bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective modulation of the interaction of α7β1 integrin with fibronectin and laminin by l-14 lectin during skeletal muscle differentiation
Authors: Gu Maojian , Wang Weigwang , Song Woo Keun , Cooper Douglas N. W. , Kaufman Stephen J.
Published: April 2021

The α7β1 integrin was originally identified and isolated from differentiating skeletal muscle and shown to be a laminin-binding protein (Song et al. (1992)J. Cell Biol. 117, 643-657). Expression of the α7 gene and protein are developmentally regulated during skeletal muscle differentiation and have been used to identify cells at distinct stages of the myogenic lineage (George-Weinstein et al. (1993)Dev. Biol. 156, 209-229). The lactoside-binding protein L-14 exists as a dimer and has been localized on a variety of cells, in association with extracellular matrix. During myogenesis in vitro, L-14 is synthesized within replicating myoblasts but it is not secreted until these cells commence terminal differentiation and fusion into multinucleate fibers (Cooper and Barondes, J. Cell Biol. (1990) 110, 1681-1691). Addition of purified L-14 to myogenic cells plated on laminin inhibits myoblast spreading and fusion, suggesting that the L-14 lectin regulates muscle cell interactions with the extracellular matrix that are germane to myogenic development (Cooper et al. (1991)J. Cell Biol. 115, 1437-1448). We demonstrate here, using affinity chromatography and immunoblots, that α7β1 also binds to fibronectin and to the L-14 lectin. L-14 binds to both laminin and to the α7β1 integrin, and it can effectively inhibit the association of laminin and this integrin. Modulation of α7β1 interaction with its ligands by L-14 is selective: L-14 does not bind to fibronectin, nor does it interfere with the binding of fibronectin to α7β1. These results are discussed in the context of the potential roles of α7β1 in its interaction with laminin and fibronectin during myogenesis.
α-melanocyte stimulating hormone and its analogue nle4dphe7α-MSH affect morphology, tyrosinase activity and melanogenesis in cultured human melanocytes
Authors: Hunt Gillian , Todd Carole , Cresswell Janet E. , Thody Anthony J.
Published: April 2021

Although melanocyte stimulating hormone (MSH) peptides are known to stimulate pigmentation in man, previous reports suggest that human melanocytes are relatively unresponsive to these peptides in vitro. This may be related to the conditions under which the melanocytes were cultured. Thus, we have re-investigated the in vitro effects of MSH peptides using human melanocytes cultured in the absence of artificial mitogens. Human melanocytes were incubated with α-MSH or its potent analogue Nle4DPhe7α-MSH for 3 days. After 18 hours, melanocyte morphology had evolved from mainly bipolar to dendritic in approxi-mately 66% of cultures. Nle4DPhe7α-MSH produced dose-related increases in both tyrosinase activity and melanin content although the degree of response was variable and tyrosinase activity was the relatively more responsive to the peptide. Similar results were obtained with α-MSH, but, although the effect on melanin content was similar to that of Nle4DPhe7α-MSH, the effect on tyrosinase activity was less marked. The preliminary EC50 values for the actions of the MSH peptides suggest that they may be equipotent in their actions on human melanocytes. In addition, we have demonstrated that the common melanocyte mitogens 12-O-tetradecanoyl phorbol-13-acetate (TPA) and cholera toxin affect basal melanogenesis and modulate the effects of the MSH peptides. However, not all melanocyte cultures showed melanogenic responses to the MSH peptides. Ability to respond was unrelated to basal levels of tyrosi-nase activity or melanin content. In at least some cultures, morphological and melanogenic responses appear to be independent of one another. For reasons that are unknown, a small number of human melanocyte cultures show neither morphological nor melanogenic responses to the MSH peptides. Neither peptide had any effect on cell numbers, regardless of whether the cultures were able to respond morphologically or melanogenically. The present findings are particularly interesting in light of recent devel-opments in the molecular biology of the MSH receptor.
Up-regulation of the connexin43+ GAP junction network in haemopoietic tissue before the growth of stem cells
Authors: Rosendaal Martin , Green Colin R. , Rahman Asaur , Morgan Deborah
Published: April 2021

The early developmental stages of haemopoiesis are thought to be regulated by paracrine growth factors and by the haemopoietic environment. Are gap junctions involved here? Gap junctions are structures in cell membranes allowing the direct transfer of ions and small molecules between adjacent cells and are known to be involved in development. We have found that although connexin43 gap junctions are rare (0.00016±0.0002/μm2 tissue) in normal adult mouse marrow their expression is 80-fold higher (0.0292±0.0147/μm2) in neonatal marrow. One difference between neonatal and adult haemopoietic tissue is that in the latter more haemopoietic cells are dividing. To test if more gap junctions were due to increased division we altered adult blood-formation by mobilizing or destroying end cells - granulocytes and red cells - or by forcing stem cells to divide by making them regenerate an ablated blood-forming system. Mobilizing end cells had no effect on the number or distribution of gap junctions in marrow but forced stem cell division caused a 100-fold increase in gap junction expression and did so before any recognizable haemopoietic cells formed. There were greater than normal numbers of gap junctions in radio-protected adult mouse marrow. The cells coupled by gap junctions are TE-7+ mesodermally derived fibroblasts, STRO-1+ stromal cells, and CD45+ and CD34+ haemopoi-etic cells. We propose that there is a latent network of C×43+ gap junctions in normal quiescent marrow. In response to events that call for active division of stem cells this network is amplified and coupled to haemopoietic stem cells, perhaps enabling them to divide.
Unorthodox male meiosis in Trichosia pubescens (sciaridae)chromosome elimination involves polar organelle degeneration and monocentric spindles in first and second division
Authors: Fuge Harald
Published: April 2021

