Showing papers in "Journal of Cell Science in 1999"
TL;DR: The involvement of nucleolin at multiple steps of this biosynthetic pathway suggests that it could play a key role in this highly integrated process of ribosome biogenesis.
Abstract: Nucleolin is an abundant protein of the nucleolus. Nucleolar proteins structurally related to nucleolin are found in organisms ranging from yeast to plants and mammals. The association of several structural domains in nucleolin allows the interaction of nucleolin with different proteins and RNA sequences. Nucleolin has been implicated in chromatin structure, rDNA transcription, rRNA maturation, ribosome assembly and nucleo-cytoplasmic transport. Studies of nucleolin over the last 25 years have revealed a fascinating role for nucleolin in ribosome biogenesis. The involvement of nucleolin at multiple steps of this biosynthetic pathway suggests that it could play a key role in this highly integrated process.
723 citations
TL;DR: It is shown that stable re-expression of epitope-tagged FAK reversed the morphological defects of the FAK- cells through the dynamic regulation of actin structures and focal contact sites in fibronectin (FN) stimulated cells.
Abstract: FAK localizes to sites of transmembrane integrin receptor clustering and facilitates intracellular signaling events. FAK-null (FAK-) fibroblasts exhibit a rounded morphology, defects in cell migration, and an elevated number of cell-substratum contact sites. Here we show that stable re-expression of epitope-tagged FAK reversed the morphological defects of the FAK- cells through the dynamic regulation of actin structures and focal contact sites in fibronectin (FN) stimulated cells. FAK re-expressing fibroblasts (clones DA2 and DP3) exhibit a characteristic fibrillar shape and display indistinguishable FN receptor-stimulated migration properties compared to normal fibroblasts. Expression of various FAK mutants in the FAK- cells showed that FAK kinase activity, the Tyr-397/SH2 domain binding site, and the first proline-rich SH3 binding region in the FAK C-terminal domain were individually needed to promote full FAK-mediated FAK- cell migration to FN whereas direct paxillin binding to FAK was not required. Expression of the FAK Phe-397 mutant did not promote FAK- cell migration and overexpression of p50(csk) in DA2 cells inhibited migration to FN suggesting that Src-family PTKs play important roles in FAK-mediated motility events. Expression of the FAK C-terminal domain, FRNK, promoted FAK dephosphorylation at Tyr-397 and potently blocked FAK-mediated cell migration. This dominant-negative effect of FRNK was reversed by a point mutation (Leu-1034 to Ser) which prevented FRNK localization to focal contact sites. Our results show that FAK functions as a key regulator of fibronectin receptor stimulated cell migration events through the recruitment of both SH2 and SH3 domain-containing signaling proteins to sites of integrin receptor clustering.
690 citations
TL;DR: Apart from providing stable membrane anchorage, GPI anchors have been implicated in the sequestration of GPI-anchored proteins into specialised membrane microdomains, known as lipid rafts, and in signal transduction events.
Abstract: The discovery of glycosylphosphatidylinositol (GPI) membrane anchors has had a significant impact on several areas of eukaryote cell biology. Studies of the African trypanosome, which expresses a dense surface coat of GPI-anchored variant surface glycoprotein, have played important roles in establishing the general structure of GPI membrane anchors and in delineating the pathway of GPI biosynthesis. The major cell-surface molecules of related parasites are also rich in GPI-anchored glycoproteins and/or GPI-related glycophospholipids, and differences in substrate specificity between enzymes of trypanosomal and mammalian GPI biosynthesis may have potential for the development of anti-parasite therapies. Apart from providing stable membrane anchorage, GPI anchors have been implicated in the sequestration of GPI-anchored proteins into specialised membrane microdomains, known as lipid rafts, and in signal transduction events.
608 citations
TL;DR: Current topics in the ADAMs field are reviewed, including the central role of fertilin in fertilization, the role of the TNF(&agr;) convertase in protein ectodomain processing, therole of Kuzbanian in Notch signaling, and links between ADAM's and processing of the amyloid-precursor protein.
Abstract: Metalloprotease-disintegrins (ADAMs) have captured our attention as key players in fertilization and in the processing of the ectodomains of proteins such as tumor necrosis factor (a) (TNF(a)), and because of their roles in Notch-mediated signaling, neurogenesis and muscle fusion. ADAMs are integral membrane glycoproteins that contain a disintegrin domain, which is related to snake-venom integrin ligands, and a metalloprotease domain (which can contain or lack a catalytic site). Here, we review and critically discuss current topics in the ADAMs field, including the central role of fertilin in fertilization, the role of the TNF(a) convertase in protein ectodomain processing, the role of Kuzbanian in Notch signaling, and links between ADAMs and processing of the amyloid-precursor protein.
568 citations
TL;DR: Using recombinant proteins, it is found that E-cadherin and lymphocyte-enhancer factor-1 (LEF-1) form mutually exclusive complexes with beta-catenin; the association of beta-Catenin with LEf-1 was competed out by the E- cadher in cytoplasmic domain.
Abstract: Beta-catenin is a multifunctional protein found in three cell compartments: the plasma membrane, the cytoplasm and the nucleus. The cell has developed elaborate ways of regulating the level and localization of beta-catenin to assure its specific function in each compartment. One aspect of this regulation is inherent in the structural organization of beta-catenin itself; most of its protein-interacting motifs overlap so that interaction with one partner can block binding of another at the same time. Using recombinant proteins, we found that E-cadherin and lymphocyte-enhancer factor-1 (LEF-1) form mutually exclusive complexes with beta-catenin; the association of beta-catenin with LEF-1 was competed out by the E-cadherin cytoplasmic domain. Similarly, LEF-1 and adenomatous polyposis coli (APC) formed separate, mutually exclusive complexes with beta-catenin. In Wnt-1-transfected C57MG cells, free beta-catenin accumulated and was able to associate with LEF-1. The absence of E-cadherin in E-cadherin-/- embryonic stem (ES) cells also led to an accumulation of free beta-catenin and its association with LEF-1, thereby mimicking Wnt signaling. beta-catenin/LEF-1-mediated transactivation in these cells was antagonized by transient expression of wild-type E-cadherin, but not of E-cadherin lacking the beta-catenin binding site. The potent ability of E-cadherin to recruit beta-catenin to the cell membrane and prevent its nuclear localization and transactivation was also demonstrated using SW480 colon carcinoma cells.
