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Results for “Bidirectional mediation”

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PubMed2002

The cytoplasmic filaments of the nuclear pore complex are dispensable for selective nuclear protein import.

The nuclear pore complex (NPC) mediates bidirectional macromolecular traffic between the nucleus and cytoplasm in eukaryotic cells. Eight filaments project from the NPC into the cytoplasm and are proposed to function in nuclear import. We investigated the localization and function of two nucleoporins on the cytoplasmic face of the NPC, CAN/Nup214 and RanBP2/Nup358. Consistent with previous data, RanBP2 was localized at the cytoplasmic filaments. In contrast, CAN was localized near the cytoplasmic coaxial ring. Unexpectedly, extensive blocking of RanBP2 with gold-conjugated antibodies failed to inhibit nuclear import. Therefore, RanBP2-deficient NPCs were generated by in vitro nuclear assembly in RanBP2-depleted Xenopus egg extracts. NPCs were formed that lacked cytoplasmic filaments, but that retained CAN. These nuclei efficiently imported nuclear localization sequence (NLS) or M9 substrates. NPCs lacking CAN retained RanBP2 and cytoplasmic filaments, and showed a minor NLS import defect. NPCs deficient in both CAN and RanBP2 displayed no cytoplasmic filaments and had a strikingly immature cytoplasmic appearance. However, they showed only a slight reduction in NLS-mediated import, no change in M9-mediated import, and were normal in growth and DNA replication. We conclude that RanBP2 is the major nucleoporin component of the cytoplasmic filaments of the NPC, and that these filaments do not have an essential role in importin alpha/beta- or transportin-dependent import.

Animals↗

PubMed1996

Bidirectional signalling through the EPH-family receptor Nuk and its transmembrane ligands.

Receptor tyrosine kinases of the EPH class have been implicated in the control of axon guidance and fasciculation, in regulating cell migration, and in defining compartments in the developing embryo. Efficient activation of EPH receptors generally requires that their ligands be anchored to the cell surface, either through a transmembrane (TM) region or a glycosyl phosphatidylinositol (GPI) group. These observations have suggested that EPH receptors can transduce signals initiated by direct cell-cell interaction. Genetic analysis of Nuk, a murine EPH receptor that binds TM ligands, has raised the possibility that these ligands might themselves have a signalling function. Consistent with this, the three known TM ligands have a highly conserved cytoplasmic region, with multiple potential sites for tyrosine phosphorylation. Here we show that challenging cells that express the TM ligands Elk-L or Htk-L with the clustered ectodomain of Nuk induces phosphorylation of the ligands on tyrosine, a process that can be mimicked both in vitro and in vivo by an activated Src tyrosine kinase. Co-culture of cells expressing a TM ligand with cells expressing Nuk leads to tyrosine phosphorylation of both the ligand and Nuk. These results suggest that the TM ligands are associated with a tyrosine kinase, and are inducibly phosphorylated upon binding Nuk, in a fashion reminiscent of cytokine receptors. Furthermore, we show that TM ligands, as well as Nuk, are phosphorylated on tyrosine in mouse embryos, indicating that this is a physiological process. EPH receptors and their TM ligands therefore mediate bidirectional cell signalling.

Amino Acid Sequence↗

PubMed2001

IgD receptor-mediated signal transduction in T cells.

