bluuebunny/crossref_metadata_2025 huggingface dataset:
Connexin46, a novel lens gap junction protein, induces voltage-gated currents in nonjunctional plasma membrane of Xenopus oocytes.
Authors: Paul D L , Ebihara L , Takemoto L J , Swenson K I , Goodenough D A
Published: May 2004

Gap junctions are composed of a family of structural proteins called connexins, which oligomerize into intercellular channels and function to exchange low molecular weight metabolites and ions between adjacent cells. We have cloned a new member of the connexin family from lens cDNA, with a predicted molecular mass of 46 kD, called rat connexin46 (Cx46). Since a full-length cDNA corresponding to the 2.8-kb mRNA was not obtained, the stop codon and surrounding sequences were confirmed from rat genomic DNA. The RNA coding for this protein is abundant in lens fibers and detectable in both myocardium and kidney. Western analysis of both rat and bovine lens membrane proteins, using the anti-MP70 monoclonal antibody 6-4-B2-C6 and three anti-peptide antibodies against Cx46 demonstrates that Cx46 and MP70 are different proteins. Immunocytochemistry demonstrates that both proteins are localized in the same lens fiber junctional maculae. Synthesis of Cx46 in either reticulocyte lysate or Xenopus oocytes yields a 46-kD polypeptide; all anti-Cx46 antisera recognize a protein in rat lens membranes 5-10 kD larger, suggesting substantive lenticular posttranslational processing of the native translation product. Oocytes that have synthesized Cx46 depolarize and lyse within 24 h, a phenomenon never observed after expression of rat connexins 32 or 43 (Cx32 and Cx43). Lysis is prevented by osmotically buffering the oocytes with 5% Ficoll. Ficoll-buffered oocytes expressing Cx46 are permeable to Lucifer Yellow but not FITC-labeled BSA, indicating the presence of selective membrane permeabilities. Cx43-expressing oocytes are impermeable to Lucifer Yellow. Voltage-gated whole cell currents are measured in oocytes injected with dilute concentrations of Cx46 but not Cx43 mRNA. These currents are activated at potentials positive to -10 mV. Unlike other connexins expressed in Xenopus oocytes, these results suggest that unprocessed Cx46 induces nonselective channels in the oolemma that are voltage dependent and opened by large depolarizations.
Immunohistochemical localization of TGF beta 1, TGF beta 2, and TGF beta 3 in the mouse embryo: expression patterns suggest multiple roles during embryonic development.
Authors: Pelton R W , Saxena B , Jones M , Moses H L , Gold L I
Published: May 2004

Isoform-specific antibodies to TGF beta 1, TGF beta 2, and TGF beta 3 proteins were generated and have been used to examine the expression of these factors in the developing mouse embryo from 12.5-18.5 d post coitum (d.p.c.). These studies demonstrate the initial characterization of both TGF beta 2 and beta 3 in mammalian embryogenesis and are compared with TGF beta 1. Expression of one or all three TGF beta proteins was observed in many tissues, e.g., cartilage, bone, teeth, muscle, heart, blood vessels, lung, kidney, gut, liver, eye, ear, skin, and nervous tissue. Furthermore, all three TGF beta proteins demonstrated discrete cell-specific patterns of expression at various stages of development and the wide variety of tissues expressing TGF beta proteins represent all three primary embryonic germ layers. For example, specific localization of TGF beta 1 was observed in the lens fibers of the eye (ectoderm), TGF beta 2 in the cortex of the adrenal gland (mesoderm), and TGF beta 3 in the cochlear epithelium of the inner ear (endoderm). Compared to the expression of TGF beta mRNA transcripts in a given embryonic tissue, TGF beta proteins were frequently colocalized within the same cell type as the mRNA, but in some cases were observed to localize to different cells than the mRNA, thereby indicating that a complex pattern of transcription, translation, and secretion for TGF beta s 1-3 exists in the mouse embryo. This also indicates that TGF beta 1, beta 2, and beta 3 act through both paracrine and autocrine mechanisms during mammalian embryogenesis.
In vivo paracrine interaction between urokinase and its receptor: effect on tumor cell invasion.
Authors: Ossowski L , Clunie G , Masucci M T , Blasi F
Published: May 2004

Numerous studies have linked the production of increased levels of urokinase type plasminogen activator (uPA) with the malignant phenotype. It has also been shown that a specific cell surface receptor can bind uPA through a domain distinct and distant from the proteolytic domain. In an in vivo model of invasion, consisting of experimentally modified chorioallantoic membrane (CAM) of a chick embryo, only cells that concurrently expressed both uPA and a receptor for uPA, and in which the receptor was saturated with uPA, were efficient in invasion. To test whether uPA produced by one cell can, in a paracrine fashion, affect the invasive capacity of a receptor-expressing cell, we transfected LB6 mouse cells with human uPA (LB6[uPA]), or human uPA-receptor cDNA (LB6[uPAR]). LB6(uPA) cells released into the medium 1-2 Ploug units of human uPA per 10(6) cells in 24 h. The LB6(uPAR) cells expressed on their surface approximately 12,000 high affinity (Kd 1.7 x 10(-10) M uPA binding sites per cell. Unlabeled LB6(uPA) and 125-IUdR-labeled LB6(uPAR) cells were coinoculated onto experimentally wounded and resealed CAMs and their invasion was compared to that of homologous mixtures of labeled and unlabeled LB6(uPAR) or LB6(uPA) cells. Concurrent presence of both cell types in the CAMs resulted in a 1.8-fold increase of invasion of the uPA-receptor expressing cells. A four-fold stimulation of invasion was observed when cells were cocultured in vitro, prior to in vivo inoculation. Enhancement of invasion was prevented in both sets of experiments by treatment with specific antihuman uPA antibodies, indicating that uPA was the main mediator of the invasion-enhancing, paracrine effect on the receptor-expressing cells.
Neurite outgrowth on immobilized axonin-1 is mediated by a heterophilic interaction with L1(G4).
Authors: Kuhn T B , Stoeckli E T , Condrau M A , Rathjen F G , Sonderegger P
Published: May 2004