Male meiosis in Trichosia pubescens (Sciaridae) was investigated by means of serial section electron microscopy and immunofluorescence light microscopy. From earlier studies of another sciarid fly, Sciara coprophila (Phillips (1967)J. Cell. Biol. 33, 73-92), it is known that the spindle poles in sciarid spermatogonia are characterized by pairs of ‘giant centrioles’, ring-shaped organelles composed of large numbers of singlet microtubules. In the present study sper-matocytes in early prophase of Trichosia were found to possess single giant centrioles at opposite sides of the nucleus. The obvious reduction in centriole number from the spermatogonial to the spermatocyte stage is suggested to be the result of a suppression of daughter centriole formation. In late prophase, a large aster is developed around the centriole at one pole. At the opposite pole no comparable aster is formed. Instead, a number of irregular centriolar components appear in this region, a process that is understood to be a degeneration of the polar organelle. The components of the degenerate pole migrate into a cyto-plasmic protrusion (‘bud’), which later is also utilized for the elimination of paternal chromosomes. The existence of only one functional polar centre is the reason for the formation of a monopolar monocentric spindle in first meiotic division, which in turn is one of the prerequisites for the elimination of paternal chromosomes. While the set of maternal and L chromosomes orientates and probably moves towards the pole, paternal chromosomes seem to be unable to contact the pole, possibly due to an inactivation of their kinetochores. Retrograde (‘away from the pole’) chromosome motion not involving kinetochores is assumed. Eventually, paternal chromosomes move into the pole-distal bud and are eliminated by casting off, together with the components of the degenerate polar organelle. Chro-mosome elimination can be delayed until the second meiotic division. The spindle of the second meiotic division is bipolar and monocentric. One spindle pole is marked by the polar centre of first division. The opposite spindle apex is devoid of a polar centre. It is assumed that spindle bipo-larity in the second division is induced by the amphi-ori-entated chromosomes themselves. The maternal and L chromosome set (except the non-disjunctional X chromo-some, which is found near the polar centre) congress in a metaphase plate, divide and segregate. Of the two daughter nuclei resulting from the second meiotic division, the one containing the X chromatids is retained as the nucleus of the future spermatozoon. The other nucleus becomes again eliminated within a second cytoplasmic bud.
Kinesin and tau bind to distinct sites on microtubules
Authors: Marya Pankaj K. , Syed Zarrin , Fraylich Paul E. , Eagles Peter A. M.
Published: April 2021

We have used a fluorescent derivative of kinesin, AF-kinesin (kinesin conjugated with 5-(iodoacetamido) fluores-cein), to investigate the binding site of kinesin on micro-tubules and to compare this site with that to which tau binds. Microtubules saturated with tau will bind AF-kinesin in the presence of the ATP analogue, 5′-[β,γ-imino]triphosphate (AdoPP[NH]P). This shows that there are distinct binding sites for the two proteins. Further evidence comes from digestion studies where taxol-sta-bilised microtubules were treated with subtilisin, resulting in the cleavage of C-terminal residues from both the α-and β-tubulin subunits. These treated microtubules can no longer bind tau, but are able to bind AF-kinesin in the presence of AdoPP[NH]P. Finally, AF-kinesin will support the gliding of subtilisin-digested microtubules in the presence of ATP at rates comparable to those obtained with non-digested microtubules. These results show directly that the binding site for kinesin is outside the C-terminal region of tubulin that is removed by subtilisin and is distinct from the binding site of tau.
Comparison of normal and tumorigenic endothelial cells: Differences in thrombospondin production and responses to transforming growth factor-beta
Authors: RayChaudhury Amlan , Frazier William A. , D’Amore Patricia A.
Published: April 2021

Cultured endothelial cells constitutively synthesize significant levels of thrombospondin, an extracellular matrix-associated protein with reported anti-angiogenic proper-ties. However, two murine endothelial cell lines, bEND.3 and Py-4-1, which have been immortalized with polyoma T oncogenes and which generate vascular malformations in vivo, produce little or no thrombospondin though bEND.3 (but not Py-4-1) growth is inhibited by the addition of exogenous thrombospondin. In addition, Py-4-1 cells are not growth-inhibited by transforming growth factor-beta, a potent endothelial inhibitor. These results indicate that these two cell lines may be useful tools in understanding the role and mechanism of action of thrombospondin and transforming growth factor-beta in endothelial cell biology. A role for thrombospondin in vascular development is further suggested by the observation of significant differ-ences in the levels of thrombospondin mRNA and protein between capillary and aortic endothelial cells. Transform-ing growth factor-beta-1 treatment of normal endothelial cells increases steady-state levels of thrombospondin mRNA and protein and results in extensive deposition of thrombospondin into the extracellular matrix. In contrast, transforming growth factor-beta-1 has little effect on thrombospondin levels in the tumorigenic endothelial cell lines. In view of our earlier finding that contact between endothelial cells and mural cells generates activated trans-forming growth factor-beta-1, and the fact that throm-bospondin is present in a fibrillar network around vascular structures in vitro, we speculate that modulation of throm-bospondin production and distribution by transforming growth factor-beta may be a physiological process to enjoin stabilization of vessels and cessation of vessel growth.
Gpi membrane anchor is determinant in intracellular accumulation of apical plasma membrane proteins in the non-polarized human colon cancer cell line HT-29 18
Authors: Nicolas F. , Tiveron M.-C. , Davoust J. , Reggio H.
Published: April 2021

