bluuebunny/crossref_metadata_2025 huggingface dataset:
Concentration of pp125 focal adhesion kinase (FAK) at the myotendinous junction
Authors: Baker Lauren P. , Daggett David F. , Peng H. Benjamin
Published: April 2021

Focal adhesion kinase is a recently characterized tyrosine kinase that is concentrated at focal contacts in cultured cells. It is thought to play an important role in the the reg- ulation of the integrin-based signal transduction mechanism involved in the assembly of this membrane spe- cialization. In this study, we examined the immunocyto- chemical distribution of focal adhesion kinase in Xenopus skeletal muscle and its role in the formation of two sar- colemmal specializations, the myotendinous junction and the neuromusuclar junction, using a monoclonal antibody (2A7) against this protein. Immunoprecipitation of Xenopus embryonic tissues with this antibody demon- strated a single band at a relative molecular mass of 116 kDa. A distinct concentration of immunolabeling for focal adhesion kinase was observed at the myotendinous junction of muscle fibers in vivo. At this site, the labeling for this protein is correlated with an accumulation of phos- photyrosine immunolabeling. Focal adhesion kinase was not concentrated at the neuromuscular junction in muscle cells either in vivo or in vitro. However, it was localized at spontaneously formed acetylcholine receptor clusters in cultured Xenopus myotomal muscle cells, although its dis- tribution was not exactly congruent with that of the receptors. In these cells, the accumulation focal adhesion kinase was induced by polystyrene microbeads. In addition, beads also induce the formation of acetylcholine receptor clusters and myotendinous junction-like special- izations. By following the appearance of the focal adhesion kinase relative to the formation of these sarcolemmal spe- cializations at bead-muscle contacts in cultured muscle cells, we conclude that the accumulation of this protein was in pace with the development of the myotendinous junction, but occurred well after the clustering of acetylcholine receptors. These results suggest that focal adhesion kinase may be involved in the development and/or maintenance of the myotendinous junction through an integrin-based signaling system. Although it can accumulate at acetyl- choline receptor clusters formed in culture, it does not appear to be involved in the development of the neuro- muscular junction.
A single p34cdc2 protein kinase (encoded by nimXcdc2) is required at G1 and G2 in Aspergillus nidulans
Authors: Osmani Aysha H. , Peij Noel van , Mischke Michelle , O’Connell Matthew J. , Osmani Stephen A.
Published: April 2021

We have cloned and sequenced a homolog of cdc2 from Aspergillus nidulans that can complement the Schizosac-charomyces pombe cdc2-33 mutation. The gene was deleted and is required for continued nuclear DNA replication but not for mitochondrial DNA replication. Three different temperature-sensitive alleles were generated by reverse genetics. All of the mutations generate the nim phenotype of A. nidulans. The new gene was designated nimXcdc2 as it is not allelic to any of the other nim genes (nimA to nimW) of A. nidulans. Reciprocal shift experiments place an essential function for nimXcdc2 in G1 and G2. Antipeptide antibodies were generated that detect NIMXcdc2, and antisera were also generated to detect NIMEcyclinB. The two p34cdc2 protein species previously detected in A. nidulans, p34 and p37, both precipitate using NIMXcdc2 C-terminus-specific antibodies but only p34 co-precipitates with NIMEcyclinB. Dephosphorylation of denatured p34 converts it to the p37 form, showing p37 to be the non-phosphory-lated form of NIMXcdc2. The phosphorylation of p34 is therefore associated with its interaction with NIMEcyclinB.
Accumulation of delta 2-tubulin, a major tubulin variant that cannot be tyrosinated, in neuronal tissues and in stable microtubule assemblies
Authors: Paturle-Lafanechère Laurence , Manier Monique , Trigault Nathalie , Pirollet Fabienne , Mazarguil Honoré , Job Didier
Published: April 2021

Tubulin is the major protein component of brain tissue. It normally undergoes a cycle of tyrosination-detyrosination on the carboxy terminus of its α-subunit and this results in subpopulations of tyrosinated tubulin and detyrosinated tubulin. Brain tubulin preparations also contain a third major tubulin subpopulation, composed of a non-tyrosinatable variant of tubulin that lacks a carboxy-terminal glutamyl-tyrosine group on its α-subunit (delta 2-tubulin). Here, the abundance of delta 2-tubulin in brain tissues, its distribution in developing rat cerebellum and in a variety of cell types have been examined and compared with that of total α-tubulin and of tyrosinated and detyrosinated tubulin. Delta 2-tubulin accounts for approximatively 35% of brain tubulin. In rat cerebellum, delta 2-tubulin appears early during neuronal differentiation and is detected only in neuronal cells. This apparent neuronal specificity of delta 2-tubulin is confirmed by examination of its distribution in cerebellar cells in primary cultures. In such cultures, neuronal cells are brightly stained with anti-delta 2-tubulin antibody while glial cells are not. Delta 2-tubulin is apparently present in neuronal growth cones. As delta 2-tubulin, detyrosinated tubulin is enriched in neuronal cells, but in contrast with delta 2-tubulin, detyrosinated tubulin is not detectable in Purkinje cells and is apparently excluded from neuronal growth cones.

In a variety of cell types such as cultured fibroblasts or primary culture of bovine adrenal cortical cells, delta 2-tubulin is confined to very stable structures such as centrosomes and primary cilia. Treatment of such cells with high doses of taxol leads to the appearence of delta 2-tubulin in microtubule bundles. Delta 2-tubulin also occurs in the paracrystalline bundles of protofilamentous tubulin formed after vinblastine treatment. Delta 2-tubulin is present in sea urchin sperm flagella and it appears in sea urchin embryo cilia during development.

