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R A Quinlan

Publications and source records attributed to R A Quinlan.

At least 55 records · Page 3Linked to original sources
PubMed1992

The 53kDa polypeptide component of the bovine fibre cell cytoskeleton is derived from the 115kDa beaded filament protein: evidence for a fibre cell specific intermediate filament protein.

The 115kDa protein found enriched in the PMCC (plasma membrane-cytoskeleton complex) fraction of the cortex in bovine lens fibre cells is proteolytically processed to a stable 53kDa product. The 115 kDa protein and the 53kDa polypeptide have been purified by a combination of ion exchange and hydroxyapatite chromatography. Tryptic peptide mapping using reverse phase HPLC and subsequent peptide sequencing confirmed that the 53kDa polypeptide is derived from the 115kDa protein. The 53kDa fragment is also a component of the PMCC as well as being a major component of the urea soluble fraction of lens plasma membranes which have been extracted with buffers containing 1M KC1. The 53kDa polypeptide has escaped identification as a breakdown product of the 115kDa protein because it is not recognised by a commonly used monoclonal antibody, R2D2, specific for the bovine 115kDa protein. This result suggests that proteolysis is important in determining the function(s) of the 115kDa protein, and that part of this function is satisfied by the 53kDa protein core. Both the purified 115kDa protein and the 53kDa polypeptide were unable to form either beaded or intermediate filaments on their own but they were able to form short 10nm rods indicative of an intermediate stage in intermediate filament assembly. Comparison ot the assembly properties of the 53 and 115kDa proteins indicate that there are sequences in the 115kDa protein which inhibit in vitro assembly. This is similar to the situation with neurofilament proteins. We suggest that the 115kDa protein is a lens-specific intermediate filament protein.

Amino Acid Sequence↗
PubMed1990

Expression and characterization of human lamin C.

We have expressed human lamin C cDNA in E. coli using a modification of the pLcII vector system. Protein produced in this way had seven additional amino acids at its N-terminus, but retained key lamin structural and assembly properties. The modified vector we produced may prove useful when difficulties are encountered in removal of the cII fusion peptide by factor X cleavage in the pLcII system. Shadowed preparations of expressed lamin C showed the presence of 50-nm rod-like particles that closely resembled those observed for native material. Isolated molecules had two globular domains at one end, indicating that they were constructed from two parallel polypeptide chains. The expressed material also formed paracrystals with a characteristic 22.5 nm axial repeat, indicating that its assembly properties had also been retained. We also used site-specific mutagenesis to engineer a lamin fragment that lacked the C-terminal non-helical domain of the molecule. This material formed paracrystals similar to those obtained with the intact molecule, indicating that the large C-terminal non-helical domain did not contain information vital for lamin assembly.

Cloning, Molecular↗
PubMed1989

Molecular interactions in paracrystals of a fragment corresponding to the alpha-helical coiled-coil rod portion of glial fibrillary acidic protein: evidence for an antiparallel packing of molecules and polymorphism related to intermediate filament structure.

We have expressed in Escherichia coli a fragment of c-DNA that broadly corresponds to the alpha-helical coiled-coil rod section of glial fibrillary acidic protein (GFAP) and have used the resultant protein to prepare paracrystals in which molecular interactions can be investigated. An engineered fragment of mouse GFAP c-DNA was inserted into a modified version of the E. coli expression vector pLcII, from which large quantities of a lambda cII-GFAP rod fusion protein were prepared. A protein fragment corresponding to the GFAP rod was then obtained by proteolysis with thrombin. Paracrystals of this material were produced using divalent cations (Mg, Ca, Ba) in the presence of a chaotrophic agent such as thiocyanate. These paracrystals showed a number of polymorphic patterns that were based on a fundamental pattern that had dyad symmetry and an axial repeat of 57 nm. Analysis of both positive and negative staining patterns showed that this fundamental pattern was consistent with a unit cell containing two 48-nm-long molecules in an antiparallel arrangement with their NH2 termini overlapping by approximately 34 nm. More complicated patterns were produced by stacking the fundamental pattern with staggers of approximately 1/5, 2/5, and 1/2 the axial repeat. The molecular packing the unit cell was consistent with a range of solution studies on intermediate filaments that have indicated that a molecular dimer (i.e., a tetramer containing four chains or two coiled-coil molecules) is an intermediate in filament assembly. Moreover, these paracrystals allow the molecular interactions involved in the tetramer to be investigated in some detail.