We have compared the intracellular localization of plasma membrane proteins anchored either with a transmembrane segment or with a glycosylphosphatidylinositol moiety to estimate the effects of membrane anchor on protein segregation in the non-polarized form of the human colon cancer cell line HT-29 18. We have monitored two endogenous proteins: the carcinoembryonic antigen, a glycosylphos-phatidylinositol protein and the transmembrane protein dipeptidyl peptidase IV, and two transfected proteins: the glycosylphosphatidylinositol protein Thy-1 and an engineered transmembrane form of Thy-1. Using immunocytochemistry on ultra-thin cryosections and confocal microscopy, we detected a carcinoembryonic antigen-rich vesicular compartment, excluding classical pre-lysosomal and lysosomal markers such as mannose 6-phosphate receptor, lamp-1 and cathepsin D. This compartment, where carcinoembryonic antigen accumulated, excluded the transmembrane protein dipeptidyl peptidase IV and was reduced during the polarization of the cells. Moreover, the glycosylphosphatidylinositol form of Thy-1 also accumulated in the carcinoembryonic antigen-rich compartment whereas the transmembrane form of Thy-1 was excluded. We proposed that, in the non-polarized HT-29 18 cells, accumulation of glycosylphosphatidylinositol proteins independently of transmembrane proteins reveals different intracellular fates for proteins according to their anchor in the plasma membrane.
Cell cycle regulation of the p34cdc2/p33cdk2-activating kinase p40MO15
Authors: Poon Randy Y. C. , Yamashita Katsumi , Howell Michael , Ershler Maxim A. , Belyavsky Alexander , Hunt Tim
Published: April 2021

A key component of Cdc2/Cdk2-activating kinase (CAK) is p40MO15, a protein kinase subunit that phosphorylates the T161/T160 residues of p34cdc2/p33cdk2. The level and activity of p40MO15 were essentially constant during cleavage of fertilised Xenopus eggs and in growing mouse 3T3 cells, but serum starvation of these cells reduced both the level and activity of p40MO15. Although the level and activity of endogenous p40MO15 did not vary in the cell cycle, we found that bacterially expressed p40MO15 was activated more rapidly by M-phase cell extracts than by interphase cell extracts. Bacterially expressed p40MO15 was phosphorylated mainly on serine 170 (a p34cdc2 phosphorylation site) by mitotic cell extracts, but mutation of S170 to alanine did not affect the activation of p40MO15, whereas mutation of T176 (the equivalent site to T161/T160 in p34cdc2/p33cdk2) abolished the activation of p40MO15. These studies suggest that the level and activity of p40MO15 is probably not a major determinant of p34cdc2/p33cdk2 activity in the cell cycle, and that the activation of p40MO15 may require phosphorylation on T176.
A Rab4-like GTPase in Dictyostelium discoideum colocalizes with V-H+-ATPases in reticular membranes of the contractile vacuole complex and in lysosomes
Authors: Bush John , Nolta Kathleen , Rodriguez-Paris Juan , Kaufmann Nancy , O’Halloran Theresa , Ruscetti Tracy , Temesvari Lesly , Steck Theodore , Cardelli James
Published: April 2021

In the course of screening a cDNA library for ras-related Dictyostelium discoideum genes, we cloned a 0.7 kb cDNA (rabD) encoding a putative protein that was 70% identical at the amino acid level to human Rab4. Rab4 is a small Mr GTPase, which belongs to the Ras superfamily and functions to regulate endocytosis in mammalian cells. Southern blot analysis indicated that the rabD cDNA was encoded by a single copy gene while Northern blot analysis revealed that the rabD gene was expressed at relatively constant levels during growth and differentiation. Affinity-purified antibodies were prepared against a RabD fusion protein expressed in bacteria; the antibodies recognized a single 23 kDa polypeptide on western blots of cell extracts. Density gradient fractionation revealed that the RabD antigen co-distributed primarily with buoyant membranes rich in vacuolar proton pumps (V-H+-ATPases) and, to a lesser extent, with lysosomes. This result was confirmed by examining cell lines expressing an epitope-tagged version of RabD. Magnetically purified early endocytic vesicles and post-lysosomal vacuoles reacted more weakly with anti-RabD antibodies than did lysosomes. Other organelles were negative for RabD. Double-label indirect immunofluorescence microscopy revealed that RabD and the 100 kDa V-H+-ATPase subunit colocalized in a fine reticular network throughout the cytoplasm. This network was reminiscent of spongiomes, the tubular elements of the contractile vacuole system. Immunoelectron microscopy confirmed the presence of RabD in lysosome fractions and in the membranes rich in V-H+-ATPase. We conclude that a Rab4-like GTPase in D. discoideum is principally associated with the spongiomes of contractile vacuole complex.
Internal structure of the 30 nm chromatin fiber
Authors: Bartolomé Salvador , Bermúdez Antonio , Daban Joan-Ramon
Published: April 2021