Axonin-1 is an axon-associated cell adhesion molecule with dualistic expression, one form being glycophosphatidylinositol-anchored to the axonal membrane, the other secreted from axons in a soluble form. When presented as a substratum for neuronal cultures it strongly promotes neurite outgrowth from chicken embryonic dorsal root ganglia neurons. In this study, the axon-associated cell adhesion molecule G4, which is identical with Ng-CAM and 8D9, and homologous or closely related to L1 of the mouse and NILE of the rat, was investigated with respect to a receptor function for axonin-1. Using fluorescent microspheres with covalently coupled axonin-1 or L1(G4) at their surface we showed that these proteins bind to each other. Within the sensitivity of this microsphere assay, no interaction of axonin-1 with itself could be detected. Axonin-1-coated microspheres also bound to the neurites of cultured dorsal root ganglia neurons. This interaction was exclusively mediated by L1(G4), as indicated by complete binding suppression by monovalent anti-L1(G4) antibodies. The interaction between neuritic L1(G4) and immobilized axonin-1 was found to mediate the promotion of neurite growth on axonin-1, as evidenced by the virtually complete arrest of neurite outgrowth in the presence of anti-L1(G4) antibodies. Convincing evidence has recently been presented that neurite growth on L1(8D9) is mediated by the homophilic binding of neuritic L1(G4) (1989. Neuron. 2: 1597-1603). Thus, both L1(G4)- and axonin-1-expressing axons may serve as "substrate pathways" for the guidance of following axons expressing L1(G4) into their target area. Conceivably, differences in the concentration of axonin-1 and L1(G4), and/or modulatory influences on their specific binding parameters in leading pathways and following axons could represent elements in the control of axonal pathway selection.
Cloning and expression of cDNAs for the alpha subunit of the murine lymphocyte-Peyer's patch adhesion molecule.
Authors: Neuhaus H , Hu M C , Hemler M E , Takada Y , Holzmann B , Weissman I L
Published: May 2004

cDNA clones encoding the alpha chain of the murine lymphocyte-Peyer's patch adhesion molecule (LPAM), which is associated with lymphocyte homing, have been isolated by screening with the human VLA-4 (alpha 4h) probe. Several alpha 4 antigenic determinants were identified on COS-7 cells after transfection. From overlapping clones, approximately 5 kb of contiguous nucleotide sequence have been determined, encoding a protein sequence of 1039 amino acids for the LPAM alpha chain (alpha 4m). LPAM is a member of the integrin family of cell-surface heterodimers, and alpha 4m is the murine homologue of the human alpha 4 h chain. The two proteins have a total sequence similarity of 84%, with an almost perfect conservation (31/32 amino acids) in the cytoplasmic domain. Like alpha 4h, alpha 4m is distinct from other integrin alpha chains because it has neither an I-domain nor a COOH-terminal cleavage site. The positions of the characteristic Cysteine residues are conserved, and a putative protease cleavage site is located near the middle of the protein sequence. The NH2-terminal part of the protein contains seven homologous repeats, and three of them include putative divalent cation-binding sites. These sites are among the most conserved between the alpha 4m sequence and other alpha chains, and may therefore be involved in the binding of integrin alpha and beta chains. An additional cDNA clone was isolated which shares a sequence of perfect homology with the alpha 4m encoding cDNAs, but has a unique 3' poly-A end. This observation correlates with the fact that three discrete murine RNA bands are observed in Northern blot experiments using alpha 4m as a probe, whereas only two human RNA species are described for alpha 4h, indicating a higher complexity for murine than for human sequences.
Elevated extracellular calcium concentrations induce type X collagen synthesis in chondrocyte cultures.
Authors: Bonen D K , Schmid T M
Published: May 2004

Chondrocytes at different stages of cellular differentiation were isolated from the tarsal element (immature chondrocytes) and zones 2 and 3 (mature chondrocytes) of 12-d chick embryo tibiotarsus. The chondrocytes from the two sources differed in their cell morphologies, growth rate and production of type X collagen. In 24 h, zone 2 and 3 chondrocytes synthesized 800 times more type X collagen than tarsal chondrocytes. The effect of exogenous CaCl2 (5 and 10 mM) on the synthesis of type X collagen by both mature and immature chondrocytes was tested. After a 72-h incubation of zone 2 and 3 chondrocytes with CaCl2 type X collagen increased 8-fold with 5 mM and 10-fold with 10 mM Ca2+. [3H]Proline incorporation into culture medium and matrix macromolecules increased 11 and 32% with 5 and 10 mM CaCl2, respectively. Type II collagen synthesis was not affected by elevated extracellular Ca2+ during this 72-h period. Similar studies with tarsal chondrocytes demonstrated a time- and dose-dependent response to CaCl2 with type X collagen levels reaching a 4-fold and 15-fold increase over controls with 5 and 10 mM Ca2+, respectively, at 48 h. Elevated extracellular Ca2+ had no effect on cell proliferation. These observations offer the first direct evidence of the induction of type X collagen synthesis with elevated extracellular Ca2+.
Vitronectin receptor has a role in bone resorption but does not mediate tight sealing zone attachment of osteoclasts to the bone surface.
Authors: Lakkakorpi P T , Horton M A , Helfrich M H , Karhukorpi E K , Väänänen H K
Published: May 2004

