Showing papers in "Journal of Cell Biology in 1994"
TL;DR: It is demonstrated that apoptosis was induced by disruption of the interactions between normal epithelial cells and extracellular matrix, and the circumvention of anoikis accompanies the acquisition of anchorage independence or cell motility.
Abstract: Cell-matrix interactions have major effects upon phenotypic features such as gene regulation, cytoskeletal structure, differentiation, and aspects of cell growth control. Programmed cell death (apoptosis) is crucial for maintaining appropriate cell number and tissue organization. It was therefore of interest to determine whether cell-matrix interactions affect apoptosis. The present report demonstrates that apoptosis was induced by disruption of the interactions between normal epithelial cells and extracellular matrix. We have termed this phenomenon "anoikis." Overexpression of bcl-2 protected cells against anoikis. Cellular sensitivity to anoikis was apparently regulated: (a) anoikis did not occur in normal fibroblasts; (b) it was abrogated in epithelial cells by transformation with v-Ha-ras, v-src, or treatment with phorbol ester; (c) sensitivity to anoikis was conferred upon HT1080 cells or v-Ha-ras-transformed MDCK cells by reverse-transformation with adenovirus E1a; (d) anoikis in MDCK cells was alleviated by the motility factor, scatter factor. The results suggest that the circumvention of anoikis accompanies the acquisition of anchorage independence or cell motility.
3,134 citations
TL;DR: The bcl-2 gene has emerged as a critical regulator of PCD in a variety of physiological and pathological contexts and can contribute to the pathogenesis of many diseases ranging from cancer to AIDS.
Abstract: T HE terms programmed cell death (PCD) 1 and apoptosis are often used interchangeably to describe a mechanism of cellular demise that is believed to play an important role in a wide variety of physiological situations, and that when dysregulated can contribute to the pathogenesis of many diseases ranging from cancer to AIDS. Recently, the bcl-2 gene has emerged as a critical regulator of PCD in a variety of physiological and pathological contexts.
2,334 citations
TL;DR: Results indicate that BMP-2 specifically converts the differentiation pathway of C2C12 myoblasts into that of osteoblast lineage cells, but that the conversion is not heritable.
Abstract: The implantation of bone morphogenetic protein (BMP) into muscular tissues induces ectopic bone formation at the site of implantation. To investigate the mechanism underlying this process, we examined whether recombinant bone morphogenetic protein-2 (BMP-2) converts the differentiation pathway of the clonal myoblastic cell line, C2C12, into that of osteoblast lineage. Incubating the cells with 300 ng/ml of BMP-2 for 6 d almost completely inhibited the formation of the multinucleated myotubes expressing troponin T and myosin heavy chain, and induced the appearance of numerous alkaline phosphatase (ALP)-positive cells. BMP-2 dose dependently induced ALP activity, parathyroid hormone (PTH)-dependent 3',5'-cAMP production, and osteocalcin production at concentrations above 100 ng/ml. The concentration of BMP-2 required to induce these osteoblastic phenotypes was the same as that required to almost completely inhibit myotube formation. Incubating primary muscle cells with 300 ng/ml of BMP-2 for 6 d also inhibited myotube formation, whereas induced ALP activity and osteocalcin production. Incubation with 300 ng/ml of BMP-2 suppressed the expression of mRNA for muscle creatine kinase within 6 h, whereas it induced mRNA expression for ALP, PTH/PTH-related protein (PTHrP) receptors, and osteocalcin within 24-48 h. BMP-2 completely inhibited the expression of myogenin mRNA by day 3. By day 3, BMP-2 also inhibited the expression of MyoD mRNA, but it was transiently stimulated 12 h after exposure to BMP-2. Expression of Id-1 mRNA was greatly stimulated by BMP-2. When C2C12 cells pretreated with BMP-2 for 6 d were transferred to a colony assay system in the absence of BMP-2, more than 84% of the colonies generated became troponin T-positive and ALP activity disappeared. TGF-beta 1 also inhibited myotube formation in C2C12 cells, and suppressed the expression of myogenin and MyoD mRNAs without inducing that of Id-1 mRNA. However, no osteoblastic phenotype was induced by TGF-beta 1 in C2C12 cells. TGF-beta 1 potentiated the inhibitory effect of BMP-2 on myotube formation, whereas TGF-beta 1 reduced ALP activity and osteocalcin production induced by BMP-2 in C2C12 cells. These results indicate that BMP-2 specifically converts the differentiation pathway of C2C12 myoblasts into that of osteoblast lineage cells, but that the conversion is not heritable.
1,410 citations
TL;DR: It is concluded that dynamin is specifically required for endocytic coated vesicle formation, and that its GTP binding and hydrolysis activities are required to form constricted coated pits and, subsequently, for coatedvesicle budding.
Abstract: Dynamin is the mammalian homologue to the Drosophila shibire gene product. Mutations in this 100-kD GTPase cause a pleiotropic defect in endocytosis. To further investigate its role, we generated stable HeLa cell lines expressing either wild-type dynamin or a mutant defective in GTP binding and hydrolysis driven by a tightly controlled, tetracycline-inducible promoter. Overexpression of wild-type dynamin had no effect. In contrast, coated pits failed to become constricted and coated vesicles failed to bud in cells overexpressing mutant dynamin so that endocytosis via both transferrin (Tfn) and EGF receptors was potently inhibited. Coated pit assembly, invagination, and the recruitment of receptors into coated pits were unaffected. Other vesicular transport pathways, including Tfn receptor recycling, Tfn receptor biosynthesis, and cathepsin D transport to lysosomes via Golgi-derived coated vesicles, were unaffected. Bulk fluid-phase uptake also continued at the same initial rates as wild type. EM immunolocalization showed that membrane-bound dynamin was specifically associated with clathrin-coated pits on the plasma membrane. Dynamin was also associated with isolated coated vesicles, suggesting that it plays a role in vesicle budding. Like the Drosophila shibire mutant, HeLa cells overexpressing mutant dynamin accumulated long tubules, many of which remained connected to the plasma membrane. We conclude that dynamin is specifically required for endocytic coated vesicle formation, and that its GTP binding and hydrolysis activities are required to form constricted coated pits and, subsequently, for coated vesicle budding.