Upregulation of immunoglobulin D-specific receptors (IgD-R) on CD4+ T cells may facilitate their interaction with specific carbohydrate moieties uniquely associated with membrane IgD on B cells. Previous studies have shown that upregulation of IgD-R facilitates cognate T-B cell interactions by mediating bidirectional signaling resulting in increased antibody responses and clonal expansion of antigen-specific T cells. Murine T hybridoma cells, 7C5, constitutively express IgD-R, as has been confirmed by staining with biotinylated IgD. Earlier studies have shown that inhibitors of protein tyrosine kinase (PTK) completely prevented upregulation of IgD-R in response to oligomeric IgD, suggesting that cross-linking of IgD-R may induce signal transduction and functional consequences through one or more PTK activation pathways, leading to upregulation of IgD-R. In the present study we show that cross-linking of IgD-R by oligomeric IgD indeed results in (a) T cell activation as seen by tyrosine phosphorylation of several intracellular proteins, (b) tyrosine phosphorylation of p56 Lck and PLC-gamma in 7C5 T hybridoma cells, and (c) phosphorylation of an approximately 29-kDa band that exhibits strong affinity for IgD. We analyzed tyrosine phosphorylation of p56 Lck and PLC-gamma in BALB/c splenic T cells that were exposed to oligomeric IgD both in vivo and in vitro. In vitro cross-linking as well as in vivo followed by in vitro cross-linking of IgD-R resulted in enhanced phosphorylation of p56 Lck and moderate tyrosine phosphorylation of PLC-gamma. These results suggest that interactions between IgD-R and IgD mediate signal transduction and support our previous findings that IgD-R+ T cells enhance cognate T cell-B cell interactions and antibody production.

Animals↗

PubMed2001

Oxidized low density lipoprotein decreases macrophage expression of scavenger receptor B-I.

Scavenger receptor class B type I (SR-BI) has recently been identified as a high density lipoprotein (HDL) receptor that mediates bidirectional flux of cholesterol across the plasma membrane. We have previously demonstrated that oxidized low density lipoprotein (OxLDL) will increase expression of another class B scavenger receptor, CD36 (Han, J., Hajjar, D. P., Febbraio, M., and Nicholson, A. C. (1997) J. Biol. Chem. 272, 21654-21659). In studies reported herein, we evaluated the effects of OxLDL on expression of SR-BI in macrophages to determine how exposure to this modified lipoprotein could alter SR-BI expression and cellular lipid flux. OxLDL decreased SR-BI expression in a dose- and time-dependent manner. Incubation with OxLDL had no effect on the membrane distribution of SB-BI, and it decreased expression of both cytosolic and membrane protein. Consistent with its effect on SR-BI protein expression, OxLDL decreased SR-BI mRNA in a dose-dependent manner. The ability of OxLDL to decrease SR-BI expression was dependent on the degree of LDL oxidation. OxLDL decreased both [(14)C]cholesteryl oleate/HDL uptake and efflux of [(14)C]cholesterol to HDL in a time-dependent manner. Incubation of macrophages with 7-ketocholesterol, but not free cholesterol, also inhibited expression of SR-BI. Finally, we demonstrate that the effect of OxLDL on SR-BI is dependent on the differentiation state of the monocyte/macrophage. These results imply that in addition to its effect in inducing foam cell formation in macrophages through increased uptake of oxidized lipids, OxLDL may also enhance foam cell formation by altering SR-BI-mediated lipid flux across the cell membrane.

Animals↗

PubMed2003

Ephs and ephrins during early stages of chick embryogenesis.

The Eph family of receptor tyrosine kinases and their ligands, the ephrins, are membrane-bound proteins that mediate bidirectional signals between adjacent cells. By modulating cytoskeleton dynamics affecting cell motility and adhesion, Ephs and ephrins orchestrate cell movements during multiple morphogenetic processes, including gastrulation, segmentation, angiogenesis, axonal pathfinding, and neural crest cell migration. The full repertoire of developmental Eph/ephrin functions remains uncertain, however, because coexpression of multiple receptor and ligand family members, and promiscuous interactions between them, can result in functional redundancy. A complete understanding of expression patterns, therefore, is a necessary prerequisite to understanding function. Here, we present a comprehensive expression overview for 10 Eph and ephrin genes during the first 48 hr of chick embryo development. First, dynamic expression domains are described for each gene between Hamburger and Hamilton stages 4 and 12; second, comparative analyses are presented of Eph/ephrin expression patterns in the primitive streak, the somites, the vasculature, and the brain. Complex spatially and temporally dynamic expression patterns are revealed that suggest novel functions for Eph and ephrin family members in both known and previously unrecognized processes. This study will provide a valuable resource for further experimental investigations of Eph and ephrin functions during early embryonic development.

Animals↗

PubMed2006

Plugs in nuclear pores: transcripts in early oocyte development identified with nanotechniques.