During bone resorption, osteoclasts form a tight attachment, the sealing zone, around resorption lacunae. Vitronectin receptor has previously been shown to be expressed in osteoclasts and it has been suggested that it mediates the tight attachment at the sealing zone. In this study we have shown that glycine-arginine-glycine-aspartic acid-serine pentapeptide inhibits bone resorption by isolated osteoclasts and drastically changes the morphology of the osteoclasts. When the vitronectin receptor was localized by immunofluorescence in rat and chicken osteoclasts cultured on bone slices, it was found to be distributed throughout the osteoclast cell membrane except in the sealing zone areas. Immunoperoxidase staining of rat bone sections at the light microscopical level also revealed intense staining of the cell membrane with occasional small unstained areas, probably corresponding to the sealing zones. Immunoelectron microscopy confirmed the results obtained by light microscopy showing specific labeling only at the ruffled borders and basolateral membranes (0.82 and 2.43 gold particles/microns of membrane, respectively), but not at the sealing zone areas (0.06 gold particles/microns of membrane). Both alpha v and beta 3 subunits of the vitronectin receptor were similarly localized. These results strongly suggest that, although the vitronectin receptor is important in the function of osteoclasts, it is not mediating the final sealing zone attachment of the osteoclasts to the mineralized bone surface.
An analysis of vertebrate mRNA sequences: intimations of translational control.
Authors: Kozak M
Published: May 2004

Five structural features in mRNAs have been found to contribute to the fidelity and efficiency of initiation by eukaryotic ribosomes. Scrutiny of vertebrate cDNA sequences in light of these criteria reveals a set of transcripts--encoding oncoproteins, growth factors, transcription factors, and other regulatory proteins--that seem designed to be translated poorly. Thus, throttling at the level of translation may be a critical component of gene regulation in vertebrates. An alternative interpretation is that some (perhaps many) cDNAs with encumbered 5' noncoding sequences represent mRNA precursors, which would imply extensive regulation at a posttranscriptional step that precedes translation.
Transcription of the muscle regulatory gene Myf4 is regulated by serum components, peptide growth factors and signaling pathways involving G proteins.
Authors: Salminen A , Braun T , Buchberger A , Jürs S , Winter B , Arnold H H
Published: May 2004

The muscle regulatory protein myogenin accumulates in differentiating muscle cells when the culture medium is depleted for serum. To investigate the regulation of myogenin gene expression, we have isolated and characterized the Myf4 gene which encodes the human homologue of murine myogenin. Serum components, basic FGF (b-FGF), transforming growth factor beta (TGF-beta), and EGF, agents which suppress differentiation of muscle cells in vitro, down-regulate the activity of the Myf4 gene, suggesting that it constitutes a nuclear target for the negative control exerted by these factors. The 5' upstream region containing the Myf4 promoter confers activity to a CAT reporter plasmid in C2C12 myotubes but not in fibroblasts and undifferentiated myoblasts. Unidirectional 5' deletions of the promoter sequence reveal that integral of 200 nucleotides upstream of the transcriptional start site are sufficient for cell type-specific expression. The forced expression of the muscle determining factors, MyoD1, Myf5, and Myf6 and to a lesser degree Myf4, results in the transactivation of the Myf4 promoter in C3H mouse 10T1/2 fibroblasts. Pathways potentially involved in conveying signals from the cell-surface receptors to the Myf4 gene were probed with pertussis- and cholera toxin, forskolin, and cAMP. Dibutyryl-cAMP and compounds that stimulate adenylate cyclase inhibit the endogenous Myf4 gene and the Myf4 promoter in CAT and LacZ reporter constructs. Conversely, pertussis toxin which modifies Gi protein stimulates Myf4 gene expression. In summary, our data provide evidence that the muscle-specific expression of the Myf4 gene is subject to negative control by serum components, growth factors and a cAMP-dependent intracellular mechanism. Positive control is exerted by a pertussis toxin-sensitive pathway that presumably involves G proteins.
Russell bodies: a general response of secretory cells to synthesis of a mutant immunoglobulin which can neither exit from, nor be degraded in, the endoplasmic reticulum.
Authors: Valetti C , Grossi C E , Milstein C , Sitia R
Published: May 2004

Dilated cisternae of the ER resembling Russell Bodies (RBs) are induced in light (L) chain producing myeloma cell lines by transfection of a mu heavy (H) chain gene lacking the first constant domain (mu delta CH1). RBs do not appear to be tissue specific, since they are also induced in a rat glioma cell line transfected with mu delta CH1 and L chain genes. Efficient RB biogenesis requires H-L assembly and polymerization. The mutant Ig is partially degraded in a pre-Golgi compartment. The remnant, however, becomes an insoluble lattice when intersubunit disulphide bonds are formed. The resulting insoluble aggregate accumulates in RBs. Replacing the COOH-terminal cysteine of mu delta CH1 chains with alanine reverses the RB-phenotype: the double mutant mu ala delta CH1 chains assemble noncovalently with L and are secreted as H2L2 complexes. Similarly, secretion of mu delta CH1 chains can be induced by culturing transfectant cells in the presence of reducing agents. The presence of RBs does not alter transport of other secretory or membrane molecules, nor does it affect cell division. Resident proteins of the ER and other secretory proteins are not concentrated in RBs, implying sorting at the ER level. Sorting could be the result of the specific molecular structure of the insoluble lattice. We propose that RBs represent a general response of the cell to the accumulation of abundant, nondegradable protein(s) that fail to exit from the ER.
Acquisition of membrane lipids by differentiating glyoxysomes: role of lipid bodies.
Authors: Chapman K D , Trelease R N
Published: May 2004

