Showing papers in "BMC Cell Biology in 2004"

Effects of "second-hand" smoke on structure and function of fibroblasts, cells that are critical for tissue repair and remodeling.

Abstract: It is known that "second-hand" cigarette smoke leads to abnormal tissue repair and remodelling but the cellular mechanisms involved in these adverse effects are not well understood. Fibroblasts play a major role in repair and remodelling. They orchestrate these processes by proliferating, migrating, and secreting proteins such as, cytokines, growth factors and extracellular matrix molecules. Therefore, we focus our studies on the effects of "second-hand" cigarette smoke on the structure and function of these cells. We used sidestream whole (SSW) smoke, a major component of "second-hand" smoke, primary embryonic fibroblasts, cells that behave very much like wound fibroblasts, and a variety of cellular and molecular approaches. We show that doses of smoke similar to those found in tissues cause cytoskeletal changes in the fibroblasts that may lead to a decrease in cell migration. In addition, we also show that these levels of cigarette smoke stimulate an increase in cell survival that is reflected in an increase and/or activation of stress/survival proteins such as cIL-8, grp78, PKB/Akt, p53, and p21. We further show that SSW affects the endomembrane system and that this effect is also accomplished by nicotine alone. Taken together, our results suggest that: (i) SSW may delay wound repair because of the inability of the fibroblasts to migrate into the wounded area, leading to an accumulation of these cells at the edge of the wound, thus preventing the formation of the healing tissue; (ii) the increase in cell survival coupled to the decrease in cell migration can lead to a build-up of connective tissue, thereby causing fibrosis and excess scarring.

The phosphatidylserine receptor from Hydra is a nuclear protein with potential Fe(II) dependent oxygenase activity

Abstract: Apoptotic cell death plays an essential part in embryogenesis, development and maintenance of tissue homeostasis in metazoan animals. The culmination of apoptosis in vivo is the phagocytosis of cellular corpses. One morphological characteristic of cells undergoing apoptosis is loss of plasma membrane phospholipid asymmetry and exposure of phosphatidylserine on the outer leaflet. Surface exposure of phosphatidylserine is recognised by a specific receptor (phosphatidylserine receptor, PSR) and is required for phagocytosis of apoptotic cells by macrophages and fibroblasts. We have cloned the PSR receptor from Hydra in order to investigate its function in this early metazoan. Bioinformatic analysis of the Hydra PSR protein structure revealed the presence of three nuclear localisation signals, an AT-hook like DNA binding motif and a putative 2-oxoglutarate (2OG)-and Fe(II)-dependent oxygenase activity. All of these features are conserved from human PSR to Hydra PSR. Expression of GFP tagged Hydra PSR in hydra cells revealed clear nuclear localisation. Deletion of one of the three NLS sequences strongly diminished nuclear localisation of the protein. Membrane localisation was never detected. Our results suggest that Hydra PSR is a nuclear 2-oxoglutarate (2OG)-and Fe(II)-dependent oxygenase. This is in contrast with the proposed function of Hydra PSR as a cell surface receptor involved in the recognition of apoptotic cells displaying phosphatidylserine on their surface. The conservation of the protein from Hydra to human infers that our results also apply to PSR from higher animals.

TMF is a golgin that binds Rab6 and influences Golgi morphology.

Abstract: Golgins are coiled-coil proteins associated with the Golgi apparatus, that are believed to be involved in the tethering of vesicles and the stacking of cisternae, as well as other functions such as cytoskeletal association Many are peripheral membrane proteins recruited by GTPases Several have been described in animal cells, and some in yeast, but the relationships between golgins from different species can be hard to define because although they share structural features, their sequences are not well conserved We show here that the yeast protein Sgm1, previously shown to be recruited to the Golgi by the GTPase Ypt6, binds to Ypt6:GTP via a conserved 100-residue coiled-coil motif that can be identified in a wide range of eukaryotes The mammalian equivalent of Sgm1 is TMF/ARA160, a protein previously identified in various screens as a putative transcription or chromatin remodelling factor We show that it is a Golgi protein, and that it binds to the three known isoforms of the Ypt6 homologue Rab6 Depletion of the protein by RNA interference in rat NRK cells results in a modest dispersal of Golgi membranes around the cell, suggesting a role for TMF in the movement or adherence of Golgi stacks We have identified TMF as an evolutionarily conserved golgin that binds Rab6 and contributes to Golgi organisation in animal cells

Functional interdependence between septin and actin cytoskeleton

Abstract: Background Septin2 is a member of a highly conserved GTPase family found in fungi and animals. Septins have been implicated in a diversity of cellular processes including cytokinesis, formation of diffusion barriers and vesicle trafficking. Septin2 partially co-localises with actin bundles in mammalian interphase cells and Septin2-filamentmorphology depends upon an intact actin cytoskeleton. How this interaction is regulated is not known. Moreover, evidence that Septin2 is remodelled or redistributed in response to other changes in actin organisation is lacking.

Cytoskeletal influences on nuclear shape in granulocytic HL-60 cells

Abstract: During granulopoiesis in the bone marrow, the nucleus differentiates from ovoid to lobulated shape. Addition of retinoic acid (RA) to leukemic HL-60 cells induces development of lobulated nuclei, furnishing a convenient model system for nuclear differentiation during granulopoiesis. Previous studies from our laboratory have implicated nuclear envelope composition as playing important roles in nuclear shape changes. Specifically noted were: 1) a paucity of lamins A/C and B1 in the undifferentiated and RA treated cell forms; 2) an elevation of lamin B receptor (LBR) during induced granulopoiesis. The present study demonstrates that perturbation of cytoskeletal elements influences nuclear differentiation of HL-60 cells. Because of cytotoxicity from prolonged exposure to cytoskeleton-modifying drugs, most studies were performed with a Bcl-2 overexpressing HL-60 subline. We have found that: 1) nocodazole prevents RA induction of lobulation; 2) taxol induces lobulation and micronuclear formation, even in the absence of RA; 3) cytochalasin D does not inhibit RA induced nuclear lobulation, and prolonged exposure induces nuclear shape changes in the absence of RA. The present results, in the context of earlier data and models, suggest a mechanism for granulocytic nuclear lobulation. Our current hypothesis is that the nuclear shape change involves factors that increase the flexibility of the nuclear envelope (reduced lamin content), augment connections to the underlying heterochromatin (increased levels of LBR) and promote distortions imposed by the cytoskeleton (microtubule motors creating tension in the nuclear envelope).

