRK line exhibiting a much more filamentous structure. In addition, nuclease treatment of NRK models causes a dramatic change in their scanning image while the 442 models are unaffected. Nuclease treated models lack microfilaments and appear to contain only intermediate filaments, although actin is still a prominent protein constituent.

Organizational differences in the membrane proteins of normal and irreversibly sickled erythrocytes.

Using two-dimensional gels, no unique membrane proteins were detected in irreversibly sickled cells. Membranes from irreversibly sickled cells were shown to cross-link much more readily with dithiobis(succinimidyl propionate) than normal erythrocyte membranes. Increased binding of band 4.5 protein and increased intra-chain disulfides were also demonstrated. These changes may correlate to enhanced cellular rigidity.