Glyoxysomes in cotyledons of cotton (Gossypium hirsutum, L.) seedlings enlarge dramatically within 48 h after seed imbibition (Kunce, C.M., R.N. Trelease, and D.C. Doman. 1984. Planta (Berl.). 161:156-164) to effect mobilization of stored cotton-seed oil. We discovered that the membranes of enlarging glyoxysomes at all stages examined contained a large percentage (36-62% by weight) of nonpolar lipid, nearly all of which were triacylglycerols (TAGs) and TAG metabolites. Free fatty acids comprised the largest percentage of these nonpolar lipids. Six uncommon (and as yet unidentified) fatty acids constituted the majority (51%) of both the free fatty acids and the fatty acids in TAGs of glyoxysome membranes; the same six uncommon fatty acids were less than 7% of the acyl constituents in TAGs extracted from cotton-seed storage lipid bodies. TAGs of lipid bodies primarily were composed of palmitic, oleic, and linoleic acids (together 70%). Together, these three major storage fatty acids were less than 10% of both the free fatty acids and fatty acids in TAGs of glyoxysome membranes. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) constituted a major portion of glyoxysome membrane phospholipids (together 61% by weight). Pulse-chase radiolabeling experiments in vivo clearly demonstrated that 14C-PC and 14C-PE were synthesized from 14C-choline and 14C-ethanolamine, respectively, in ER of cotyledons, and then transported to mitochondria; however, these lipids were not transported to enlarging glyoxysomes. The lack of ER involvement in glyoxysome membrane phospholipid synthesis, and the similarities in lipid compositions between lipid bodies and membranes of glyoxysomes, led us to formulate and test a new hypothesis whereby lipid bodies serve as the dynamic source of nonpolar lipids and phospholipids for membrane expansion of enlarging glyoxysomes. In a cell-free system, 3H-triolein (TO) and 3H-PC were indeed transferred from lipid bodies to glyoxysomes. 3H-PC, but not 3H-TO, also was transferred to mitochondria in vitro. The amount of lipid transferred increased linearly with respect to time and amount of acceptor organelle protein, and transfer occurred only when lipid body membrane proteins were associated with the donor lipid bodies. 3H-TO was transferred to and incorporated into glyoxysome membranes, and then hydrolyzed to free fatty acids. 3H-PC was transferred to and incorporated into glyoxysome and mitochondria membranes without subsequent hydrolysis. Our data are inconsistent with the hypothesis that ER contributes membrane lipids to glyoxysomes during postgerminative seedling growth.(ABSTRACT TRUNCATED AT 400 WORDS)
Intracellular transport and localization of major histocompatibility complex class II molecules and associated invariant chain.
Authors: Pieters J , Horstmann H , Bakke O , Griffiths G , Lipp J
Published: May 2004

The intracellular transport and location of major histocompatibility complex (MHC) class II molecules and associated invariant chain (Ii) were investigated in a human melanoma cell line. In contrast to the class II molecules, which remain stable for greater than 4 h after synthesis, the associated Ii is proteolytically processed within 2 h. During or shortly after synthesis the NH2-terminal cytoplasmic and membrane-spanning segment is in some of the Ii molecules cleaved off; during intracellular transport, class II associated and membrane integrated Ii is processed from its COOH terminus in distinct steps in endocytic compartments. Immunocytochemical studies at the light and electron microscopic level revealed the presence of class II molecules, but not of Ii on the cell surface. Intracellularly both Ii and class II molecules were localized in three morphologically and kinetically distinct compartments, early endosomes, multivesicular bodies, and prelysosomes. This localization in several distinct endosomal compartments contrasts with the localization of class II molecules in mainly one endocytic compartment in B lymphoblastoid cell lines. As in these lymphoblastoid cell lines Ii is known to be rapidly degraded it is conceivable that the rate of proteolysis of the class II associated Ii and its dissociation from class II molecules modulates the retention of the oligomeric complex in endocytic compartments, and as a consequence the steady-state distribution of these molecules within the endosomal system.
Uncoupled cell cycle without mitosis induced by a protein kinase inhibitor, K-252a.
Authors: Usui T , Yoshida M , Abe K , Osada H , Isono K , Beppu T
Published: May 2004

The staurosporine analogues, K-252a and RK-286C, were found to cause DNA re-replication in rat diploid fibroblasts (3Y1) without an intervening mitosis, producing tetraploid cells. Analysis of cells synchronized in early S phase in the presence of K-252a revealed that initiation of the second S phase required a lag period of 8 h after completion of the previous S phase. Reinitiation of DNA synthesis was inhibited by cycloheximide, actinomycin D, and serum deprivation, but not by Colcemid, suggesting that a functional G1 phase dependent on de novo synthesis of protein and RNA is essential for entry into the next S phase. In a src-transformed 3Y1 cell line, as well as other cell lines, giant cells containing polyploid nuclei with DNA contents of 16C to 32C were produced by continuous treatment with K-252a, indicating that the agent induced several rounds of the incomplete cell cycle without mitosis. Although the effective concentration of K-252a did not cause significant inhibition of affinity-purified p34cdc2 protein kinase activity in vitro, in vivo the full activation of p34cdc2 kinase during the G2/M was blocked by K-252a. On the other hand, the cyclic fluctuation of partially activated p34cdc2 kinase activity peaking in S phase still continued. These results suggest that a putative protein kinase(s) sensitive to K-252a plays an important role in the mechanism for preventing over-replication after completion of previous DNA synthesis. They also suggest that a periodic activation of p34cdc2 is required for S phases in the cell cycle without mitosis.
Variations in the cytoskeletal interaction and posttranslational modification of the CD44 homing receptor in macrophages.
Authors: Camp R L , Kraus T A , Puré E
Published: May 2004

