Surface particle transport mechanism independent of myosin II in Dictyostelium.

Cellular locomotion could be driven by the rearward transport of membrane-bound particles observed on motile fibroblasts, keratinocytes and neuronal growth cones. A force propelling free surface particles backwards could move the cell forwards if the particles were anchored to a rigid substratum. During capping, myosin II ('double-headed' myosin) draws crosslinked membrane proteins to the rear of a cell. The mhcA- mutant of the amoebal stage of the slime mould Dictyostelium discoideum, in which the myosin II gene has been deleted, cannot cap surface particles but can crawl along the substratum. Thus, the mechanism driving capping is not essential for locomotion. We show here that the null mutant is capable of a different type of active rearward transport, independent of myosin II and distinct from capping. The transported particles on mhcA- cells follow parallel paths. In the wild-type Ax2 strain, myosin II causes particles to converge towards a focal point and significantly increases the velocity of transport behind the leading edge of the cell.     

SLAC1 is required for plant guard cell S-type anion channel function in stomatal signalling

Stomatal pores, formed by two surrounding guard cells in the epidermis of plant leaves, allow influx of atmospheric carbon dioxide in exchange for transpirational water loss. Stomata also restrict the entry of ozone--an important air pollutant that has an increasingly negative impact on crop yields, and thus global carbon fixation and climate change. The aperture of stomatal pores is regulated by the transport of osmotically active ions and metabolites across guard cell membranes. Despite the vital role of guard cells in controlling plant water loss, ozone sensitivity and CO2 supply, the genes encoding some of the main regulators of stomatal movements remain unknown. It has been proposed that guard cell anion channels function as important regulators of stomatal closure and are essential in mediating stomatal responses to physiological and stress stimuli. However, the genes encoding membrane proteins that mediate guard cell anion efflux have not yet been identified. Here we report the mapping and characterization of an ozone-sensitive Arabidopsis thaliana mutant, slac1. We show that SLAC1 (SLOW ANION CHANNEL-ASSOCIATED 1) is preferentially expressed in guard cells and encodes a distant homologue of fungal and bacterial dicarboxylate/malic acid transport proteins. The plasma membrane protein SLAC1 is essential for stomatal closure in response to CO2, abscisic acid, ozone, light/dark transitions, humidity change, calcium ions, hydrogen peroxide and nitric oxide. Mutations in SLAC1 impair slow (S-type) anion channel currents that are activated by cytosolic Ca2+ and abscisic acid, but do not affect rapid (R-type) anion channel currents or Ca2+ channel function. A low homology of SLAC1 to bacterial and fungal organic acid transport proteins, and the permeability of S-type anion channels to malate suggest a vital role for SLAC1 in the function of S-type anion channels.     

Mode of action of yeast killer toxins: channel formation in lipid bilayer membranes

The toxic action of yeast killer proteins seems to involve selective functional damage to the plasma membrane of the sensitive cell. Physiological effects include leakage of K+ (refs 1, 2), inhibition of active transport of amino acids and acidification of the cell interior. These effects are strikingly similar to the effects of certain bacterial colicins which have been demonstrated previously to form channels in membranes. Proposed mechanisms of action have usually postulated a limited permeability change induced by the toxin in the plasma membrane. We report here that a killer toxin from the yeast Pichia kluyveri forms ion-permeable channels in phospholipid bilayer membranes, and we propose that the in vitro electrophysiological properties of these channels account for the morbid effects observed in intoxicated cells. A preliminary account of this work has appeared elsewhere.     

Resemblance of actin-binding protein/actin gels to covalently crosslinked networks

The maintainance of the shape of cells is often due to their surface elasticity, which arises mainly from an actin-rich cytoplasmic cortex. On locomotion, phagocytosis or fission, however, these cells become partially fluid-like. The finding of proteins that can bind to actin and control the assembly of, or crosslink, actin filaments, and of intracellular messages that regulate the activities of some of these actin-binding proteins, indicates that such 'gel-sol' transformations result from the rearrangement of cortical actin-rich networks. Alternatively, on the basis of a study of the mechanical properties of mixtures of actin filaments and an Acanthamoeba actin-binding protein, alpha-actinin, it has been proposed that these transformations can be accounted for by rapid exchange of crosslinks between actin filaments: the cortical network would be solid when the deformation rate is greater than the rate of crosslink exchange, but would deform or 'creep' when deformation is slow enough to permit crosslinker molecules to rearrange. Here we report, however, that mixtures of actin filaments and actin-binding protein (ABP), an actin crosslinking protein of many higher eukaryotes, form gels rheologically equivalent to covalently crosslinked networks. These gels do not creep in response to applied stress on a time scale compatible with most cell-surface movements. These findings support a more complex and controlled mechanism underlying the dynamic mechanical properties of cortical cytoplasm, and can explain why cells do not collapse under the constant shear forces that often exist in tissues.     