Calcineurin activation influences muscle phenotype in a muscle-specific fashion.

Abstract: The calcium activated protein phosphatase 2B, also known as calcineurin, has been implicated as a cell signaling molecule involved with transduction of physiological signals (free cytosolic Ca2+) into molecular signals that influence the expression of phenotype-specific genes in skeletal muscle. In the present study we address the role of calcineurin in mediating adaptations in myosin heavy chain (MHC) isoform expression and muscle mass using 3-month old wild-type (WT) and transgenic mice displaying high-level expression of a constitutively active form of calcineurin (MCK-CN* mice). Slow muscles, e.g., soleus, were significantly larger (by ~24%), whereas fast muscles, e.g., medial gastrocnemius (MG) and tibialis anterior were significantly smaller (by ~26 and ~16%, respectively) in MCK-CN* mice compared to WT. The masses of mixed phenotype muscles, such as the plantaris and the extensor digitorum longus, were not significantly changed from WT. The soleus, plantaris, MG and diaphragm displayed shifts toward slower MHC isoforms, e.g., soleus from WT mice contained ~52% MHC-I, ~39% MHC-IIa, and ~9% MHC-IIx, whereas MCK-CN* mice had ~67% MHC-I, ~26% MHC-IIa, and ~7% MHC-IIx. The specific isoforms that were either up or down-regulated were muscle-specific. For instance, the proportion of MHC-IIa was decreased in the soleus and diaphragm, but increased in the plantaris and MG of MCK-CN* mice. Also, the proportion of MHC-IIx was unchanged in the soleus, decreased in the diaphragm and increased in the plantaris and MG of MCK-CN* relative to WT mice. Fast to slow shifts in fiber type proportions were evident for the plantaris, but not the soleus. Fast, but not slow, plantaris fibers of MCK-CN* mice had higher oxidative and lower glycolytic properties than WT. These data suggest that calcineurin activation can influence muscle phenotype and that the specific influence of calcineurin activation on the phenotypic and mass characteristics of a muscle is dependent upon the original phenotypic state of the muscle.

Two Drosophila suppressors of cytokine signaling (SOCS) differentially regulate JAK and EGFR pathway activities

Abstract: The Janus kinase (JAK) cascade is an essential and well-conserved pathway required to transduce signals for a variety of ligands in both vertebrates and invertebrates. While activation of the pathway is essential to many processes, mutations from mammals and Drosophila demonstrate that regulation is also critical. The SOCS (S uppressor O f C ytokine S ignaling) proteins in mammals are regulators of the JAK pathway that participate in a negative feedback loop, as they are transcriptionally activated by JAK signaling. Examination of one Drosophila SOCS homologue, Socs36E, demonstrated that its expression is responsive to JAK pathway activity and it is capable of downregulating JAK signaling, similar to the well characterized mammalian SOCS. Based on sequence analysis of the Drosophila genome, there are three identifiable SOCS homologues in flies. All three are most similar to mammalian SOCS that have not been extensively characterized: Socs36E is most similar to mammalian SOCS5, while Socs44A and Socs16D are most similar to mammalian SOCS6 and 7. Although Socs44A is capable of repressing JAK activity in some tissues, its expression is not regulated by the pathway. Furthermore, Socs44A can enhance the activity of the EGFR/MAPK signaling cascade, in contrast to Socs36E. Two Drosophila SOCS proteins have some overlapping and some distinct capabilities. While Socs36E behaves similarly to the canonical vertebrate SOCS, Socs44A is not part of a JAK pathway negative feedback loop. Nonetheless, both SOCS regulate JAK and EGFR signaling pathways, albeit differently. The non-canonical properties of Socs44A may be representative of the class of less characterized vertebrate SOCS with which it shares greatest similarity.

Sam68 exerts separable effects on cell cycle progression and apoptosis

Abstract: The RNA-binding protein Sam68 has been implicated in a number of cellular processes, including transcription, RNA splicing and export, translation, signal transduction, cell cycle progression and replication of the human immunodeficiency virus and poliovirus. However, the precise impact it has on essential cellular functions remains largely obscure. In this report we show that conditional overexpression of Sam68 in fibroblasts results in both cell cycle arrest and apoptosis. Arrest in G1 phase of the cell cycle is associated with decreased levels of cyclins D1 and E RNA and protein, resulting in dramatically reduced Rb phosphorylation. Interestingly, cell cycle arrest does not require the specific RNA binding ability of Sam68. In marked contrast, induction of apoptosis by Sam68 absolutely requires a fully-functional RNA binding domain. Moreover, the anti-cancer agent trichostatin A potentiates Sam68-driven apoptosis. For the first time we have shown that Sam68, an RNA binding protein with multiple apparent functions, exerts functionally separable effects on cell proliferation and survival, dependent on its ability to bind specifically to RNA. These findings shed new light on the ability of signal transducing RNA binding proteins to influence essential cell function. Moreover, the ability of a class of anti-cancer therapeutics to modulate its ability to promote apoptosis suggests that Sam68 status may impact some cancer treatments.

