Cytoskeleton Preparations Research Articles

Interaction of two brain annexins, CaBP33 and CaBP37, with membrane-skeleton proteins

CaPB33 and CaPB37, two annexins purified from bovine brain, interact with a Triton X-100-resistant fraction (cytoskeleton) from bovine brain membranes in a Ca 2+-dependent way in vitro. The binding is saturable with respect to the CaBP33–CaBP37 concentration, half-maximal binding occurring at ~15 μg of the CaBP33–CaBP37 mixture/ml. The binding of these two annexins to the crude cytoskeleton preparation as a function of free Ca 2+ concentration is biphasic, with half-maximal binding at ~ 50 μM and ~ 400 μM free Ca 2+ for the first and the second component, respectively. By an overlay technique, CaBP33 and CaBP37 bind to a set of low M r polypeptides (10–20 kDa) in the crude cytoskeleton preparation, with formation of an 85–90 kDa complex as investigated in cross-linking experiments. No binding of the CaBP33–CaBP37 mixture to either G-or F-actin has been observed. Identification of the CaBP33–CaBP37-binding proteins in cytoskeletons would help elucidating the function(s) of these annexins in the brain.

An Erythrocyte Glycoprotein of Apparent Mr 60,000 Expresses the Sc1 and Sc2 Antigens

Immunoblotting with human anti-Sc1 and anti-Sc2 locates the Sc1 and Sc2 antigens to an erythrocyte membrane glycoprotein of apparent Mr 60,000. The antigens are destroyed by pronase, and require intact disulphide bonds for expression. A proportion of the molecules carrying the Sc1 and Sc2 antigens are associated with red cell cytoskeleton preparations. Treatment of intact cells with an Endo F preparation resulted in the loss of the Sc2 antigen but not the Sc1 antigen, suggesting that the Sc2 antigen is dependent on the presence of one or more complex N-glycans for its expression.

An Erythrocyte Glycoprotein of Apparent M(r) 60,000 Expresses the Sc1 and Sc2 Antigens

Immunoblotting with human anti-Sc1 and anti-Sc2 locates the Sc1 and Sc2 antigens to an erythrocyte membrane glycoprotein of apparent M(r) 60,000. The antigens are destroyed by pronase, and require intact disulphide bonds for expression. A proportion of the molecules carrying the Sc1 and Sc2 antigens are associated with red cell cytoskeleton preparations. Treatment of intact cells with an Endo F preparation resulted in the loss of the Sc2 antigen but not the Sc1 antigen, suggesting that the Sc2 antigen is dependent on the presence of one or more complex N-glycans for its expression.

Association of type 3 protein kinase C with focal contacts in rat embryo fibroblasts.

We have used immunocytofluorescence techniques to determine the subcellular distribution of the Ca2+, phospholipid-dependent protein kinase, protein kinase C (PKC). Using monoclonal antibodies that are specific for Type 3 (alpha) PKC, we have determined that there are least two pools of PKC in normal rat embryo fibroblasts (REF52 cells): diffuse cytoplasmic and fiber-associated. Extraction with chelators and detergent before fixing and staining removes the cytoplasmic PKC. The fiber-associated staining remains in these cytoskeleton preparations. The cytoskeleton Type 3 PKC staining closely resembles that of the focal contact protein vinculin and colocalizes with another focal contact protein, talin. Cytochalasin, but not colchicine, coordinately disrupts the staining pattern of vinculin and PKC. Activation of PKC by treatment with phorbol esters causes depolymerization of microfilaments and reorganization of vinculin staining. We propose that Type 3 PKC is a modulatory component of the focal contact and has a primary role in regulation of the association of microfilament bundles with the plasma membrane.