Thus, delta 2-tubulin is apparently a marker of very long-lived microtubules. It might represent the final stage of α-tubulin maturation in long-lived polymers.
Primary sequence of paxillin contains putative SH2 and SH3 domain binding motifs and multiple LIM domains: identification of a vinculin and pp125Fak- binding region
Authors: Turner* Christopher E. , Miller John T.
Published: April 2021

Paxillin is a cytoskeletal protein involved in actin- membrane attachment at sites of cell adhesion to the extra- cellular matrix. Extensive tyrosine phosphorylation of this protein occurs during integrin-mediated cell adhesion, embryonic development, fibroblast transformation and following stimulation of cells by mitogens that operate through the family of seven membrane-spanning G- protein-coupled receptors. Paxillin binds in vitro to the focal adhesion protein vinculin as well as to the SH3 domain of c-src and, when tyrosine phosphorylated, to the SH2 domain of v-crk. Here, we report the complementary DNA, and derived amino acid sequence, that codes for approximately 90% of the paxillin protein. We have iden- tified a region in the amino-terminal half of the protein that supports the binding of both vinculin and the focal adhesion tyrosine kinase, pp125Fak. Although there is no significant overall homology with other identified proteins, the carboxyl third of paxillin contains one LIM domain and three LIM-like sequences. The LIM motif is common to a number of transcription factors and to two other focal adhesion proteins, zyxin and cysteine-rich protein. In addition to several potential tyrosine phosphorylation sites there are five tyrosine-containing sequences that conform to SH2-binding motifs. The protein also contains a short proline-rich region indicative of a SH3-binding domain. Taken together, these data suggest that paxillin is a unique cytoskeletal protein capable of interaction with a variety of intracellular signaling, and structural, molecules important in growth control and the regulation of cytoskeletal organization. The homology with zyxin and cysteine-rich protein defines a novel family of LIM-con- taining molecules associated with sites of actin-membrane attachment.
Immunolocalization of tropomodulin, tropomyosin and actin in spread human erythrocyte skeletons
Authors: Ursitti Jeanine A. , Fowler Velia M.
Published: April 2021

The human erythrocyte membrane skeleton consists of a network of short actin filaments cross-linked into a hexagonal network by long, flexible spectrin molecules. The lengths of the short actin filaments (33±5 nm) at the central junctions are proposed to be stabilized and limited by asso- ciation with tropomyosin and the tropomyosin-binding protein, tropomodulin. Here, we use immunogold labelling followed by negative staining to specifically localize tropo- modulin, tropomyosin and actin to the sites of the central junctions in spread membrane skeletons. In addition to negative staining, immunogold labelling for tropomodulin at the sites of the central junctions was also visualized by a quick-freeze, deep-etch, rotary-replication technique. These experiments confirm previous indirect evidence that the short filaments at the central junctions are indeed actin filaments and provide the first direct evidence that tropo- modulin and tropomyosin are associated with the erythro- cyte actin filaments in situ.
Probing the action of Clostridium difficile toxin B in Xenopus laevis oocytes
Authors: Just Ingo , Richter Hans-Peter , Prepens Ulrike , Eichel-Streiber Christoph von , Aktories Klaus
Published: April 2021

Clostridium difficile toxin B and Clostridium botulinum C3 exoenzyme caused comparable morphological alteration of CHO cells, which was accompanied by disaggregation of the microfilamental cytoskeleton. The cytotoxic effect of toxin B was correlated with a decrease in C3-catalyzed ADP-ribosylation of the low-molecular-mass GTP-binding protein Rho, which is involved in the regulation of the actin cytoskeleton. We used Xenopus laevis oocytes as a model to study the toxin effect on Rho in more detail. Toxin B treatment of oocytes caused a decrease in subsequent ADP-ribosylation of cytoplasmic Rho by C3. This decrease was observed when toxin B was applied externally or after microinjection. Besides endogenous Rho, microinjected recombinant Rho-glutathione S-transferase fusion protein was affected. Impaired ADP-ribosylation of Rho was neither due to altered guanine nucleotide binding nor to complexation with the guanine nucleotide dissociation inhibitor, which is known to inactivate Rho and to prevent Rho modification by C3. Proteolytical degradation of Rho was excluded by immunoblot analysis. In intact oocytes toxin B caused neither ADP-ribosylation nor phosphorylation of Rho. The data indicate that C. difficile toxin B acts on Rho proteins in Xenopus oocytes to inhibit ADP-ribosylation by C3. It is suggested that toxin B mediates its cytotoxic effect via functional inactivation of Rho.
Evidence that the 16 kDa proteolipid (subunit c) of the vacuolar H+-ATPase and ductin from gap junctions are the same polypeptide in Drosophila and Manduca: molecular cloning of the Vha16k gene from Drosophila
Authors: Finbow M. E. , Goodwin S. F. , Meagher L. , Lane N. J. , Keen J. , Findlay J. B. C. , Kaiser K.
Published: April 2021