4-D single particle tracking of synthetic and proteinaceous microspheres reveals preferential movement of nuclear particles along chromatin – poor tracks

Abstract: The dynamics of nuclear organization, nuclear bodies and RNPs in particular has been the focus of many studies. To understand their function, knowledge of their spatial nuclear position and temporal translocation is essential. Typically, such studies generate a wealth of data that require novel methods in image analysis and computational tools to quantitatively track particle movement on the background of moving cells and shape changing nuclei. We developed a novel 4-D image processing platform (TIKAL) for the work with laser scanning and wide field microscopes. TIKAL provides a registration software for correcting global movements and local deformations of cells as well as 2-D and 3-D tracking software. With this new tool, we studied the dynamics of two different types of nuclear particles, namely nuclear bodies made from GFP-NLS-vimentin and microinjected 0.1 μm – wide polystyrene beads, by live cell time-lapse microscopy combined with single particle tracking and mobility analysis. We now provide a tool for the automatic 3-D analysis of particle movement in parallel with the acquisition of chromatin density data. Kinetic analysis revealed 4 modes of movement: confined obstructed, normal diffusion and directed motion. Particle tracking on the background of stained chromatin revealed that particle movement is directly related to local reorganization of chromatin. Further a direct comparison of particle movement in the nucleoplasm and the cytoplasm exhibited an entirely different kinetic behaviour of vimentin particles in both compartments. The kinetics of nuclear particles were slightly affected by depletion of ATP and significantly disturbed by disruption of actin and microtubule networks. Moreover, the hydration state of the nucleus had a strong impact on the mobility of nuclear bodies since both normal diffusion and directed motion were entirely abolished when cells were challenged with 0.6 M sorbitol. This effect correlated with the compaction of chromatin. We conclude that alteration in chromatin density directly influences the mobility of protein assemblies within the nucleus.

Mig12, a novel Opitz syndrome gene product partner, is expressed in the embryonic ventral midline and co-operates with Mid1 to bundle and stabilize microtubules

Abstract: Opitz G/BBB syndrome is a genetic disorder characterized by developmental midline abnormalities, such as hypertelorism, cleft palate, and hypospadias. The gene responsible for the X-linked form of this disease, MID1, encodes a TRIM/RBCC protein that is anchored to the microtubules. The association of Mid1 with the cytoskeleton is regulated by dynamic phosphorylation, through the interaction with the α4 subunit of phosphatase 2A (PP2A). Mid1 acts as an E3 ubiquitin ligase, regulating PP2A degradation on microtubules. In spite of these findings, the biological role exerted by the Opitz syndrome gene product is still unclear and the presence of other potential interacting moieties in the Mid1 structure prompted us to search for additional cellular partners. Through a yeast two-hybrid screening approach, we identified a novel gene, MIG12, whose protein product interacts with Mid1. We confirmed by immunoprecipitation that this interaction occurs in vivo and that it is mediated by the Mid1 coiled-coil domain. We found that Mig12 is mainly expressed in the neuroepithelial midline, urogenital apparatus, and digits during embryonic development. Transiently expressed Mig12 is found diffusely in both nucleus and cytoplasm, although it is enriched in the microtubule-organizing center region. Consistently with this, endogenous Mig12 protein is partially detected in the polymerized tubulin fraction after microtubule stabilization. When co-transfected with Mid1, Mig12 is massively recruited to thick filamentous structures composed of tubulin. These microtubule bundles are resistant to high doses of depolymerizing agents and are composed of acetylated tubulin, thus representing stabilized microtubule arrays. Our findings suggest that Mig12 co-operates with Mid1 to stabilize microtubules. Mid1-Mig12 complexes might be implicated in cellular processes that require microtubule stabilization, such as cell division and migration. Impairment in Mig12/Mid1-mediated microtubule dynamic regulation, during the development of embryonic midline, may cause the pathological signs observed in Opitz syndrome patients.

The architecture of chicken chromosome territories changes during differentiation

Abstract: Between cell divisions the chromatin fiber of each chromosome is restricted to a subvolume of the interphase cell nucleus called chromosome territory. The internal organization of these chromosome territories is still largely unknown. We compared the large-scale chromatin structure of chromosome territories between several hematopoietic chicken cell types at various differentiation stages. Chromosome territories were labeled by fluorescence in situ hybridization in structurally preserved nuclei, recorded by confocal microscopy and evaluated visually and by quantitative image analysis. Chromosome territories in multipotent myeloid precursor cells appeared homogeneously stained and compact. The inactive lysozyme gene as well as the centromere of the lysozyme gene harboring chromosome located to the interior of the chromosome territory. In further differentiated cell types such as myeloblasts, macrophages and erythroblasts chromosome territories appeared increasingly diffuse, disaggregating to separable substructures. The lysozyme gene, which is gradually activated during the differentiation to activated macrophages, as well as the centromere were relocated increasingly to more external positions. Our results reveal a cell type specific constitution of chromosome territories. The data suggest that a repositioning of chromosomal loci during differentiation may be a consequence of general changes in chromosome territory morphology, not necessarily related to transcriptional changes.