1,234 citations
TL;DR: A clinical case reconstructed by Guido Majno (1975) from the Hippocratic records describes a wrestler who visits the iatreion (out-patient clinic) to be treated for a shoulder dislocation, and a drastic measure of inducing wound contraction is adopted by poking a hot needle through the skin of the armpit.
Abstract: clinical case reconstructed by Guido Majno (1975) from the Hippocratic records describes a wrestler who visits the iatreion (out-patient clinic) to be treated for a shoulder dislocation. With less invasive procedures no longer working to tighten the dislocation, the clinic adopts the drastic measure of inducing wound contraction by poking a hot needle through the skin of the armpit, and \"in this way the cavity, into which the humerous is mostly displaced, is best scarred over and cut off\" In Greek medicine circa 400 B.C.E., familiarity with wound contraction after burn injury already was commonplace. Closure of cutaneous wounds involves three processes: epithelization, connective tissue deposition, and contraction. The contribution of each process varies according to the type of wound. In general, epithelization results in resurfacing of the wound; connective tissue deposition results in replacement of damaged dermis; and contraction brings the margins of open wounds together (Peacock, 1984; Clark, 1988; Mast, 1992). In mammals with loose skin (meaning loosely attached to the underlying tissue layer), wound contraction leads to wound closure with little scarring or loss of function. In humans, whose skin is more firmly attached to underlying tissues, the consequences of contraction are less beneficial, ranging from minimal cosmetic scar in some cases to loss of joint motion or major body deformation in others. Consequently, a distinction has been made between contraction as a normal process of wound closure, and contracture as the abnormal result of the contraction process where signifcant scarring or loss of function occurs (Hunt and Dunphy, 1979). The pathologic consequences of tissue contraction include a variety of conditions ranging from contracture of the fibrous capsule surrounding breast implants to constricture of hollow organs (e.g., the esophagus) after injury (Skalli and Gabbiani, 1988; Rudolph et al., 1992). In contemporary cell biology, research on wound contraction focuses on the wound fibroblast. Skin fibroblasts normally are sessile and quiescent, but shortly after cutaneous wounding, they become activated. Activated fibroblasts migrate to the fibronectin-fibrin wound interface, proliferate, and synthesize a new collagen-containing matrix called granulation tissue. Around the same time, wound contraction begins. Once the wound defect is replaced, the expanded fibroblast population stops dividing and regresses and extracellular matrix remodeling commences (Peacock, 1984; Clark, 1993). Despite the importance of wound contraction for wound
1,099 citations
TL;DR: The properties of these cells after transplantation--the stability of resulting hybrid myofibers without immune suppression, the persistence of transgene expression, and the lack of tumorigenicity-- suggest that studies of cell-mediated gene therapy using primary myoblasts can now be broadly applied to mouse models of human muscle and non-muscle diseases.
Abstract: The transplantation of cultured myoblasts into mature skeletal muscle is the basis for a new therapeutic approach to muscle and non-muscle diseases: myoblast-mediated gene therapy. The success of myoblast transplantation for correction of intrinsic muscle defects depends on the fusion of implanted cells with host myofibers. Previous studies in mice have been problematic because they have involved transplantation of established myogenic cell lines or primary muscle cultures. Both of these cell populations have disadvantages: myogenic cell lines are tumorigenic, and primary cultures contain a substantial percentage of non-myogenic cells which will not fuse to host fibers. Furthermore, for both cell populations, immune suppression of the host has been necessary for long-term retention of transplanted cells. To overcome these difficulties, we developed novel culture conditions that permit the purification of mouse myoblasts from primary cultures. Both enriched and clonal populations of primary myoblasts were characterized in assays of cell proliferation and differentiation. Primary myoblasts were dependent on added bFGF for growth and retained the ability to differentiate even after 30 population doublings. The fate of the pure myoblast populations after transplantation was monitored by labeling the cells with the marker enzyme beta-galactosidase (beta-gal) using retroviral mediated gene transfer. Within five days of transplantation into muscle of mature mice, primary myoblasts had fused with host muscle cells to form hybrid myofibers. To examine the immunobiology of primary myoblasts, we compared transplanted cells in syngeneic and allogeneic hosts. Even without immune suppression, the hybrid fibers persisted with continued beta-gal expression up to six months after myoblast transplantation in syngeneic hosts. In allogeneic hosts, the implanted cells were completely eliminated within three weeks. To assess tumorigenicity, primary myoblasts and myoblasts from the C2 myogenic cell line were transplanted into immunodeficient mice. Only C2 myoblasts formed tumors. The ease of isolation, growth, and transfection of primary mouse myoblasts under the conditions described here expand the opportunities to study muscle cell growth and differentiation using myoblasts from normal as well as mutant strains of mice. The properties of these cells after transplantation--the stability of resulting hybrid myofibers without immune suppression, the persistence of transgene expression, and the lack of tumorigenicity--suggest that studies of cell-mediated gene therapy using primary myoblasts can now be broadly applied to mouse models of human muscle and non-muscle diseases.
975 citations
TL;DR: It is concluded that occludin itself can localize at TJ and directly associate with ZO-1, and the association with underlying cytoskeletons through Zo-1 is required for occluda to be localized at TJ.
Abstract: Occludin is an integral membrane protein localizing at tight junctions (TJ) with four transmembrane domains and a long COOH-terminal cytoplasmic domain (domain E) consisting of 255 amino acids. Immunofluorescence and laser scan microscopy revealed that chick full-length occludin introduced into human and bovine epithelial cells was correctly delivered to and incorporated into preexisting TJ. Further transfection studies with various deletion mutants showed that the domain E, especially its COOH-terminal approximately 150 amino acids (domain E358/504), was necessary for the localization of occludin at TJ. Secondly, domain E was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and this fusion protein was shown to be specifically bound to a complex of ZO-1 (220 kD) and ZO-2 (160 kD) among various membrane peripheral proteins. In vitro binding analyses using glutathione-S-transferase fusion proteins of various deletion mutants of domain E narrowed down the sequence necessary for the ZO-1/ZO-2 association into the domain E358/504. Furthermore, this region directly associated with the recombinant ZO-1 produced in E. coli. We concluded that occludin itself can localize at TJ and directly associate with ZO-1. The coincidence of the sequence necessary for the ZO-1 association with that for the TJ localization suggests that the association with underlying cytoskeletons through ZO-1 is required for occludin to be localized at TJ.