566 citations
TL;DR: Findings suggest a role for contractility in the generation of matrix adhesion diversity, and particularly informative was fluorescence ratio imaging, comparing the local labeling intensities of different plaque molecules, including vinculin, paxillin, tensin and phosphotyrosine-containing proteins.
Abstract: In this study we have examined for molecular heterogeneity of cell-matrix adhesions and the involvement of actomyosin contractility in the selective recruitment of different plaque proteins. For this purpose, we have developed a novel microscopic approach for molecular morphometry, based on automatic identification of matrix adhesions, followed by quantitative immunofluorescence and morphometric analysis. Particularly informative was fluorescence ratio imaging, comparing the local labeling intensities of different plaque molecules, including vinculin, paxillin, tensin and phosphotyrosine-containing proteins. Ratio imaging revealed considerable molecular heterogeneity between and within adhesion sites. Most striking were the differences between focal contacts, which are vinculin- and paxillin-rich and contain high levels of phosphotyrosine, and fibrillar adhesions, which are tensin-rich and contain little or no phosphotyrosine. Ratio imaging also revealed considerable variability in the molecular substructure of individual focal contacts, pointing to a non-uniform distribution of phosphotyrosine and the different plaque constituents. Studying the quantitative relationships between the various components of the submembrane plaque indicated that the levels of vinculin, paxillin and phosphotyrosine in adhesion sites are positively correlated with each other and negatively correlated with the levels of tensin. Tyrosine phosphorylation of focal contacts was highly sensitive to cellular contractility, and was diminished within 5 minutes after treatment with the kinase inhibitor H-7, an inhibitor of actomyosin contractility. This was followed by the loss of paxillin and vinculin from the focal adhesions. Tensin-rich fibrillar adhesions were relatively insensitive to H-7 treatment. These findings suggest a role for contractility in the generation of matrix adhesion diversity.
484 citations
TL;DR: The results suggest that corneal cells, like lens cells, may preferentially express water-soluble proteins, often enzymes, for controlling their optical properties.
Abstract: In vivo corneal light scattering measurements using a novel confocal microscope demonstrated greatly increased backscatter from corneal stromal fibrocytes (keratocytes) in opaque compared to transparent corneal tissue in both humans and rabbits. Additionally, two water-soluble proteins, transketolase (TKT) and aldehyde dehydrogenase class 1 (ALDH1), isolated from rabbit keratocytes showed unexpectedly abundant expression ( approximately 30% of the soluble protein) in transparent corneas and markedly reduced levels in opaque scleral fibroblasts or keratocytes from hazy, freeze injured regions of the cornea. Together these data suggest that the relatively high expressions of TKT and ALDH1 contribute to corneal transparency in the rabbit at the cellular level, reminiscent of enzyme-crystallins in the lens. We also note that ALDH1 accumulates in the rabbit corneal epithelial cells, rather than ALDH3 as seen in other mammals, consistent with the taxon-specificity observed among lens enzyme-crystallins. Our results suggest that corneal cells, like lens cells, may preferentially express water-soluble proteins, often enzymes, for controlling their optical properties.
472 citations
TL;DR: The data establish the endothelial cell spheroid-based three-dimensional angiogenic technique as a standardized, highly reproducible quantitative assay for in vitro angiogenesis studies and demonstrate that integrin-dependent matrix tensional forces control directional capillary sprouting and network formation.
Abstract: During angiogenesis, anastomosing capillary sprouts align to form complex three-dimensional networks of new blood vessels. Using an endothelial cell spheroid model that was developed to study endothelial cell differentiation processes, we have devised a novel collagen gel-based three-dimensional in vitro angiogenesis assay. In this assay, cell number-defined, gel-embedded endothelial cell spheroids act as a cellular delivery device, which serves as a focal starting point for the sprouting of lumenized capillary-like structures that can be induced to form complex anastomosing networks. Formation of capillary anastomoses is associated with tensional remodeling of the collagen matrix and directional sprouting of outgrowing capillaries towards each other. To analyze whether directional sprouting is dependent on cytokine gradients or on endothelial cell-derived tractional forces transduced through the extracellular matrix, we designed a matrix tension generator that enables the application of defined tensional forces on the extracellular matrix. Using this matrix tension generator, causal evidence is presented that tensional forces on a fibrillar extracellular matrix such as type I collagen, but not fibrin, are sufficient to guide directional outgrowth of endothelial cells. RGD peptides but not control RAD peptides disrupted the integrity of sprouting capillary-like structures and induced detachment of outgrowing endothelial cells cultured on top of collagen gels, but did not inhibit primary outgrowth of endothelial cells. The data establish the endothelial cell spheroid-based three-dimensional angiogenesis technique as a standardized, highly reproducible quantitative assay for in vitro angiogenesis studies and demonstrate that integrin-dependent matrix tensional forces control directional capillary sprouting and network formation.
456 citations
TL;DR: It is demonstrated that activated satellite cells, which express either Myf5 or MyoD, do not accumulate selectively on fast or slow muscle fibres, and all proliferating myoblasts express MyfD and part of the population is (beta)-galactosidase (Myf5) positive.
