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R D Goldman

Publications and source records attributed to R D Goldman.

At least 127 records · Page 7Linked to original sources
PubMed1982

Dynamic aspects of the supramolecular organization of intermediate filament networks in cultured epidermal cells.

We have shown, by indirect immunofluorescence microscopy using an antiserum against the mouse keratin subunit K2 and by electron microscopy, that transformed (PAM) and primary (PME) mouse epidermal cells possess extensive networks of IF bundles. Following trypsinization and replating of PAM cells, IF bundles are seen to move as a continuous network from a perinuclear zone into the peripheral cytoplasmic regions. In PAM cells lysed in high-ionic-strength solutions containing Triton X-100 and DNAase-1, IF bundles appear to be closely associated with nuclear envelope remnants and, in some cases, appear to be attached to nuclear pore complexes. PME cells cultivated in low Ca2+-containing medium possess perinuclear birefringent arrays of IF bundles. Within 2 hours of switching the cells to normal Ca2+ levels, the PME IF bundle network moves towards and establishes contact with the cell surface as desmosomes form. Live cells observed by phase contrast and fixed cells observed by immunofluorescence microscopy demonstrate that desmosomes can be distinguished as dark bands separating neighboring cells. There is little difference between the major proteins seen in SDS-polyacrylamide gel profiles of isolated IF bundle networks from PME cells before and after the Ca2+ switch. Therefore, a reorganization of relatively insoluble membrane-associated protein following the Ca2+ switch may be involved in desmosome formation. The isolated IF networks from PAM cells differ in protein composition compared to the PME IF networks. This may be related to the greatly reduced number of desmosomes in PAM cells. The IF bundle system in epidermal cells appears to be involved in shape formation, shape maintenance, the establishment of desmosomes, nuclear centration, and cell-cell contact.

Animals↗
PubMed1982

In vitro reconstitution of intermediate filaments form mammalian neurofilament triplet polypeptides.

Intermediate filaments (IF) were reconstituted in vitro from bovine neurofilament triplet polypeptides. Neural IF, solubilized in either low salt or 8 M urea solution, assembled into IF when returned to near-physiological solution conditions. The 68,000-dalton component of the triplet, purified to homogeneity by preparative NaDodSO4 electrophoresis, was renatured and reassembled into short (approximatley 0.05-micrometer) approximatley 10 nm-diameter filaments. These results demonstrate that the triplet polypeptides are components of neural IF and that the 68,000-dalton polypeptide is an IF structural protein.

Animals↗
PubMed1981

In vitro assembly of homopolymer and copolymer filaments from intermediate filament subunits of muscle and fibroblastic cells.

This paper presents evidence that the intermediate filament (IF) subunits of muscle cells (skeletin or desmin) and fibroblastic cells (decamin or vimentin) separately form homopolymer IF in vitro and, when mixed, prefer to form copolymer IF in vitro. Because they coexist in cells, they may also form copolymers in vivo. The IFs of baby hamster kidney fibroblasts (BHK-21) consist of a major subunit, decamin, and two minor subunits which, on the basis of two-dimensional gel and peptide mapping criteria, are identical to the alpha and beta subunits of smooth muscle desmin. The subunits differ only in their degrees of phosphorylation: alpha desmin contained 2 mol/mol of O-phosphoserine whereas beta desmin contained none. The decamin and desmin subunits assembled into homopolymer IF in vitro in high yield from purified denatured subunits under identical conditions of pH and ionic strength. However, homopolymer decamin IF disassembled into soluble protofilaments in solutions of ionic strength less than 0.05 mol/liter whereas homopolymer desmin IF disassembled at ionic strength less than 0.03 mol/liter. When decamin and desmin were mixed together as denatured subunits or as soluble protofilaments, the IF assembled in vitro had solubility properties intermediate between those of the homopolymer IFs, indicating that the two subunits had formed copolymer IF. The stoichiometry of copolymerization as determined in mixtures in which one subunit was present in excess was suggestive of the formation of three-chain units. The possibility of nonspecific aggregation was eliminated by isolation of stable three-chain alpha-helix-enriched particles from such IF. When tracer amounts of [35S]methionine-labeled decamin were mixed with desmin, labeled IFs were obtained under conditions in which homopolymer decamin IFs were soluble. These in vitro findings may be of physiological significance because native BHK-21 IF also had solubility properties similar to those of the copolymer IF.