Murine CD44 is a cell surface glycoprotein that is thought to play a role in leukocyte migration. We studied the structure and expression of CD44 on two populations of macrophages: those that reside in the peritoneum of unprimed mice, and those that have been elicited to migrate into the peritoneum by the intraperitoneal injection of agents that cause localized inflammatory responses. Our studies reveal structural variations in both the extracellular and intracellular domains of CD44 expressed by these two macrophage populations. The form of CD44 in elicited macrophages has an apparent molecular mass that is approximately 5 kD greater and more heterogenous than that in resident macrophages. This structural changes is posttranslational, extracellular, and apparently reflects increases in N-linked glycosylation. It is also specific for CD44 and does not occur with several other glycoproteins examined. This novel regulation of glycosylation may play an important role in the ability of CD44 to bind to different substrates, particularly lectin-like ligands. In addition, we demonstrate that CD44 in resident macrophages is divided into two pools, one containing nonphosphorylated, cytoskeletally associated CD44, and one containing phosphorylated, unassociated CD44. In contrast, CD44 on the surface of elicited macrophages does not associate with the cytoskeleton. The attachment of CD44 to the cytoskeleton involves either direct or indirect association with actin. The regulated association of CD44 with the cytoskeleton suggests that it may influence or be influenced by macrophage mobility.
Intermediate filaments formed de novo from tail-less cytokeratins in the cytoplasm and in the nucleus.
Authors: Bader B L , Magin T M , Freudenmann M , Stumpp S , Franke W W
Published: May 2004

The roles of the different molecular domains of intermediate filament (IF) proteins in the assembly and higher order organization of IF structures have recently been studied by various groups but with partially controversial results. To examine the requirement of the aminoterminal (head) and the carboxyterminal (tail) domain of cytokeratins (CKs) for de novo IF formation in the living cell, we have constructed cDNAs coding for intact as well as head- and/or tail-less human CKs 8 and 18 and the naturally tail-less human CK 19, all under the control of the human beta-actin promoter. After transient and stable transfections of mouse 3T3-L1 cells, which are devoid of any CKs, we have studied, with such constructs, the resulting gene products by gel electrophoresis and immunolocalization techniques. By light and electron microscopy we show that extended cytoplasmic IF meshworks are formed from pairs of the type II CK 8 with the type I CKs 18 or 19 as well as from pairs of tail-less CK 8 with tail-less CKs 18 or 19 in the transfected cells, proving that the absence of the tail domain in both types of CKs does not prevent the de novo formation of regular IFs. Most surprisingly, however, we have observed spectacular alterations in the nucleocytoplasmic distribution of the IFs formed from tail-less CKs. In many of the transfected cells, a large part, or all, of the detectable CKs was found to occur in extensive IF bundles in the nucleoplasm. Intranuclear accumulations of CK deposits, however mostly nonfibrillar, were also observed when the cells had been transfected with cDNAs encoding tail-less CKs also lacking their head domains, whereas CKs deleted only in the head domain were found exclusively in the cytoplasm. The specific domain requirements for the assembly of cytoplasmic IF bundles are discussed and possible mechanisms of intranuclear accumulation of IFs are proposed.
Dihydropyridine receptor alpha subunits in normal and dysgenic muscle in vitro: expression of alpha 1 is required for proper targeting and distribution of alpha 2.
Authors: Flucher B E , Phillips J L , Powell J A
Published: May 2004

We have studied the subcellular distribution of the alpha 1 and alpha 2 subunits of the skeletal muscle dihydropyridine (DHP) receptor with immunofluorescence labeling of normal and dysgenic (mdg) muscle in culture. In normal myotubes both alpha subunits were localized in clusters associated with the T-tubule membranes of longitudinally as well as transversely oriented T-tubules. The DHP receptor-rich domains may represent the sites where triad junctions with the sarcoplasmic reticulum are being formed. In cultures from dysgenic muscle the alpha 1 subunit was undetectable and the distribution patterns of the alpha 2 subunit were abnormal. The alpha subunit did not form clusters nor was it discretely localized in the T-tubule system. Instead, alpha 2 was found diffusely distributed in parts of the T-system, in structures in the perinuclear region and in the plasma membrane. These results suggest that an interaction between the two alpha subunits is required for the normal distribution of the alpha 2 subunit in the T-tubule membranes. Spontaneous fusion of normal non-muscle cells with dysgenic myotubes resulted in a regional expression of the alpha 1 polypeptide near the foreign nuclei, thus defining the nuclear domain of a T-tubule membrane protein in multi-nucleated muscle cells. Furthermore, the normal intracellular distribution of the alpha 2 polypeptide was restored in domains containing a foreign "rescue" nucleus; this supports the idea that direct interactions between the DHP receptor alpha 1 and alpha 2 subunits are involved in the organization of the junctional T-tubule membranes.
Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques.
Authors: Musil L S , Goodenough D A
Published: May 2004