Novel form of growth cone motility involving site-directed actin filament assembly.

Regulation of cytoskeletal structure and motility by extracellular signals is essential for all directed forms of cell movement and underlies the developmental process of axonal guidance in neuronal growth cones. Interaction with polycationic microbeads can trigger morphogenic changes in neurons and muscle cells normally associated with formation of pre- and postsynaptic specializations. Furthermore, when various types of microscopic particles are applied to the lamellar surface of a neuronal growth cone or motile cell they often exhibit retrograde movement at rates of 1-6 microns min-1 (refs 3-6). There is strong evidence that this form of particle movement results from translocation of membrane proteins associated with cortical F-actin networks, not from bulk retrograde lipid flow and may be a mechanism behind processes such as cell locomotion, growth cone migration and capping of cell-surface antigens. Here we report a new form of motility stimulated by polycationic bead interactions with the growth-cone membrane surface. Bead binding rapidly induces intracellular actin filament assembly, coincident with a production of force sufficient to drive bead movements. These extracellular bead movements resemble intracellular movements of bacterial parasites known to redirect host cell F-actin assembly for propulsion. Our results suggest that site-directed actin filament assembly may be a widespread cellular mechanism for generating force at membrane-cytoskeletal interfaces.     

Leukocytes express a novel gene encoding a putative transmembrane protein-kinase devoid of an extracellular domain

Tyrosine-specific phosphorylation of proteins is a key to the control of diverse pathways leading to cell growth and differentiation. The protein-tyrosine kinases described to date are either transmembrane proteins having an extracellular ligand binding domain or cytoplasmic proteins related to the v-src oncogene. Most of these proteins are expressed in a wide variety of cells and tissues; few are tissue-specific. Previous studies have suggested that lymphokines could mediate haematopoietic cell survival through their action on glucose transport, regulated in some cells through the protein-tyrosine kinase activity of the insulin receptor. We have investigated the possibility that insulin receptor-like genes are expressed specifically in haematopoietic cells. Using the insulin receptor-related avian sarcoma oncogene v-ros as a probe, we have isolated and characterized the complementary DNA of a novel gene, ltk (leukocyte tyrosine kinase). The ltk gene is expressed mainly in leukocytes, is related to several tyrosine kinase receptor genes of the insulin receptor family and has unique structural properties: it apparently encodes a transmembrane protein devoid of an extracellular domain. Two candidate ltk proteins have been identified with antibodies in the mouse thymus, and have properties indicating that they are integral membrane proteins. These features suggest that ltk could be a signal transduction subunit for one or several of the haematopoietic receptors.     

Altered Gs and adenylate cyclase activity in human GH-secreting pituitary adenomas

Gs and Gi are guanine nucleotide-binding, heterotrimer proteins that regulate the activity of adenylate cyclase, and are responsible for transferring stimulatory and inhibitory hormonal signals, respectively, from cell surface receptors to the enzyme catalytic unit. These proteins can be directly activated by agents such as GTP and analogues, fluoride and magnesium. Decreased amounts of Gs and Gi, and even the absence of Gs, have been described, whereas an altered Gs has been reported in a cultured cell line (UNC variant of S49 lymphoma cells), but has never been observed in human disease states. We have found a profoundly altered Gs protein in a group of human growth hormone-secreting pituitary adenomas, characterized by high secretory activity and intracellular cyclic AMP levels. In the membranes from these tumours no stimulation of adenylate cyclase activity by growth hormone-releasing hormone, by GTP or by fluoride was observed. Indeed, the last two agents caused an inhibition, probably mediated by Gi. In contrast, adenylate cyclase stimulation by Mg2+ was enormously increased. This altered pattern of adenylate cyclase regulation was reproduced when a cholate extract of the tumour membranes (which contains G proteins) was reconstituted with Gs-free, cyc- S49 cell membranes. Inasmuch as secretion from somatotrophic cells is known to be a cAMP-dependent function, the alteration of Gs could be the direct cause of the high secretory activity of the tumours in which it occurs.     