The 16 kDa proteolipid (subunit c) of the eukaryotic vacuolar H+-ATPase (V-ATPase) is closely related to the ductin polypeptide that forms the connexon channel of gap junctions in the crustacean Nephrops norvegicus. Here we show that the major protein component of Manduca sexta gap junction preparations is a 16 kDa polypeptide whose N-terminal sequence is homologous to ductin and is identical to the deduced sequence of a previously cloned cDNA from Manduca (Dow et al., Gene, 122, 355-360, 1992). We also show that a Drosophila melanogaster cDNA, highly homologous to the Manduca cDNA, can rescue Saccharomyces cerevisiae, defective in V-ATPase function, in which the corresponding yeast gene, VMA3, has been inactivated. Evidence is presented for a single genetic locus (Vha16) in Drosophila, which in adults at least contains a single transcriptional unit. Taken together, the data suggest that in Drosophila and Manduca, the same polypeptide is both the proteolipid subunit c component of the V-ATPase and the ductin component of gap junctions. The intron/exon structure of the Drosophila Vha16 is identical to that of a human Vha16 gene, and is consistent with an ancient duplication of an 8 kDa domain. A pilot study for gene inactivation shows that transposable P-elements can be easily inserted into the Drosophila ductin Vha16 gene. Although without phenotypic consequences, these can serve as a starting point for generation of null alleles.
Molecular characterization of two functional domains of CLIP-170 in vivo
Authors: Pierre Philippe , Pepperkok Rainer , Kreis Thomas E.
Published: April 2021

CLIP-170 is a microtubule-binding protein isolated from HeLa cells that is involved in the interaction of endosomes with microtubules. The basic N-terminal domain of CLIP-170 binds to microtubules in vitro. To characterize further the functional domains of this cytoplasmic linker protein, we have transiently expressed intact and mutant forms of CLIP-170 in mammalian cells (HeLa and Vero cells) and show that the tandem repeat present in the N-terminal domain is essential for its binding to microtubules in vivo as previously found in vitro. With increasing levels of expression of CLIP-170, the sites with which the peripheral ends of microtubules interact enlarge, eventually forming large patches, which finally lead to the apparent bundling of microtubules. These patches do not form when the C-terminal domain is absent from the transfected protein. Modification of the microtubule-binding region, particularly of the tandem repeat motif, modulates the binding of CLIP-170 to microtubules. Overexpressed CLIP-170 appears neither to interact with nor to influence the organization of the intermediate filaments, and collapsing the network of intermediate filaments with microinjected anti-bodies against vimentin has no effect on the distribution of CLIP-170. These data suggest that CLIP-170 has at least two functional domains in vivo, an N-terminal micro-tubule-binding domain, and a C-terminal domain that is involved in the anchoring of microtubules to peripheral cytoplasmic structures.
Lineage-specific regulation of cell cycle gene expression in differentiating myeloid cells
Authors: Bürger Christiane , Wick Maresa , Müller Rolf
Published: April 2021

We have analysed the expression of 7 cyclin and cyclin-associated kinase (cdk) genes in the human myeloid cell line HL-60 at different stages of the cell cycle in non-synchronised cells and during terminal differentiation. A clear cell cycle-dependent expression was found with cyclins A (S+G2), B (G2) and E (late G1 and S), while other cell cycle genes showed only very weak (cdk2) or no periodic expression (cyclin D1, cyclin D2 and cdk4). Induction of macrophage-like differentiation by TPA or granulocytic differentiation by retinoic acid or DMSO was accompanied by a block in G1 and resulted in distinct patterns of gene expression that were lineage- and inducer-specific. These included: (i) a dramatic decrease in the expression of cyclin A, cyclin B and cdk2, and surprisingly an up-regulation of cyclin D1 in TPA-induced macrophage-like cells; (ii) a down-regulation of cyclin E in retinoic acid-induced granulocytic cells; and (iii) a decreased abundance of cyclin D1 and D2, but high levels of cyclin A, B and E RNA in DMSO-induced granulocytic cells. These observations suggest that the mechanisms leading to a differentiation-associated cell cycle arrest are lineage-specific, and that the sustained expression of cyclin and cdk genes does not interfere with the induction of terminal differentiation.
Mutations in the Caenorhabditis elegans p-tubulin gene mec-7: effects on microtubule assembly and stability and on tubulin autoregulation
Authors: Savage Cathy , Xue Yingzi , Mitani Shohei , Hall David , Zakhary Randa , Chalfie Martin
Published: April 2021

We have sequenced 45 mutations in mec-7, a β-tubulin gene required for the production of 15-protofilament microtubules in the nematode Caenorhabditis elegans, and have correlated sequence alterations with mutant phenotypes. The expression patterns of most alleles have also been determined by in situ hybridization and immunocytochemistry. Most (12/16) complete loss-of-function alleles, which are recessive, result from nonsense mutations, insertions, or deletions; three others disrupt a putative GTP-binding domain. Three of the four loss-of-function, missense mutations result in elevated mec-7 message levels, suggesting a defect in tubulin autoregulation that may be attributable to a loss in the ability to form heterodimers.