Insulin and Insulin-like Growth Factor I (IGF I) Stimulate Phosphorylation of a Mr 175,000 Cytoskeleton-associated Protein in Intact FRTL5 Cells

FRTL5 rat thyroid cells possess separate high affinity receptors for insulin and insulin-like growth factor I (IGF I) that undergo beta-subunit phosphorylation upon interaction with the specific ligand. Phosphorylation is rapid and dose-dependent and occurs primarily on tyrosine residues. Within 2 min, both insulin and IGF I also give rise to a Mr 175,000 phosphoprotein (pp175) that can be immunoprecipitated by anti-phosphotyrosine antibody (alpha-Tyr(P]. Phosphorylation of pp175 occurs on serine and threonine as well as tyrosine residues. When FRTL5 cells are solubilized with 1% Triton X-100, alpha-Tyr(P) immunoprecipitates phosphorylated insulin and IGF I receptors but little pp175 from the Triton-soluble fraction. After treatment of the Triton-insoluble portion with 1% sodium dodecyl sulfate at 100 degrees C, pp175 can be identified by immunoprecipitation with alpha-Tyr(P). The fraction of FRTL5 cells that remains after extraction of an attached monolayer with 1% Triton for 5 min at 22 degrees C contains most of the cytoskeleton and also nuclei. Extraction of this 32P-labeled cytoskeleton preparation with sodium dodecyl sulfate followed by alpha-Tyr(P) immunoprecipitation results in almost complete recovery of the pp175 content of the cells. When a nuclear fraction was prepared from FRTL5 cells by differential centrifugation, pp175 was not found in the nuclear pellet from labeled cells, but greater than 80% of pp175 was recovered in the supernatant. We conclude that pp175 is a common substrate for insulin and IGF I receptor kinases which, in FRTL5 cells, is associated with the cytoskeleton. It is suggested that phosphorylation of proteins associated with cytoskeletal elements could be involved in insulin and IGF I action in cells.

Partial characterisation of the human erythrocyte antigen Pta.

Ghosts were prepared from erythrocytes positive for the rare blood group antigen Pta. Immunoblotting of the solubilised ghosts with anti-Pta located the antigen on a band with an Mr of 31,600, about 1,100 higher than that of sialoglycoprotein gamma. Binding to the same band was also observed when cytoskeleton preparations from Pt(a+) erythrocytes were immunoblotted with the antibody. Haemagglutination and immunoblotting experiments were consistent in demonstrating that the Pta antigen is not inactivated by treatment of intact erythrocytes with neuraminidase or trypsin but is destroyed by treatment with alpha-chymotrypsin, papain or pronase. The data indicate that the Pta antigen is carried on a 'new' erythrocyte membrane protein.

Partial Characterisation of the Human Erythrocyte Antigen Pt^a

Ghosts were prepared from erythrocytes positive for the rare blood group antigen Pt^a. Immunoblotting of the solubilised ghosts with anti-Pt^a located the antigen on a band with an M(r)r of 31,600, about 1,100 higher than that of sialoglycoprotein γ. Binding to the same band was also observed when cytoskeleton preparations from Pt(a+) erythrocytes were immunoblotted with the antibody. Haemagglutination and immunoblotting experiments were consistent in demonstrating that the Pt^a antigen is not inactivated by treatment of intact erythrocytes with neuraminidase or trypsin but is destroyed by treatment with α-chymotrypsin, papain or pronase. The data indicate that the Pt^a antigen is carried on a ‘new’ erythrocyte membrane protein.

Phosphorylation of vimentin in mitotically selected cells. In vitro cyclic AMP-independent kinase and calcium-stimulated phosphatase activities.