Abstract: Regeneration of adult skeletal muscle is an asynchronous process requiring the activation, proliferation and fusion of satellite cells, to form new muscle fibres. This study was designed to determine the pattern of expression in vivo of the two myogenic regulatory factors, Myf5 and MyoD during this process. Cardiotoxin was used to induce regeneration in the gastrocnemius and soleus muscles of heterozygous Myf5-nlacZ mice, and the muscles were assayed for the presence of (beta)-galactosidase (Myf5) and MyoD. Adult satellite cells identified by M-cadherin labelling, when activated, initially express either MyoD or Myf5 or both myogenic factors. Subsequently all proliferating myoblasts express MyoD and part of the population is (beta)-galactosidase (Myf5) positive. Furthermore, we demonstrate that activated satellite cells, which express either Myf5 or MyoD, do not accumulate selectively on fast or slow muscle fibres.
451 citations
TL;DR: TGF-(beta)1 signals potently and passes the activation threshold to evoke NMuMG cell transdifferentiation and induced the nuclear accumulation of Smad2 and/or Smad3, and the TGF-( beta)1 type I receptor (ALK-5) and its effectors mediate the epithelial to mesenchymal transition.
Abstract: The capacities of different transforming growth factor-(beta) (TGF-(beta)) superfamily members to drive epithelial to mesenchymal transdifferentiation of the murine mammary epithelial cell line NMuMG were investigated. TGF-(beta)1, but not activin A or osteogenic protein-1 (OP-1)/bone morphogenetic protein-7 (BMP-7), was able to induce morphological transformation of NMuMG cells as shown by reorganisation of the actin cytoskeleton and relocalisation/downregulation of E-cadherin and (beta)-catenin, an effect that was abrogated by the more general serine/threonine kinase and protein kinase C inhibitor, staurosporine. TGF-(beta)1 bound to TGF-(beta) type I receptor (T(beta)R-I)/ALK-5 and T(beta)R-II, but not to activin type I receptor (ActR-I)/ALK-2. Activin A bound to ActR-IB/ALK-4 and ActR-II, and BMP-7 bound to ActR-I/ALK-2, BMP type I receptor (BMPR-I)/ALK-3, ActR-II and BMPR-II. TGF-(beta)1 and BMP-7 activated the Smad-binding element (SBE)(4) promoter with equal potency, whereas activin A had no effect. Transfection of constitutively active (CA)-ALK-4 activated the 3TP promoter to the same extent as TGF-(beta)1 and CA-ALK-5 indicating that activin signalling downstream of type I receptors was functional in NMuMG cells. In agreement with this, activin A induced low levels of plasminogen activator inhibitor I expression compared to the high induction by TGF-(beta)1. In contrast to activin A and BMP-7, TGF-(beta)1 strongly induced Smad2 phosphorylation. Consistent with these findings, TGF-(beta)1 induced the nuclear accumulation of Smad2 and/or Smad3. In addition, NMuMG cells transiently infected with adenoviral vectors expressing high level CA-ALK-5 exhibited full transdifferentiation. On the other hand, infections with low level CA-ALK-5, which alone did not result in transdifferentiation, together with Smad2 and Smad4, or with Smad3 and Smad4 led to transdifferentiation. In conclusion, TGF-(beta)1 signals potently and passes the activation threshold to evoke NMuMG cell transdifferentiation. The TGF-(beta) type I receptor (ALK-5) and its effector Smad proteins mediate the epithelial to mesenchymal transition. Activin A does not induce mesenchymal transformation, presumably because the number of activin receptors is limited, while BMP-7-initiated signalling cannot mediate transdifferentiation.
435 citations
TL;DR: Results presented here show that EH domains are required for correct coated pit targeting of Eps15 and strongly suggest a role for Eps15 in coated pit assembly and more precisely a role in the docking of AP-2 onto the plasma membrane.
Abstract: Recent data have shown that Eps15, a newly identified component of clathrin-coated pits constitutively associated with the AP-2 complex, is required for receptor-mediated endocytosis. However, its precise function remains unknown. Interestingly, Eps15 contains three EH (Eps15-Homology) domains also found in proteins required for the internalization step of endocytosis in yeast. Results presented here show that EH domains are required for correct coated pit targeting of Eps15. Furthermore, when cells expressed an Eps15 mutant lacking EH domains, the plasma membrane punctate distribution of both AP-2 and clathrin was lost, implying the absence of coated pits. This was further confirmed by the fact that dynamin, a GTPase found in coated pits, was homogeneously redistributed on the plasma membrane and that endocytosis of transferrin, a specific marker of clathrin-dependent endocytosis, was strongly inhibited. Altogether, these results strongly suggest a role for Eps15 in coated pit assembly and more precisely a role for Eps15 in the docking of AP-2 onto the plasma membrane. This hypothesis is supported by the fact that a GFP fusion protein encoding the ear domain of (alpha)-adaptin, the AP-2 binding site for Eps15, was efficiently targeted to plasma membrane coated pits.
TL;DR: TNFalpha impairs epithelial barrier function by altering structure and function of the tight junction, which could be of pathogenic relevance in intestinal inflammation.
Abstract: Cytokines are supposed to be mediators in diarrhoeal diseases. The aim of this study is to characterize the effect of tumor necrosis factor-alpha (TNFalpha) on epithelial barrier function in the colonic epithelial cell line HT-29/B6. Active ion transport and barrier function were measured as short-circuit current and transepithelial electrical resistance (Rt), respectively. In parallel, freeze-fracture electron microscopy (EM) of tight junctions (TJ) and immunofluorescence microscopy of the zonula occludens protein-1 (ZO-1) were performed. Serosal addition of TNF(alpha) (100 ng/ml) decreased Rt by 81%. This effect was dose-dependent and could be mimicked by antibodies against the p55 form of the TNF receptor. Cytotoxic effects were excluded by a negative lactate dehydrogenase (LDH) assay. Immunofluorescence localization with anti-ZO-1 antibodies revealed no evidence for disruption of the monolayer after TNFalpha treatment. In freeze-fracture EM, TJ complexity was decreased by TNFalpha, as indicated by a decrease in the number of strands from 4.7 to 3.4. The tyrosine kinase blocker genistein and the protein kinase A inhibitor H-8 reduced the effect of TNFalpha. A combination of TNFalpha with interferon-gamma acted synergistically on the epithelial barrier. In conclusion, TNFalpha impairs epithelial barrier function by altering structure and function of the tight junction, which could be of pathogenic relevance in intestinal inflammation.