Most (8/9) mild alleles are caused by missense mutations. Two mild alleles appear to increase microtubule stability and lead to the elaboration of ectopic neuronal processes in mec-7-expressing cells. Most (15/23) mutations that cause severe dominant or semidominant phenotypes are clustered into three discrete domains; four others occur in putative GTP-binding regions. Many of these dominant mutations appear to completely disrupt microtubule assembly.
Quantification of low density lipoprotein and transferrin endocytic sorting in HEp2 cells using confocal microscopy
Authors: Ghosh Richik N. , Gelman Dana L. , Maxfield Frederick R.
Published: April 2021

Numerous experiments on CHO cells have shown that endosomes are composed of separate vesicular and tubulo-vesicular compartments, such as the sorting endosome, the recycling compartment, and the late endosome. However, Hopkins et al. (Nature 346, 335–339, 1990) have reported that endosomes in HEp2 human carcinoma cells form an extensive tubular reticulum. To resolve their observations with previous results from CHO and other cells, we examined the sorting and intracellular transport of endo-cytosed macromolecules in HEp2 cells, using low density lipoprotein (LDL) and transferrin (Tf) to probe the lyso-somally directed and recycling pathways, respectively. Fluorescent LDL and Tf were observed with laser scanning confocal microscopy to visualize simultaneously both probes’ sorting and subsequent post-sorting behavior in HEp2 cells. Quantifying the 3-dimensional cellular distributions of fluorescent LDL and Tf, after a variety of pulsechase schemes, gave the ligands’ trafficking rates.

Initially, both ligands appear in the same punctate sorting endosomes, and fingers of Tf start extending from these sorting endosomes. Tf rapidly leaves dual-labeled sorting endosomes ( 5 minutes) and enters a postsorting recycling compartment from which it is recycled out of the cell ( minutes). We present both morphological and kinetic data supporting the existence of these two separate compartments along the recycling pathway in HEp2 cells. LDL remains in punctate sorting endosomes that eventually lose the ability to receive newly endocytosed LDL, and mature into late endosomes. The trafficking and sorting of Tf and LDL in HEp2 cells follow the same general scheme as in CHO cells, indicating that the tubular endosomes previously seen may be the tubular parts of the sorting endosomes and recycling compartments in these cells. We propose that the endosomes in the recycling pathway of HEp2 cells, as in CHO cells, are composed of short-lived sorting endosomes, accessible to both Tf and LDL, and long-lived post-sorting recycling compartments, which contain Tf and recycling receptors but not LDL.
Cell kinetic characterization of growth arrest in cultured human keratinocytes
Authors: Ruissen Fred van , Erp Piet E. J. van , Jongh Gijs J. de , Boezeman Jan B. M. , van de Kerkhof Peter C. M. , Schalkwijk Joost
Published: April 2021

In this study we have performed a cell kinetic characterization of growth and growth arrest of keratinocytes derived from normal human skin. Proliferative activity of the cell cultures was analysed with a flow cytometric technique, measuring relative DNA content and iododeoxyuridine (IdUrd) incorporation simultaneously. Normal human keratinocytes were grown in keratinocyte growth medium (KGM) and growth arrest was induced by using either keratinocyte basal medium (KBM) or KGM supplemented with TGF-β1. It was found that human keratinocytes grown in KGM plus TGF-β1 were growth-arrested within 52 hours. The rate of IdUrd incorporation into DNA decreased by more than 95% after 52 hours and parallelled the decrease of cells in S-phase. Within 52 hours after addition of TGF-β1, 79% of the growth-arrested cells were in the G0/G1-phase of the cell cycle, a situation that approaches that of the normal epidermis. Growth arrest of human keratinocytes in KBM showed a similar decrease in the rate of IdUrd incorporation. However, the decrease in IdUrd incorporation was not reflected in a decrease in cells in S-phase, suggesting that the cells were blocked in G0/G1, S or G2/M-phase rather than selectively in the physiological growth arrest state of G0/G1. Secondly, we investigated the kinetics of the cells when they were restimulated after growth arrest. We found that after termination of the growth arrest in KGM supplemented with TGF-β1 the cells require 6 to 8 hours to initiate DNA synthesis, with a continued decrease in the G0/G1 population, suggesting that the cells are recruited as a cohort. After growth arrest induced by KBM, cells also require 6 to 8 hours in KGM to initiate DNA synthesis, but the cells are not recruited as a cohort. We conclude that growth arrest induced by TGF-β1 is the preferred system in which to study induction of keratinocyte proliferation, since it induces a state of quiescence that approaches that of normal human epidermis.
Tenascin-R (J1 160/180) inhibits fibronectin-mediated cell adhesion-functional relatedness to tenascin-C
Authors: Pesheva Penka , Probstmeier Rainer , Skubitz Amy P. N. , McCarthy James B. , Furcht Leo T. , Schachner Melitta
Published: April 2021

Cell adhesion and neurite outgrowth on fibronectin is a multistep process modulated by different extra- and intra-cellular signals. Fibronectin-mediated cell attachment and spreading can be affected in a negative way by tenascin-C, an extracellular matrix glycoprotein expressed in a tempo-rally and spacially restricted manner during early mor-phogenesis. Tenascin-R (J1-160/180), consisting of two major isoforms of 160 kDa (tenascin-R 160) and 180 kDa (tenascin-R 180) in mammals, is an extracellular matrix glycoprotein of the central nervous system that shares high structural homologies with tenascin-C. Here we show that in relation to fibronectin-mediated adhesion, the two extra-cellular matrix molecules are also functionally closely related. When offered as mixed substrata with other extra-cellular matrix molecules, the two tenascin-R isoforms and tenascin-C derived from mouse brain selectively inhibit fibronectin-dependent cell adhesion and neurite outgrowth, and affect cell morphology of different mesenchymal and neural cells. This effect is partially due to interactions at the substrate level that result in a steric hindrance and/or conformational change of the cell binding sites of the fibronectin molecule. In addition, tenascin-R 180 and tenascin-C interact with cells by an RGD- and β1 integrin-independent mechanism, leading to cell rounding and detachment from such substrata. The expression of tenascin-R and tenascin-C in the nervous system at times and locations where fibronectin-mediated cellular processes take place may be related to the role of inhibitory signals in the extracellular matrix in the regulation of cell migration and differentiation in general.
Fast myosin heavy chains expressed in secondary mammalian muscle fibers at the time of their inception
Authors: Cho Mildred , Hughes Simon M. , Karsch-Mizrachi Ilene , Travis Marilyn , Leinwand Leslie A. , Blau Helen M.
Published: April 2021