Animals↗
PubMed1980

In vitro reassembly of squid brain intermediate filaments (neurofilaments): purification by assembly-disassembly.

Intermediate filaments from squid brain tissue were reassembled in vitro and purified by two cycles of assembly and disassembly. Purified squid brain filaments contained one major polypeptide (60,000 daltons), which constituted about 70 percent of the total protein, and three minor polypeptides (74,000, 100,000, and 220,000 daltons). Squid brain intermediate filaments were reconstituted from rod-shaped protofilamentous subunits. In addition to the intermediate filaments, dense bodies which may function in intermediate filament nucleation or organization were retained through two purification cycles.

Animals↗
PubMed1980

Intermediate filaments of baby hamster kidney (BHK-21) cells and bovine epidermal keratinocytes have similar ultrastructures and subunit domain structures.

Structural features of the intermediate filaments (IF) of bovine epidermal keratinocytes (keratin filaments) and of baby hamster kidney (BHK-21) cells were studied to define the molecular basis of the similarities and differences in their properties. Purified subunits of the IF were specifically cleaved at their methionine and tryptophan residues. Peptide maps indicated that the IF subunits of BHK-21 cells were similar but not identical to each other and quite different from each of the keratin IF subunits. Intact IF were subjected to limited tryptic digestion, and structurally identical alpha-helix-enriched particles were released. Their properties indicated these IF were composed of a similar three-chain unit which contained regions of coiled-coil alpha-helix interspersed with region of non-alpha-helix. These two types of experiments permitted the construction of subunit domain maps which revealed a common structure: all subunits possessed two alpha-helical domains of the same size that were adjoined by non-alpha-helical domains of variable size. We propose that the reported solubility and immunological differences in these IF and perhaps those of other types of cells are due largely to variations in the size, configuration, and amino acid sequence of the non-alpha-helical regions of the subunits in the IF.

Amino Acid Sequence↗
PubMed1980

Microfilaments and tropomyosin of cultured mammalian cells: isolation and characterization.

Microfilaments were isolated from cultured mammalian cells, utilizing procedures similar to those for isolation of "native" thin filaments from muscle. Isolated microfilaments from rat embryo, baby hamster kidney (BHK- 21), and Swiss mouse 3T3 cells appeared structurally similar to muscle thin filaments, exhibiting long, 6 nm Diam profiles with a beaded, helical substructure. An arrowhead pattern was observed after reaction of isolated microfilaments with rabbit skeletal muscle myosin subfragment 1. Under appropriate conditions, isolated microfilaments will aggregate into a form that resembles microfilament bundles seen in situ cultured cells. Isolated microfilaments represent a complex of proteins including actin. Some of these components have been tentatively identified, based on coelectrophoresis with purified proteins, as myosin, tropomyosin, and a high molecular weight actin-binding protein. The tropomyosin components of isolated microfilaments were unexpected; polypeptides comigrated on SDS-polyacrylamide gels with both muscle and nonmuscle types of tropomyosin. In order to identify more specifically these subunits, we isolated and partially characterized tropomyosin from three cell types. BHK-21 cell tropomyosin was similar to other nonmuscle tropomyosins, as judged by several criteria. However, tropomyosin isolated from rate embryo and 3T3 cells contained subunits that comigrated with both skeletal muscle and nonmuscle types of myosin, whereas the BHK cell protein consistently contained a minor muscle-like subunit. The array of tropomyosin subunits present in a cell culture was reflected in the polypeptide chain pattern seen on SDS-polyacrylamide gels of microfilaments isolated from that culture. These studies provide a starting point for correlating changes in the ultrastructural organization of microfilaments with alterations in their protein composition.

Actins↗
PubMed1979

Isolation and characterization of baby hamster kidney (BHK-21) cell modulator protein.