In the presence of 1.7 mM Mg2+, the diameter of the circular structures produced by small chromatin fragments isolated from chicken erythrocytes remains essentially unchanged when the number of nucleosomes in these fragments increases from 10 to 36. In contrast, the results obtained in unidirectional shadowing experiments show that under the same conditions the height of the chromatin fragments increases with the number of nucleosomes. These observations indicate that the electron microscope images studied in this work correspond to a top view of small chromatin fragments. Rotary-shadowed chromatin fragments show three parts: (a) a contour with a heavy deposition of platinum; (b) an annular zone between the central region and the periphery; and (c) a central hole. The heterogeneous ring generated by the deposition of platinum in the periphery suggests that nucleosomes form a one-start helix (5–7 nucleosomes per turn) that apparently can be left- or right-handed. The annular region (thickness of about 11 nm) shows spokes probably due to flat faces and core DNA of radially oriented nucleosomes. The central hole (8-12 nm) is clearly seen in many images but it is not empty because some deformed fragments show coated material (probably linker DNA) that protrudes from this central depression. We have observed that these structural elements directly detected in short chromatin fragments are also present in long chromatin fibers. This allows us to conclude that these elements are basic structural components of the 30 nm chromatin fiber.
Meiosis-specific cell cycle regulation in maturing Xenopus oocytes
Authors: Ohsumi Keita , Sawada Wako , Kishimoto Takeo
Published: April 2021

Meiotic cell cycles differ from mitotic cell cycles in that the former lack S-phase in the interphase between meiosis I and meiosis II. To obtain clues for mechanisms involved in the cell cycle regulation unique to meiosis, we have examined changes in chromosomal morphology and H1 kinase activity during a meiotic period from metaphase I (MI) to metaphase II (MII) in Xenopus oocytes. Using populations of oocytes that underwent germinal vesicle breakdown (GVBD) within a 10 minute interval, we found that the kinase activity declined gradually during the 60 minute period after GVBD and then increased steadily during the following 80 minute interval, showing remarkable differences from the rapid drop and biphasic increase of the kinase activity in intermitotic periods (Solomon et al. (1990)Cell 63, 1013-1024; Dasso and Newport (1990)Cell 61, 811-823). We also found that the exit from MI lagged, by more than 30 minutes, behind the time of lowest H1 kinase activity, whereas the two events took place concomitantly at the end of meiosis II and mitosis. Conse quently, the H1 kinase activity was already increasing during the first meiotic division. When H1 kinase activation at MII was delayed by a transient inhibition of protein synthesis after GVBD, oocytes were able to support formation of interphase nuclei and DNA replication between the first meiotic division and the MII arrest, indicating that the cell cycle entered S-phase between meiosis I and meiosis II. These results strongly suggest that the machinery required for entering S-phase has been established in maturing oocytes by the end of meiosis I. The lack of S-phase in oocyte meiotic interphase, therefore, should be ascribed to cell cycle regulation that enables the transition from meiosis I to meiosis II without S-phase. The asynchrony between the inactivation of H1 kinase activity and the completion of meiosis I may be involved in the regulation of this unique feature of the meiotic cell cycle.
Initiation of DNA synthesis by nuclei from scrape-ruptured quiescent mammalian cells in high-speed supernatants of Xenopus egg extracts
Authors: Hola M. , Castleden S. , Howard M. , Brooks R. F.
Published: April 2021

Demembranated sperm heads, detergent-isolated somatic nuclei and even naked DNA are efficiently replicated in cytoplasmic extracts of activated amphibian eggs, but only after nuclear assembly and the formation of an intact nuclear envelope. DNA synthesis has not previously been shown to be initiated in high-speed (200,000 g) supernatants of egg cytoplasm because they are depleted of the vesicular material required to support nuclear envelope formation. Here we show that mammalian nuclei prepared by scrape-rupture are able to initiate DNA replication in such high-speed supernatants. These nuclei begin DNA synthesis asynchronously. This asynchrony cannot be attributed to differences in the time taken for nuclear assembly. Instead, we suggest that the asynchrony reflects intrinsic differences between nuclei and that these differences are a major cause of cell cycle variability. Our demonstration of initiation in high-speed supernatants now enables the initiation of eukaryotic DNA synthesis to be studied independently of nuclear assembly.
Differential localization of myosin-II isozymes in human cultured cells and blood cells
Authors: Maupin Pamela , Phillips Carrie L. , Adelstein Robert S. , Pollard Thomas D.
Published: April 2021

We used purified polyclonal antibodies to human cytoplasmic myosin-IIA and myosin-IIB directly labeled with fluorescent dyes to localize these myosin-II isozymes in HeLa cells, melanoma cells and blood cells. Both antibodies react strongly with myosin-II isozymes in HeLa cells, melanoma cells and blood eosinophils, but only anti-myosin-IIA antibodies stain platelets, lymphocytes, neutrophils and monocytes in smears of human blood. Both antibodies stain small spots along the stress fibers of interphase HeLa cells and melanoma cells, but double staining revealed that the detailed distributions of myosin-IIA and myosin-IIB differ. A low concentration of diffuse myosin-IIB is present in the cortex, both in lamellar regions around the periphery of the cell and over the free surface. Myosin-IIB is also concentrated in spots along perinuclear stress fibers. Myosin-IIA is absent from the cortex but is concentrated in spots along stress fibers located near the basal surface of cultured cells.