Animals↗
PubMed1989

Expression in Escherichia coli of fragments of glial fibrillary acidic protein: characterization, assembly properties and paracrystal formation.

We have expressed in Escherichia coli a 1258 bp cDNA fragment corresponding to 97% of mouse glial fibrillary acidic protein (GFAP), the principal intermediate filament protein of astrocytes. High levels of expression were obtained, as a fusion protein with 32 residues of the bacteriophage lambda cII protein, using the pLcII expression vector system of K. Nagai and H.-C. Thogersen. Although removal of the cII protein fragment by proteolysis using factor X proved difficult, a protein corresponding to most of the cDNA fragment was obtained by cleaving at the endogenous thrombin site near the middle of the N-terminal non-helical domain of GFAP. A shorter 1047 bp fragment, in which the C-terminal non-helical domain of GFAP was deleted, was also produced using oligonucleotide-directed site-specific mutagenesis of the original cDNA clone. After proteolysis with thrombin, this material gave a fragment that corresponded to the alpha-helical coiled-coil rod region of the GFAP molecule, together with a portion of the non-helical N-terminal domain. The fragments produced were characterized both biochemically and ultrastructurally, and appeared to retain the conformation of native GFAP. Crosslinking showed that all fragments formed molecules containing two chains ('dimers') that associated to form four-chain molecular dimers ('tetramers') analogous to those formed by intact intermediate filament proteins. Shadowed preparations showed the presence of rod-like particles that closely resembled those observed for other intermediate filament proteins and proteolytically prepared rod domains. Remarkably, the fusion protein produced from the entire 1258 bp cDNA fragment and the cII peptide was able to form filaments that closely resembled those produced by native GFAP. However, fragments in which either the cII peptide or the C-terminal non-helical domain were removed, or in which both were removed, failed to form filaments under standard assembly conditions. Although preliminary in nature, these results suggest that both N- and C-terminal non-helical domains may have a role in intermediate filament formation. Moreover, the fragment corresponding approximately to the GFAP rod formed paracrystals similar to those observed with other coiled-coil proteins. The molecules in these paracrystals were arranged antiparallel with the two molecules in the unit cell, which may correspond to the four-chain molecular dimer (tetramer), overlapping by approximately two-thirds of their length.

Amino Acid Sequence↗
PubMed1987

Structural differences between blood-platelet tubulin and other mammalian tubulins.

Carboxymethylated pig blood-platelet tubulin subunits have been characterized by comparing their electrophoretic mobilities and two-dimensional tryptic peptide maps with those of tubulin subunits isolated from four other mammalian tissues. These were pig brain, pig lung, pig sperm and bovine kidney. In addition to the major blood-platelet alpha- and beta-tubulin subunits, minor alpha- and beta-components could be resolved on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The major blood-platelet beta-tubulin, as well as the minor brain beta 2-tubulin subunit, were clearly tissue specific. In both cases, their two-dimensional peptide maps contained unique heavily labelled peptides. Distinct peptide maps were also observed for the major blood-platelet alpha-tubulin subunit as well as for sperm tail alpha-tubulin, both of which were missing peptides (different in each case) that were present in all of the other alpha-tubulin maps. Under our conditions, a protein with a molecular weight of 70,000 and an isoelectric point of pH 6.4 co-purified with blood-platelet microtubules.