A Ca2+-dependent modulator protein has been isolated from BHK-21 cells. The purification requires heat treatment, ion-exchange chromatography, and gel filtration. The protein appears homogenous on sodium dodecyl sulfate--polyacrylamide and isoelectric focusing gels. The protein comigrates with purified smooth muscle and brain modulators. BHK-21 modulator is characterized by a high content of aspartic and glutamic acids and by a high phenylalanine/tyrosine ratio. It lacks both cysteine and tryptophan. The protein is effective in activating brain-modulator-deficient phosphodiesterase. It can also be used in assay systems to generate Ca2+-sensitive actin activation of both BHK-21 and smooth muscle myosins. Therefore, it is proposed that the BHK-21 modulator protein is a component of the Ca2+-dependent mechanism involved in the regulation of actin--myosin interactions in BHK-21 cells.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1979

Calcium-sensitive regulation of actin-myosin interactions in baby hamster kidney (BHK-21) cells.

A fraction has been obtained from baby hamster kidney (BHK-21) cells that will stimulate the actin-moderated ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity of both BHK-21 myosin and gizzard smooth muscle myosin. This activation is associated with the specific phosphorylation of the myosin 20,000-dalton light chain. The BHK-21 myosin light chain kinase preparation contains a major protein of approximately 105,000 molecular weight as determined by sodium dodecyl sulfate gel electrophoresis. Both the actin activation and phosphorylation events require the presence of Ca2+ and the so-called modulator or calcium-dependent regulator protein that has been isolated from smooth muscle, brain, and other tissues. On the basis of these results we propose that this kinase system constitutes a Ca2+-dependent regulatory mechanism for myosin-actin interactions in nonmuscle mammalian cells.

Actins↗
PubMed1979

In vitro assembly of intermediate filaments from baby hamster kidney (BHK-21) cells.

Intermediate filaments (IF) from baby hamster kidney (BHK-21) cells can be disassembled at low ionic strength and reassembled upon addition of salt. Turbidimetric analyses show that reassembled IF exhibit the light scattering properties of long rods under physiological conditions (5 mM Na+/K+ phosphate, pH 7.2/170 mM NaCl at 21 degrees C). IF weight concentration, determined by centrifugation, is directly proportional to the optical density at 3000 nm. Thus, turbidity can be used as a quantitative assay for IF assembly. Turbidimetric and centrifugation analyses both indicate that IF assembly exhibits a critical protein concentration of 0.05-0.15 mg/ml. Above the critical concentration, IF weight concentration at steady-state is a linear function of the total protein concentration. Negative stain observations at early stages of the assembly process suggest lateral association of protofilaments to form short IF. This lateral association is accompanied by a rapid turbidity increase which is then followed by IF elongation and a slower turbidity increase to plateau. Further purification of IF by low/high-NaCl-induced cycles of disassembly/reassembly results in retention of 54- and 55-kilodalton (decamin) polypeptides. These results constitute a quantitative description of in vitro reassembly of IF from homogeneous cultures of nonkeratinizing cells and establish conditions for further studies on the regulation of IF assembly.

Animals↗
PubMed1979

Isolation of a high molecular weight actin-binding protein from baby hamster kidney (BHK-21) cells.

A high molecular weight protein (HMWP) with properties similar to those of both actin-binding protein (ABP) and filamin has been isolated from cultured baby hamster kidney (BHK-21) cells. The protein was present in an actomyosin-depleted sucrose extract of the cells and was eluted, upon gel chromatography on Sepharose 4B, near the void volume. The subunit migration on sodium dodecyl sulfate/polyacrylamide gels and the amino acid composition of HMWP were similar to those of ABP and filamin. HMWP bound to and crosslinked F-actin from rabbit muscle, as shown by the formation of a gel that was sedimented with low-speed centrifugation. This interaction was insensitive to temperature and low concentrations of calcium ions, although it may depend on the presence of myosin. Observation of thin sections of the actin-HMWP gel revealed crosslinked complexes of laterally aggregated actin filaments. The axial period of the dense crosslinks was 34 nm. The HMWP may be involved in regulation of microfilament organization.

Actins↗
PubMed1979

Ultrastructure of microfilament bundles in baby hamster kidney (BHK-21) cells. The use of tannic acid.