A plasmid-based system for expressing small interfering RNA libraries in mammalian cells

Abstract: RNA interference (RNAi) is an evolutionarily conserved process that functions to inhibit gene expression. The use of RNAi in mammals as a tool to study gene function has rapidly developed in the last couple of years since the discovery that the function-inhibiting units of RNAi are short 21–25 nt double-stranded RNAs (siRNAs) derived from their longer template. The use of siRNAs allows for gene-specific knock-down without induction of the non-specific interferon response in mammalian cells. Multiple systems have been developed to introduce siRNAs into mammals. One of the most appealing of these techniques is the use of vectors containing polymerase III promoters to drive expression of hairpin siRNAs. However, there are multiple limitations to using hairpin siRNA vectors including the observation that some are unstable in bacteria and are difficult to sequence. To circumvent the limitation of hairpin siRNA vectors we have developed a convergent opposing siRNA expression system called pHippy. We have generated pHippy vectors or expression cassettes that knock down the expression of both reporter and endogenous genes. As a proof of principle that pHippy can be used to generate random siRNA libraries, we generated a small siRNA library against PGL3 luciferase and demonstrated that we could recover functional siRNAs that knock down PGL3 luciferase. siRNA is a powerful tool to study gene function. We have developed a new vector with opposing convergent promoters for the expression of siRNAs, which can be used to knock down endogenous genes in a high throughput manner or to perform functional screening with random or cDNA-derived siRNA libraries.

Isoform-specific expression of the Coxsackie and adenovirus receptor (CAR) in neuromuscular junction and cardiac intercalated discs.

Abstract: The Coxsackie and adenovirus receptor (CAR) has a restricted expression pattern in the adult. In skeletal muscle, although CAR is expressed in immature fibers, its transcript levels are barely detectable in mature muscle. This is in contrast to the robust expression observed in the heart. However, both heart and skeletal muscle are susceptible to infection with the Coxsackie B virus which utilizes primarily CAR for cellular internalization. The specific point of viral entry in skeletal and heart muscle remains unknown. Using antibodies directed against the extracellular and the cytoplasmic domains of CAR, we show CAR in normal human and mouse skeletal muscle to be a novel component of the neuromuscular junction. In cardiac muscle, CAR immunoreactivity is observed at the level of intercalated discs. We demonstrate a single isoform of CAR to be expressed exclusively at the human neuromuscular junction whereas both predominant CAR isoforms are expressed at the intercalated discs of non-diseased human heart. The localization of CAR to these important junctional complexes suggests that CAR may play both a structural and a regulatory role in skeletal and cardiac muscle, and that these complexes may serve as a point of entry for Coxsackie B virus.

The two neutrophil members of the formylpeptide receptor family activate the NADPH-oxidase through signals that differ in sensitivity to a gelsolin derived phosphoinositide-binding peptide.

Abstract: The formylpeptide receptor family members FPR and FPRL1, expressed in myeloid phagocytes, belong to the G-protein coupled seven transmembrane receptor family (GPCRs). They share a high degree of sequence similarity, particularly in the cytoplasmic domains involved in intracellular signaling. The established model of cell activation through GPCRs states that the receptors isomerize from an inactive to an active state upon ligand binding, and this receptor transformation subsequently activates the signal transducing G-protein. Accordingly, the activation of human neutrophil FPR and FPRL1 induces identical, pertussis toxin-sensitive functional responses and a transient increase in intracellular calcium is followed by a secretory response leading to mobilization of receptors from intracellular stores, as well as a release of reactive oxygen metabolites. We report that a cell permeable ten amino acid peptide (PBP10) derived from the phosphatidylinositol 4,5-bisphosphate (PIP2) binding region of gelsolin (an uncapper of actin filaments) blocks granule mobilization as well as secretion of oxygen radicals. The inhibitory effect of PBP10 is, however, receptor specific and affects the FPRL1-, but not the FPR-, induced cellular response. The transient rise in intracellular calcium induced by the active receptors is not affected by PBP10, suggesting that the blockage occurs in a parallel, novel signaling pathway used by FPRL1 to induce oxygen radical production and secretion. Also the FPR can activate neutrophils through a PBP10-sensitive signaling pathway, but this signal is normally blocked by the cytoskeleton. This study demonstrates that the two very closely related chemoattractant receptors, FPR and FPRL1, use distinct signaling pathways in activation of human neutrophils. The PIP2-binding peptide PBP10 selectively inhibits FPRL1-mediated superoxide production and granule mobilization. Furthermore, the activity of this novel PBP10 sensitive pathway in neutrophils is modulated by the actin cytoskeleton network.

Altered protein dynamics of disease-associated lamin A mutants

Abstract: Background Recent interest in the function of the nuclear lamina has been provoked by the discovery of lamin A/C mutations in the laminopathy diseases. However, it is not understood why mutations in lamin A give such a range of tissue-specific phenotypes. Part of the problem in rationalising genotype-phenotype correlations in the laminopathies is our lack of understanding of the function of normal and mutant lamin A. To investigate this we have used photobleaching in human cells to analyse the dynamics of wild-type and mutant lamin A protein at the nuclear periphery.

Concentration-dependent effects of native and polymerised α1-antitrypsin on primary human monocytes, in vitro

Abstract: α1-antitrypsin (AAT) is one of the major serine proteinase inhibitors controlling proteinases in many biological pathways. There is increasing evidence that AAT is able to exert other than antiproteolytic effects. To further examine this question we compared how various doses of the native (inhibitory) and the polymerised (non-inhibitory) molecular form of AAT affect pro-inflammatory responses in human monocytes, in vitro. Human monocytes isolated from different donors were exposed to the native or polymerised form of AAT at concentrations of 0.01, 0.02, 0.05, 0.1, 0.5 and 1 mg/ml for 18 h, and analysed to determine the release of cytokines and to detect the activity of NF-κB. We found that native and polymerised AAT at lower concentrations, such as 0.1 mg/ml, enhance expression of TNFα (10.9- and 4.8-fold, p < 0.001), IL-6 (22.8- and 23.4-fold, p < 0.001), IL-8 (2.4- and 5.5-fold, p < 0.001) and MCP-1 (8.3- and 7.7-fold, p < 0.001), respectively, compared to buffer exposed cells or cells treated with higher doses of AAT (0.5 and 1 mg/ml). In parallel to increased cytokine levels, low concentrations of either conformation of AAT (0.02–0.1 mg/ml) induced NF-κB p50 activation, while 1 mg/ml of either conformation of AAT suppressed the activity of NF-κB, compared to controls. The observations reported here provide further support for a central role of AAT in inflammation, both as a regulator of proteinase activity, and as a signalling molecule for the expression of pro-inflammatory molecules. This latter role is dependent on the concentration of AAT, rather than on its proteinase inhibitory activity.