TL;DR: A model is proposed for an amplification step in chemotactically sensitive cells or growth cones that accounts for their extraordinary directional sensitivity and accounts for the generation of a stable cell polarity.
Abstract: A model is proposed for an amplification step in chemotactically sensitive cells or growth cones that accounts for their extraordinary directional sensitivity. It is assumed that cells have an intrinsic pattern forming system that generates the signals for extension of filopods and lamellipods. An external signal such as a graded cue is assumed to impose some directional preference onto the pattern formed. According to the model, a saturating, self-enhancing reaction is coupled with two antagonistic reactions. One antagonist equilibrates rapidly over the whole cell, causing competition between different surface elements of the cell cortex for activation. It will be won by those cortical regions of the cell that are exposed to the highest concentrations of the external graded cues. The second antagonistic reaction is assumed to act more locally and has a longer time constant. It causes a destabilization of peaks after they have formed. While the total activated area on the cell surface is maintained, the disappearance of some hot spots allows the formation of new ones, preferentially at positions specified by the actual external guiding signal. Computer simulations show that the model accounts for the highly dynamic behaviour of chemotactic cells and growth cones. In the absence of external signals, maxima of the internal signals emerge at random positions and disappear after some time. Travelling waves or oscillations in counter phase can emerge on the cell cortex, in agreement with observations reported in the literature. In other ranges of parameters, the model accounts for the generation of a stable cell polarity.
TL;DR: It is reported that HSP27 like alphaB-crystallin is associated with glial fibrillary acidic protein and vimentin intermediate filament networks in unstressed U373MG astrocytoma cells, suggesting that one of the major functions of the association of small heat shock proteins with intermediate filaments is to help manage the interactions that occur between filaments in their cellular networks.
Abstract: HSP27 and alphaB-crystallin are both members of the small heat shock protein family. alphaB-crystalllin has been proposed to modulate intermediate filaments and recently a mutation in alphaB-crystallin has been identified as the genetic basis of desmin related myopathy. This disease is characterised in its pathology by aggregates of intermediate filaments associated with alphaB-crystallin. Here we report that HSP27 like alphaB-crystallin is associated with glial fibrillary acidic protein and vimentin intermediate filament networks in unstressed U373MG astrocytoma cells. HSP27 is also associated with keratin filaments in MCF7 cells, indicating that this association is not restricted to a particular intermediate filament type. The association of sHSPs with both the soluble and filamentous intermediate filament fractions of U373 cells was demonstrated biochemically. Heat shock or drug treatments induced a co-collapse of intermediate filaments and associated small heat shock proteins. These data show that the presence of HSP27 or alphaB-crystallin could not prevent filament collapse and suggest that the purpose of this association is more than just filament binding. Indeed, in U373MG cells the intermediate filament association with small heat shock proteins is similar to that observed for another protein chaperone, HSC70. In order to discern the effect of different chaperone classes on intermediate filament network formation and maintenance, several in vitro assays were assessed. Of these, falling ball viscometry revealed a specific activity of small heat shock proteins compared to HSC70 that was apparently inactive in this assay. Intermediate filaments form a gel in the absence of small heat shock proteins. In contrast, inclusion of alphaB-crystallin or HSP27 prevented gel formation but not filament assembly. The transient transfection of GFAP into MCF7 cells was used to show that the induction of a completely separate network of intermediate filaments resulted in the specific association of the endogenous HSP27 with these new GFAP filaments. These data lead us to propose that one of the major functions of the association of small heat shock proteins with intermediate filaments is to help manage the interactions that occur between filaments in their cellular networks. This is achieved by protecting filaments against those non-covalent interactions that result when they come into very close proximity as seen from the viscosity experiments and which have the potential to induce intermediate filament aggregation as seen in some disease pathologies.
TL;DR: The data suggest that tau can control intracellular trafficking by affecting the attachment and detachment cycle of the motors, in particular by reducing the attachment of kinesin to microtubules, whereas the movement itself is unaffected.
Abstract: We have performed a real time analysis of fluorescence-tagged vesicle and mitochondria movement in living CHO cells transfected with microtubule-associated protein tau or its microtubule-binding domain. tau does not alter the speed of moving vesicles, but it affects the frequencies of attachment and detachment to the microtubule tracks. Thus, tau decreases the run lengths both for plus-end and minus-end directed motion to an equal extent. Reversals from minus-end to plus-end directed movement of single vesicles are strongly reduced by tau, but reversals in the opposite direction (plus to minus) are not. Analogous effects are observed with the transport of mitochondria and even with that of vimentin intermediate filaments. The net effect is a directional bias in the minus-end direction of microtubules which leads to the retraction of mitochondria or vimentin IFs towards the cell center. The data suggest that tau can control intracellular trafficking by affecting the attachment and detachment cycle of the motors, in particular by reducing the attachment of kinesin to microtubules, whereas the movement itself is unaffected.
TL;DR: Observations suggest that ARF6, a non-Rho family GTPase, can, by itself, alter cortical actin and can influence the ability of Rac1 to form lamellipodia, in part, by regulating its trafficking to the plasma membrane.