Animals↗
PubMed1987

Crystalline tubes of myosin subfragment-2 showing the coiled-coil and molecular interaction geometry.

We have produced crystalline tubes of chicken breast myosin long subfragment-2 that show order to resolutions better than 2 nm. The tubes were formed from a thin sheet in which the myosin long subfragment-2 molecules were arranged on an approximately rectangular crystalline lattice with a = 14.1 +/- 0.2 nm and b = 3.9 +/- 0.1 nm in projection. Shadowing indicated that the tube wall was approximately 7 nm thick and that the sheets from which it was formed followed a right-handed helix. Superposition of the lattices from the top and bottom of the tube produced a moire pattern in negatively stained material, but images of single sheets were easily obtained by computer image processing. Although several molecules were superimposed perpendicular to the plane of the sheet, the modulation in density due to the coiled-coil envelope was clear, indicating that the coiled-coils in these molecules were in register (or staggered by an even number of quarter pitches). In projection the coiled-coil had an apparent pitch of 14.1 nm (the axial repeat of the unit cell), but the small number of molecules (probably four) superimposed perpendicular to the plane of the sheet meant that pitches within approximately 1 nm of this value could have shown a modulation. Therefore, a more precise determination of the coiled-coil pitch must await determination of the sheet's three-dimensional structure. The coiled-coils of adjacent molecules within the plane of the sheet were staggered by an odd number of quarter pitches. This arrangement was similar to that between paramyosin molecules in molluscan thick filaments and may have features in common with other coiled-coil protein assemblies, such as intermediate filaments. Each molecule in the crystal had two types of neighbor: one staggered by an odd number of quarter pitches and the other by an even number of quarter pitches, as has been proposed for the general packing of coiled-coils (Longley, W., 1975, J. Mol. Biol., 93:111-115). We propose a model for the detailed packing within the sheet whereby molecules are inclined slightly to the plane of the sheet so that its thickness is determined by the molecular length.

Animals↗
PubMed1986

Characterization of dimer subunits of intermediate filament proteins.

The fundamental subunit of the various types of intermediate-sized filaments (IF) has been shown to be a tetramer that is thought to represent a double dimer, i.e. an array of two laterally packed coiled-coils of alpha-helices. The two-chain state of intact IF proteins had up to this point not been isolated and characterized as has been done for other fibrous alpha-helical coiled-coil proteins. Using buffers containing 3 M-guanidinium hydrochloride we prepared dimers by depolymerization of IF or by reconstitution from fully denatured molecules. Dimers of desmin (from chicken gizzard), vimentin (from bovine lens tissue and cultured human fibroblasts) and the neurofilament protein NF-L (from bovine brain) as well as in vitro formed homodimers of human and rat cytokeratins numbers 8 (A), 18 (D) and 19 ("40K"), are characterized by ultracentrifugation techniques (sedimentation velocity and equilibrium), electron microscopy and chemical cross-linking. The results show that IF proteins from discrete complexes of two polypeptide chains in parallel orientation and probably in coiled-coil configuration, which apparently have a high tendency to further associate into double dimers. Implications of these results for concepts of IF organization and IF protein assembly are discussed.

Animals↗
PubMed1985

Identification of a distinct soluble subunit of an intermediate filament protein: tetrameric vimentin from living cells.