Throughout oogenesis, huge amounts of RNA are produced that are needed for early development. Early stages of oocyte development are characterized by high transcriptional activity whereas translation of maternal RNA dominates late stages. Nuclear pore complexes (NPCs), located in the nuclear envelope (NE), mediate bidirectional macromolecule exchange between the nuclear and cytosolic compartments including RNA export. Here, we report on structural correlates of this transport pathway at single NPC level. Using atomic force microscopy (AFM), we imaged the nucleoplasmic ("inner") surface of the NE of Xenopus laevis oocytes in different stages of development. We found that NPC frequency per nucleus increases with maturation. However, individual NPCs are more active in immature stages. In early stages, known for high transcriptional activity, we found nearly 10% of NPC central channels plugged with a 400-800 kDa mass. In contrast, the incidence of plugged NPCs was below 1% in late oocyte stages. On-site RNA digestion led to a change in plug shape from prominent to flat while plug mass decreased by almost 20%. Quantitative AFM analysis revealed that RNase exposure reduced total nucleoplasmic NPC mass by about 58 and 25% in early and late stage oocytes, respectively. We conclude: (i) NPCs of immature oocytes are more active in RNA transport, (ii) Plugs identified at the nucleoplasmic entrance of NPC central channels represent ribonucleoproteins exiting the nucleus, (iii) RNA is a structural component of the NPC nanomachine.

Animals↗

PubMed1985

Adenosine receptor-mediated changes in cyclic AMP production and DNA synthesis in cultured arterial smooth muscle cells.

The effects of adenosine and two analogs, L-phenylisopropyladenosine (L-PIA) and 5'-N-ethylcarboxamidoadenosine (NECA), on cAMP production and on platelet-derived growth factor (PDGF)-stimulated initiation of DNA synthesis in growth-arrested cultures of rat arterial smooth muscle cells (SMC) were studied. The intracellular cAMP concentration was dose-dependently enhanced by micromolar concentrations of adenosine and its analogs, with the potency order NECA greater than adenosine greater than L-PIA. The effect was antagonized, in a competitive manner, by the adenosine receptor antagonist 8-phenyltheophylline (8-PT). The stimulatory effect of adenosine was enhanced by 3 microM dipyridamole an adenosine-uptake blocker. DNA synthesis was inhibited in a parallel manner, showing the same potency order. The inhibition was antagonized by 8-PT. Forskolin, a diterpene with the ability to stimulate the catalytic unit of adenylate cyclase and thereby cAMP formation, potentiated the effects of micromolar concentrations of NECA and L-PIA. Forskolin, by itself, stimulated cAMP production and inhibited DNA synthesis. The forskolin-stimulated increase in cAMP was inhibited by L-PIA at nanomolar concentrations. L-PIA in the nanomolar concentration range also stimulated DNA synthesis when initiation was stimulated with suboptimal concentrations of PDGF. These findings suggest the presence of adenosine receptors of both the A1- and A2-subtype on SM-mediating bidirectional changes of cAMP and DNA synthesis.

Adenine↗

PubMed2005

Proprotein convertases furin and PC5: targeting atherosclerosis and restenosis at multiple levels.

Several growth factors, chemokines, adhesion molecules, and proteolytic enzymes important for cell-cell/cell-matrix interactions in atherosclerosis and restenosis are initially synthesized as inactive precursor proteins. Activation of proproteins to biologically active molecules is regulated by limited endoproteolytic cleavage at dibasic amino acid residues. This type of activation typically requires the presence of suitable proprotein convertases (PCs). The PC-isozymes furin and PC5 are expressed in human atherosclerotic lesions and have been found to be up-regulated, following vascular injury in animal models in vivo. In vitro, these PCs can regulate vascular smooth muscle cell and macrophage functions and signaling events, through activation of pro-alpha-integrins and/or pro-membrane-type matrix metalloproteinases. Integrins link the cytoskeleton with the extracellular matrix and mediate bidirectional signaling and mechanotransduction, whereas matrix metalloproteinases are the major matrix-degrading enzymes. Both activities are required for cell recruitment to the intima. Furthermore, cleavage of extracellular matrix molecules by matrix metalloproteinases potentially contributes to weakening of the fibrous cap, promoting plaque rupture. Based on these recent in vitro and in vivo data, furin and PC5 are potential contributors to the initiation, progression, and complications of atherosclerosis and restenosis. Targeting these PCs may provide future anti-atherosclerotic therapies.