Mammalian skeletal muscle is generated by two waves of fiber formation, resulting in primary and secondary fibers. These fibers mature to give rise to several classes of adult muscle fibers with distinct contractile properties. Here we describe fast myosin heavy chain (MyHC) isoforms that are expressed in nascent secondary, but not primary, fibers in the early development of rat and human muscle. These fast MyHCs are distinct from previously described embryonic and neonatal fast MyHCs. To identify these MyHCs, mon-oclonal antibodies were used whose specificity was determined in western blots of MyHCs on denaturing gels and reactivity with muscle tissue at various stages of development. To facilitate a comparison of our results with those of others obtained using different antibodies or species, we have identified cDNAs that encode the epitopes recognized by our antibodies wherever possible. The results suggest that epitopes characteristic of adult fast MyHCs are expressed very early in muscle fiber development and distinguish newly formed secondary fibers from primary fibers. This marker of secondary fibers, which is detectable at the time of their inception, should prove useful in future studies of the derivation of primary and secondary fibers in mammalian muscle development.
Association of the Dictyostelium 30 kDa actin bundling protein with contact regions
Authors: Fechheimer Marcus , Ingalls Hilary M. , Furukawa Ruth , Luna Elizabeth J.
Published: April 2021

‘Contact regions’ are plasma membrane domains derived from areas of intercellular contact between aggregating Dictyostelium amebae (H. M. Ingalls et al. (1986). Proc. Nat. Acad. Sci. USA 83, 4779). Purified contact regions contain a prominent actin-binding protein with an Mr of 34,000. Immunoblotting with monoclonal antibodies identifies this polypeptide as a 34,000 Mr actin-bundling protein (known as 30 kDa protein), previously shown to be enriched in filopodia (M. Fechheimer (1987). J. Cell Biol. 104, 1539). About four times more 30 kDa protein by mass is associated with contact regions than is found in total plasma membranes isolated from aggregating cells. In agreement with these observations, immunostaining of the 30 kDa protein in aggregating cells reveals a prominent localization along the plasma membrane at sites of intercellular contact. By contrast, alpha-actinin does not appear to be significantly enriched at sites of cell to cell contact. Binding experiments using purified plasma membranes, actin and 30 kDa protein indicate that the 30 kDa protein is associated with the plasma membrane primarily through interactions with actin filaments. Calcium ions are known to decrease the interaction of actin with 30 kDa protein in solution. Surprisingly, membrane-associated complexes of actin and the 30 kDa protein are much less sensitive to dissociation by micromolar levels of free calcium ions than are complexes in solutions lacking membranes. These results suggest that the interaction of the 30 kDa protein with F-actin at regions of cell-cell contact may be less sensitive to disruption by free calcium ions than elsewhere in the cell cortex. The positively cooperative assembly of stable complexes of actin and the 30 kDa protein at contact regions may be an important factor in the organization of both the cortex and these membrane domains that are spe-cialized for intercellular adhesion.
Repression of a malignant cell-substratum adhesion phenotype by inhibiting the production of the anti-adhesive proteoglycan, PG-M/versican
Authors: Yamagata Masahito , Kimata Koji
Published: April 2021

Malignantly transformed cells usually display a rosette-like morphology of substratum adhesions (called podosomes) and disorganized microfilaments, and are often associated with elevated production of chondroitin sulphate. We pre-viously showed that many tissues and cells express alter-natively spliced multiforms of the large chondroitin sulphate proteoglycan termed PG-M (versican is one of the short transcripts). Since PG-M/versican inhibits many types of cell-substratum adhesion and is found to be excluded from focal contacts of cultured fibroblasts, it is likely that this proteoglycan is generally involved in regulating cell-substratum adhesion. We report here that PG-M/versican is selectively excluded from podosomes of human osteosarcoma cells and that specific inhibition of its biosynthesis by an antisense method suppresses such a malignant cell-adhesive phenotype. The results support the idea that PG-M/versican acts as an anti-adhesive molecule and raise the possibility that PG-M/versican controls one type of cancer cell behaviour.
Drosophila mutants in the 55 kDa regulatory subunit of protein phosphatase 2A show strongly reduced ability to dephosphorylate substrates of p34cdc2
Authors: Mayer-Jaekel Regina E. , Ohkura Hiroyuki , Ferrigno Paul , Andjelkovic Natasa , Shiomi Kensuke , Uemura Tadashi , Glover David M. , Hemmings Brian A.
Published: April 2021