We previously demonstrated that the gap junction protein connexin43 is translated as a 42-kD protein (connexin43-NP) that is efficiently phosphorylated to a 46,000-Mr species (connexin43-P2) in gap junctional communication-competent, but not in communication-deficient, cells. In this study, we used a combination of metabolic radiolabeling and immunoprecipitation to investigate the assembly of connexin43 into gap junctions and the relationship of this event to phosphorylation of connexin43. Examination of the detergent solubility of connexin43 in communication-competent NRK cells revealed that processing of connexin43 to the P2 form was accompanied by acquisition of resistance to solubilization in 1% Triton X-100. Immunohistochemical localization of connexin43 in Triton-extracted NRK cells demonstrated that connexin43-P2 (Triton-insoluble) was concentrated in gap junctional plaques, whereas connexin43-NP (Triton-soluble) was predominantly intracellular. Using either a 20 degrees C intracellular transport block or cell-surface protein biotinylation, we determined that connexin43 was transported to the plasma membrane in the Triton-soluble connexin43-NP form. Cell-surface biotinylated connexin43-NP was processed to Triton-insoluble connexin43-P2 at 37 degrees C. Connexin43-NP was also transported to the plasma membrane in communication defective, gap junction-deficient S180 and L929 cells but was not processed to Triton-insoluble connexin43-P2. Taken together, these results demonstrate that gap junction assembly is regulated after arrival of connexin43 at the plasma membrane and is temporally associated with acquisition of insolubility in Triton X-100 and phosphorylation to the connexin43-P2 form.
Dihydrofolate reductase gene amplification-associated shift of differentiation in methotrexate-adapted HT-29 cells.
Authors: Lesuffleur T , Barbat A , Luccioni C , Beaumatin J , Clair M , Kornowski A , Dussaulx E , Dutrillaux B , Zweibaum A
Published: May 2004

Postconfluent cultures of HT-29 cells form a heterogeneous multilayer of which greater than 95% of the cells are undifferentiated. In contrast, when stably adapted to normally lethal concentrations of methotrexate (10(-6)-10(-5) M), they form a monolayer of gobletlike cells (Lesuffleur et al., 1990) which secrete large quantities of mucins and display a discrete brush border with the presence of villin, dipeptidylpeptidase-IV, and carcinoembryonic antigen. When adapted to even higher concentrations of methotrexate (10(-4) and 10(-3) M) there is a shift in the pattern of differentiation from gobletlike to dome-forming absorptive-like cells. These cells still display an apical brush border which expresses villin and dipeptidylpeptidase-IV, but no longer express significant levels of mucins and carcinoembryonic antigen. This shift of differentiation coincides with a sudden amplification of the gene coding for dihydrofolate reductase and an increased activity of the enzyme.
Integrin alpha 2 beta 1 is upregulated in fibroblasts and highly aggressive melanoma cells in three-dimensional collagen lattices and mediates the reorganization of collagen I fibrils.
Authors: Klein C E , Dressel D , Steinmayer T , Mauch C , Eckes B , Krieg T , Bankert R B , Weber L
Published: May 2004

The ability of cultured human fibroblasts to reorganize and contract three dimensional collagen I gels is regarded as an in vitro model for the reorganization of connective tissue during wound healing. We investigated whether adhesion receptors of the integrin family are involved. It was found that synthesis and transcription of the alpha 2 beta 1 integrin (but not of alpha 1 beta 1 or alpha 3 beta 1) is selectively upregulated when fibroblasts are seeded into type I collagen gels. Time course experiments revealed that high synthetic levels of alpha 2 beta 1 parallel the gel contraction process and return to "baseline" levels after the contraction has subsided. Furthermore, function-blocking mAbs directed to the alpha 2 and beta 1 chain of integrins inhibited gel contraction. Remodelling of connective tissue can be important for tumor cells during invasion and formation of metastases. Therefore, we tested human melanoma cell lines for this function. Five out of nine melanoma lines contracted collagen gels in vitro. Among these, two highly aggressive melanoma cell lines (MV3 and BLM) most efficiently contracted gels almost reaching the rate of normal adult fibroblasts. In these cells, synthesis of alpha 2 beta 1 was also significantly upregulated when seeded into collagen I gels. Moreover, function blocking anti-alpha 2 in conjunction with anti-beta 1 chain mAbs completely inhibited gel contraction for several days. Other melanoma cells (530) with lower metastatic potential which were not able to contract gels, showed no induction of alpha 2 beta 1 synthesis in gel culture. Our results suggest an important role of integrin alpha 2 beta 1 in the contraction of collagen I by normal diploid fibroblasts during wound healing and in the reorganization of collagen matrices by highly aggressive human melanoma cells.
A 220-kD undercoat-constitutive protein: its specific localization at cadherin-based cell-cell adhesion sites.
Authors: Itoh M , Yonemura S , Nagafuchi A , Tsukita S , Tsukita S
Published: May 2004