Atherosclerosis↗

PubMed1992

Cytokines and neuropathology.

Inflammatory processes in the brain require the cooperation of immunocompetent cells and glial cells, which communicate by secreting bidirectional mediators. Resident cells within the nervous system can synthesize and secrete inflammatory cytokines, as well as neuropeptides, contributing to the response within the CNS to injury or immunological challenge. Although the mechanisms of cell activation and immune interaction are poorly understood, accumulating evidence implicates these pathways in neuropathogenesis, as described here by Sharon Wahl and colleagues. For example, in the acquired immune deficiency syndrome (AIDS), HIV-1-induced nervous system dysfunction and dementia are associated with the presence of infiltrating leukocytes and the release of inflammatory cytokines. Defining the pathways of cytokine dysregulation and neurotoxicity invoked by the infiltrating leukocytes, as well as the contribution of the neural cells themselves, may help to identify mechanisms of intervention in this and other debilitating CNS diseases.

Acquired Immunodeficiency Syndrome↗

PubMed1999

Symmetrical mutant phenotypes of the receptor EphB4 and its specific transmembrane ligand ephrin-B2 in cardiovascular development.

Ephrin-B2 is a transmembrane ligand that is specifically expressed on arteries but not veins and that is essential for cardiovascular development. However, ephrin-B2 is also expressed in nonvascular tissues and interacts with multiple EphB class receptors expressed in both endothelial and nonendothelial cell types. Thus, the identity of the relevant receptor for ephrin-B2 and the site(s) where these molecules interact to control angiogenesis were not clear. Here we show that EphB4, a specific receptor for ephrin-B2, is exclusively expressed by vascular endothelial cells in embryos and is preferentially expressed on veins. A targeted mutation in EphB4 essentially phenocopies the mutation in ephrin-B2. These data indicate that ephrin-B2-EphB4 interactions are intrinsically required in vascular endothelial cells and are consistent with the idea that they mediate bidirectional signaling essential for angiogenesis.

Animals↗

PubMed2003

Structural determinants of integrin recognition by talin.

The binding of cytoplasmic proteins, such as talin, to the cytoplasmic domains of integrin adhesion receptors mediates bidirectional signal transduction. Here we report the crystal structure of the principal integrin binding and activating fragment of talin, alone and in complex with fragments of the beta 3 integrin tail. The FERM (four point one, ezrin, radixin, and moesin) domain of talin engages integrins via a novel variant of the canonical phosphotyrosine binding (PTB) domain-NPxY ligand interaction that may be a prototype for FERM domain recognition of transmembrane receptors. In combination with NMR and mutational analysis, our studies reveal the critical interacting elements of both talin and the integrin beta 3 tail, providing structural paradigms for integrin linkage to the cell interior.

Amino Acid Sequence↗

PubMed2003

A time-dependent phase shift in the mammalian unfolded protein response.

Unfolded or misfolded proteins in the endoplasmic reticulum (ER) must be refolded or degraded to maintain homeostasis of the ER. The ATF6 and IRE1-XBP1 pathways are important for the refolding process in mammalian cells; activation of these transcriptional programs culminates in induction of ER-localized molecular chaperones and folding enzymes. We show here that degradation of misfolded glycoprotein substrates requires transcriptional induction of EDEM (ER degradation-enhancing alpha-mannosidase-like protein), and that this is mediated specifically by IRE1-XBP1 and not by ATF6. As XBP1 is produced after ATF6 activation, our results reveal a time-dependent transition in the mammalian unfolded protein response: an ATF6-mediated unidirectional phase (refolding only) is followed by an XBP1-mediated bidirectional phase (refolding plus degradation) as the response progresses.

Activating Transcription Factor 6↗

PubMed2004

Imaging of single-molecule translocation through nuclear pore complexes.