Control and maintenance of mammalian cell size

Abstract: Conlon and Raff propose that mammalian cells grow linearly during the division cycle. According to Conlon and Raff, cells growing linearly do not need a size checkpoint to maintain a constant distribution of cell sizes. If there is no cell-size-control system, then exponential growth is not allowed, as exponential growth, according to Conlon and Raff, would require a cell-size-control system. A reexamination of the model and experiments of Conlon and Raff indicates that exponential growth is fully compatible with cell size maintenance, and that mammalian cells have a system to regulate and maintain cell size that is related to the process of S-phase initiation. Mammalian cell size control and its relationship to growth rate–faster growing cells are larger than slower growing cells–is explained by the initiation of S phase occurring at a relatively constant cell size coupled with relatively invariant S- and G2-phase times as interdivision time varies. This view of the mammalian cell cycle, the continuum model, explains the mass growth pattern during the division cycle, size maintenance, size determination, and the kinetics of cell-size change following a shift-up from slow to rapid growth.

Phosphorylated guanine nucleotide exchange factor C3G, induced by pervanadate and Src family kinases localizes to the Golgi and subcortical actin cytoskeleton

Abstract: The guanine nucleotide exchange factor C3G (RapGEF1) along with its effector proteins participates in signaling pathways that regulate eukaryotic cell proliferation, adhesion, apoptosis and embryonic development. It activates Rap1, Rap2 and R-Ras members of the Ras family of GTPases. C3G is activated upon phosphorylation at tyrosine 504 and therefore, determining the localization of phosphorylated C3G would provide an insight into its site of action in the cellular context. C3G is phosphorylated in vivo on Y504 upon coexpression with Src or Hck, two members of the Src family tyrosine kinases. Here we have determined the subcellular localization of this protein using antibodies specific to C3G and Tyr 504 phosphorylated C3G (pY504 C3G). While exogenously expressed C3G was present mostly in the cytosol, pY504 C3G formed upon Hck or Src coexpression localized predominantly at the cell membrane and the Golgi complex. Tyrosine 504-phosphorylated C3G showed colocalization with Hck and Src. Treatment of Hck and C3G transfected cells with pervanadate showed an increase in the cytosolic staining of pY504 C3G suggesting that tyrosine phosphatases may be involved in dephosphorylating cytosolic phospho-C3G. Expression of Src family kinases or treatment of cells with pervanadate resulted in an increase in endogenous pY504 C3G, which was localized predominantly at the Golgi and the cell periphery. Endogenous pY504 C3G at the cell periphery colocalized with F-actin suggesting its presence at the subcortical actin cytoskeleton. Disruption of actin cytoskeleton by cytochalasin D abolished phospho-C3G staining at the periphery of the cell without affecting its Golgi localization. These findings show that tyrosine kinases involved in phosphorylation of C3G are responsible for regulation of its localization in a cellular context. We have demonstrated the localization of endogenous C3G modified by tyrosine phosphorylation to defined subcellular domains where it may be responsible for restricted activation of signaling pathways.

Endoplasmic reticulum degradation impedes olfactory G-protein coupled receptor functional expression

Abstract: Research on olfactory G-protein coupled receptors (GPCRs) has been severely impeded by poor functional expression in heterologous systems. Previously, we demonstrated that inefficient olfactory receptor (OR) expression at the plasma membrane is attributable, in part, to degradation of endoplasmic reticulum (ER)-retained ORs by the ubiquitin-proteasome system and sequestration of ORs in ER aggregates that are degraded by autophagy. Thus, experiments were performed to test the hypothesis that attenuation of ER degradation improves OR functional expression in heterologous cells. To develop means to increase the functional expression of ORs, we devised an approach to measure activation of the mOREG OR (Unigene # Mm.196680; Olfr73) through coupling to an olfactory cyclic nucleotide-gated cation channel (CNG). This system, which utilizes signal transduction machinery coupled to OR activation in native olfactory sensory neurons, was used to demonstrate that degradation, both by the ubiquitin-proteasome system and autophagy, limits mOREG functional expression. The stimulatory effects of proteasome and autophagy inhibitors on mOREG function required export from the ER and trafficking through the biosynthetic pathway. These findings demonstrate that poor functional expression of mOREG in heterologous cells is improved by blocking proteolysis. Inhibition of ER degradation may improve the function of other ORs and assist future efforts to elucidate the molecular basis of odor discrimination.

Immortalization of mouse myogenic cells can occur without loss of p16INK4a, p19ARF, or p53 and is accelerated by inactivation of Bax.

Abstract: Upon serial passaging of mouse skeletal muscle cells, a small number of cells will spontaneously develop the ability to proliferate indefinitely while retaining the ability to differentiate into multinucleate myotubes. Possible gene changes that could underlie myogenic cell immortalization and their possible effects on myogenesis had not been examined. We found that immortalization occurred earlier and more frequently when the myogenic cells lacked the pro-apoptotic protein Bax. Furthermore, myogenesis was altered by Bax inactivation as Bax-null cells produced muscle colonies with more nuclei than wild-type cells, though a lower percentage of the Bax-null nuclei were incorporated into multinucleate myotubes. In vivo, both the fast and slow myofibers in Bax-null muscles had smaller cross-sectional areas than those in wild-type muscles. After immortalization, both Bax-null and Bax-positive myogenic cells expressed desmin, retained the capacity to form multinucleate myotubes, expressed p19 ARF protein, and retained p53 functions. Expression of p16 INK4a , however, was found in only about half of the immortalized myogenic cell lines. Mouse myogenic cells can undergo spontaneous immortalization via a mechanism that can include, but does not require, loss of p16 INK4a , and also does not require inactivation of p19 ARF or p53. Furthermore, loss of Bax, which appears to be a downstream effector of p53, accelerates immortalization of myogenic cells and alters myogenesis.