The 55 kDa regulatory subunit of Drosophila protein phos-phatase 2A is located in the cytoplasm at all cell cycle stages, by the criterion of immunofluorescence. We are unable to detect significant change in protein phosphatase activity during the nuclear division cycle of syncytial embryos. However, cell cycle function of the enzyme is suggested by the mitotic defects exhibited by two Drosophila mutants, aar1 and twinsP, defective in the gene encoding the 55 kDa subunit. The reduced levels of the 55 kDa subunit correlate with the loss of protein phosphatase 2A-like, okadaic acid-sensitive phosphatase activity of brain extracts against caldesmon and histone H1 phospho-rylated by p34cdc2/cyclin B kinase, but not against phos-phorylase a. Thus the mitotic defects of aar1 and twinsP are likely to result from the lack of dephosphorylation of specific substrates by protein phosphatase 2A.
Inhibition of 3T3-L1 adipose differentiation by 2,3,7,8-tetrachlorodibenzo-p-dioxin
Authors: Phillips Marjorie , Enan Essam , Liu Phillip C. C. , Matsumura Fumio
Published: April 2021

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-induced toxicity is particularly striking in adipose tissue, where it causes severe wasting. This phenomenon suggests that TCDD could have effects on adipocyte differentiation, now demonstrated using 3T3-L1 cells as a model system. When cells were treated with 10 nM TCDD before differentiation or during the first two days of induction in the presence of dexamethasone (dex) and isobutylmethylxanthine (IBMX), a reduction occurred in the number of fat cell colonies measured 7-10 days later by Oil Red O staining. Northern blotting showed an accompanying reduction in amounts of mRNA encoding several adipocyte markers. In contrast, when TCDD was added after differentiation, it had no effect on the maintenance of the adipose phenotype. Dose-response and structure-activity relationships were consistent with a process mediated by interaction of TCDD with the Ah receptor. The possibility that TCDD acts by inhibiting the signaling pathways activated by dex and IBMX was investigated. TCDD did not interfere with glucocorticoid-inducible transcription as judged by the unimpaired responsiveness of a transfected reporter construct. Treatment of cells with TCDD augmented the increase in protein kinase A (PKA) activity elicited by either IBMX or forskolin; therefore, if TCDD disrupts the cAMP signaling pathway, it must do so at a step after activation of PKA.
Down regulation of talin alters cell adhesion and the processing of the α5β1 integrin
Authors: Albigès-Rizo Corinne , Frachet Philippe , Block Marc R.
Published: April 2021

The role of talin was addressed by down regulating its expression using an antisense RNA strategy. HeLa cells were transfected with a talin 5′ cDNA fragment under the control of the inducible human metallothionein promotor. Isolated clones displayed a decrease in talin level down to 10% of control. The reduction in talin expression dramatically slowed down the kinetics of cell spreading. Mocktransfected cells, spread out onto fibronectin, exhibited large peripheral adhesion plaques. In contrast, cells with reduced talin expression showed smaller focal contacts localized all over the ventral face, and displayed a marked decrease in the number of stress fibers. Immunoprecipitation experiments carried out with a polyclonal antibody on surface-labeled receptor indicated a shift in the mobility for both α5 and β1 subunits. Surprisingly, β1 integrin chains could not be detected by indirect immunofluorescence using monoclonal antibodies in talin deficient clones. Western blot analysis indicated the presence of two forms of β1. We analyzed the processing of β1 in normal and talin deficient cells using pulse chase experiments. Normal cells required a minimum of 5 hours for the processing of mature β1, while the talin deficient AT22 clone showed that the β1 precursor was slowly converted into a very low molecular mass product. Our data demonstrate that talin plays a central role in the establishment of cell-matrix contacts. In addition, down regulation of talin impairs the folding and processing of β1 integrins.
Identification and cDNA cloning of a Xenopus nucleolar phosphoprotein, xNopp180, that is the homolog of the rat nucleolar protein Nopp140
Authors: Cairns Carol , McStay Brian
Published: April 2021

The monoclonal antibody G1C7, recognises both Xenopus nucleolin and a protein of 180 kDa present in Xenopus oocyte nucleoli. This antibody was used to obtain a cDNA clone encoding the 180 kDa protein now called xNopp180 (Xenopus nucleolar phosphoprotein of 180 kDa). Analysis of the deduced amino acid sequence from this cDNA shows that xNopp180 is almost entirely composed of alternating acidic and basic domains. We show that xNopp180 is heavily phosphorylated and that it contains multiple consensus sites for phosphorylation by casein kinase II and cdc2 kinase. In addition we show that xNopp180 is the 180 kDa antigen recognised by the monoclonal antibody No-114, thus allowing reinterpretation of previous work with this antibody. xNopp180 appears to be the Xenopus homolog of the rat nucleolar protein Nopp140. Nopp140 is a nuclear localisation signal binding protein that shuttles on curvilinear tracks between the nucleolus and the cytoplasm. Possible roles for xNopp180/Nopp140 in ribosome biogenesis are discussed.
Integrin and spectrin homologues, and cytoplasm-wall adhesion in tip growth
Authors: Kaminskyj Susan G. W. , Heath I. Brent
Published: April 2021