Intermediate-sized filaments (IF) are among the most insoluble intracellular protein polymer structures. We have analyzed the small amounts of soluble vimentin, an IF protein, present in cytosol fractions obtained from lysis of cultured cells [rat RVF-SM cells, simian virus 40-transformed human fibroblasts, and human rhabdomyosarcoma (RD line) cells]. The molecular form of this soluble vimentin was determined by sucrose density gradient centrifugation, using vimentin-specific antibodies for subsequent ELISA and immunoblotting analyses. The majority of the soluble vimentin appeared in a distinct form indistinguishable in its sedimentation behavior from reconstituted tetrameric subunits of purified vimentin arrested at low ionic strength. The tetrameric coiled-coil nature of the soluble form of vimentin was indicated by the digestion pattern with chymotrypsin and by chemical crosslinking with copper-1,10-phenanthroline and dimethylsuberimidate. The competence of this soluble vimentin to assemble into IF at higher salt concentrations was demonstrated by electron microscopy. Pulse-chase experiments showed that the soluble form was not an exclusively posttranslational intermediate. We propose that in the living cell a small pool of a distinct soluble tetrameric form of vimentin exists which may exchange with polymeric IF vimentin.

Animals↗
PubMed1985

Patterns of expression and organization of cytokeratin intermediate filaments.

Cytokeratins are a large multigene family comprising two polypeptide types, i.e. acidic (type I) and basic (type II) ones, which are distinguished on the basis of immunological, peptide mapping, mRNA hybridization, and primary amino acid sequence data. The acidic (type I) cytokeratins can be subdivided into at least two different subtypes on the basis of their carboxy-terminal sequences. Considerable interspecies conservation of sequences exists, even extending to the 3'-non-coding mRNA regions. Different pairs of type I and II cytokeratins show different resistance to dissociation in urea. Sequence differences of the type I cytokeratins containing functional domains may be an explanation of the observed preference of co-expression with certain type II cytokeratins. The distribution of the different type I and II cytokeratins in normal epithelia and in carcinomas is differentiation related and can be used for cell typing and identification. The cell type-specific expression of cytokeratin polypeptides is recognized at both the protein and the mRNA level. The building block of cytokeratin IFs is a heterotypic tetramer, consisting of two type I and two type II polypeptides arranged in pairs of laterally aligned coiled coils. This principle of tetrameric organization is thought to be generally applicable to IFs.

Amino Acid Sequence↗
PubMed1984

Heterotypic tetramer (A2D2) complexes of non-epidermal keratins isolated from cytoskeletons of rat hepatocytes and hepatoma cells.

Cytoskeletal residues obtained after extraction of rat liver and cultured rat hepatoma cells (line MH1C1) were used to isolate cytokeratin subunit complexes by solubilization in low salt buffer containing 4 M-urea. Alternatively, the complexes were prepared by solubilization of total cytoskeletal proteins in 9.5 M-urea or 6 M-guanidinium hydrochloride (Gu . HCl), followed by separation using reversed phase high pressure liquid chromatography and dialysis first against either 9.5 M-urea or 6 M-Gu . HCl and then against buffers containing either 4 M-urea or 2 M-Gu . HCl, respectively. The complexes contained only two cytokeratin polypeptides in a 1 : 1 ratio as demonstrated by electrophoresis and isoelectric focusing, i.e. components A (Mr 55,000; isoelectric point in 9.5 M-urea, pH 6.4) and D (Mr 49,000; isoelectric point, pH 5.38) which were separated from each other at urea concentrations higher than 7 M. The complex had a sedimentation coefficient S25,w of 4.96 S in 2 M-Gu . HCl. Sedimentation equilibrium analysis gave an average Mr value of 207,000 which was interpreted as a tetramer containing two chains each of A and D. This complex was also directly demonstrated by gel electrophoresis under non-dissociating conditions. Using dimethyl suberimidate to cross-link the complex in solution of 4 M-urea or 2 M-Gu . HCl, we identified covalently linked heterodimers of A and D, and a tetrameric unit containing equal amounts of A and D which was the largest cross-link product obtained. This complex was similar to the tetrameric complex of rat and human vimentin formed under the same conditions. The constituents of the cross-linked products were identified by two-dimensional ("diagonal") gel electrophoresis, involving the cleavage of the bis(amidine) cross-links after the initial separation in the first dimension. Identical cross-link products were recognized when cytokeratin filaments were used. By electron microscopy the complexes appeared as threads of 2 to 3 nm diameter with a mean length of approximately 48 nm. On dialysis to low salt buffer, the complexes formed 2 to 3 nm protofilaments, intertwisted 3 to 4 nm protofilaments and typical 7 to 11 nm intermediate-sized filaments. Complexes formed from equivalent cytokeratins of other species such as man and cow, as well as heterologous recombinations such as human component A mixed with bovine component D and vice versa, showed the same characteristics.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Proteolytic modification of acidic and basic keratins during terminal differentiation of mouse and human epidermis.