Direct interaction between Smad3, APC10, CDH1 and HEF1 in proteasomal degradation of HEF1.

Abstract: The Transforming Growth Factor-β (TGF-β) regulates myriad cellular events by signaling through members of the Smad family signal transducers. As a key signal transducer of TGF-β, Smad3 exhibits the property of receptor-activated transcriptional modulator and also the novel ability of regulating the proteasomal degradation of two Smad3 interacting proteins, SnoN and HEF1. It has been shown that Smad3 recruits two types of Ub E3 ligases, Smurf2 and the Anaphase Promoting Complex (APC), to mediate SnoN ubiquitination, thereby enhancing SnoN degradation. The molecular mechanisms underlying Smad3-regulated HEF1 degradation are not well understood. Furthermore, it is not clear how Smad3 recruits the APC complex. We detected physical interaction between Smad3 and an APC component APC10, as well as the interaction between HEF1 and CDH1, which is the substrate-interacting component within APC. Detailed domain mapping studies revealed distinct subdomains within the MH2 domain of Smad3 for binding to APC10 and HEF1 and suggests the formation of a complex of these four proteins (Smad3, HEF1, APC10 and CDH1). In addition, the protein levels of HEF1 are subjected to the regulation of overexpressed APC10 and CDH1. Our data suggests that Smad3 may recruit the APC complex via a direct interaction with the APC subunit APC10 to regulate the ubiquitination and degradation of its interactor HEF1, which is recognized as an ubiquitination substrate by the CDH1 subunit of the APC complex.

Expression and localization of nuclear proteins in autosomal-dominant Emery-Dreifuss muscular dystrophy with LMNA R377H mutation

Abstract: The autosomal dominant form of Emery-Dreifuss muscular dystrophy (AD-EDMD) is caused by mutations in the gene encoding for the lamins A and C (LMNA). Lamins are intermediate filament proteins which form the nuclear lamina underlying the inner nuclear membrane. We have studied the expression and the localization of nuclear envelope proteins in three different cell types and muscle tissue of an AD-EDMD patient carrying a point mutation R377H in the lamin A/C gene. Lymphoblastoid cells, skin fibroblasts, primary myoblasts and muscle thin sections were studied by immunocytochemistry and electron microscopy. Cellular levels of A-type lamins were reduced compared to control cells. In contrast, the amount of emerin and lamin B appeared unaltered. Cell synchronization experiments showed that the reduction of the cellular level of A-type lamin was due to instability of lamin A. By electron microscopy, we identified a proportion of nuclei with morphological alterations in lymphoblastoid cells, fibroblasts and mature muscle fibres. Immunofluorescence microscopy showed that a major population of the lamin B receptor (LBR), an inner nuclear membrane protein, was recovered in the cytoplasm in association with the ER. In addition, the intranuclear organization of the active form of RNA polymerase II was markedly different in cells of this AD-EDMD patient. This aberrant intranuclear distribution was specifically observed in muscle cells where the pathology of EDMD predominates. From our results we conclude: Firstly, that structural alterations of the nuclei which are found only in a minor fraction of lymphoblastoid cells and mature muscle fibres are not sufficient to explain the clinical pathology of EDMD; Secondly, that wild type lamin A is required not only for the retention of LBR in the inner nuclear membrane but also for a correct localization of the transcriptionally active RNA pol II in muscle cells. We speculate that a rearrangement of the internal chromatin could lead to muscle-specific disease symptoms by interference with proper mRNA transcription.

MAB21L2, a vertebrate member of the Male-abnormal 21 family, modulates BMP signaling and interacts with SMAD1

Abstract: Through in vivo loss-of-function studies, vertebrate members of the Male abnormal 21 (mab-21) gene family have been implicated in gastrulation, neural tube formation and eye morphogenesis. Despite mounting evidence of their considerable importance in development, the biochemical properties and nature of MAB-21 proteins have remained strikingly elusive. In addition, genetic studies conducted in C. elegans have established that in double mutants mab-21 is epistatic to genes encoding various members of a Transforming Growth Factor beta (TGF-beta) signaling pathway involved in the formation of male-specific sensory organs. Through a gain-of-function approach, we analyze the interaction of Mab21l2 with a TGF-beta signaling pathway in early vertebrate development. We show that the vertebrate mab-21 homolog Mab21l2 antagonizes the effects of Bone Morphogenetic Protein 4 (BMP4) overexpression in vivo, rescuing the dorsal axis and restoring wild-type distribution of Chordin and Xvent2 transcripts in Xenopus gastrulae. We show that MAB21L2 immunoprecipitates in vivo with the BMP4 effector SMAD1, whilst in vitro it binds SMAD1 and the SMAD1-SMAD4 complex. Finally, when targeted to an heterologous promoter, MAB21L2 acts as a transcriptional repressor. Our results provide the first biochemical and cellular foundation for future functional studies of mab-21 genes in normal neural development and its pathological disturbances.