This population of peripheral stress fibers is highly enriched in myosin-IIA relative to myosin-IIB, but both are found together in centrally located stress fibers. In prophase and metaphase both isozymes are concentrated in the cortex in small spots less than 04.µm in size, similar to those in stress fibers. As the chromosomes begin the separate at anaphase, most of the myosin-II spots become concentrated in the outer 0.7 µm of the equatorial cortex in 100% of cells. This concentration of myosin-II isozymes in the cleavage furrow is maintained until the daughter cells separate. The superimposition of these small spots concentrated in the cleavage furrow produces the intense, uniform staining observed in conventional micrographs of whole cells.
Approaching microtubule structure with the scanning tunneling microscope (STM)
Authors: Maaloum M. , Chrétien D. , Karsenti E. , Hörber J. K. H.
Published: April 2021

We demonstrate that the scanning tunneling microscope can be used to obtain information about arrangement of tubulin subunits in the microtubule wall. Long rows of subunits with a periodicity of 3.8±0.4 nm were clearly visible in the images of microtubules. The separation between the rows of subunits was 4.8±0.4 nm. Close inspection of two images revealed another periodicity of 7.8±0.4 nm in the contour levels of the protofilaments. This indicates that α and β tubulin monomers can be resolved. In these areas the monomers were arranged according to a ‘B-type’ lattice. Scanning tunneling microscope images confirm that the lateral contacts between tubulin monomers in adjacent protofilaments are compatible with a three-start, left-handed helix model. This study demonstrates that scanning tunneling microscopy can give direct information on the structure and organization of macromolecular assemblies and can complement the classical methods of electron microscopy and X-ray scattering.
A specifically apical sub-membrane intermediate filament cytoskeleton in non-brush-border epithelial cells
Authors: Rodriguez Marcelo L. , Brignoni Mirtha , Salas Pedro J. I.
Published: April 2021

Although many pieces of evidence support the notion of a role for the cytoskeleton in epithelial polarization, no cytoskeletal component has been found to be specifically apical, except for some actin-binding proteins. Here we report the apical distribution of a 53 kDa cytokeratin. Furthermore, this cytokeratin co-purified with biotinylated apical plasma membrane proteins in high density complexes. Differential biotinylation of the basolateral domain showed that the 53 kDa protein is mainly attached to the apical membrane, although a companion 58 kDa protein attaches to both apical and basolateral membrane proteins. Immunoprecipitation experiments indicated that a number of apical components are directly or indirectly linked to the 53 kDa protein. These results indicate the existence of a terminal web-like structure in non-brushborder cells, which attaches to the apical domain and may play a role in apical polarization, especially during the acquisition of polarity from non-polarized cellular stages.
Integrin α 6β 4 mediates dynamic interactions with laminin
Authors: Tözeren Aydin , Kleinman Hynda K. , Wu Stephen , Mercurio Arthur M. , Byers Stephen W.
Published: April 2021

We present here a novel form of dynamic adhesion in which both the integrin receptor and the ligand supporting dynamic adhesion have been identified. Laminar flow assays showed that laminin supported attachment of α 6β 4-positive cells in the presence of fluid shear stress (τ≤2 dyn/cm2), indicating that these cells adhered to laminin within a fraction of a second. Further increases in flow rate (3.5 dyn/cm2≤τ≤100 dyn/cm2) initiated rolling of attached cells in the direction of flow, suggesting that rapidly formed adhesion is reversible and repeatable. Laminin fragment E8, which interacts with α6 integrins, supported dynamic attachment and rolling but extracellular matrix glycoprotein fibronectin did not. In cell lines that express α 6β 4 but not α 6β 1an anti-α 6 monoclonal antibody inhibited attachment to laminin in the presence of flow and following 5 minutes of static incubation. Infusion of this antibody onto cells adherent to laminin-coated slides led to rapid detachment of cells from the substratum. An anti-β1 monoclonal antibody diminished adhesion strength following static incubation but did not inhibit rapid attachment and flow-initiated rolling. These results indicate that in some α 6β 4-expressing epithelial and carcinoma cell lines, integrin α 6β 4mediates rapidly formed dynamic interactions with laminin.
Epican, a heparan/chondroitin sulfate proteoglycan form of CD44, mediates cell-cell adhesion
Authors: Milstone Leonard M. , Hough-Monroe Lynne , Kugelman Lisa C. , Bender Jeffrey R. , Haggerty John G.
Published: April 2021

Epican is a heparan/chondroitin sulfate proteoglycan form of CD44 and is expressed on the surface of keratinocytes from the basal layer to the granular layer of the epidermis. To analyze the adhesive properties of epican apart from the influence of other adhesive molecules found on keratinocytes, mouse L cell fibroblasts were transfected with CD44Epican cDNA. The epican expressed on the surface of transfected L cells was predominantly a heparan or chondroitin sulfate proteoglycan.

The CD44Epican-transfected L cells acquired: (a) a selfaggregating phenotype that required hyaluronan but was calcium-independent; and (b) a new capacity to adhere to keratinocytes, a property that was blocked by an antiepican antibody. Both aggregation and adhesion of CD44Epican-transfected cells were completely prevented by pretreatment with hyaluronidase, but were totally restored by the addition of exogenous hyaluronan. Aggregation of transfected L cells was minimally influenced by other glycosaminoglycans, but adhesion of transfected L cells to keratinocytes was substantially inhibited by heparin.

The ability of epican to mediate adhesion between keratinocytes was evaluated in a newly developed adhesion assay. In the presence of 0.03 mM calcium, a monoclonal antibody against epican inhibited keratinocyte adhesion to keratinocyte monolayers by 80%.