Abstract: The ARF6 GTPase regulates a novel endosomal-plasma membrane recycling pathway and influences cortical actin remodeling. Here we examined the relationship between ARF6 and Rac1, a Rho family GTPase, implicated in cortical actin rearrangements. Endogenous Rac1 colocalized with ARF6 at the plasma membrane and on the ARF6 recycling endosome in untransfected HeLa and primary human fibroblast cells. In transfected HeLa cells Rac1 and ARF6 also colocalized. Cells expressing wild-type ARF6 or Rac1 formed actin-containing surface protrusions and membrane ruffles, respectively, upon treatment with the G protein activator aluminum fluoride. Aluminum fluoride-treatment of cells transfected with equivalent amounts of plasmid resulted in enhanced membrane ruffling, with protrusions appearing as Rac expression was lowered. Co-expression of the dominant negative, GTP binding-defective ARF6 T27N mutant inhibited the aluminum fluoride-induced ruffling observed in cells expressing Rac1, and the constitutive ruffling observed in cells expressing the activated Rac1 Q61L mutant. In contrast, co-expression of the GTP-binding-defective, T17N mutant of either Rac1 or Cdc42 with ARF6 did not inhibit the aluminum fluoride-induced surface protrusions, nor did inactivation of Rho with C3-transferase. These observations suggest that ARF6, a non-Rho family GTPase, can, by itself, alter cortical actin and can influence the ability of Rac1 to form lamellipodia, in part, by regulating its trafficking to the plasma membrane.
TL;DR: It is shown here the first demonstration of uniform differentiation of ES cells in response to biological factors, which appears to be an obligatory step in the differentiation of the inner cell mass or ES cells into cell lineages of the embryonic germ layers.
Abstract: The primitive ectoderm of the mouse embryo arises from the inner cell mass between 4.75 and 5.25 days post coitum, around the time of implantation. Positioned at a pivotal time in development, just prior to formation of the three germ layers of the embryo proper, the primitive ectoderm responds directly to the signals generated during gastrulation. We have identified a conditioned medium, MEDII, which caused the homogeneous conversion of ES cells to a morphologically distinct cell population, termed early primitive ectoderm-like (EPL) cells. EPL cells expressed the pluripotent cell markers Oct4, SSEA1 and alkaline phosphatase. However, the formation of EPL cells was accompanied by alterations in Fgf5, Gbx2 and Rex1 expression, a loss in chimaera forming ability, changes in factor responsiveness and modified differentiation capabilities, all consistent with the identification of EPL cells as equivalent to the primitive ectoderm population of the 5.5 to 6.0 days post coitum embryo. EPL cell formation could be reversed in the presence of LIF and withdrawal of MEDII, which suggested that EPL cell formation was not a terminal differentiation event but reflected the ability of pluripotent cells to adopt distinct cell states in response to specific factors. Partial purification of MEDII revealed the presence of two separable biological activities, both of which were required for the induction and maintenance of EPL cells. We show here the first demonstration of uniform differentiation of ES cells in response to biological factors. The formation of primitive ectoderm, both in vivo and in vitro, appears to be an obligatory step in the differentiation of the inner cell mass or ES cells into cell lineages of the embryonic germ layers. EPL cells potentially represent a model for the development of lineage specific differentiation protocols and analysis of gastrulation at a molecular level. An understanding of the active components of MEDII may provide a route for the identification of factors which induce primitive ectoderm formation in vivo.
TL;DR: This work has made enormous progress in elucidating the mechanisms by which successful intracellular infection occurs, and characterization of immunological pathways that are responsible for resistance or susceptibility to Leishmania has given rise to the Th1/Th2 paradigm of cellular/humoral dominance of the immune response.
Abstract: Leishmania species are obligate intracellular parasites of cells of the macrophage-dendritic cell lineage. Indeed, the ability to survive and multiply within macrophages is a feature of a surprising number of infectious agents of major importance to public health, including Mycobacterium tuberculosis, Mycobacterium leprae, Listeria monocytogenes, Salmonella typhimurium, Toxoplasma gondii and Trypanosoma cruzi. The relationship between such organisms and their host cells is particularly intriguing because, not only are macrophages capable of potent microbicidal activity, but in their antigen-presenting capacity they can orchestrate the developing immune response. Thus, to initiate a successful infection parasites must gain entry into macrophages, and also withstand or circumvent their killing and degradative functions. However, to sustain a chronic infection, parasites must also subvert macrophage-accessory-cell activities and ablate the development of protective immunity. The leishmanias produce a wide spectrum of disease in mice, and as such they have provided excellent models for studying problems associated with intracellular parasitism. In recent years, largely using these organisms, we have made enormous progress in elucidating the mechanisms by which successful intracellular infection occurs. Furthermore, characterization of immunological pathways that are responsible for resistance or susceptibility to Leishmania has given rise to the Th1/Th2 paradigm of cellular/humoral dominance of the immune response.
TL;DR: The findings indicate that in the process of differentiation to osteoblasts, BMP-6 binds to ALK-2 as well as other type I receptors, and transduces signals mainly through Smad5 and possibly throughSmad1.
Abstract: Bone morphogenetic protein (BMP)-6 is a member of the transforming growth factor (TGF)-(b) superfamily, and is most similar to BMP-5, osteogenic protein (OP)-1/BMP-7, and OP-2/BMP-8. In the present study, we characterized the endogenous BMP-6 signaling pathway during osteoblast differentiation. BMP-6 strongly induced alkaline phosphatase (ALP) activity in cells of osteoblast lineage, including C2C12 cells, MC3T3-E1 cells, and ROB-C26 cells. The profile of binding of BMP-6 to type I and type II receptors was similar to that of OP-1/BMP-7 in C2C12 cells and MC3T3-E1 cells; BMP-6 strongly bound to activin receptor-like kinase (ALK)-2 (also termed ActR-I), together with type II receptors, i.e. BMP type II receptor (BMPR-II) and activin type II receptor (ActR-II). In addition, BMP-6 weakly bound to BMPR-IA (ALK-3), to which BMP-2 also bound. In contrast, binding of BMP-6 to BMPR-IB (ALK-6), and less efficiently to ALK-2 and BMPR-IA, together with BMPR-II was detected in ROB-C26 cells. Intracellular signalling was further studied using C2C12 and MC3T3-E1 cells. Among the receptor-regulated Smads activated by BMP receptors, BMP-6 strongly induced phosphorylation and nuclear accumulation of Smad5, and less efficiently those of Smad1. However, Smad8 was constitutively phosphorylated, and no further phosphorylation or nuclear accumulation of Smad8 by BMP-6 was observed. These findings indicate that in the process of differentiation to osteoblasts, BMP-6 binds to ALK-2 as well as other type I receptors, and transduces signals mainly through Smad5 and possibly through Smad1.