Cloning of a novel signaling molecule, AMSH-2, that potentiates transforming growth factor β signaling

Abstract: Transforming growth factor-βs (TGF-βs), bone morphogenetic proteins (BMPs) and activins are important regulators of developmental cell growth and differentiation Signaling by these factors is mediated chiefly by the Smad family of latent transcription factors There are a large number of uncharacterized cDNA clones that code for novel proteins with homology to known signaling molecules We have identified a novel molecule from the HUGE database that is related to a previously known molecule, AMSH (a ssociated m olecule with the SH 3 domain of STAM), an adapter shown to be involved in BMP signaling Both of these molecules contain a coiled-coil domain located within the amino-terminus region and a JAB (Domain in J un kinase a ctivation domain b inding protein and proteasomal subunits) domain at the carboxy-terminus We show that this novel molecule, which we have designated AMSH-2, is widely expressed and its overexpression potentiates activation of TGF-β-dependent promoters Coimmunoprecipitation studies indicated that Smad7 and Smad2, but not Smad3 or 4, interact with AMSH-2 We show that overexpression of AMSH-2 decreases the inhibitory effect of Smad7 on TGF-β signaling Finally, we demonstrate that knocking down AMSH-2 expression by RNA interference decreases the activation of 3TP-lux reporter in response to TGF-β This report implicates AMSH and AMSH-2 as a novel family of molecules that positively regulate the TGF-β signaling pathway Our results suggest that this effect could be partially explained by AMSH-2 mediated decrease of the action of Smad7 on TGF-β signaling pathway

Subcellular distribution of the V-ATPase complex in plant cells, and in vivo localisation of the 100 kDa subunit VHA-a within the complex

Abstract: Vacuolar H+-ATPases are large protein complexes of more than 700 kDa that acidify endomembrane compartments and are part of the secretory system of eukaryotic cells. They are built from 14 different (VHA)-subunits. The paper addresses the question of sub-cellular localisation and subunit composition of plant V-ATPase in vivo and in vitro mainly by using colocalization and fluorescence resonance energy transfer techniques (FRET). Focus is placed on the examination and function of the 95 kDa membrane spanning subunit VHA-a. Showing similarities to the already described Vph1 and Stv1 vacuolar ATPase subunits from yeast, VHA-a revealed a bipartite structure with (i) a less conserved cytoplasmically orientated N-terminus and (ii) a membrane-spanning C-terminus with a higher extent of conservation including all amino acids shown to be essential for proton translocation in the yeast. On the basis of sequence data VHA-a appears to be an essential structural and functional element of V-ATPase, although previously a sole function in assembly has been proposed. To elucidate the presence and function of VHA-a in the plant complex, three approaches were undertaken: (i) co-immunoprecipitation with antibodies directed to epitopes in the N- and C-terminal part of VHA-a, respectively, (ii) immunocytochemistry approach including co-localisation studies with known plant endomembrane markers, and (iii) in vivo-FRET between subunits fused to variants of green fluorescence protein (CFP, YFP) in transfected cells. All three sets of results show that V-ATPase contains VHA-a protein that interacts in a specific manner with other subunits. The genomes of plants encode three genes of the 95 kDa subunit (VHA-a) of the vacuolar type H+-ATPase. Immuno-localisation of VHA-a shows that the recognized subunit is exclusively located on the endoplasmic reticulum. This result is in agreement with the hypothesis that the different isoforms of VHA-a may localize on distinct endomembrane compartments, as it was shown for its yeast counterpart Vph1.

Xenopus Cdc14α/β are localized to the nucleolus and centrosome and are required for embryonic cell division

Abstract: The dual specificity phosphatase Cdc14 has been shown to be a critical regulator of late mitotic events in several eukaryotes, including S. cerevisiae, S. pombe. C. elegans and H. sapiens. However, Cdc14 homologs have clearly evolved to regulate distinct cellular processes and to respond to regulatory signals important for these processes. The human paralogs hCdc14A and B are the only vertebrate Cdc14 homologues studied to date, but their functions are not well understood. Therefore, it is of great interest to examine the function Cdc14 homologs in other vertebrate species. We identified two open reading frames from Xenopus laevis closely related to human Cdc14A, called XCdc14α and XCdc14β, although no obvious paralog of the hCdc14B was found. To begin a functional characterization of Xcdc14α and XCdc14β, we raised polyclonal antibodies against a conserved region. These antibodies stained both the nucleolus and centrosome in interphase Xenopus tissue culture cells, and the mitotic centrosomes. GFP-tagged version of XCdc14α localized to the nucleulus and GFP-XCdc14β localized to the centrosome, although not exclusively. XCdc14α was also both meiotically and mitotically phosphorylated. Injection of antibodies raised against a conserved region of XCdc14/β into Xenopus embryos at the two-cell stage blocked division of the injected blastomeres, suggesting that activities of XCdc14α/β are required for normal cell division. These results provide evidence that XCdc14α/β are required for normal cellular division and are regulated by at least two mechanisms, subcellular localization and possibly phosphorylation. Due to the high sequence conservation between Xcdc14α and hCdc14A, it seems likely that both mechanisms will contribute to regulation of Cdc14 homologs in vertebrates.

The XMAP215-family protein DdCP224 is required for cortical interactions of microtubules

Abstract: Interactions of peripheral microtubule tips with the cell cortex are of crucial importance for nuclear migration, spindle orientation, centrosome positioning and directional cell movement. Microtubule plus end binding proteins are thought to mediate interactions of microtubule tips with cortical actin and membrane proteins in a dynein-dependent manner. XMAP215-family proteins are main regulators of microtubule plus end dynamics but so far they have not been implicated in the interactions of microtubule tips with the cell cortex. Here we show that overexpression of an N-terminal fragment of DdCP224, the Dictyostelium XMAP215 homologue, caused a collapse of the radial microtubule cytoskeleton, whereby microtubules lost contact with the cell cortex and were dragged behind like a comet tail of an unusually motile centrosome. This phenotype was indistinguishable from mutants overexpressing fragments of the dynein heavy chain or intermediate chain. Moreover, it was accompanied by dispersal of the Golgi apparatus and reduced cortical localization of the dynein heavy chain indicating a disrupted dynein/dynactin interaction. The interference of DdCP224 with cortical dynein function is strongly supported by the observations that DdCP224 and its N-terminal fragment colocalize with dynein and coimmunoprecipitate with dynein and dynactin. Our data show that XMAP215-like proteins are required for the interaction of microtubule plus ends with the cell cortex in interphase cells and strongly suggest that this function is mediated by dynein.