Recently we developed an isolation procedure for the cell-to-cell adherens junctions (AJ; cadherin-based junctions) from rat liver (Tsukita, Sh. and Sa. Tsukita. 1989. J. Cell Biol. 108:31-41). In this study, using the isolated AJ, we have obtained two mAbs specific to the 220-kD undercoat-constitutive protein. Immunofluorescence and immunoelectron microscopy with these mAbs showed that this 220-kD protein was highly concentrated at the undercoat of cell-to-cell AJ in various types of tissues and that this protein was located in the immediate vicinity of the plasma membrane in the undercoat of AJ. In the cells lacking typical cell-to-cell AJ, such as fibroblasts, the 220-kD protein was immunofluorescently shown to be coconcentrated with cadherin molecules at cell-cell adhesion sites. These localization analyses appeared to indicate the possible direct or indirect association of the 220-kD protein with cadherin molecules. Furthermore, it was revealed that the 220-kD protein and alpha-spectrin were coimmunoprecipitated with the above mAbs in both the isolated AJ and the brain. The affinity-purified 220-kD protein molecule looked like a spherical particle, and its binding site on the spectrin molecule was shown to be in the position approximately 10-20 nm from the midpoint of spectrin tetramer by low-angle rotary-shadowing electron microscopy. Taking all these results together with biochemical and immunological comparisons, we are persuaded to speculate that the 220-kD protein is a novel member of the ankyrin family. However, the possibility cannot be excluded that the 220-kD protein is an isoform of beta-spectrin. The possible roles of this 220-kD protein in the association of cadherin molecules with the spectrin-based membrane skeletons at the cadherin-based cell-cell adhesion sites are discussed.
Distribution of newly synthesized lysosomal enzymes in the endocytic pathway of normal rat kidney cells.
Authors: Ludwig T , Griffiths G , Hoflack B
Published: May 2004

We have investigated the distribution of newly synthesized lysosomal enzymes in endocytic compartments of normal rat kidney (NRK) cells. The mannose-6-phosphate (Man6-P) containing lysosomal enzymes could be iodinated in situ after internalization of lactoperoxidase (LPO) by fluid phase endocytosis and isolated on CI-MPR affinity columns. For EM studies, the ectodomain of the CI-MPR conjugated to colloidal gold was used as a probe specific for the phosphomannosyl marker of the newly synthesized hydrolases. In NRK cells, approximately 20-40% of the phosphorylated hydrolases present in the entire pathway were found in early endocytic structures proximal to the 18 degrees C temperature block including early endosomes. These structures were characterized by a low content of endogenous CI-MPR and were accessible to fluid phase markers internalized for 5-15 min at 37 degrees C. The bulk of the phosphorylated lysosomal enzymes was found in late endocytic structures distal to the 18 degrees C block, rich in endogenous CI-MPR and accessible to endocytic markers internalized for 30-60 min at 37 degrees C. The CI-MPR negative lysosomes were devoid of phosphorylated hydrolases. This distribution was unchanged in cells treated with Man6-P to block recapture of secreted lysosomal enzymes. However, lysosomal enzymes were no longer detected in the early endosomal elements of cells treated with cycloheximide. Immunoprecipitation of cathepsin D from early endosomes of pulse-labeled cells showed that this hydrolase is a transient component of this compartment. These data indicate that in NRK cells, the earliest point of convergence of the lysosomal biosynthetic and the endocytic pathways is the early endosome.
An endogenous MDCK lysosomal membrane glycoprotein is targeted basolaterally before delivery to lysosomes.
Authors: Nabi I R , Le Bivic A , Fambrough D , Rodriguez-Boulan E
Published: May 2004

Using surface immunoprecipitation at 37 degrees C to "catch" the transient apical or basolateral appearance of an endogenous MDCK lysosomal membrane glycoprotein, the AC17 antigen, we demonstrate that the bulk of newly synthesized AC17 antigen is polarly targeted from the Golgi apparatus to the basolateral plasma membrane or early endosomes and is then transported to lysosomes via the endocytic pathway. The AC17 antigen exhibits very similar properties to members of the family of lysosomal-associated membrane glycoproteins (LAMPs). Parallel studies of an avian LAMP, LEP100, transfected into MDCK cells revealed colocalization of the two proteins to lysosomes, identical biosynthetic and degradation rates, and similar low levels of steady-state expression on both the apical (0.8%) and basolateral (2.1%) membranes. After treatment of the cells with chloroquine, newly synthesized AC17 antigen, while still initially targeted basolaterally, appears stably in both the apical and basolateral domains, consistent with the depletion of the AC17 antigen from lysosomes and its recycling in a nonpolar fashion to the cell surface.
Fusion of sequence elements from non-anchored proteins to generate a fully functional signal for glycophosphatidylinositol membrane anchor attachment.
Authors: Moran P , Caras I W
Published: May 2004

Glycophosphatidylinositol (GPI) membrane anchor attachment is directed by a cleavable signal at the COOH terminus of the protein. The complete lack of homology among different GPI-anchored proteins suggests that this signal is of a general nature. Previous analysis of the GPI signal of decay accelerating factor (DAF) suggests that the minimal requirements for GPI attachment are (a) a hydrophobic domain and (b) a cleavage/attachment site consisting of a pair of small residues positioned 10-12 residues NH2-terminal to a hydrophobic domain. As an ultimate test of these rules we constructed four synthetic GPI signals, meeting these requirements but assembled entirely from sequence elements not normally involved in GPI attachment. We show that these synthetic signals are able to direct human growth hormone (hGH), a secreted protein, to the plasma membrane via a GPI anchor. Our results indicate that different hydrophobic sequences, derived from either the prolactin or hGH NH2-terminal signal peptide, can be linked to different cleavage sites via different hydrophilic spacers to produce a functional GPI signal. These data confirm that the only requirements for GPI-anchoring are a pair of small residues positioned 10-12 residues NH2 terminal to a hydrophobic domain, no other structural motifs being necessary.
The actin filament severing protein actophorin promotes the formation of rigid bundles of actin filaments crosslinked with alpha-actinin.
Authors: Maciver S K , Wachsstock D H , Schwarz W H , Pollard T D
Published: May 2004