TL;DR: A growing number of serine-threonine kinases highly homologous to the Saccharomyces cerevisiae Ipl1p kinase have been isolated in various organisms.
Abstract: During the past five years, a growing number of serine-threonine kinases highly homologous to the Saccharomyces cerevisiae Ipl1p kinase have been isolated in various organisms. A Drosophila melanogaster homologue, aurora, was the first to be isolated from a multicellular organism. Since then, several related kinases have been found in mammalian cells. They localise to the mitotic apparatus: in the centrosome, at the poles of the bipolar spindle or in the midbody. The kinases are necessary for completion of mitotic events such as centrosome separation, bipolar spindle assembly and chromosome segregation. Extensive research is now focusing on these proteins because the three human homologues are overexpressed in various primary cancers. Furthermore, overexpression of one of these kinases transforms cells. Because of the myriad of kinases identified, we suggest a generic name: Aurora/Ipl1p-related kinase (AIRK). We denote AIRKs with a species prefix and a number, e.g. HsAIRK1.
TL;DR: It is shown that SUMO-1 covalently modifies PML both in vivo and in vitro and that the modification is mediated either directly or indirectly by the interaction of UBC9 with PML through the RING finger domain and the modifications are dependant on the correct subcellular compartmentalisation of target proteins.
Abstract: PML is a nuclear phosphoprotein that was first identified as part of a translocated chromosomal fusion product associated with acute promyelocytic leukaemia (APL). PML localises to distinct nuclear multi-protein complexes termed ND10, Kr bodies, PML nuclear bodies and PML oncogenic domains (PODs), which are disrupted in APL and are the targets for immediate early viral proteins, although little is known about their function. In a yeast two-hybrid screen, we first identified a ubiquitin-like protein named PIC1 (now known as SUMO-1), which interacts and co-localises with PML in vivo. More recent studies have now shown that SUMO-1 covalently modifies a number of target proteins including PML, RanGAP1 and IkappaBalpha and is proposed to play a role in either targeting modified proteins and/or inhibiting their degradation. The precise molecular role for the SUMO-1 modification of PML is unclear, and the specific lysine residues within PML that are targeted for modification and the PML sub-domains necessary for mediating the modification in vivo are unknown. Here we show that SUMO-1 covalently modifies PML both in vivo and in vitro and that the modification is mediated either directly or indirectly by the interaction of UBC9 with PML through the RING finger domain. Using site-specific mutagenesis, we have identified the primary PML-SUMO-1 modification site as being part of the nuclear localisation signal (Lys487 or Lys490). However SUMO-1 modification is not essential for PML nuclear localisation as only nuclear PML is modified. The sequence of the modification site fits into a consensus sequence for SUMO-1 modification and we have identified several other nuclear proteins which could also be targets for SUMO-1. We show that SUMO-1 modification appears to be dependant on the correct subcellular compartmentalisation of target proteins. We also find that the APL-associated fusion protein PML-RARA is efficiently modified in vitro, resulting in a specific and SUMO-1-dependent degradation of PML-RARA. Our results provide significant insights into the role of SUMO-1 modification of PML in both normal cells and the APL disease state.
TL;DR: Observations demonstrate that vimentin is transiently associated with, and could be functionally involved in, the migratory status of human epithelial cells.
Abstract: Vimentin expression in human mammary epithelial MCF10A cells was examined as a function of their migratory status using an in vitro wound-healing model. Analysis of the trajectories of the cells and their migratory speeds by time lapse-video microscopy revealed that vimentin mRNA and protein expression were exclusively induced in cells at the wound's edge which were actively migrating towards the center of the lesion. Actin labeling showed the reorganization of actin filaments in cells at the wound's edge which confirmed the migratory phenotype of this cell subpopulation. Moreover, the vimentin protein disappeared when the cells became stationary after wound closure. Using cells transfected with the vimentin promoter controlling the green fluorescent protein gene, we also demonstrated the specific activation of the vimentin promoter in the migratory cells at the wound's edge. Transfection of the antisense vimentin cDNA into MCF10A cells clearly reduced both their ability to express vimentin and their migratory speed. Taken together, these observations demonstrate that vimentin is transiently associated with, and could be functionally involved in, the migratory status of human epithelial cells.
TL;DR: Evidence is presented that P23 has properties of a tubulin binding protein that associates with microtubules in a cell cycle-dependent manner and elevation of P23 levels leads to microtubule rearrangements and to an increase in micro Tubulin mass and stability.
Abstract: The translationally controlled protein P23 was discovered by the early induction of its rate of synthesis after mitogenic stimulation of mouse fibroblasts. P23 is expressed in almost all mammalian tissues and it is highly conserved between animals, plants and yeast. Based on its amino acid sequence, P23 cannot be attributed to any known protein family, and its cellular function remains to be elucidated. Here, we present evidence that P23 has properties of a tubulin binding protein that associates with microtubules in a cell cycle-dependent manner. (1) P23 is a cytoplasmic protein that occurs in complexes of 100-150 kDa, and part of P23 can be immunoprecipitated from HeLa cell extracts with anti-tubulin antibodies. (2) In immunolocalisation experiments we find P23 associated with microtubules during G1, S, G2 and early M phase of the cell cycle. At metaphase, P23 is also bound to the mitotic spindle, and it is detached from the spindle during metaphase-anaphase transition. (3) A GST-P23 fusion protein interacts with alpha- and beta-tubulin, and recombinant P23 binds to taxol-stabilised microtubules in vitro. The tubulin binding domain of P23 was identified by mutational analysis; it shows similarity to part of the tubulin binding domain of the microtubule-associated protein MAP-1B. (4) Overexpression of P23 results in cell growth retardation and in alterations of cell morphology. Moreover, elevation of P23 levels leads to microtubule rearrangements and to an increase in microtubule mass and stability.