Receptor binding and internalization of immobilized transcobalamin II by mouse leukaemia cells

Membrane transport of vitamin B12 (cyanocobalamin; Cbl) into mammalian cells is mediated by the serum protein transcobalamin II (TCII). In mouse leukaemia L1210 cells, TCII-Cbl binds to membrane receptors in a rapid, temperature-independent step and is internalized by a slow, temperature-dependent process. To delineate the location of receptors on these cells, we have constructed a visual probe by covalently coupling purified TCII-Cbl to submicrometre latex particles (minibeads). We report here that when L1210 cells are incubated with minibeads containing TCII-Cbl at 4 degrees C and examined by scanning electron microscopy (SEM), the particles are found attached predominantly to microvilli. Incubation of the cells at 37 degrees C results in the internalization of the minibeads. As visualized by transmission electron microscopy (TEM), this endocytotic process seems to occur in clathrin-coated pits and vesicles at the cell surface.     

The RickA protein of Rickettsia conorii activates the Arp2/3 complex

Actin polymerization, the main driving force for cell locomotion, is also used by the bacteria Listeria and Shigella and vaccinia virus for intracellular and intercellular movements. Seminal studies have shown the key function of the Arp2/3 complex in nucleating actin and generating a branched array of actin filaments during membrane extension and pathogen movement. Arp2/3 requires activation by proteins such as the WASP-family proteins or ActA of Listeria. We previously reported that actin tails of Rickettsia conorii, another intracellular bacterium, unlike those of Listeria, Shigella or vaccinia, are made of long unbranched actin filaments apparently devoid of Arp2/3 (ref. 4). Here we identify a R. conorii surface protein, RickA, that activates Arp2/3 in vitro, although less efficiently than ActA. In infected cells, Arp2/3 is detected on the rickettsial surface but not in actin tails. When expressed in mammalian cells and targeted to the membrane, RickA induces filopodia. Thus RickA-induced actin polymerization, by generating long actin filaments reminiscent of those present in filopodia, has potential as a tool for studying filopodia formation.     

白芷细胞外CaM结合位点的扫描电镜定位研究

利用胶体金标记的具有生物活性的钙调素（Ｃａｌｍｏｄｕｌｉｎ,ＣａＭ）,对白芷愈伤组织培养细胞胞外钙调素结合位点进行了扫描电镜定位,发现白芷愈伤组织培养细胞表面有不同密度的金颗粒,证明其细胞表面存在着ＣａＭ结合位点．     

Chloride/proton antiporter activity of mammalian CLC proteins ClC-4 and ClC-5

ClC-4 and ClC-5 are members of the CLC gene family, with ClC-5 mutated in Dent's disease, a nephropathy associated with low-molecular-mass proteinuria and eventual renal failure. ClC-5 has been proposed to be an electrically shunting Cl- channel in early endosomes, facilitating intraluminal acidification. Motivated by the discovery that certain bacterial CLC proteins are secondary active Cl-/H+ antiporters, we hypothesized that mammalian CLC proteins might not be classical Cl- ion channels but might exhibit Cl(-)-coupled proton transport activity. Here we report that ClC-4 and ClC-5 carry a substantial amount of protons across the plasma membrane when activated by positive voltages, as revealed by measurements of pH close to the cell surface. Both proteins are able to extrude protons against their electrochemical gradient, demonstrating secondary active transport. H+, but not Cl-, transport was abolished when a pore glutamate was mutated to alanine (E211A). ClC-0, ClC-2 and ClC-Ka proteins showed no significant proton transport. The muscle channel ClC-1 exhibited a small H+ transport that might be physiologically relevant. For ClC-5, we estimated that Cl- and H+ transport contribute about equally to the total charge movement, raising the possibility that the coupled Cl-/H+ transport of ClC-4 and ClC-5 is of significant magnitude in vivo.     

Cloning and sequencing of human cholesteryl ester transfer protein cDNA

The transfer of insoluble cholesteryl esters among lipoprotein particles is a vital step in normal cholesterol homeostasis and may be involved in the development of atherosclerosis. Extrahepatic tissues lack the enzymes required for the degradation of sterols to the excretable form of bile acids. Cholesterol synthesized in these tissues in excess of that needed for the synthesis of cell membranes or steroid hormones must accordingly be returned through the plasma to the liver for catabolism. The series of reactions involved has been termed reverse cholesterol transport. Catalysed steps of this pathway are believed to include an efflux from peripheral cells, which generates a diffusion gradient between these membranes and extracellular fluid; esterification of this cholesterol by lecithin-cholesterol acyltransferase (LCAT) (phosphatidylcholine-sterol acyltransferase) acting on species of high-density lipoproteins; transfer of the cholesteryl esters formed (largely to low- and very low-density lipoproteins) (LDL and VLDL) by a cholesteryl ester transfer protein (CETP); and removal of these lipoproteins, together with their cholesteryl ester content, by the liver through receptor-mediated and nonspecific endocytosis. Of these steps, the CETP reaction is the least characterized. Several laboratories have reported the purification from human plasma of proteins active on cholesteryl ester transfer between lipoprotein particles and possibly between cells and plasma. However, the reported relative molecular mass (Mr), abundance and specificity of the purified activities have differed considerably. We have recently described the preparation of a highly active CETP of Mr 74,000 purified about 100,000-fold from human plasma, which may represent the functional component of earlier preparations. Using a partial amino-acid sequence from this purified protein, CETP complementary DNA derived from human liver DNA has been cloned and sequenced and the cloned DNA used to detect CETP messenger RNA in a number of human tissues.     