946 citations
TL;DR: It is concluded that TGF-beta has the ability to modulate E-cadherin expression and induce a reversible epithelial to mesenchymal transdifferentiation in epithelial cells.
Abstract: The secreted polypeptide transforming growth factor-beta (TGF-beta) exerts its multiple activities through type I and II cell surface receptors. In epithelial cells, activation of the TGF-beta signal transduction pathways leads to inhibition of cell proliferation and an increase in extracellular matrix production. TGF-beta is widely expressed during development and its biological activity has been implicated in epithelial-mesenchymal interactions, e.g., in branching morphogenesis of the lung, kidney, and mammary gland, and in inductive events between mammary epithelium and stroma. In the present study, we investigated the effects of TGF-beta on mouse mammary epithelial cells in vitro. TGF-beta reversibly induced an alteration in the differentiation of normal mammary epithelial NMuMG cells from epithelial to fibroblastic phenotype. The change in cell morphology correlated with (a) decreased expression of the epithelial markers E-cadherin, ZO-1, and desmoplakin I and II; (b) increased expression of mesenchymal markers, such as fibronectin; and (c) a fibroblast-like reorganization of actin fibers. This phenotypic differentiation displays the hallmarks of an epithelial to mesenchymal transdifferentiation event. Since NMuMG cells make high levels of the type I TGF-beta receptor Tsk7L, yet lack expression of the ALK-5/R4 type I receptor which has been reported to mediate TGF-beta responsiveness, we evaluated the role of the Tsk7L receptor in TGF-beta-mediated transdifferentiation. We generated NMuMG cells that stably overexpress a truncated Tsk7L type I receptor that lacks most of the cytoplasmic kinase domain, thus function as a dominant negative mutant. These transfected cells no longer underwent epithelial to mesenchymal morphological change upon exposure to TGF-beta, yet still displayed some TGF-beta-mediated responses. We conclude that TGF-beta has the ability to modulate E-cadherin expression and induce a reversible epithelial to mesenchymal transdifferentiation in epithelial cells. Unlike other transdifferentiating growth factors, such as bFGF and HGF, these changes are accompanied by growth inhibition. Our results also implicate the Tsk7L type I receptor as mediating the TGF-beta-induced epithelial to mesenchymal transition.
940 citations
TL;DR: Characterization of these complexes by micro-sequencing and immuno- blotting reveals known receptors for modified forms of LDL, multiple GPI-linked proteins, an anion transporter, cytoskeletal elements, and cytoplasmic signaling molecules--including Src-like kinases, hetero- trimeric G-proteins, and three members of the Rap family of small GTPases.
Abstract: Caveolae are 50-100-nm membrane microdomains that represent a subcompartment of the plasma membrane. Previous morphological studies have implicated caveolae in (a) the transcytosis of macromolecules (including LDL and modified LDLs) across capillary endothelial cells, (b) the uptake of small molecules via a process termed potocytosis involving GPI-linked receptor molecules and an unknown anion transport protein, (c) interactions with the actin-based cytoskeleton, and (d) the compartmentalization of certain signaling molecules, including G-protein coupled receptors. Caveolin, a 22-kD integral membrane protein, is an important structural component of caveolae that was first identified as a major v-Src substrate in Rous sarcoma virus transformed cells. This finding initially suggested a relationship between caveolin, transmembrane signaling, and cellular transformation. We have recently developed a procedure for isolating caveolin-rich membrane domains from cultured cells. To facilitate biochemical manipulations, we have applied this procedure to lung tissue--an endothelial and caveolin-rich source-allowing large scale preparation of these complexes. These membrane domains retain approximately 85% of caveolin and approximately 55% of a GPI-linked marker protein, while they exclude > or = 98% of integral plasma membrane protein markers and > or = 99.6% of other organelle-specific membrane markers tested. Characterization of these complexes by micro-sequencing and immuno-blotting reveals known receptors for modified forms of LDL (scavenger receptors: CD 36 and RAGE), multiple GPI-linked proteins, an anion transporter (plasma membrane porin), cytoskeletal elements, and cytoplasmic signaling molecules--including Src-like kinases, hetero-trimeric G-proteins, and three members of the Rap family of small GTPases (Rap 1--the Ras tumor suppressor protein, Rap 2, and TC21). At least a fraction of the actin in these complexes appeared monomeric (G-actin), suggesting that these domains could represent membrane bound sites for microfilament nucleation/assembly during signaling. Given that the majority of these proteins are known molecules, our current studies provide a systematic basis for evaluating these interactions in vivo.
926 citations
TL;DR: It is shown that the ability of sterol binding agents such as filipin to disassemble endothelial noncoated but not coated plasmalemmal vesicles selectively inhibits caveolae-mediated intracellular and transcellular transport of select macromolecules in endothelium.
Abstract: Caveolae or noncoated plasmalemmal vesicles found in a variety of cells have been implicated in a number of important cellular functions including endocytosis, transcytosis, and potocytosis. Their function in transport across endothelium has been especially controversial, at least in part because there has not been any way to selectively inhibit this putative pathway. We now show that the ability of sterol binding agents such as filipin to disassemble endothelial noncoated but not coated plasmalemmal vesicles selectively inhibits caveolae-mediated intracellular and transcellular transport of select macromolecules in endothelium. Filipin significantly reduces the transcellular transport of insulin and albumin across cultured endothelial cell monolayers. Rat lung microvascular permeability to albumin in situ is significantly decreased after filipin perfusion. Conversely, paracellular transport of the small solute inulin is not inhibited in vitro or in situ. In addition, we show that caveolae mediate the scavenger endocytosis of conformationally modified albumins for delivery to endosomes and lysosomes for degradation. This intracellular transport is inhibited by filipin both in vitro and in situ. Other sterol binding agents including nystatin and digitonin also inhibit this degradative process. Conversely, the endocytosis and degradation of activated alpha 2-macroglobulin, a known ligand of the clathrin-dependent pathway, is not affected. Interestingly, filipin appears to inhibit insulin uptake by endothelium for transcytosis, a caveolae-mediated process, but not endocytosis for degradation, apparently mediated by the clathrin-coated pathway. Such selective inhibition of caveolae not only provides critical evidence for the role of caveolae in the intracellular and transcellular transport of select macromolecules in endothelium but also may be useful for distinguishing transport mediated by coated versus noncoated vesicles.