Nuclear pore complexes (NPCs) mediate bidirectional transport of proteins, RNAs, and ribonucleoprotein complexes across the double-membrane nuclear envelope. In vitro studies with purified transport cofactors have revealed a general scheme of cofactor-dependent transport energetically driven by the G protein Ran. However, the size and complexity of NPCs have made it difficult to clearly define the loci and kinetics of the cofactor-NPC interactions required for transport. We now report the use of single-molecule fluorescence microscopy to directly monitor a model protein substrate undergoing transport through NPCs in permeabilized cells. This substrate, NLS-2xGFP, interacts with NPCs for an average of 10 +/- 1 ms during transport. However, because the maximum nuclear accumulation rate of NLS-2xGFP was measured to be at least approximately 10(3) molecules per NPC per s, NPCs must be capable of transporting at least approximately 10 substrate molecules simultaneously. Molecular tracking reveals that substrate molecules spend most of their transit time randomly moving in the central pore of the NPC and that the rate-limiting step is escape from the central pore.

Antigens, Viral, Tumor↗

PubMed2005

Regulation of heterogenous nuclear ribonucleoprotein A1 transport by phosphorylation in cells stressed by osmotic shock.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is an alternative splicing factor that is mainly nuclear, although it shuttles rapidly between nuclear and cytoplasmic compartments. Cells stressed by osmotic shock (OSM) activate the mitogen-activated protein kinase kinase(3/6)-p38 signaling pathway, which in turn results in accumulation of hnRNP A1 in the cytoplasm. This effect modulates alternative splicing regulation in vivo and correlates with increased hnRNP A1 phosphorylation. We have characterized the molecular mechanism involved in the cytoplasmic accumulation of hnRNP A1 in NIH 3T3 cells subjected to OSM. This treatment results in serine-specific phosphorylation within a C-terminal peptide, dubbed the "F-peptide," which is adjacent to the M9 motif that mediates bidirectional transport of hnRNP A1. Analysis of mutants in which the F-peptide serines were replaced by aspartic acids or alanines showed that F-peptide phosphorylation is required for the subcellular redistribution of hnRNP A1 in cells subjected to OSM. Furthermore, F-peptide phosphorylation modulates the interaction of hnRNP A1 with transportin Trn1. Our findings suggest that the phosphorylation of F-peptide by cell-signaling pathways regulates the rate of hnRNP A1 nuclear import.

Active Transport, Cell Nucleus↗

PubMed2005

Membrane-proximal {alpha}/{beta} stalk interactions differentially regulate integrin activation.

The affinity of integrin-ligand interaction is regulated extracellularly by divalent cations and intracellularly by inside-out signaling. We report here that the extracellular, membrane-proximal alpha/beta stalk interactions not only regulate cation-induced integrin activation but also play critical roles in propagating inside-out signaling. Two closely related integrins, alphaIIbbeta3 and alphaVbeta3, share high structural homology and bind to similar ligands in an RGD-dependent manner. Despite these structural and functional similarities, they exhibit distinct responses to Mn(2+). Although alphaVbeta3 showed robust ligand binding in the presence of Mn(2+), alphaIIbbeta3 showed a limited increase but failed to achieve full activation. Swapping alpha stalk regions between alphaIIb and alphaV revealed that the alpha stalk, but not the ligand-binding head region, was responsible for the difference. A series of alphaIIb/alphaV domain-swapping chimeras were constructed to identify the responsible domain. Surprisingly, the minimum component required to render alphaIIbbeta3 susceptible to Mn(2+) activation was the alphaV calf-2 domain, which does not contain any divalent cation-binding sites. The calf-2 domain makes interface with beta epidermal growth factor 4 and beta tail domain in three-dimensional structure. The effect of calf-2 domain swapping was partially reproduced by mutating the specific amino acid residues in the calf-2/epidermal growth factor 4-beta tail domain interface. When this interface was constrained by an artificially introduced disulfide bridge, the Mn(2+)-induced alphaVbeta3-fibrinogen interaction was significantly impaired. Notably, a similar disulfide bridge completely abrogated fibrinogen binding to alphaIIbbeta3 when alphaIIbbeta3 was activated by cytoplasmic tail truncation to mimic inside-out signaling. Thus, disruption/formation of the membrane-proximal alpha/beta stalk interface may act as an on/off switch that triggers integrin-mediated bidirectional signaling.