After standard glutaraldehyde-osmium tetroxide fixation procedures, the majority of microfilament bundles in BHK-21 cells exhibit relatively uniform electron density along their long axes. The inclusion of tannic acid in the glutaraldehyde fixation solution results in obvious electron density shifts along the majority of microfilament bundles. Striated patterens are frequently observed which consist of regularly spaced electron dense (D) and electron lucid (L) bands. A striated pattern is also observed along many BHK-21 stress fibers after processing for indirect immunofluorescence utilizing BHK-21 myosin antiserum. A direct correlation of these periodicities seen by light and electron microscope techniques is impossible at the present time. However, comparative measurements indicate that the overall patterns seen in the immunofluorescence and electron microscope preparations are similar. The ultrastructural results provide an initial clue for the ultimate determination of the supramolecular organization of contracile proteins other than actin within the microfilament bundles of non-muscle cells.

Animals↗
PubMed1978

Ten-nanometer filaments of hamster BHK-21 cells and epidermal keratin filaments have similar structures.

The 10-nm filaments of baby hamster kidney (BHK-21) cells, when examined either in the form of native filament caps or polymerized in vitro, are long tubes of protein 8--10 nm in diameter. They contain about 42% alpha-helix, which, on the basis of x-ray diffraction data, is arranged in a coiled-coil conformation characteristic of proteins of the alpha type. The known structural properties such as morphology, dimensions, subunit composition, and ultrastructure of this fibrous protein are very similar to those of the mammalian epidermal keratin filament, to which it may therefore be related.

Cell Line↗
PubMed1978

Biochemical and immunological analysis of rapidly purified 10-nm filaments from baby hamster kidney (BHK-21) cells.

Juxtanuclear birefringent caps (FC) containing 10-nm filaments form during the early stages of baby hamster kidney (BHK-21) cell spreading. FC are isolated from spreading cells after replating by treatment with 0.6 M KCl, 1% Triton X-100 (Rohm & Haas Co., Philadelphia, Pa.) and DNase I in phosphate-buffered saline. Purified FC are birefringent and retain the pattern of distribution of 10-nm filaments that is seen in situ. Up to 90% of the FC protein is resolved as two polypeptides of approximately 54,000 and 55,000 molecular weight on sodium dodecyl sulfate (SDS) polyacrylamide gels. The protein is immunologically and biochemically distinct from tubulin as determined by indirect immunofluorescence, double immunodiffusion, one-dimensional peptide mapping by limited proteolysis in SDS gels, and amino acid analysis. The BHK-21 FC amino acid composition, however, is very similar to that obtained for 10-nm filament protein derived from other sources including brain and smooth muscle. Partial disassembly of 10-nm filaments has been achieved by treatment of FC with 6 mM sodium-potassium phosphate buffer, pH 7.4. The solubilized components assemble into distinct 10-nm filaments upon the addition of 0.171 M sodium chloride.

Amino Acids↗
PubMed1978

Functions of cytoplasmic fibers in intracellular movements in BHK-21 cells.

After trypsinization and replating, BHK-21 cells spread and change shape from a rounded to a fibroblastic form. Time-lapse movies of spreading cells reveal that organelles are redistributed by saltatory movements from a juxtanuclear position into the expanding regions of cytoplasm. Bidirectional saltations are seen along the long axes of fully spread cells. As the spreading process progresses, the pattern of saltatory movements changes and the average speed of saltations increases from 1.7 MICROMETER/S during the early stages of spreading to 2.3 micrometer/s in fully spread cells. Correlative electron microscope studies indicate that the patterns of saltatory movements that lead to the redistribution of organelles during spreading are closely related to changes in the degree of assembly, organization, and distribution of microtubules and 10-nm filaments. Colchicine (10 microgram/ml of culture medium) reversibly disassembles the microtubule-10-nm filament complexes which form during cell spreading. This treatment results in the disappearance of microtubules and the appearance of a juxtanuclear accumulation of 10-nm filaments. These changes closely parallel an inhibition of saltatory movements. Within 30 min after the addition of the colchicine, pseudopod-like extensions form rapidly at the cell periphery, and adjacent organelles are seen to stream into them. The pseudopods contain extensive arrays of actinlike microfilament bundles which bind skeletal-muscle heavy meromyosin (HMM). Therefore, in the presence of colchicine, intracellular movements are altered from a normal saltatory pattern into a pattern reminiscent of the type of cytoplasmic streaming seen in amoeboid organisms. The streaming may reflect either the activity or the contractility of submembranous microfilament bundles. Streaming activity is not seen in cells containing well-organized microtubule-10-nm filament complexes.

Cell Line↗