913 citations
TL;DR: Data from a PCR-based technique and in situ hybridization revealed that while c-jun was induced in most neurons, c-fos induction was restricted to neurons undergoing chromatin condensation, a hallmark of apoptosis, and these data may outline a genetic program responsible for neuronal programmed cell death.
Abstract: We have examined the hypothesis that neuronal programmed cell death requires a genetic program; we used a model wherein rat sympathetic neurons maintained in vitro are deprived of NGF and subsequently undergo apoptosis. To evaluate gene expression potentially necessary for this process, we used a PCR-based technique and in situ hybridization; patterns of general gene repression and selective gene induction were identified in NGF-deprived neurons. A temporal cascade of induced genes included "immediate early genes," which were remarkable in that their induction occurred hours after the initial stimulus of NGF removal and the synthesis of some required ongoing protein synthesis. The cascade also included the cell cycle gene c-myb and the genes encoding the extracellular matrix proteases transin and collagenase. Concurrent in situ hybridization and nuclear staining revealed that while c-jun was induced in most neurons, c-fos induction was restricted to neurons undergoing chromatin condensation, a hallmark of apoptosis. To evaluate the functional role of the proteins encoded by these genes, neutralizing antibodies were injected into neurons. Antibodies specific for either c-Jun or the Fos family (c-Fos, Fos B, Fra-1, and Fra-2) protected NGF-deprived neurons from apoptosis, whereas antibodies specific for Jun B, Jun D, or three nonimmune antibody preparations had no protective effect. Because these induced genes encode proteins ranging from a transcription factor necessary for death to proteases likely involved in tissue remodeling concurrent with death, these data may outline a genetic program responsible for neuronal programmed cell death.
TL;DR: It is concluded that ERM family members work as molecular linkers between the cytoplasmic domain of CD44 and actin-based cytoskeletons.
Abstract: The ERM family members, ezrin, radixin, and moesin, localizing just beneath the plasma membranes, are thought to be involved in the actin filament/plasma membrane association. To identify the integral membrane protein directly associated with ERM family members, we performed immunoprecipitation studies using antimoesin mAb and cultured baby hamster kidney (BHK) cells metabolically labeled with [35S]methionine or surface-labeled with biotin. The results indicated that moesin is directly associated with a 140-kD integral membrane protein. Using BHK cells as antigens, we obtained a mAb that recognized the 140-kD membrane protein. We next cloned a cDNA encoding the 140-kD membrane protein and identified it as CD44, a broadly distributed cell surface glycoprotein. Immunoprecipitation with various anti-CD44 mAbs showed that ezrin and radixin, as well as moesin, are associated with CD44, not only in BHK cells, but also in mouse L fibroblasts. Furthermore, immunofluorescence microscopy revealed that in both BHK and L cells, the Triton X-100-insoluble CD44 is precisely colocalized with ERM family members. We concluded that ERM family members work as molecular linkers between the cytoplasmic domain of CD44 and actin-based cytoskeletons.
TL;DR: Although the exact mechanism of internalization remains unknown, the results show that caveolae are dynamic structures which can be internalized into the cell and may be regulated by kinase activity and require an intact actin network.
Abstract: Caveolae are specialized invaginations of the plasma membrane which have been proposed to play a role in diverse cellular processes such as endocytosis and signal transduction. We have developed an assay to determine the fraction of internal versus plasma membrane caveolae. The GPI-anchored protein, alkaline phosphatase, was clustered in caveolae after antibody-induced crosslinking at low temperature and then, after various treatments, the relative amount of alkaline phosphatase on the cell surface was determined. Using this assay we were able to show a time- and temperature-dependent decrease in cell-surface alkaline phosphatase activity which was dependent on antibody-induced clustering. The decrease in cell surface alkaline phosphatase activity was greatly accelerated by the phosphatase inhibitor, okadaic acid, but not by a protein kinase C activator. Internalization of clustered alkaline phosphatase in the presence or absence of okadaic acid was blocked by cytochalasin D and by the kinase inhibitor staurosporine. Electron microscopy confirmed that okadaic acid induced removal of caveolae from the cell surface. In the presence of hypertonic medium this was followed by the redistribution of groups of caveolae to the center of the cell close to the microtubule-organizing center. This process was reversible, blocked by cytochalasin D, and the centralization of the caveolar clusters was shown to be dependent on an intact microtubule network. Although the exact mechanism of internalization remains unknown, the results show that caveolae are dynamic structures which can be internalized into the cell. This process may be regulated by kinase activity and require an intact actin network.
TL;DR: It is suggested that catenins represent an important link between EGF-induced signal transduction and cadherin function.
Abstract: Catenins mediate the linkage of classical cadherins with actin microfilaments and are part of a higher order protein structure by which cadherins are connected to other cytoplasmic and transmembrane proteins. The ratio of actin-bound to free cadherin-catenin complex, which varies depending on the type and growth rate of cells, is thought to be altered by cellular signals, such as those associated with mitosis, polarization of cells and growth factors during development. EGF induces an immediate tyrosine phosphorylation of beta-catenin and gamma-catenin (plakoglobin). We show here an association of the EGF-receptor with the cadherin-catenin complex. Using recombinant proteins we demonstrate the interaction of EGF-receptor and beta-catenin in in vitro kinase assays. This interaction is mediated by the evolutionarily conserved central "core" region of beta-catenin. These results suggest that catenins represent an important link between EGF-induced signal transduction and cadherin function.
TL;DR: Both cytoplasmic domains are required for energy-dependent, cell type-specific affinity modulation and mutations that disrupted a highly conserved alpha subunit GFFKR motif, resulted in high affinity binding of ligands to alpha IIb beta 3.