Keratins from the living cell layers of human and neonatal mouse epidermis (prekeratins) have been compared to those from the stratum corneum (SC keratins). Human and mouse epidermis contained four prekeratins, two of each keratin subfamily: type II basic (pI 6.5-8.5; human 68 kDa, 60.5 kDa and mouse 67 kDa, 60 kDa) and type I acidic (pI 4.7-5.7; human 57 kDa, 51 kDa and mouse 58 kDa, 53 kDa,). While all four were present in equal amounts in adult human epidermis, two (67 kDa basic, 58 kDa acidic) were more prominent in neonatal mouse epidermis. Preliminary results with cell fractions (basal, spinous and granular) indicated that quantitative differences were a function of morphology, basal cells containing the smaller member of each subfamily and granular cells the larger. Mouse stratum corneum extracts contained four keratins (three in human): type II neutral-acidic (pI 5.7-6.7; human 65 kDa and mouse 64 kDa, 62 kDa) and type I acidic (pI 4.9-5.4; human 57.5 kDa, 55 kDa and mouse 58.5 kDa, 57.5 kDa). In both species, one-dimensional and two-dimensional peptide mapping (with V8 protease and trypsin respectively) indicated that while all four prekeratins were distinct gene products, similarities existed in the type II basic and the type I acidic keratin subfamilies. A strong homology also existed between type II SC keratins and the larger basic (type II) prekeratin (human 68 kDa and mouse 67 kDa) and between type I SC keratins and the larger acidic (type I) prekeratin (human 57 kDa and mouse 58 kDa). These results indicate a precursor-product relationship within each keratin subfamily, between SC keratins and the prekeratins abundant in the adjacent granular layer. This differentiation-related keratin processing was similar in mouse and human epidermis, and may represent a widespread phenomenon amongst keratinising epithelia.

Animals↗
PubMed1983

Molecular interactions in intermediate-sized filaments revealed by chemical cross-linking. Heteropolymers of vimentin and glial filament protein in cultured human glioma cells.

Certain glia cells, notably astrocytes and tumor cells derived therefrom, express simultaneously two types of proteins of intermediate-sized filaments, vimentin and glia filament protein (GFP). We have used an established human glioma (astrocytoma) cell culture line (U 333 CG/343 MG) in which both proteins are seen in partly overlapping fibrillar structures by immunofluorescence microscopy, to examine the possible existence of heteropolymer filaments of these two proteins by using reversible oxidative cross-linking facilitated by the 1,10-phenanthroline-cupric ion complex. Dimeric cross-link products are characterized by one-dimensional and two-dimensional gel electrophoresis under non-reducing and reducing conditions as well as by peptide mapping. The relatively large proportions of heterodimers of vimentin and GFP obtained in cytoskeletal filaments cross-linked in this way, demonstrate the frequency of heteropolymer filaments in this cell as well as the frequency of face-to-face 'pairs' of GFP and vimentin in such filaments. Together with our related observations on heteropolymer filaments between vimentin and desmin in some smooth muscle cells [Quinlan, R. A. and Franke, W. W. (1982) Proc. Natl Acad. Sci. USA, 79, 3452-3456], we discuss this as evidence for common principles of molecular arrangements of vimentin, GFP and desmin, at least in the cysteine-containing surface domains. The results are also discussed in relation to cytoskeletal changes during glial differentiation.