The actin filament severing protein, Acanthamoeba actophorin, decreases the viscosity of actin filaments, but increases the stiffness and viscosity of mixtures of actin filaments and the crosslinking protein alpha-actinin. The explanation of this paradox is that in the presence of both the severing protein and crosslinker the actin filaments aggregate into an interlocking meshwork of bundles large enough to be visualized by light microscopy. The size of these bundles depends on the size of the containing vessel. The actin filaments in these bundles are tightly packed in some areas while in others they are more disperse. The bundles form a continuous reticulum that fills the container, since the filaments from a particular bundle may interdigitate with filaments from other bundles at points where they intersect. The same phenomena are seen when rabbit muscle aldolase rather than alpha-actinin is used as the crosslinker. We propose that actophorin promotes bundling by shortening the actin filaments enough to allow them to rotate into positions favorable for lateral interactions with each other via alpha-actinin. The network of bundles is more rigid and less thixotropic than the corresponding network of single actin filaments linked by alpha-actinin. One explanation may be that alpha-actinin (or aldolase) normally in rapid equilibria with actin filaments may become trapped between the filaments increasing the effective concentration of the crosslinker.
Uncoupling of Chlamydomonas flagellar gene expression and outgrowth from flagellar excision by manipulation of Ca2+.
Authors: Cheshire J L , Keller L R
Published: May 2004

Chlamydomonas cells respond to certain environmental stimuli by shedding their flagella. Flagellar loss induces a rapid, transient increase in expression of a specific set of genes encoding flagellar proteins, and assembly of a new flagellar pair. While flagellar gene expression and initiation of flagellar outgrowth are normally tightly coupled to flagellar excision, our results demonstrate that these processes can be uncoupled by manipulating Ca2+ levels or calmodulin activity. In our experiments, wild-type cells were stimulated to excise their flagella using mechanical shearing, and at times after deflagellation, flagellar lengths were measured and flagellar mRNA abundance changes were determined by S1 nuclease protection analysis. When extracellular Ca2+ was lowered by addition of EGTA to cultures before excision, flagellar mRNA abundance changes and flagellar outgrowth were temporally uncoupled from flagellar excision. When extracellular Ca2+ was lowered immediately after excision or when calmodulin activity was inhibited with W-7, flagellar outgrowth was uncoupled from flagellar excision and flagellar mRNA abundance changes. Whenever events in the process of flagellar regeneration were temporally uncoupled, the magnitude of the flagellar mRNA abundance change was reduced. These results suggest that flagellar gene expression may be regulated by multiple signals generated from these events, and implicate Ca2+ as a factor in the mechanisms controlling flagellar regeneration.
Polarized and functional epithelia can form after the targeted inactivation of both mouse keratin 8 alleles.
Authors: Baribault H , Oshima R G
Published: May 2004

We have tested the requirement of keratin intermediate filaments for the formation and function of a simple epithelium. We disrupted both alleles of the mouse keratin 8 (mK8) gene in embryonic stem cells, and subsequently analyzed the phenotype in developing embryoid bodies in suspension culture. After the inactivation of the mouse keratin 8 (mK8) gene by a targeted insertion, mK8 protein synthesis was undetectable. In the absence of mK8 its complementary partners mK18 and mK19 were unable to form filaments within differentiated cells. Surprisingly, these ES cells differentiate to both simple and cystic embryoid bodies with apparently normal epithelia. Ultrastructural analysis shows an apparently normal epithelium with microvilli on the apical membrane, tight junctions and desmosomes on the lateral membrane, and an underlying basal membrane. No significant differences in the synthesis or secretion of alpha 1-fetoprotein and laminin were observed between the mK8- or wild-type embryoid bodies. Our data show that mK8 is not required for simple epithelium formation of extraembryonic endoderm.
Dystrophin-associated proteins are greatly reduced in skeletal muscle from mdx mice.
Authors: Ohlendieck K , Campbell K P
Published: May 2004

Dystrophin, the protein product of the human Duchenne muscular dystrophy gene, exists in skeletal muscle as a large oligomeric complex that contains four glycoproteins of 156, 50, 43, and 35 kD and a protein of 59 kD. Here, we investigated the relative abundance of each of the components of the dystrophin-glycoprotein complex in skeletal muscle from normal and mdx mice, which are missing dystrophin. Immunoblot analysis using total muscle membranes from control and mdx mice of ages 1 d to 30 wk found that all of the dystrophin-associated proteins were greatly reduced (80-90%) in mdx mouse skeletal muscle. The specificity of the loss of the dystrophin-associated glycoproteins was demonstrated by the finding that the major glycoprotein composition of skeletal muscle membranes from normal and mdx mice was identical. Furthermore, skeletal muscle membranes from the dystrophic dy/dy mouse exhibited a normal density of dystrophin and dystrophin-associated proteins. Immunofluorescence microscopy confirmed the results from the immunoblot analysis and showed a drastically reduced density of dystrophin-associated proteins in mdx muscle cryosections compared with normal and dy/dy mouse muscle. Therefore, our results demonstrate that all of the dystrophin-associated proteins are significantly reduced in mdx skeletal muscle and suggest that the loss of dystrophin-associated proteins is due to the absence of dystrophin and not due to secondary effects of muscle fiber degradation.