These data demonstrate that epican causes adhesion and aggregation in the transfected L cell model system, that the adhesive function of epican is hyaluronan-dependent, and that epican could have an adhesive function in intact epidermis.
Biosynthesis of glycosphingolipids is reduced in the absence of a vimentin intermediate filament network
Authors: Gillard Baiba K. , Thurmon Lisa T. , Harrell Rhonda G. , Capetanaki Yassemi , Saito Megumi , Yu Robert K. , Marcus Donald M.
Published: April 2021

Our previous observations on the immunocytochemical colocalization of intermediate filaments and glycosphingolipids led us to analyze the role of filaments in the biosynthesis and intracellular transport of glycosphingolipids. Cells with (vim+) and without (vim−) vimentin intermediate filaments were cloned from the adrenal carcinoma cell line SW13. There was no difference between vim+ and vim− cells in the proportion of newly synthesized C6-NBD-glucosylceramide transported to the plasma membrane. The vim+ cells synthesized glycosphingolipids, especially lactosylceramide and globotriosylceramide, and to a lesser extent GM3 ganglioside, more rapidly than vim− cells. The altered rate of biosynthesis did not result from differences in the levels of the glycosyltransferases that synthesize those compounds. To determine whether the presence of a vimentin network was responsible for the differences in biosynthesis, mouse vimentin cDNA was transfected into vim− cells. Transfected cells that expressed a mouse vimentin network demonstrated a twofold or greater increase in the rate of biosynthesis of neutral glycosphingolipids and gangliosides. There was no difference between vim+ and vim− cells in the synthesis of ceramide or sphingomyelin, or in their content of phospholipids or cholesterol. The nature of the biochemical defect(s) underlying the diminished incorporation of radiolabeled sugars into glycosphingolipids is unclear. Possibilities include alterations in the ultrastructure of the Golgi and/or abnormalities in a portion of the endocytic pathway.
Tyrosine phosphorylation is involved in reorganization of the actin cytoskeleton in response to serum or LPA stimulation
Authors: Chrzanowska-Wodnicka Magdalena , Burridge Keith
Published: April 2021

Tyrosine phosphorylation is known to regulate the formation of focal adhesions in cells adhering to extracellular matrix (ECM). We have investigated the possible involvement of tyrosine phosphorylation and the focal adhesion kinase (FAK) in the cytoskeletal changes induced by serum or lysophosphatidic acid (LPA) in quiescent Swiss 3T3 fibroblasts. As shown previously by others, quiescent cells stimulated with serum or LPA reveal a rapid reappearance of focal adhesions and stress fibers. Here we show that this is accompanied by an increase in phosphotyrosine in focal adhesions and specifically an increase in the tyrosine phosphorylation of FAK. The LPA-stimulated reappearance of focal adhesions and stress fibers is blocked by inhibitors of phospholipase C but not by pertussis toxin (PTX), indicating that this LPA signaling pathway is mediated by phospholipase C activation and does not involve PTX-sensitive G proteins. In the absence of serum or LPA, these cytoskeletal effects and the tyrosine phosphorylation of FAK can be mimicked by sodium orthovanadate in conjunction with hydrogen peroxide, agents that inhibit protein tyrosine phosphatases and thereby elevate levels of phosphotyrosine. Two tyrosine kinase inhibitors, erbstatin and genistein block both the serum-induced tyrosine phosphorylation of FAK and the assembly of focal adhesions and stress fibers. Two other tyrosine kinase inhibitors, tyrphostins 47 and 25, previously shown to inhibit FAK, failed to prevent FAK phos-phorylation or the reassembly of focal adhesions and stress fibers in response to serum. However, these inhibitors did prevent FAK phosphorylation and cytoskeletal assembly in response to lysophosphatidic acid (LPA), one component of serum previously shown to stimulate assembly of focal adhesions and stress fibers. Our findings suggest that the response to serum is complex and that although FAK phos-phorylation is important, other tyrosine kinases may also be involved.
Assembly of the cadherin-catenin complex in vitro with recombinant proteins
Authors: Aberle Hermann , Butz Stefan , Stappert Jörg , Weissig Helge , Kemler Rolf , Hoschuetzky Heinz
Published: April 2021

The cytoplasmic domain of classical cadherins is tightly associated with three proteins termed α-, β- and γ-catenin. These accessory proteins are of central importance for the adhesive properties of this class of cell adhesion molecules. In order to examine the molecular architecture of the cadherin-catenin complex in more detail we have expressed the catenins and the cytoplasmic domain of E-cadherin as fusion proteins in Escherichia coli, and analyzed the interaction of purified recombinant cadherin and catenins in combinatorial protein-protein interaction experiments. The cytoplasmic domain of E-cadherin cannot directly associate with α-catenin but interacts with high affinity with β-catenin, whereas the binding of γ-catenin (plakoglobin) to E-cadherin is less efficient. α- and β-catenin assemble into a 1:1 heterodimeric complex. The analysis of various truncated β-catenins revealed that an α-catenin binding site in β-catenin is localized between amino acid positions 120 and 151. The central role of β-catenin for the assembly of the heterotrimeric E-cadherin/α-catenin/β-catenin complex in mixing experiments with all components was demonstrated. The reconstitution in vitro of the cadherin-catenin complex should allow the study of the interaction with signalling molecules and with the actinbased cytoskeleton.
Coiled bodies in the nucleolus of breast cancer cells
Authors: Ochs Robert L. , Stein Thomas W. , Tan Eng M. , Keck W. M.
Published: April 2021