Animals↗

PubMed1998

Molecular architecture of the yeast nuclear pore complex: localization of Nsp1p subcomplexes.

The nuclear pore complex (NPC), a supramolecular assembly of approximately 100 different proteins (nucleoporins), mediates bidirectional transport of molecules between the cytoplasm and the cell nucleus. Extensive structural studies have revealed the three- dimensional (3D) architecture of Xenopus NPCs, and eight of the approximately 12 cloned and characterized vertebrate nucleoporins have been localized within the NPC. Thanks to the power of yeast genetics, 30 yeast nucleoporins have recently been cloned and characterized at the molecular level. However, the localization of these nucleoporins within the 3D structure of the NPC has remain elusive, mainly due to limitations of preparing yeast cells for electron microscopy (EM). We have developed a new protocol for preparing yeast cells for EM that yielded structurally well-preserved yeast NPCs. A direct comparison of yeast and Xenopus NPCs revealed that the NPC structure is evolutionarily conserved, although yeast NPCs are 15% smaller in their linear dimensions. With this preparation protocol and yeast strains expressing nucleoporins tagged with protein A, we have localized Nsp1p and its interacting partners Nup49p, Nup57p, Nup82p, and Nic96p by immuno-EM. Accordingly, Nsp1p resides in three distinct subcomplexes which are located at the entry and exit of the central gated channel and at the terminal ring of the nuclear basket.

Animals↗

PubMed2005

Interaction of organic cations with a newly identified plasma membrane monoamine transporter.

Many endogenous compounds and xenobiotics are organic cations that rely on polyspecific organic cation transporters (OCTs) to traverse cell membranes. We recently cloned a novel human plasma membrane monoamine transporter (PMAT) that belongs to the equillibrative nucleoside transporter (ENT) family. We have reported previously that, unlike other ENTs, PMAT (also known as ENT4) is a Na+-independent and membrane potential-sensitive transporter that transports monoamine neurotransmitters and the neurotoxin 1-methyl-4-phenylpyridinium (MPP+). These compounds are the known substrates for OCTs, which raises the possibility that PMAT functions as a polyspecific transporter like the OCTs. In the present study, we analyzed the interaction of PMAT with a series of structurally diverse organic cations using MDCK cells stably expressing human PMAT. Our study showed that PMAT interacts with many organic cations that have heterogeneous chemical structures. PMAT transports classic OCT substrates, such as tetraethylammonium, guanidine, and histamine. Prototype OCT inhibitors, including cimetidine, and type II cations (e.g., quinidine, quinine, verapamil, and rhodamine123) are also PMAT inhibitors. An analysis of molecular structures and apparent binding affinities revealed that charge and hydrophobicity are the principal determinants for transporter-substrate/inhibitor interaction. A planar aromatic mass seems to be important for high affinity interaction. trans-Stimulation and efflux studies demonstrate that PMAT is able to mediate bidirectional transport. These functional properties of PMAT are strikingly similar to those of the OCTs. We therefore conclude that PMAT can function as a polyspecific organic cation transporter, which may play a role in organic cation transport in vivo.

1-Methyl-4-phenylpyridinium↗

PubMed2000

New steps toward the nucleocytoplasmic traffic of macromolecules.

The nucleocytoplasmic transport of functional molecules is mediated bidirectionally through the nuclear pore complex (NPC), which spans the double membranes of the nuclear envelope. It has recently been shown that signaling between the nucleus and the cytoplasm plays a key role in coordinating the cellular processes such as the cell cycle and cell differentiation (Yoneda, 2000). As the result of recent extensive analysis, significant progress has been made in our understanding of the fundamental mechanism of nuclear transport of proteins and RNAs and numerous transport factors have now been identified. In this special issue of review articles, we focus on our rapid growing knowledge of nucleocytoplasmic transport, especially the translocation of proteins through the NPC and mRNA export, and review this exciting field from various points of view including cell biology, structural biology and yeast genetics.

Active Transport, Cell Nucleus↗