Abstract: We analyzed the binding of fibronectin to integrin alpha 5 beta 1 in various cells; in some cells fibronectin bound with low affinity (e.g., K562 cells) whereas in others (e.g., CHO), it bound with high affinity (Kd approximately 100 nM) in an energy-dependent manner. We constructed chimeras of the extracellular and transmembrane domains of alpha IIb beta 3 joined to the cytoplasmic domains of alpha 5 beta 1. The affinity state of these chimeras was assessed by binding of fibrinogen or the monoclonal antibody, PAC1. The cytoplasmic domains of alpha 5 beta 1 conferred an energy-dependent high affinity state on alpha IIb beta 3 in CHO but not K562 cells. Three additional alpha cytoplasmic domains (alpha 2, alpha 6A, alpha 6B) conferred PAC1 binding in CHO cells, while three others (alpha M, alpha L, alpha v) did not. In the high affinity alpha chimeras, cotransfection with a truncated (beta 3 delta 724) or mutated (beta 3(S752-->P)) beta 3 subunit abolished high affinity binding. Thus, both cytoplasmic domains are required for energy-dependent, cell type-specific affinity modulation. In addition, mutations that disrupted a highly conserved alpha subunit GFFKR motif, resulted in high affinity binding of ligands to alpha IIb beta 3. In contrast to the chimeras, the high affinity state of these mutants was independent of cellular metabolism, cell type, and the bulk of the beta subunit cytoplasmic domain. Thus, integrin cytoplasmic domains mediate inside-out signaling. Furthermore, the highly conserved GFFKR motif of the alpha subunit cytoplasmic domain maintains the default low affinity state.
TL;DR: Observations suggest that phagolysosome formation is a highly dynamic process that involves the gradual and regulated acquisition of markers from endocytic organelles.
Abstract: We have examined the modifications occurring during the transformation of phagosomes into phagolysosomes in J-774 macrophages. The use of low density latex beads as markers of phagosomes (latex bead compartments, LBC) allowed the isolation of these organelles by flotation on a simple sucrose gradient. Two-dimensional gel electrophoresis, immunocytochemistry, and biochemical assays have been used to characterize the composition of LBC at different time points after their formation, as well as their interactions with the organelles of the endocytic pathway. Our results show that LBC acquire and lose various markers during their transformation into phagolysosomes. Among these are members of the rab family of small GTPases as well as proteins of the lamp family. The transfer of the LBC of lamp 2, a membrane protein associated with late endocytic structures, was shown to be microtubule dependent. Video-microscopy showed that newly formed phagosomes were involved in rapid multiple contacts with late components of the endocytic pathway. Collectively, these observations suggest that phagolysosome formation is a highly dynamic process that involves the gradual and regulated acquisition of markers from endocytic organelles.
TL;DR: It is shown that the APC tumor suppressor gene product forms strikingly similar associations as found in cell junctions and suggested that beta-catenin and plakoglobin are central regulators of cell adhesion, cytoskeletal interaction, and tumor suppression.
Abstract: beta-Catenin is involved in the formation of adherens junctions of mammalian epithelia. It interacts with the cell adhesion molecule E-cadherin and also with the tumor suppressor gene product APC, and the Drosophila homologue of beta-catenin, armadillo, mediates morphogenetic signals. We demonstrate here that E-cadherin and APC directly compete for binding to the internal, armadillo-like repeats of beta-catenin; the NH2-terminal domain of beta-catenin mediates the interaction of the alternative E-cadherin and APC complexes to the cytoskeleton by binding to alpha-catenin. Plakoglobin (gamma-catenin), which is structurally related to beta-catenin, mediates identical interactions. We thus show that the APC tumor suppressor gene product forms strikingly similar associations as found in cell junctions and suggest that beta-catenin and plakoglobin are central regulators of cell adhesion, cytoskeletal interaction, and tumor suppression.
TL;DR: It is concluded that only one complex exists which contains cadherin and all of the catenins, and this complex is likely to be the E-cadherin/catenin complex.
Abstract: Calcium-dependent cell-cell adhesion is mediated by the cadherin family of cell adhesion proteins. Transduction of cadherin adhesion into cellular reorganization is regulated by cytosolic proteins, termed alpha-, beta-, and gamma-catenin (plakoglobin), that bind to the cytoplasmic domain of cadherins and link them to the cytoskeleton. Previous studies of cadherin/catenin complex assembly and organization relied on the coimmunoprecipitation of the complex with cadherin antibodies, and were limited to the analysis of the Triton X-100 (TX-100)-soluble fraction of these proteins. These studies concluded that only one complex exists which contains cadherin and all of the catenins. We raised antibodies specific for each catenin to analyze each protein independent of its association with E-cadherin. Extracts of Madin-Darby canine kidney epithelial cells were sequentially immunoprecipitated and immunoblotted with each antibody, and the results showed that there were complexes of E-cadherin/alpha-catenin, and either beta-catenin or plakoglobin in the TX-100-soluble fraction. We analyzed the assembly of cadherin/catenin complexes in the TX-100-soluble fraction by [35S]methionine pulse-chase labeling, followed by sucrose density gradient fractionation of proteins. Immediately after synthesis, E-cadherin, beta-catenin, and plakoglobin cosedimented as complexes. alpha-Catenin was not associated with these complexes after synthesis, but a subpopulation of alpha-catenin joined the complex at a time coincident with the arrival of E-cadherin at the plasma membrane. The arrival of E-cadherin at the plasma membrane coincided with an increase in its insolubility in TX-100, but extraction of this insoluble pool with 1% SDS disrupted the cadherin/catenin complex. Therefore, to examine protein complex assembly in both the TX-100-soluble and -insoluble fractions, we used [35S]methionine labeling followed by chemical cross-linking before cell extraction. Analysis of cross-linked complexes from cells labeled to steady state indicates that, in addition to cadherin/catenin complexes, there were cadherin-independent pools of catenins present in both the TX-100-soluble and -insoluble fractions. Metabolic labeling followed by chase showed that immediately after synthesis, cadherin/beta-catenin, and cadherin/plakoglobin complexes were present in the TX-100-soluble fraction. Approximately 50% of complexes were titrated into the TX-100-insoluble fraction coincident with the arrival of the complexes at the plasma membrane and the assembly of alpha-catenin. Subsequently, > 90% of labeled cadherin, but no additional labeled catenin complexes, entered the TX-100-insoluble fraction.(ABSTRACT TRUNCATED AT 400 WORDS)
TL;DR: RhoA is essential for receptor-mediated force generation and ensuing neurite retraction in N1E-115 and PC12 cells, and that inactivation of RhoA by ADP-ribosylation abolishes actomyosin contractility and promotes neurite outgrowth.