The phosphorylation of the intermediate filament protein vimentin was examined under in vitro conditions. Cell cytosol and Triton-insoluble cytoskeleton preparations from nonmitotic and mitotically selected mouse L-929 cells exhibited vimentin kinase activity that is apparently cAMP and Ca2+ independent. The level of vimentin kinase activity was greater in preparations from mitotically selected cells than nonmitotic cells. Addition of Ca2+ to mitotic cytosol decreased net vimentin phosphorylation. Dephosphorylation experiments indicated that there is phosphatase activity in these preparations which is stimulated by addition of Ca2+. Fractionation of cytosol from nonmitotic cells on DEAE-Sephacel and phosphocellulose revealed a single major vimentin kinase activity (peak I). Fractionation of cytosol from mitotically selected cells yielded a similar activity (peak I) and an additional vimentin kinase activity (peak II) that was not found in nonmitotic preparations. Based on substrate specificity and lack of inhibition to characteristic inhibitors, the semipurified peak I and II vimentin kinase activities appear to be cAMP-independent enzymes that are distinct from casein kinases I and II. Phosphopeptide mapping studies indicated that both peak I and peak II vimentin kinases phosphorylate tryptic peptides in the NH2-terminal region of vimentin that are phosphorylated in intact cells. Electron microscopic examination of reconstituted vimentin filaments phosphorylated with both semipurified kinases indicated that phosphorylation induced filament disassembly. These experiments indicate that the increased phosphorylation of vimentin during mitosis may be catalyzed by a discrete cAMP-independent protein kinase. In addition, preparations from mitotic cells exhibited a Ca2+-stimulated phosphatase activity, suggesting that Ca2+ may play a regulatory role in vimentin dephosphorylation during mitosis.

Evidence for heterogeneity in the 160/165 x 10(3) Mr glycoprotein components of desmosomes.

We have prepared both monoclonal and polyclonal antibody preparations directed against the 160/165 x 10(3) Mr glycoproteins (desmogleins) of bovine tongue epithelial desmosomes. The polyclonal antibody preparation recognizes desmosomes in a number of mouse tissues, e.g. mouse skin, heart, bladder and trachea, as determined by immunofluorescence microscopy. Furthermore, the polyclonal antibodies recognize polypeptide(s), present in the high salt, Triton-insoluble residues ('cytoskeleton preparations') of mouse skin, heart, bladder and trachea, which comigrate with the 160/165 x 10(3) Mr glycoproteins of bovine tongue epithelial desmosomes as determined by 'Western' immunoblotting. Conversely, the monoclonal 160/165 x 10(3) Mr antibody preparation recognizes desmosomes of stratified squamous epithelial tissues but not desmosomes in other tissue types. Moreover, whereas the monoclonal antibodies recognize 160/165 x 10(3) Mr polypeptides in mouse skin cell cytoskeletons they show no immunoreactivity with the cytoskeleton preparations of mouse bladder, trachea and heart following immunoblotting. These results suggest therefore that although there are conserved epitopes of the 160/165 x 10(3) Mr glycoproteins there are also epitopes of these molecules which vary from tissue to tissue. Double label immunofluorescence observations of cryostat sections of mouse skin using the monoclonal antibodies and antibodies directed against desmoplakin, a plaque component of desmosomes, reveal that the monoclonal antibodies do not recognize certain desmosomes in basal cells which are recognized by desmoplakin antibodies. Indeed, double label observations of cryostat sections of mouse skin using the monoclonal antibodies and human autoantibodies which react with hemidesmosomal components suggest that the monoclonal antibodies stain desmosomes located along the apical surfaces of basal cells but fail to recognize desmosomes along the lateral surfaces of these same cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Chapter 14 Biochemical and Genetic Approaches to Microtubule Function in Dictyostelium discoideum