Biopolymers↗
PubMed1983

Identification and characterization of axopodial tubulins from Echinosphaerium nucleofilum.

Isolated microtubule protein from axopodia of the heliozoan Echinosphaerium nucleofilum, consisting of two major bands on SDS-polyacrylamide gel electrophoresis (SDS-PAGE), has been compared to axonemal and cytoplasmic tubulins from both animal and non-animal sources. The upper E. nucleofilum protein band migrated faster than the alpha-tubulins of bovine brain and sea anemone sperm tails but with approximately the same electrophoretic mobility as the axonemal alpha-tubulins of Tetrahymena pyriformis and the alga Chlorogonium elongatum and cytoplasmic alpha-tubulin from the slime mold Physarum polycephalum. The lower E. nucleofilum protein band, however, had a higher electrophoretic mobility than all the beta-tubulins which we have so far examined. It was, nevertheless, a true beta-tubulin as shown by its migration on two-dimensional gel electrophoresis and the general resemblance of its one- and two-dimensional peptide maps to those of other beta-tubulins. The Staphylococcus aureus protease cleavage pattern of the upper axopodial protein band was similar to those of other non-animal alpha-tubulins but quite different from those of the animal alpha-tubulins. In contrast, the two-dimensional tryptic peptide map of axopodial alpha-tubulin was distinct from all of them. For example, a characteristic constellation of peptides common to the peptide maps of the other alpha-tubulins was absent from that of E. nucleofilum. In contrast to Physarum and metazoan tubulins but similar to Tetrahymena tubulin, the axopodial alpha-tubulin had a more basic isoelectric point than the beta-subunit as shown by two dimensional gel electrophoresis. Some of the unusual characteristics of E. nucleofilum axopodial tubulin may not only reflect phylogenetic variation, but also the different functional requirements of axopodial microtubules.

Animals↗
PubMed1982

Heteropolymer filaments of vimentin and desmin in vascular smooth muscle tissue and cultured baby hamster kidney cells demonstrated by chemical crosslinking.

Certain smooth muscle cells of blood vessel walls as well as cultured baby hamster kidney cells contain simultaneously two different intermediate-sized filament (IF) proteins, desmin and vimentin. We have examined the question of the occurrence of both proteins in the same IF by chemically crosslinking the single cysteine group present in each of them. Oxidative crosslinking of filaments present in cytoskeletal preparations with cupric ion complexes of 1,10-phenanthroline resulted in formation of three types of dimers: vimentin-vimentin, desmin-desmin, and vimentin-desmin. These dimers were separated by NaDodSO4/polyacrylamide gel electrophoresis and characterized by binding of specific antibodies, by one- and two-dimensional gel electrophoresis of monomers obtained after cleavage of the disulfide bond by thiol agents, and by mapping of radioiodinated tryptic peptides. The demonstration of heterodimers of vimentin and desmin in vascular smooth muscle tissue of cow and chicken and in baby hamster kidney cells shows that the two proteins can be integrated in the same IF and can be nearest neighbors, oriented with their cysteine residues in a mirror-image symmetry. The possible existence of heteropolymer IF in other cell types is discussed.

Animals↗
PubMed1981

A correlation between in vivo and in vitro effects of the microtubule inhibitors colchicine, parbendazole and nocodazole on myxamoebae of Physarum polycephalum.

The effects of the microtubule inhibitors colchicine, parbendazole and nocodazole on the growth of myxamoebae of Physarum polycephalum were closely paralleled by the effects of these drugs on the assembly in vitro of purified amoebal microtubule protein. Colchicine at 100 microM did not inhibit amoebal growth and did not inhibit formation, or depolymerization, of amoebal microtubules. The benzimidazole carbamate derivatives nocodazole and parbendazole were very effective in both inhibiting growth and inhibiting the assembly in vitro of amoebal microtubule protein. Parbendazole was the most effective.

Benzimidazoles↗