Abstract: Addition of the bioactive phospholipid lysophosphatidic acid (LPA) or a thrombin receptor-activating peptide (TRP) to serum-starved N1E-115 or NG108-15 neuronal cells causes rapid growth cone collapse, neurite retraction, and transient rounding of the cell body. These shape changes appear to be driven by receptor-mediated contraction of the cortical actomyosin system independent of classic second messengers. Treatment of the cells with Clostridium botulinum C3 exoenzyme, which ADP-ribosylates and thereby inactivates the Rho small GTP-binding protein, inhibits LPA- and TRP-induced force generation and subsequent shape changes. C3 also inhibits LPA-induced neurite retraction in PC12 cells. Biochemical analysis reveals that the ADP-ribosylated substrate is RhoA. Prolonged C3 treatment of cells maintained in 10% serum induces the phenotype of serum-starved cells, with initial cell flattening being followed by neurite outgrowth; such C3-differentiated cells fail to retract their neurites in response to agonists. We conclude that RhoA is essential for receptor-mediated force generation and ensuing neurite retraction in N1E-115 and PC12 cells, and that inactivation of RhoA by ADP-ribosylation abolishes actomyosin contractility and promotes neurite outgrowth.
TL;DR: It is concluded that vertebrate somatic cells possess a metaphase-anaphase checkpoint control that monitors sister kinetochore attachment to the spindle, and that the chromosomes move poleward during anaphase.
Abstract: To test the popular but unproven assumption that the metaphase-anaphase transition in vertebrate somatic cells is subject to a checkpoint that monitors chromosome (i.e., kinetochore) attachment to the spindle, we filmed mitosis in 126 PtK1 cells. We found that the time from nuclear envelope breakdown to anaphase onset is linearly related (r2 = 0.85) to the duration the cell has unattached kinetochores, and that even a single unattached kinetochore delays anaphase onset. We also found that anaphase is initiated at a relatively constant 23-min average interval after the last kinetochore attaches, regardless of how long the cell possessed unattached kinetochores. From these results we conclude that vertebrate somatic cells possess a metaphase-anaphase checkpoint control that monitors sister kinetochore attachment to the spindle. We also found that some cells treated with 0.3-0.75 nM Taxol, after the last kinetochore attached to the spindle, entered anaphase and completed normal poleward chromosome motion (anaphase A) up to 3 h after the treatment--well beyond the 9-48-min range exhibited by untreated cells. The fact that spindle bipolarity and the metaphase alignment of kinetochores are maintained in these cells, and that the chromosomes move poleward during anaphase, suggests that the checkpoint monitors more than just the attachment of microtubules at sister kinetochores or the metaphase alignment of chromosomes. Our data are most consistent with the hypothesis that the checkpoint monitors an increase in tension between kinetochores and their associated microtubules as biorientation occurs.
TL;DR: It is demonstrated here that EGP40 is an epithelium-specific intercellular adhesion molecule that resembles to some extent the molecules of the immunoglobulin superfamily of cell adhesion molecules (CAMs), although no immunoglobeulin-like repeats are present in the EGP30 molecule.
Abstract: The epithelial glycoprotein 40 (EGP40, also known as GA733-2, ESA, KSA, and the 17-1A antigen), encoded by the GA-733-2 gene, is expressed on the baso-lateral cell surface in most human simple epithelia. The protein is also expressed in the vast majority of carcinomas and has attracted attention as a tumor marker. The function of the protein is unknown. We demonstrate here that EGP40 is an epithelium-specific intercellular adhesion molecule. The molecule mediates, in a Ca(2+)-independent manner, a homophilic cell-cell adhesion of murine cells transfected with the complete EGP40 cDNA. Two murine cell lines were tested for the effects of EGP40 expression: fibroblastic L cells and dedifferentiated mammary carcinoma L153S cells. The expression of the EGP40 protein causes morphological changes in cultures of transfected cells--increasing intercellular adhesion of the transfectants--and has a clear effect on cell aggregating behavior in suspension aggregation assays. EGP40 directs sorting in mixed cell populations, in particular, causes segregation of the transfectants from the corresponding parental cells. EGP40 expression suppresses invasive colony growth of L cells in EHS-matrigel providing tight adhesions between cells in growing colonies. EGP40 can thus be considered a new member of the intercellular adhesion molecules. In its biological behavior EGP40 resembles to some extent the molecules of the immunoglobulin superfamily of cell adhesion molecules (CAMs), although no immunoglobulin-like repeats are present in the EGP40 molecule. Certain structural similarities in general organization of the molecule exist between EGP40 and the lin-12/Notch proteins. A possible role of this adhesion molecule in formation of architecture of epithelial tissues is discussed. To reflect the function of the molecule the name Ep-CAM for EGP40 seems appropriate.
TL;DR: Dramatic change in lamellipodia structure, and actin stress fiber organization was observed using the same antizipcode oligonucleotides which delocalized the mRNA, suggesting discrete 3'UTR sequences direct beta-actin isoform synthesis to the leading lamellae and affect cell morphology, presumably through the actin cytoskeleton.
Abstract: We have characterized the structure and function of RNA sequences that direct beta-cytoplasmic actin mRNA to the cell periphery were mapped to two segments of 3'-untranslated region by expression of LacZ/beta-actin chimeric mRNAs in chicken embryo fibroblasts (CEFs). A 54-nt segment, the "RNA zipcode," and a homologous but less active 43-nt segment each localized beta-galactosidase activity to the leading lamellae. This zipcode contains the full activity, and mutations or deletions within it reduce, but do not eliminate, its activity, indicating that several motifs contribute to the activity. Two of these motifs, when multimerized, can regenerate almost full activity. These sequences are highly conserved in evolution, since the human beta-actin zipcode, positioned identically in the 3'UTR localizes equally well in chicken cells. Complementary phosphorothioate oligonucleotides against the zipcode delocalized endogenous beta-actin mRNA, whereas those complementary to the region just outside the zipcode, or sense oligonucleotides, did not. Actin mRNA or protein levels were unaffected by the antisense treatments, but a dramatic change in lamellipodia structure, and actin stress fiber organization was observed using the same antizipcode oligonucleotides which delocalized the mRNA. Hence, discrete 3'UTR sequences direct beta-actin isoform synthesis to the leading lamellae and affect cell morphology, presumably through the actin cytoskeleton.
TL;DR: Four polypeptides of 47, 44, 40, and 35 kD that bind to profilin-Sepharose and elute with high salt are identified and appear to be novel proteins.