Methods have been developed for analyzing tubulin and microtubules from the cellular slime mold D. discoideum. alpha- and beta-tubulin have been identified on high-resolution 2D gels, and microtubules have been isolated in cytoskeleton preparations from amoebae (White et al., 1983). These studies have revealed properties unique to Dictyostelium tubulin. Amoebal microtubules can be visualized by indirect immunofluorescence, which has aided in the identification of inhibitors which specifically depolymerize microtubules and block amoebae in mitosis. The mitotic inhibitors CIPC, NOC, and TBZ have been used to select resistant mutants which are currently the subjects of biochemical, morphological, and genetic analysis (Katz et al., 1982; White, 1983). One mitotic inhibitor-resistant mutant, CIPC 6, was found to be temperature-sensitive for growth at 27 degrees C as well as CIPC-resistant. At the restrictive temperature amoebae from this mutant are deficient in the passage through mitosis. After incubation for 12 hours at the restrictive temperature, 20% of the CIPC 6 amoebae displayed condensed chromosomes, compared to 2% at the permissive temperature, as determined by Giemsa staining. Examination of the microtubules of this mutant by indirect immunofluorescence showed abnormal spindle microtubule formation at the restrictive temperature, which is the likely cause of the mitotic arrest (White, 1983). Cytoplasmic microtubules were also disrupted in nonmitotic amoebae of CIPC 6 at 27 degrees C. This temperature-sensitive loss of microtubule function suggested the possibility that tubulin from CIPC 6 might be altered. When tubulin from CIPC 6 was examined on 2D gels, no reproducible electrophoretic change was observed from that of the wild type. Through further characterization of mitotic inhibitor-resistant mutants like CIPC 6, more mitotic or microtubule mutants will be identified. Among these mutants, some should contain electrophoretically altered tubulin, microtubule-associated proteins, or components of the amoebal cytoskeleton. Possessing Dictyostelium mutants with known biochemical alterations in cytoskeletal proteins should reveal significant information regarding the function of these proteins in eukaryotic growth and development.

Connections of intermediate filaments with the nuclear lamina and the cell periphery

We investigated the relationship between intermediate filaments (IFs) and other detergent- and nuclease-resistant filamentous structures of cultured liver epithelial cells (T51B cell line) using whole mount unembedded preparations which were sequentially extracted with Triton X-100 and nucleases. Immunogold labelling and stereoscopic observation facilitated the examination of each filamentous structure and their three-dimensional relationships to each other. After solubilizing phospholipid, nucleic acid and soluble cellular protein, the resulting cytoskeleton preparation consisted of a network of cytokeratin and vimentin IFs linked by 3 nm filaments. The IFs were anchored to and determined the position of the nuclear lamina filaments (NLF) network and the centrioles. The NLF was composed of the nuclear lamina filaments measuring 3-6 nm in diameter which radiated from and anchored to the skeleton of the nuclear pores. The IFs located in the nuclear region appeared to be interwoven with the NLF. At the cell surface, the IFs seemed to be attached to the putative actin filament network. They formed a focally interrupted plexus-like structure at the cell periphery. Fragments of vimentin filaments were found among the filamentous network located at the cell surface, and some filaments terminated blindly there.

Cytoskeletons from a mutant of Dictyostelium discoideum with flattened cells.

Development of a mutant of Dictyostelium discoideum, HG403, is described whose cells spread strongly on a substratum. Although the mutant cells were less clearly polarized into the front and rear ends, and usually less extensively elongated than wild-type cells, their aggregation pattern was only slightly less regular. Cells of the mutant responded well to cyclic AMP by chemotaxis, although their capability of stabilizing cell shape and maintaining dominance of a single moving front appeared to be reduced. Mutant HG403 proved to be ideal for the preparation of cytoskeletons in which the organization of the microtubular system, the network of filaments between them, the dense texture of the microfilament network at the periphery of the cells, as well as the bundling of microfilaments in spike-like extensions, could be observed.

The characterization of an acidic calmodulin-binding protein in brain cytoskeleton and membrane fractions.

One of the most abundant acidic proteins in rat brain has an Mr of 68,000 and a pI of 5.6 (68K 5.6 protein) when analysed by two-dimensional gel electrophoresis. The 68K 5.6 protein was found in large relative amounts in brain cytoskeleton preparations and in membrane and supernatant fractions. High-salt washing and proteolytic digestion did not remove this protein from the membrane elements. The 68K 5.6 protein was also found in the microtubule-associated protein fraction of purified microtubules and was present in large relative amounts in preparations of intermediate-filament proteins. The 68K 5.6 protein binds to calmodulin in the presence of Ca2+ ions, and we found it to be an abundant acidic calmodulin-binding protein in brain tissue.