Saprolegnia ferax contains an integrin homologue, identified by crossreactivity with antiserum to the consensus sequence of human/chick/Xenopus cytoplasmic domain β1-integrin, which is highly conserved. In non-reduced samples, this integrin was larger than the reported size range for β1-integrins, at 178 kDa. In reduced samples, there was a reducing agent-concentration-dependent conversion from 178 kDa to 120 kDa, well within the reported size range for β1-integrins in other organisms. The integrin antiserum stained plasma membrane-associated patches, which had a shallow tip-high gradient. This population was reduced and its distribution perturbed in hyphae whose growth rate was reduced by half with tetrapentyl ammonium chloride. The expected integrin function in cytoplasm-cell wall attachment was shown by differential resistance to plasmolysis-induced separation, which positively correlated with integrin abundance. However, when there was separation, remnants of cytoplasm stayed attached to the wall. These were enriched in actin and integrin. Saprolegnia also has a spectrin homologue identified by crossreactivity with an erythrocyte spectin antibody, which has a size (246 kDa) similar to other organisms. This spectrin had a superficially similar distribution to that of integrin, but it did not participate in cytoplasm-wall anchoring. These data suggest that Saprolegnia hyphae have a plasma membrane which is strengthened by spectrin, and cytoplasm which is attached to the cell wall by integrin.
Transmembrane domain of CD44 is required for its detergent insolubility in fibroblasts
Authors: Perschl Astrid , Lesley Jayne , English Nicole , Hyman Robert , Trowbridge Ian S.
Published: April 2021

The hyaluronan receptor CD44 is an abundant glycopro-tein expressed on a variety of different cell types. In fibrob-lasts a significant portion of receptor molecules remain in the detergent-insoluble fraction after Triton X-100 extrac-tion. Detergent insolubility of these CD44 molecules has been interpreted to reflect their association with the cytoskeleton. In this study we examined the structural features of CD44 required for its Triton X-100 insolubility in murine fibroblasts. We expressed in L cells the wild-type hematopoietic form of CD44, a mutant CD44 lacking the cytoplasmic domain, and two mutant CD44 molecules with substituted transmembrane domains. Immunofluorescence and cell surface iodination were performed and the detergent extraction profile of the transfected CD44 molecules was determined. No difference in detergent sol-ubility was observed between wild-type and tailless mutant-transfected molecules. However, both CD44 mutants with a heterologous transmembrane domain, derived from either the CD3 ζ chain or CD45, were completely soluble in Triton X-100. These results demonstrate that the transmembrane region but not the cytoplasmic domain of CD44 is required for the detergent-insolubility in these cells. No obvious colocalization of CD44 and actin stress fibers was observed before or after treatment with cytochalasin D, and no change in the detergent extraction profile of wild-type and mutant CD44 molecules was effected by cytochalasin D. In equilibrium density sucrose gradients the Triton-insoluble CD44 component was found in the low density fractions, indicating an association with Triton X-100-insoluble lipids. Together, these results suggest that Triton X-100 insolubility of a fraction of the CD44 molecules in fibroblasts is not the result of interac-tion with the actin-based cytoskeleton but rather due to an association with Triton-insoluble lipids, which is, in turn, dependent on the transmembrane domain of CD44.
Molecular genetic analysis of myoc, a Dictyostelium myosin I
Authors: Peterson Michelle D. , Novak Kristine D. , Reedy Mary C. , Ruman Jane I. , Titus Margaret A.
Published: April 2021

The protozoan myosin Is are widely expressed actin-based motors, yet their in vivo roles remain poorly understood. Molecular genetic studies have been carried out to determine their in vivo function in the simple eukaryote Dictyostelium, an organsim that contains a family of four myosin Is. Here we report the characterization of myoC, a gene that encodes a fifth member of this family. Analysis of the deduced amino acid sequence reveals that the myoC gene encodes a myosin that is homologous to the well-described Acanthamoeba myosin Is as well as to Dic-tyostelium myoB and-D. The expression pattern of the myoC mRNA is similar to that of myoB and myoD, with a peak of expression at times of maximal cell migration, around 6 hours development. Deletion of the myoB gene has been previously shown to result in mutant cells that are defective in pseudopod extension and phagocytosis. However, no obvious differences in cell growth, develop-ment, phagocytosis or motility were detected in cells in which the myoC gene had been disrupted by homologous recombination. F-actin localization and ultrastructural organization also appeared unperturbed in myoC− cells. This apparent ‘lack’ of phenotype in a myosin I single knockout cannot be simply explained by redundancy of function. Our results rather suggest that the present means of assessing myosin I function in vivo are insufficient to identify the unique roles of these actin-based motors.
Change in intracellular K+ concentration caused by external osmolality change regulates sperm motility of marine and freshwater teleosts
Authors: Takai Hiroyuki , Morisawa Masaaki
Published: April 2021

We previously demonstrated that osmolality isotonic to the seminal plasma suppresses sperm motility in marine and freshwater teleosts, and exposure of sperm to hypertonicity of sea water or hypotonicity of fresh water, respectively, induces the initiation of sperm motility at spawning. The motile sperm became immotile by return of osmolality to the isotonic osmolality both in a marine teleost, the puffer fish, and a freshwater teleost, the zebrafish. The initiation and termination of sperm motility could be repeated several times by changing surrounding osmolality in both species. In demembranated sperm, motility was suppressed by a K+ concentration equivalent to the seminal salt concentration in both puffer and zebrafish. Demembranated puffer sperm were reactivated when K+ concentration of the reactivating solution increased. Conversely, initiation of motility in the demembranated zebrafish sperm was induced by decreasing K+ concentration. The initiation and termination of the demembranated sperm were alternately repeated by changing K+ concentration of the reactivation solution in both species. Furthermore, intracellular K+ concentration rose when sperm motility of the puffer was initiated in hypertonic solutions. These results suggest that change in external osmolality is converted into change in intracellular K+ concentration, and that the change affects the fiagellar axoneme as a signal to initiate or terminate sperm motility. The initiation and termination of motility in the demembranated puffer sperm were caused at a high pH and a low pH of the reactivating solution, respectively, suggesting the contribution of intracellular pH in the regulation of fiagellar motility.
Increased axon regeneration in astrocytes grown in the presence of proteoglycan synthesis inhibitors
Authors: Smith-Thomas Linda C. , Stevens James , Fok-Seang Juin , Faissner Andreas , Rogers John H. , Fawcett James W.
Published: April 2021