TL;DR: Data indicate a regulation of keratinocyte growth by a double paracrine mechanism through release of IL-1 which induces KGF in cocultured fibroblasts, suggesting IL- 1, in addition to its proinflammatory function in skin, may play an essential role in regulating tissue homeostasis.
Abstract: Epithelial-mesenchymal interactions play an important role in regulating tissue homeostasis and repair. For skin, the regulatory mechanisms of epidermal-dermal interactions were studied in cocultures of normal human epidermal keratinocytes (NEK) and dermal fibroblasts (HDF) rendered postmitotic by alpha-irradiation (HDFi). The expression kinetics of different cytokines and their receptors with presumed signalling function in skin were determined at the RNA and protein level in mono- and cocultured NEK and HDFi. In cocultured HDFi, mRNA and protein synthesis of keratinocyte growth factor (KGF) (FGF-7) was strongly enhanced, whereas in cocultured keratinocytes interleukin (IL)-1alpha and -1beta mRNA expression increased compared to monocultures. Thus we postulated that IL-1, which had no effect on keratinocyte proliferation, induced in fibroblasts the expression of factors stimulating keratinocyte proliferation, such as KGF. The functional significance of this reciprocal modulation was substantiated by blocking experiments. Both IL-1alpha and -1beta-neutralizing antibodies and IL-1 receptor antagonist significantly reduced keratinocyte proliferation supposedly through abrogation of KGF production, because IL-1 antibodies blocked the induced KGF production. These data indicate a regulation of keratinocyte growth by a double paracrine mechanism through release of IL-1 which induces KGF in cocultured fibroblasts. Thus IL-1, in addition to its proinflammatory function in skin, may play an essential role in regulating tissue homeostasis.
TL;DR: Time-lapse video microscopic analysis of macrophages containing rhodamine-labeled actin and fluorescein dextran showed that actin was concentrated at the distal margins of closing phagosomes, suggesting that myosin IC mediates the purse-string-like contraction that closes phagosome.
Abstract: Studies of Fc-mediated phagocytosis by mouse macrophages identified a contractile activity at the distal margins of forming phagosomes. Time-lapse video microscopic analysis of macrophages containing rhodamine-labeled actin and fluorescein dextran showed that actin was concentrated at the distal margins of closing phagosomes. Phagocytosis-related contractile activities were observed when one IgG-opsonized erythrocyte was engaged by two macrophages. Both cells extended pseudopodia until they met midway around the erythrocyte. It was then constricted and pulled into two phagosomes, which remained interconnected by a string of erythrocyte membrane. Butanedione monoxime, an uncompetitive inhibitor of class II and perhaps other myosins, and wortmannin and LY294002, inhibitors of phosphoinositide 3-kinase, prevented the constrictions without inhibiting the initial pseudopod extension. Immunofluorescence microscopy showed the presence of myosins IC, II, V and IXb in phagosomes. Of these, only myosin IC was concentrated around the strings connecting shared erythrocytes, suggesting that myosin IC mediates the purse-string-like contraction that closes phagosomes. The sequential processes of pseudopod extension and contraction can explain how macropinosomes and spacious phagosomes form without guidance from a particle surface.
TL;DR: It is shown that COS cells, transiently transfected with both MAP1B and GSK3beta, express high levels of the phosphorylated isoform of MAP 1B (MAP1B-P), suggesting that a primary role of MAP1b-P in growing axons may be to maintain growth cone microtubules in a dynamically unstable state, a known requirement of growth conemicrotubules during pathfinding.
Abstract: We have recently shown that glycogen synthase kinase 3beta (GSK3beta) phosphorylates the microtubule-associated protein (MAP) 1B in an in vitro kinase assay and in cultured cerebellar granule cells. Mapping studies identified a region of MAP1B high in serine-proline motifs that is phosphorylated by GSK3beta. Here we show that COS cells, transiently transfected with both MAP1B and GSK3beta, express high levels of the phosphorylated isoform of MAP1B (MAP1B-P) generated by GSK3beta. To investigate effects of MAP1B-P on microtubule dynamics, double transfected cells were labelled with antibodies to tyrosinated and detyrosinated tubulin markers for stable and unstable microtubules. This showed that high levels of MAP1B-P expression are associated with the loss of a population of detyrosinated microtubules in these cells. Transfection with MAP1B protected microtubules in COS cells against nocodazole depolymerisation, confirming previous studies. However, this protective effect was greatly reduced in cells containing high levels of MAP1B-P following transfection with both MAP1B and GSK3beta. Since we also found that MAP1B binds to tyrosinated, but not to detyrosinated, microtubules in transfected cells, we propose that MAP1B-P prevents tubulin detyrosination and subsequent conversion of unstable to stable microtubules and that this involves binding of MAP1B-P to unstable microtubules. The highest levels of MAP1B-P are found in neuronal growth cones and therefore our findings suggest that a primary role of MAP1B-P in growing axons may be to maintain growth cone microtubules in a dynamically unstable state, a known requirement of growth cone microtubules during pathfinding. To test this prediction, we reduced the levels of MAP1B-P in neuronal growth cones of dorsal root ganglion cells in culture by inhibiting GSK3beta with lithium. In confirmation of the proposed role of MAP1B-P in maintaining microtubule dynamics we found that lithium treatment dramatically increased the numbers of stable (detyrosinated) microtubules in the growth cones of these neurons.
TL;DR: It is demonstrated that exocytic cargo moves along microtubule tracks and reveals that carriers are capable of switching between tracks.