A Monoclonal antibody against mouse oocyte cytoskeleton recognizing cytokeratin-type filaments

Monoclonal antibodies were raised to detergent-extracted cytoskeleton preparations of mouse oocytes. In immunofluorescence microscopy, one of the antibodies, OCS-1, localizes exclusively to epithelial cells in frozen tissue sections, including various simple and stratified epithelia. The antibody decorates a keratin-type of fibrillar, vinblastine-resistant network in various cultured, epithelial-type cells, but not in myoid or fibroblastoid cells. In mouse oocytes and cleavage-stage embryos, the OCS-1 antibody gives a diffuse, spotty staining pattern. In blastocyst-stage embryos, the antibody reveals a keratin-type filamentous organization in the trophectoderm cells. In immunoelectron microscopy, the OCS-1 antibody decorates 10 nm-thick filaments, often identifiable as desmosome-attached tonofilaments, in detergent-treated trophectoderm cells. The antigen(s) recognized by the OCS-1 antibody is apparently present in, or closely associated with, cytokeratin filaments. In addition to mouse oocytes and early embryos, a wide variety of epithelial cells in various species seem to share this antigen(s). The present results suggest that at the early stages, the cytokeratin-related antigen(s) defined by the OCS-1 antibody are stored in a non-fibrillar form which is then converted into a fibrillar network at the blastocyst stage. A pre-existing supply of cytokeratin-related protein may be essential for the development of the blastocyst.

Effects of cytochalasin B on cell movements and chemoattractant-elicited actin changes of Dictyostelium

The actin-binding drug cytochalasin B (CB) was employed to study the stability and role of cytoskeletal actin following chemotactic stimulation of Dictyostelium discoideum. Intact amoebae were found to be impermeable to this drug, as shown by lack of inhibition of chemotactic movement in its presence and failure of [ 3H]CB to bind to intact amoebae. However, there were approx. 150 000 high affinity CB-binding sites per cell detectable after cell breakage and preparation of Triton-insoluble cytoskeletons. The effect of CB on cytoskeletons was to destabilize the second (25–45 sec) and third (60 sec) chemotactically-induced peaks of cytoskeletal actin accumulation and to reduce the actin levels to the low prestimulus amount. In contrast, the drug had no such action on the rapid (3–5 sec) actin peak. This peak appeared to be stable in the presence of CB added before or simultaneously with lysis of the cell. It was also observed that the instability of the second and third peaks to CB gradually decreased after cell lysis (as did the number of CB binding sites) such that if CB was added 5 min after lysis of the chemotactically stimulated amoebae it had no destabilizing effect. Evidence was obtained from experiments employing centrifugation of cytoskeletons at 100 000 g and from the use of the DNase I inhibition assay for G-actin, that the first (3–5 sec) actin peak of accumulation involved polymerization rather than just cross-linking of short filamentous actin fragments. The significance of these actin accumulation peaks is discussed and their timing correlated with events involved in chemotaxis.

Cellular distribution of three mammalian Ca2+-binding proteins related to Torpedo calelectrin.

Addition of Ca2+ to post-microsomal fractions of bovine adrenal or liver produced a sedimentable complex of membrane vesicles and cytoplasmic proteins. Proteins with apparent mol. wts. 70 000, 36 000 and 32 500 were solubilized from this complex by Ca2+ chelation. The 36 000 mol. wt. protein (p36) was immunoprecipitated by an antiserum specific for pp36, a major substrate for Rous sarcoma virus src-gene tyrosine kinase. This protein was present in many mesenchymal cells and associated with membrane cytoskeleton of bovine fibroblasts in a Ca2+-dependent manner. The 70 000 and 32 500 mol. wt. proteins were widely distributed in established cell lines, but were not clearly associated with cell organelles in tissue sections, nor retained in cytoskeleton preparations. On immunoblots p36 reacted strongly with antibodies produced against the electric fish protein Torpedo calelectrin and the similar Ca2+-binding properties and subunit mol. wts. of these proteins suggests that they might be functionally related. Since Torpedo calelectrin, p70, p36 and p32.5 were bound by lipid vesicles or microsomal membranes at micromolar free Ca2+ concentrations, regulated association with intrinsic membrane components may be involved in the functions of these widespread proteins.