Abstract: We identified four polypeptides of 47, 44, 40, and 35 kD that bind to profilin-Sepharose and elute with high salt. When purified by conventional chromatography using an antibody to the 47-kD polypeptide, these four polypeptides copurified as a stoichiometric complex together with three additional polypeptides of 19, 18, and 13 kD that varied in their proportions to the other polypeptides. Partial protein sequences showed that the 47-kD polypeptide is a homologue of S. pombe act2 and the 44-kD polypeptide is a homologue of S. cerevisiae ACT2, both unconventional actins. The 40-kD polypeptide contains a sequence similar to the WD40 motif of the G beta subunit of a trimeric G-protein from Dictyostelium discoideum. From partial sequences, the 35-, 19-, and 18-kD polypeptides appear to be novel proteins. On gel filtration the complex of purified polypeptides cochromatograph with a Stokes' radius of 4.8 nm, a value consistent with a globular particle of 220 kD containing one copy of each polypeptide. Cell extracts also contain components of the complex that do not bind the profilin column. Affinity purified antibodies localize 47- and 18/19-kD polypeptides in the cortex and filopodia of Acanthamoeba. Antibodies to the 47-kD unconventional actin cross-react on immunoblots with polypeptides of similar size in Dictyostelium, rabbit muscle, and conventional preparations of rabbit muscle actin but do not react with actin.
TL;DR: Data indicate that cell attachment to matrix or integrin binding per se is not sufficient for maintaining cell viability, and that cells need to undergo some minimal degree of shape change to survive.
Abstract: When cultivated on substrates that prevent cell adhesion (the polymer polyhydroxyethylmethacrylate, bovine serum albumin, and Teflon), human endothelial cells (EC) rapidly lost viability with a half-life of approximately 10 h. Dying EC showed the morphological and biochemical characteristics of apoptosis. The apoptotic process of suspended EC was delayed by the protein synthesis inhibitor cycloheximide. To obtain information as to the mechanism involved in the apoptosis of suspended EC, we investigated whether adhesion to matrix proteins or integrin occupancy in EC retaining a round shape may affect EC suicide. EC bound to low coating concentration of either fibronectin or vitronectin, retaining a round shape and failing to organize actin microfilaments, underwent to rapid cell death; by contrast, cells on high substrate concentrations became flattened, showed actin microfilament organization, and retained viability. Addition of saturating amounts of soluble vitronectin to suspended round-shaped EC did not reduce the process of apoptosis. Finally, when suspended EC bound Gly-Arg-Gly-Asp-Ser-coated microbeads (approximately 10 microbeads/cell), yet retaining a round shape, the apoptotic process was not affected. Oncogene-transformed EC in suspension were less susceptible to cell death and apoptosis than normal EC. Overall, these data indicate that cell attachment to matrix or integrin binding per se is not sufficient for maintaining cell viability, and that cells need to undergo some minimal degree of shape change to survive. Modulation of interaction with the extracellular matrix can, therefore, be an important target for the control of angiogenesis.
TL;DR: Findings suggest that H2O2 acts as a mediator for the TGF-beta 1-induced transcription of egr-1 gene.
Abstract: TGF-beta 1 controls the expression of numerous genes, including early response and cellular matrix genes. However, the signal-transducing mechanism underlying this regulation of gene expression is not fully understood. In this study, we investigated whether redox regulation plays a role in the TGF-beta 1 signal transduction in the mouse osteoblastic cell line (MC3T3-E1). The overall intracellular oxidized state of the cells, when measured using 29,79-dichlorofluorescin diacetate by laser-scanning confocal microscopy, was increased transiently after the addition of TGF-beta 1. This increase was abolished by the addition of oxygen radical scavengers such as catalase and N-acetylcysteine. In a variant cell line lacking the TGF-beta 1 receptor, the intracellular oxidized state was not modulated by treatment with TGF-beta 1. We then examined the expression of early growth response-1 (egr-1) gene, which is inducible by TGF-beta 1 and H2O2. Radical scavengers inhibited the induction of egr-1 by TGF-beta 1, but not that by 12-O-tetradecanoylphorbol-13 acetate. A nuclear run-on assay indicated that this inhibition was at the transcriptional level. From transient expression experiments using chloramphenicol acetyltransferase gene linked to serially deleted egr-1 gene 59-upstream region, the CArG element in the 59 flanking region of egr-1 was identified as an essential sequence in the transcriptional activation for both TGF-beta 1 and H2O2 stimulation. These findings suggest that H2O2 acts as a mediator for the TGF-beta 1-induced transcription of egr-1 gene.
TL;DR: The results suggest that the latent form of TGF-beta 1 associates with the extracellular matrix via LTBP, and that the release of latent TGF -beta 1 from the matrix is a consequence of proteolytic cleavage(s) of LTBP.
Abstract: The role of latent transforming growth factor-beta (TGF-beta) binding protein (LTBP) in the association of TGF-beta 1 to the extracellular matrix of cultured fibroblasts and HT-1080 fibrosarcoma cells was studied by immunochemical methods. The matrices were isolated from the cells, and the levels of LTBP and TGF-beta 1 were estimated by immunoblotting and immunoprecipitation. LTBP, TGF-beta 1, and its propeptide (latency-associated peptide, LAP) were found to associate to the extracellular matrix. Immunoblotting analysis indicated that treatment of the cells with plasmin resulted in a concomitant time and dose dependent release of both LTBP and TGF-beta 1 from the extracellular matrix to the supernatant. Comparison of molecular weights suggested that plasmin treatment resulted in the cleavage of LTBP from the high molecular weight fibroblast form to a form resembling the low molecular weight LTBP found in platelets. Pulse-chase and immunoprecipitation analysis indicated that both the free form of LTBP and LTBP complexed to latent TGF-beta were efficiently incorporated in the extracellular matrix, from where both complexes were slowly released to the culture medium. Addition of plasmin to the chase solution resulted, however, in a rapid release of LTBP from the matrix. Fibroblast derived LTBP was found to associate to the matrix of HT-1080 cells in a plasmin sensitive manner as shown by immunoprecipitation analysis. These results suggest that the latent form of TGF-beta 1 associates with the extracellular matrix via LTBP, and that the release of latent TGF-beta 1 from the matrix is a consequence of proteolytic cleavage(s) of LTBP.