We have recently reported that the critical difference between astrocytic cell lines that are good or poor promoters of axon growth lies in the extracellular matrix. We demonstrated that much of this difference between matrix produced by permissive and non-permissive cell lines could be ascribed to one or more dermatan/keratan sulphate proteoglycans and that these proteoglycans are able to block the neurite-promoting effect of laminin. These proteoglycans are also produced by cultures of primary astrocytes. In the present study, we have demonstrated that treatment of both astrocytic cell lines and primary astro-cytes with inhibitors of proteoglycan synthesis, β-D-xylosides and sodium chlorate, can strongly influence the axon growth promoting properties of both matrix and whole cells. Dorsal root ganglia grown on matrix or in con-ditioned medium from cultures treated with β-D-xylosides or sodium chlorate had twice as many axons and the axons grew to twice the length as in control cultures. Following treatment of Neu7 cells with proteoglycan synthesis inhibitors there was also a significant reduction in the ability of Neu7 conditioned medium to block the neurite-promoting effect of laminin. Dorsal root ganglia grown on Neu7 cells treated with sodium chlorate extended 2 to 3 times the number of axons for approximately 300 mm longer distance than on control cultures. Treatment of Neu7 cells with β-D-xylosides, however, did not make the cells less inhibitory to axon growth. We have also examined the effects of proteoglycan synthesis inhibitors on three-dimensional primary astrocyte cultures, which closely mimic the in vivo effects of astrocytes on axon growth. Here again, dorsal root ganglia extended many more axons for a longer distance than in control cultures. We conclude from these results that interfering with astrocyte proteo-glycan synthesis makes them better able to support axon regeneration.
The topogenic fate of the polytopic transmembrane proteins, synaptophysin and connexin, is determined by their membrane-spanning domains
Authors: Leube Rudolf E.
Published: April 2021

The synaptophysins and connexins are polytopic trans-membrane proteins of similar secondary structure that accumulate as multiple homo-oligomers in specialized membrane regions, the presynaptic transmitter vesicles or gap junctions. Transfection and expression of the respective genes in cultured epithelial cells results in the de novo formation of either small cytoplasmic, synaptophysin-rich vesicles, or functional gap junctions consisting of clustered connexin molecules. To examine the molecular requirements for the specific enrichment and topogenesis of both types of molecule, chimeric cDNAs were constructed composed of different parts of the rat synaptophysin and rat liver connexin32 genes. Expression of the encoded chimeric polypeptides in hepatocellular carcinoma-derived cells showed that only chimeras with all four transmem-brane domains from either parent molecule were delivered to their specific destination. In contrast, chimeras with transmembrane domains from both connexin32 and synap-tophysin were always retained in the endoplasmic reticulum. The topogenic nature of the transmembrane domains was further demonstrated by deletion mutagen-esis, indicating that removal of cytoplasmic end domains or intravesicular loops does not abolish targeting. On the other hand, excision of individual transmembrane domains or introduction of point mutations in transmembrane segments resulted in retention in the endoplasmic reticulum.
Role of the carboxyl-terminal Fib2 domain in fibronectin matrix assembly
Authors: Ichihara-Tanaka Keiko , Titani Koiti , Sekiguchi Kiyotoshi
Published: April 2021

A truncated form of fibronectin consisting of the N-terminal 70 kDa and C-terminal 37 kDa regions, desig-nated r70F2, retained the ability to assemble into the extra-cellular matrix when expressed in cultured fibroblasts (Ichihara-Tanaka et al. (1992)FEBS Lett. 299, 155-158). To elucidate the role of the C-terminal 37 kDa region in fibronectin matrix assembly, we expressed a panel of mutant forms of r70F2 with various deletions and amino acid substitutions in mouse L cells. Although substitution of Ser for two Cys residues in the C-terminal dimer-forming segment led to a marked reduction in the matrix assembly activity of r70F2, the resulting monomeric r70F2 still retained a low, but significant activity to assemble into the matrix. Neither the N-terminal 70 kDa nor the C-terminal 37 kDa regions, when expressed as monomeric forms, exhibited any residual activity, suggesting that the core domain of the 37 kDa region consisting of III15 and I10 through I12 modules, termed Fib2 domain, is actively involved in the matrix assembly of r70F2. In support of the role of Fib2 domain, the proteolytic fragment derived from the 37 kDa region inhibited the assembly of r70F2. Fur-thermore, en bloc deletion of the Fib2 domain or deletion of the I10 through I12 modules from r70F2 resulted in a marked decrease of the matrix assembly activity. Since deletion of any one of the three type I modules led to a much lesser decrease in activity, it seems likely that a cluster of the three type I modules in the Fib2 domain, but not any one in particular, serves as a functional unit for the matrix assembly of r70F2. Further supporting the active role of the Fib2 domain, a recombinant homodimer of the 37 kDa region was found to be incorporated into the deoxy-cholate-insoluble matrix. These results, taken together, indicate that the Fib2 domain per se has an intrinsic ability to assemble into the matrix and is actively involved in the matrix assembly of fibronectin.