Coiled bodies are a special type of small round nuclear body, composed of coiled fibers and granules, especially prominent in the nucleoplasm of highly active cells (Brasch and Ochs (1992)Exp. Cell Res. 202, 211-223). Although no specific function has been assigned to coiled bodies, they contain spliceosome snRNAs and proteins, as well as the nucleolar U3 RNA-associated protein fibrillarin. In the present study, we have used antibodies to the coiled body-specific protein p80-coilin, together with double-label immunofluorescence, confocal microscopy and immuno-electron microscopy, to examine the distribution of coiled bodies in a number of different breast cancer cell lines. By immunofluorescence, all cell lines had prominent coiled bodies in the nucleoplasm and several cell lines appeared to have coiled bodies within the nucleolus itself. Double-label immunofluorescence and confocal laser scanning microscopy confirmed the nucleolar localization of coiled bodies. Besides containing p80-coilin, nucleoplasmic and nucleolar coiled bodies contained fibrillarin and Sm proteins. By conventional and immunoelectron microscopy, nucleolar coiled bodies appeared as discrete structures within the nucleolus in a number of different morphotypes, distinct from the normal nucleolar domains of granular component, dense fibrillar component, and fibrillar centers. While the significance of finding coiled bodies in the nucleolus of certain breast cancer cell lines is at present unknown, this represents the first report of coiled bodies and Sm staining in the nucleolus of mammalian cells.
Heparin stimulates the proliferation of intestinal epithelial cells in primary culture
Authors: Flint N. , Cove F. L. , Evans G. S.
Published: April 2021

Heparin is a sulphated glycosaminoglycan derived from mast cells and has a number of functions including the inhibition of proliferation in several cell types and interactions with a range of heparin-binding growth factors. We report that heparin is a trophic factor in primary cultures of rat small intestinal epithelium. Heparin elicits a dose-dependent increase in epithelial proliferation and inhibits the growth of associated mesenchyme. The trophic effect of this molecule is not reproduced by other glycosaminogly-cans including heparan sulphate but is dependent upon extensive molecular sulphation. Highly sulphated polysac-charides that are structurally unrelated to heparin (e.g. dextran sulphate and pentosan polysulphate) also stimulate epithelial proliferation in primary cultures. Heparin may act by the potentiation of mesenchyme-derived heparin-binding growth factors and these data suggest an in vivo role for mast cell-derived heparin in mucosal wound regeneration.
The effects of age on the response of rabbit periosteal osteoprogenitor cells to exogenous transforming growth factor-β2
Authors: Critchlow Matthew A. , Bland Yvette S. , Ashhurst Doreen E.
Published: April 2021

Additional bone and cartilage are formed if transforming growth factor-β is injected into the periosteum of calvariae or long bones. To investigate this further, transforming growth factor-β2 was injected into the periosteum of the tibia of 3-day-old, 3-month-old and 2-year-old rabbits. In all instances, there was an increase in proliferation of the cells of the cambial layer of the periosteum, that is, the osteoprogenitor cells, and breakdown of the fibrous layer. Oedema was induced in the surrounding connective tissues.

Over the experimental period the normal neonatal tibia is undergoing rapid growth; there is periosteal bone formation and endosteal resorption. In the experimental neonatal tibiae, an increase in periosteal bone formation is seen after three injections of 20 ng of transforming growth factor-β2, which is accompanied by cartilage after five injections; the amounts of induced bone and cartilage increase with the number of injections. The chondrocytes hypertrophy after 4 days and the cartilage is replaced by bone endochondrally.

In contrast, after seven injections of 20 ng transforming growth factor-β2, there is only a small amount of new bone on the 3-month-old tibia and none on the 2-year-old tibia. One day after seven injections of 200 ng transforming growth factor-β2, there is a small amount of bone formation, while seven days after cartilage is found as small discrete nodules on the 3-month-old tibia, but as small areas within the bone on the 2-year-old tibia.

It is concluded that the primary effect of transforming growth factor-β2 in this experimental model is to increase the proliferative rate of the osteoprogenitor cells in the periosteum. It is argued that transforming growth factor-β2 does not initiate osteoblastic or chondrocytic differen-tiation of osteoprogenitor cells. It is suggested that their differentiation is controlled by the local environment, in particular, the vascularity and locally circulating growth factors.
Lamellipod extension and K+current in osteoclasts are regulated by different types of G proteins
Authors: Arkett Stuart A. , Dixon S. Jeffrey , Sims Stephen M.
Published: April 2021

Osteoclasts are the cells responsible for the resorption of bone and other mineralized tissues. GTP-binding proteins (G proteins) play important roles in regulating the activity of many cell types; however, there is limited knowledge of their functions in osteoclasts. We used the patch-clamp technique in the whole-cell configuration to introduce either hydrolysis-resistant guanosine triphosphate analogues or fluoroaluminate into single rat osteoclasts, and examined the effects of G protein activation on cell morphology and ionic conductances. Guanosine 5′-O-(3-thiotriphosphate) or 5′-guanylyl-imidodiphosphate, but not the control compounds adenosine 5′-O-(3-thiotriphos-phate) or guanosine 5′-O-(2-thiodiphosphate), induced: (1) prompt spreading due to extension of lamellipodia; and (2) after a latency of several minutes, complete suppression of the inwardly rectifying K+ current. Pertussis toxin did not alter either spreading or suppression of K+ current induced by guanosine 5′-O-(3-thiotriphosphate). Cytochalasin D, but not colchicine, prevented guanosine 5′-O-(3-thio-triphosphate)-induced spreading, consistent with actin polymerization underlying lamellipod extension. Whole-cell capacitance did not change during guanosine 5′-O-(3-thiotriphosphate)-induced spreading, which is consistent with a lack of change in total plasma membrane area. Flu-oroaluminate did not induce spreading, but it did suppress the K+ current. The differential effects of fluoroaluminate and guanosine 5′-O-(3-thiotriphosphate) suggest that lamellipod extension is regulated by a small molecular mass, monomeric G protein, whereas the inwardly rectify-ing K+ current is regulated by a large molecular mass, het-erotrimeric G protein. Thus, osteoclast motility and ion transport are regulated by separate G protein-coupled pathways.
Reorganization of the centrosome and associated microtubules during the morphogenesis of a mouse cochlear epithelial cell
Authors: Henderson Craig G. , Tucker John B. , Chaplin Mark A. , Mackie John B. , Maidment Steven N. , Mogensen Mette M. , Paton Calum C.
Published: April 2021