TL;DR: Cloned a human cDNA that encodes the 17-kD histone-like centromere antigen, CENP-A, indicates that the assembly of centromeres is driven, at least in part, by the incorporation of a novel core histone into centromeric chromatin.
Abstract: Centromeres are the differentiated chromosomal domains that specify the mitotic behavior of chromosomes. To examine the molecular basis for the specification of centromeric chromatin, we have cloned a human cDNA that encodes the 17-kD histone-like centromere antigen, CENP-A. Two domains are evident in the 140 aa CENP-A polypeptide: a unique NH2-terminal domain and a 93-amino acid COOH-terminal domain that shares 62% identity with nucleosomal core protein, histone H3. An epitope tagged derivative of CENP-A was faithfully targeted to centromeres when expressed in a variety of animal cells and this targeting activity was shown to reside in the histone-like COOH-terminal domain of CENP-A. These data clearly indicate that the assembly of centromeres is driven, at least in part, by the incorporation of a novel core histone into centromeric chromatin.
TL;DR: CD44-9v and CD44-6v were the main variants that were transiently upregulated on T cells after mitogenic stimulation and on myelomonocytic cell lines by TNF alpha and IFN gamma treatment.
Abstract: CD44 is a ubiquitous surface molecule that exists as a number of isoforms, generated by alternative splicing of 10 "variant" exons. Little is known about the expression and function of the variant isoforms, except that certain isoforms may play a role in cancer metastasis. We produced mAbs against CD44 variant regions encoded by exons 4v, 6v, and 9v, by immunizing mice with a fusion protein spanning variant exons 3v to 10v. A comprehensive analysis of human tissues revealed that CD44 variant isoforms were expressed widely throughout the body, principally by epithelial cells. However there was differential expression of CD44 variant exons by different epithelia. Most epithelia expressed exon 9v, but much fewer expressed 6v or 4v. The regions of epithelia that expressed the highest levels of the variant isoforms were the generative cells, particularly the basal cells of stratified squamous epithelium, and of glandular epithelium. CD44 variant isoforms were also expressed differentially by leukocytes, with CD44-9v expressed at very low levels and CD44-6v and 4v virtually absent. However, CD44-9v and CD44-6v were the main variants that were transiently upregulated on T cells after mitogenic stimulation and on myelomonocytic cell lines by TNF alpha and IFN gamma treatment. Some epithelial cell lines could preferentially upregulate CD44-6v upon IFN gamma incubation. These results show that CD44 variant isoforms are expressed much more widely than first appreciated, and that expression of the variant isoforms on some cell types can be modulated by particular cytokines.
TL;DR: P appears to modulate both the proliferation and differentiation of chondrocytes and its absence alters the temporal and spatial sequence of epiphyseal cartilage development and of subsequent endochondral bone formation necessary for normal elongation of long bones.
Abstract: To elucidate the role of PTHrP in skeletal development, we examined the proximal tibial epiphysis and metaphysis of wild-type (PTHrP-normal) 18-19-d-old fetal mice and of chondrodystrophic litter mates homozygous for a disrupted PTHrP allele generated via homologous recombination in embryonic stem cells (PTHrP-depleted). In the PTHrP-normal epiphysis, immunocytochemistry showed PTHrP to be localized in chondrocytes within the resting zone and at the junction between proliferative and hypertrophic zones. In PTHrP-depleted epiphyses, a diminished [3H]thymidine-labeling index was observed in the resting and proliferative zones accounting for reduced numbers of epiphyseal chondrocytes and for a thinner epiphyseal plate. In the mutant hypertrophic zone, enlarged chondrocytes were interspersed with clusters of cells that did not hypertrophy, but resembled resting or proliferative chondrocytes. Although the overall content of type II collagen in the epiphyseal plate was diminished, the lacunae of these non-hypertrophic chondrocytes did react for type II collagen. Moreover, cell membrane-associated chondroitin sulfate immunoreactivity was evident on these cells. Despite the presence of alkaline phosphatase activity on these nonhypertrophic chondrocytes, the adjacent cartilage matrix did not calcify and their persistence accounted for distorted chondrocyte columns and sporadic distribution of calcified cartilage. Consequently, in the metaphysis, bone deposited on the irregular and sparse scaffold of calcified cartilage and resulted in mixed spicules that did not parallel the longitudinal axis of the tibia and were, therefore, inappropriate for bone elongation. Thus, PTHrP appears to modulate both the proliferation and differentiation of chondrocytes and its absence alters the temporal and spatial sequence of epiphyseal cartilage development and of subsequent endochondral bone formation necessary for normal elongation of long bones.
TL;DR: Results indicated that spherical structures completely enclosed in a double membrane were found near the vacuoles of protease-deficient mutant cells when the cells were shifted to nutrient-starvation media, suggesting that these structures are precursors of autophagic bodies, "autophagosomes" in yeast.
Abstract: Under nutrient-deficient conditions, the yeast S. cerevisiae sequesters its own cytoplasmic components into vacuoles in the form of "autophagic bodies" (Takeshige, K., M. Baba, S. Tsuboi, T. Noda, and Y. Ohsumi. 1992. J. Cell Biol. 119:301-311). Immunoelectron microscopy showed that two cytosolic marker enzymes, alcohol dehydrogenase and phosphoglycerate kinase, are present in the autophagic bodies at the same densities as in the cytosol, but are not present in vacuolar sap, suggesting that cytosolic enzymes are also taken up into the autophagic bodies. To understand this process, we performed morphological analyses by transmission and immunological electron microscopies using a freeze-substitution fixation method. Spherical structures completely enclosed in a double membrane were found near the vacuoles of protease-deficient mutant cells when the cells were shifted to nutrient-starvation media. Their size, membrane thickness, and contents of double membrane-structures corresponded well with those of autophagic bodies. Sometimes these double membrane structures were found to be in contact with the vacuolar membrane. Furthermore their outer membrane was occasionally seen to be continuous with the vacuolar membrane. Histochemical staining of carbohydrate strongly suggested that the structures with double membranes fused with the vacuoles. These results indicated that these structures are precursors of autophagic bodies, "autophagosomes" in yeast. All the data obtained suggested that the autophagic process in yeast is essentially similar to that of the lysosomal system in mammalian cells.