Down-Regulation of the M6P/IGF-II Receptor Increases Cell Proliferation and Reduces Apoptosis in Neonatal Rat Cardiac Myocytes

Abstract: The mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF2R) is a multi-functional protein that has been implicated in regulation of cell growth and apoptosis. Cardiac myocytes express relatively high levels of M6P/IGF2R, and cardiomyocyte apoptosis has been identified in a variety of cardiovascular disorders, such as myocardial infarction and heart failure. However, involvement of M6P/IGF2R in the pathogenesis of these conditions has not been determined. Thus, the objective of this study was to determine the role of M6P/IGF2R in regulation of cardiac myocyte growth and apoptosis. We down-regulated the expression of M6P/IGF2R in neonatal rat cardiac myocytes and examined the effect on cell proliferation and apoptosis. Infection of neonatal cardiomyocytes with an adenovirus expressing a ribozyme targeted against the M6P/IGF2R significantly reduced the level of M6P/IGF2R mRNA, as determined by RT-PCR and Ribonuclease Protection Assay (RPA). M6P-containing protein binding and endocytosis as well as the M6P/IGF2R-mediated internalization of 125I-IGF-II were lower in the ribozyme-treated cells than the control myocytes, indicating that the number of functional M6P/IGF2R in the ribozyme treated cells was reduced. Accordingly, a marked increase in cell proliferation and a reduced cell susceptibility to hypoxia- and TNF-induced apoptosis were observed in the ribozyme-treated cells. These findings suggest that M6P/IGF2R may play a role in regulation of cardiac myocyte growth and apoptosis. Down regulation of this gene in cardiac tissues might be a new approach to prevention of cell death or promotion of mitogenesis for certain heart diseases.

Cytochalasin B triggers a novel pertussis toxin sensitive pathway in TNF-alpha primed neutrophils.

Abstract: Cytochalasin B does not directly activate the oxygen-radical-producing NADPH oxidase activity of neutrophils but transfers desensitized G-protein coupled receptors (GPCR) into an active signaling state by uncoupling GCPR from the cytoskeleton. The receptor uncoupling results in respiratory burst activity when signals generated by reactivated formyl peptide receptors trigger the NADPH-oxidase to produce superoxide anions. Tumor necrosis factor alpha (TNF-alpha) primes neutrophils for subsequent activation by cytochalasin B. Pretreatment with TNF-alpha induced mobilization of receptor-storing neutrophil organelles, suggesting that receptor up-regulation significantly contributes to the response, but the receptor mobilization was not sufficient for induction of the cytochalasin B sensitive state. The TNF-alpha primed state resembled that of the desensitized non-signaling state of agonist-occupied neutrophil formyl peptide receptors. The fact that the TNF-alpha primed, cytochalasin B-triggered activation process was pertussis toxin sensitive suggests that the activation process involves a GPCR. Based on desensitization experiments the unidentified receptor was found to be distinct from the C5a receptor as well as the formyl peptide receptor family members FPR and FPRL1. Based on the fact the occupied and desensitized receptors for interleukin-8 and platelet activating factor could not be reactivated by cytochalasin B, also these could be excluded as receptor candidates involved in the TNF-alpha primed state. The TNF-alpha-induced priming signals could possibly trigger a release of an endogenous GPCR-agonist, amplifying the response to the receptor-uncoupling effect of cytochalasin B. However, no such substance could be found, suggesting that TNF-alpha can transfer G-protein coupled receptors to a signaling state independently of agonist binding.

Systemic endopolyploidy in Spathoglottis plicata (Orchidaceae) development.

Abstract: Endopolyploidy is developmentally regulated. Presence of endopolyploidy as a result of endoreduplication has been characterized in insects, mammals and plants. The family Orchidaceae is the largest among the flowering plants. Many of the members of the orchid family are commercially micropropagated. Very little has been done to characterize the ploidy variation in different tissues of the orchid plants during development. The DNA contents and ploidy level of nuclei extracted from various tissues of a tropical terrestrial orchid Spathoglottis plicata were examined by flow cytometry. Sepals, petals and ovary tissues were found to have only a 2C (C, DNA content of the unreplicated haploid chromosome complement) peak. Columns, floral pedicels of newly open flowers and growing flower stems were observed to have an endopolyploid 8C peak in addition to 2C and 4C peaks. In developing floral pedicels, four peaks were observed for 2C, 4C, 8C and 16C. In root tips, there were 2C, 4C and 8C peaks. But in the root tissues at the region with root hairs, only a 2C peak was observed. Nuclei extracted from young leaves shown three peaks for 2C, 4C and 8C. A similar pattern was found in the vegetative tissues of both greenhouse-grown plants and tissue-cultured plantlets. In mature leaves, a different pattern of ploidy level was found at different parts of the leaves. In the leaf tips and middle parts, there were 2C and 4C peaks. Only at the basal part of the leaves, there were three peaks for 2C, 4C and 8C. Systemic variation of cellular endopolyploidy in different tissues during growth and development of Spathoglottis plicata from field-grown plants and in vitro cultures was identified. The implication of the findings was discussed.