The Rhesus (D) polypeptide is linked to the human erythrocyte cytoskeleton

Cytoskeleton preparations derived from lactoperoxidase-radioiodinated human erythrocytes were found to be enriched in a labelled component with the same apparent molecular mass as the Rhesus (D) (Rh(D)) antigen polypeptide. Immune precipitation from the cytoskeleton preparations confirmed that this component is the Rh(D) polypeptide. The results suggest that the Rh(D) polypeptide may be linked to the erythrocyte skeletal matrix. The possibility that the Rh(D) antigen is involved in maintaining the shape and viability of the erythrocyte is discussed.

Cytoplasmic microtubule-associated proteins: Phosphorylation at novel sites is correlated with their incorporation into assembled microtubules

We have analyzed the detailed structure and cytoplasmic distribution of cytoplasmic microtubule-associated proteins. The procedure used to identify these proteins, based on preparation of detergent-extracted cytoskeletons, permits separation of fractions containing assembled and unassembled microtubule proteins. We show that two of these proteins, 69 and 80 kd, are closely related to one another and that each protein is present as a set of structurally related polypeptides with differing isoelectric points. In both neuroblastoma and pheochromocytoma cells, several of the isoelectric variants are greatly enriched in the fraction containing assembled microtubule components. Their differential distribution is correlated with phosphorylation at novel sites on the protein. These results support the possibility that covalent modification of a cytoskeletal component may specify its functional state.

Intermediate filament systems are collapsed onto the nuclear surface after isolation of nuclei from tissue culture cells

Standard procedures for the biochemical isolation and purification of tissue culture cell nuclei are very similar to recently described methods for the preparation of detergent-resistant cytoskeletons. But the latter in addition to extracted nuclei contain cytoplasmic filament systems. We, therefore, investigated the distribution of these filamentous systems during nuclear isolation both by immunofluorescence microscopy and by SDS-gel electrophoresis. The intermediate filaments copurify with each single isolated nucleus. The majority of the filaments is collapsed onto isolated nuclei and still constitutes a filamentous system. This can be shown by partially unfolding the collapsed filament systems.

The cytoskeleton of primary astrocytes in culture contains actin, glial fibrillary acidic protein, and the fibroblast-type filament protein, vimentin.

Primary astrocytes were cultured from forebrains of 1-day-old rats. Immunofluorescence microscopy showed that approximately 80% of the cells were positive for glial fibrillary acidic protein (GFAP) and greater than 80% were stained with an antiserum to the molecular weight 58,000 fibroblast intermediate filament protein (vimentin). Gel electrophoresis of Triton-insoluble cytoskeleton preparations from these cultures revealed three major bands having molecular weights of 58,000, 51,000, and 42,000, together with some prominent lower-molecular-weight species. The protein of molecular weight 51,000 was not present in preparations from fibroblasts. Each of the three major astrocyte proteins was subjected to limited proteolysis, while two of the proteins were cleaved by cyanogen bromide. The electrophoretic peptide patterns of the 58,000 protein were similar to those of vimentin isolated from NIL-8 fibroblasts, and the patterns of the 51,000 protein were similar to those of GFAP isolated from rat spinal cord. The patterns of the protein of molecular weight 42,000 resembled those of muscle action. Rocket immunoelectrophoresis showed that the 51,000 astrocyte protein reacted with an antiserum to bovine GFAP, but the 58,000 and 42,000 proteins failed to react. We conclude that the major proteins of cytoskeleton preparations from cultured primary astrocytes are vimentin (58,000), GFAP (51,000), and actin (42,000), and that our data show no obvious structural relationship among them.