Reorganization of centrosomal microtubule-organizing centres and the minus ends of microtubules occurs as the centrosomal ends of large microtubule bundles are reposi-tioned and anchored to cell junctions in certain epithelial cells called inner pillar cells in the mouse organ of Corti.

The microtubule bundle that assembles in each cell consists of two distinct microtubule arrays that run closely alongside each other. Both arrays are attached to the cell surface at their upper and lower ends. One of the arrays spans the entire length of a cell but the other is confined to its lower portion.

Initially, about 3,000 microtubules elongate downwards from an apically situated centrosome in each cell. Subse-quently, the minus ends of these microtubules, and the cen-trosome and its two centrioles, migrate for about 12 μm to the tip of a laterally directed projection. Then, a meshwork of dense material accumulates to link microtubule minus ends and the centrosome to cell junctions at the tip of the projection. Pericentriolar satellite bodies, which form after the initial burst of microtubule nucleation, may represent a condensed and inactive concentration of microtubule-nucleating elements.

Suprisingly, as a cell matures, about 2,000 microtubules are eliminated from the centrosomal end of the micro-tubule bundle. However, about 2,000 microtubules are added to the basal portion of each bundle at levels that are remote with respect to the location of the centrosome. Possibly, these microtubules have escaped from the cen-trosome. If this is the case, then both the plus and minus ends of most of the errant microtubules are captured by sites at the cell surface where the ends are finally anchored. Alternatively, each cell possesses at least one other major microtubule-nucleating site (which does not possess centri-oles) in addition to its centrosome.
Cytochemical features common to nucleoli and cytoplasmic nucleoloids of Olea europaea meiocytes: detection of rRNA by in situ hybridization
Authors: Alché J. D. , Fernández M. C. , Rodríguez-García M. I.
Published: April 2021

We used light and electron microscopic techniques to study the composition of cytoplasmic nucleoloids during meiotic division in Olea europaea. Nucleoloids were found in two clearly distinguishable morphological varieties: one similar in morphology to the nucleolus, and composed mainly of dense fibrillar component, and another surrounded by many ribosome-like particles. Cytochemical and immuno- cytochemical techniques showed similar reactivities in nucleoloids and the nucleolus: both are ribonucleoproteic in nature, and possess argyrophillic, argentaffinic and highly phosphorylated proteins. Immunohistochemical techniques failed to detect DNA in either structure. In situ hybridization to a 18 S rRNA probe demonstrated the presence of ribosomal transcripts in both the nucleolus and nucleoloids. These similarities in morphology and compo- sition may reflect similar functionalities.
Site of transcription of ribosomal RNA and intranucleolar structure in HeLa cells
Authors: Hozák Pavel , Cook Peter R. , Schöfer Christian , Mosgöller Wilhelm , Wachtler Franz
Published: April 2021

Sites of transcription of ribosomal RNA in HeLa cells were visualized by electron microscopy. Cells were either incubated with Br-uridine, or permeabilized and then incubated with BrUTP, before sites containing Br-RNA were immunolabelled with gold particles. Short incuba-tions ensured that most incorporated analogue remained at synthetic sites. Fibrillar centres were unlabelled except at their periphery; label was concentrated over certain regions of the surrounding dense fibrillar component. These results suggest that the dense fibrillar component is the site of rRNA transcription. After dispersing the granular component and the dense fibrillar component by a hypotonic treatment, removal of most chromatin and preparation of resinless sections, fibrillar centres remained fixed to a nucleoskeleton. These structural and functional features are incorporated into a model for rRNA tran-scription.
Tissue-specific expression of the fibril-associated collagens XII and XIV
Authors: Wälchli Chantal , Koch Manuel , Chiquet Matthias , Odermatt Bernhard F. , Trueb Beat
Published: April 2021

Interstitial collagen fibrils form the supporting scaffold of all connective tissues. The synthesis of this framework is subject to a precise spatial and temporal regulation in order to meet the mechanical needs of every tissue type. A subgroup of non-fibrillar collagens termed FACIT seems to play a role in this regulation by providing specific molecular bridges between fibrils and other matrix com-ponents. Collagens XII and XIV represent such FACIT molecules and occur preferentially in tissues containing banded type I collagen fibrils. We have used the techniques of indirect immunofluorescence and in situ hybridization to investigate the expression patterns of the two molecules during chicken embryonic development. We detected specific differences in these patterns, which may be related to the respective functions of the two proteins within the connective tissues. Collagen XIV was expressed at very few sites in the 6-day-old embryo, but occurred in virtually every collagen I-containing tissue (skeletal muscle, cardiac muscle, gizzard, tendon, periosteum, nerve) by the end of embryonic development. In contrast, collagen XII was fairly abundant in the 6-day-old embryo but was, at later stages, restricted to only a few dense connective tissue structures (bone, tendon, gizzard). Thus, our results suggest that collagen XII and collagen XIV serve different functions during embryonic development although their structures are highly similar.