Abstract: The mechanisms and carriers responsible for exocytic protein trafficking between the trans-Golgi network (TGN) and the plasma membrane remain unclear. To investigate the dynamics of TGN-to-plasma membrane traffic and role of the cytoskeleton in these processes we transfected cells with a GFP-fusion protein, vesicular stomatitis virus G protein tagged with GFP (VSVG3-GFP). After using temperature shifts to block VSVG3-GFP in the endoplasmic reticulum and subsequently accumulate it in the TGN, dynamics of TGN-to-plasma membrane transport were visualized in real time by confocal and video microscopy. Both small vesicles ( 1.5 microm long) are used as transport containers (TCs). These TCs rapidly moved out of the Golgi along curvilinear paths with average speeds of approximately 0.7 micrometer/second. Automatic computer tracking objectively determined the dynamics of different carriers. Fission and fusion of TCs were observed, suggesting that these late exocytic processes are highly interactive. To directly determine the role of microtubules in post-Golgi traffic, rhodamine-tubulin was microinjected and both labeled cargo and microtubules were simultaneously visualized in living cells. These studies demonstrated that exocytic cargo moves along microtubule tracks and reveals that carriers are capable of switching between tracks.
TL;DR: Using alpha3 laminin subunit and integrin function-inhibiting antibodies, evidence is provided that LN5 and its two integrin receptors appear necessary for wound healing to occur in MCF-10A cell culture wounds.
Abstract: Previously, we demonstrated that proteolytic processing within the globular domain of the alpha3 subunit of laminin-5 (LN5) converts LN5 from a cell motility-inducing factor to a protein complex that can trigger the formation of hemidesmosomes, certain cell-matrix attachment sites found in epithelial cells. We have prepared a monoclonal antibody (12C4) whose epitope is located toward the carboxy terminus of the globular domain of the alpha3 laminin subunit. This epitope is lost from the alpha3 subunit as a consequence of proteolytic processing. Antibody 12C4 stains throughout the matrix of cells that fail to process the alpha3 laminin subunit, but does not recognize the matrix of confluent cultures of MCF-10A cells, which efficiently process their alpha3 laminin chain. In subconfluent populations of MCF-10A cells, 12C4 only stains matrix deposited at the outer edges of cell colonies. In these cells, integrin alpha3beta1 occasionally colocalizes with the staining generated by the 12C4 antibody but alpha6beta4 integrin does not. In wounded MCF-10A cell cultures, the 12C4 antibody stains the extracellular matrix beneath those cells at the very edge of the cellular sheet that moves to cover the wound site. A similar phenomenon is observed in human skin wounds, since we also detect expression of the unprocessed alpha3 laminin subunit at the leading tip of the sheet of epidermal cells that epithelializes skin wounds in vivo. In addition, using alpha3 laminin subunit and integrin function-inhibiting antibodies, we provide evidence that LN5 and its two integrin receptors (alpha6beta4 and alpha3beta1) appear necessary for wound healing to occur in MCF-10A cell culture wounds. We propose a model for healing of wounded epithelial tissues based on these results.
TL;DR: The actin-based motility of R. conorii is analysed in comparison to that of L. monocytogenes and S. flexneri to suggest that it may use a different mechanism of actin polymerization.
Abstract: Listeria monocytogenes, Shigella flexneri, and Rickettsia conorii are three bacterial pathogens that are able to polymerize actin into ‘comet tail’ structures and move within the cytosol of infected cells. The actin-based motilities of L. monocytogenes and S. flexneri are known to require the bacterial proteins ActA and IcsA, respectively, and several mammalian cytoskeleton proteins including the Arp2/3 complex and VASP (vasodilator-stimulated phosphoprotein) for L. monocytogenes and vinculin and N-WASP (the neural Wiskott-Aldrich syndrome protein) for S. flexneri. In contrast, little is known about the motility of R. conorii. In the present study, we have analysed the actin-based motility of this bacterium in comparison to that of L. monocytogenes and S. flexneri. Rickettsia moved at least three times more slowly than Listeria and Shigella in both infected cells and Xenopus laevis egg extracts. Decoration of actin with the S1 subfragment of myosin in infected cells showed that the comet tails of Rickettsia have a structure strikingly different from those of L. monocytogenes or S. flexneri. In Listeria and Shigella tails, actin filaments form a branching network while Rickettsia tails display longer and not cross-linked actin filaments. Immunofluorescence studies revealed that the two host proteins, VASP and (α)-actinin colocalized with actin in the tails of Rickettsia but neither the Arp2/3 complex which we detected in the Shigella actin tails, nor N-WASP, were detected in Rickettsia actin tails. Taken together, these results suggest that R. conorii may use a different mechanism of actin polymerization.
TL;DR: These findings show for the first time that the newly discovered lamin A 10 protein is localized to the nuclear membrane.
Abstract: The behavior of chimeric proteins consisting of A-type lamins and green fluorescent protein (GFP) was studied to investigate the localization and dynamics of nuclear lamins in living cells. Cell line CHO-K1 was transfected with cDNA constructs encoding fusion proteins of lamin A-GFP, lamin Adelta10-GFP, or lamin C-GFP. In the interphase nucleus lamin-GFP fluorescence showed a perinuclear localization and incorporation into the lamina for all three constructs. Our findings show for the first time that the newly discovered lamin A 10 protein is localized to the nuclear membrane. The GFP-tagged lamins were processed and behaved similarly to the endogenous lamin molecules, at least in cells that expressed physiological levels of the GFP-lamins. In addition to the typical perinuclear localization, in the majority of transfected cells each individual A-type lamin-GFP revealed an extensive collection of branching intra- and trans-nuclear tubular structures, which showed a clear preference for a vertical orientation. Time-lapse studies of 3-D reconstructed interphase cells showed a remarkable stability in both number and location of these structures over time, while the lamina showed considerable dynamic movements, consisting of folding and indentation of large parts of the lamina. Fluorescence recovery after bleaching studies revealed a low protein turnover of both tubular and lamina-associated lamins. Repetitive bleaching of intranuclear areas revealed the presence of an insoluble intranuclear fraction of A-type lamins. Time-lapse studies of mitotic cells showed that reformation of the lamina and the tubular structures consisting of A-type lamins did not occur until after cytokinesis was completed.