Function of DnaJ and DnaK as chaperones in origin-specific DNA binding by RepA

Heat-shock proteins are normal constituents of cells whose synthesis is increased on exposure to various forms of stress. They are interesting because of their ubiquity and high conservation during evolution. Two families of heat-shock proteins, hsp60s and hsp70s, have been implicated in accelerating protein folding and oligomerization and also in maintaining proteins in an unfolded state, thus facilitating membrane transport. The Escherichia coli hsp70 analogue, DnaK, and two other heat-shock proteins, DnaJ and GrpE, are required for cell viability at high temperatures and are involved in DNA replication of phage lambda and plasmids P1 and F. These three proteins are involved in replication in vitro of P1 DNA along with many host replication proteins and the P1 RepA initiator protein. RepA exists in a stable protein complex with DnaJ containing a dimer each of RepA and DnaJ. We report here that DnaK and DnaJ mediate an alteration in the P1 initiator protein, rendering it much more active for oriP1 DNA binding.     

Germ-line transmission of a disrupted beta 2-microglobulin gene produced by homologous recombination in embryonic stem cells

Major histocompatibility complex (MHC) class I molecules are integral membrane proteins present on virtually all vertebrate cells and consist of a heterodimer between the highly polymorphic alpha-chain and the beta 2-microglobulin (beta 2-m) protein of relative molecular mass 12,000 (ref. 1). These cell-surface molecules play a pivotal part in the recognition of antigens, the cytotoxic response of T cells, and the induction of self tolerance. It is possible, however, that the function of MHC class I molecules is not restricted to the immune system, but extends to a wide variety of biological reactions including cell-cell interactions. For example, MHC class I molecules seem to be associated with various cell-surface proteins, including the receptors for insulin, epidermal growth factor, luteinizing hormone and the beta-adrenergic receptor. In mice, class I molecules are secreted in the urine and act as highly specific olfactory cues which influence mating preference. The beta 2-m protein has also been identified as the smaller component of the Fc receptor in neonatal intestinal cells, and it has been suggested that the protein induces collagenase in fibroblasts. Cells lacking beta 2-m are deficient in the expression of MHC class I molecules, indicating that the association with beta 2-m is crucial for the transport of MHC class I molecules to the cell surface. The most direct means of unravelling the many biological functions of beta 2-m is to create a mutant mouse with a defective beta 2-m gene. We have now used the technique of homologous recombination to disrupt the beta 2-m gene. We report here that introduction of a targeting vector into embryonic stem cells resulted in beta 2-m gene disruption with high frequency. Chimaeric mice derived from blastocysts injected with mutant embryonic stem cell clones transmit the mutant allele to their offspring.     

Functional proteomic identification of DNA replication proteins by induced proteolysis in vivo

Evolutionarily diverse eukaryotic cells share many conserved proteins of unknown function. Some are essential for cell viability, emphasising their importance for fundamental processes of cell biology but complicating their analysis. We have developed an approach to the large-scale characterization of such proteins, based on conditional and rapid degradation of the target protein in vivo, so that the immediate consequences of bulk protein depletion can be examined. Budding yeast strains have been constructed in which essential proteins of unknown function have been fused to a 'heat-inducible-degron' cassette that targets the protein for proteolysis at 37 degrees C (ref. 4). By screening the collection for defects in cell-cycle progression, here we identify three DNA replication factors that interact with each other and that have uncharacterized homologues in human cells. We have used the degron strains to show that these proteins are required for the establishment and normal progression of DNA replication forks. The degron collection could also be used to identify other, essential, proteins with roles in many other processes of eukaryotic cell biology.     

Human immunodeficiency virus induces phosphorylation of its cell surface receptor

AIDS is an immunoregulatory disorder characterized by depletion of the CD4+, helper/inducer lymphocyte population. The causative agent of this disease is the human immunodeficiency virus, HIV, which infects CD4+ cells and leads to cytopathic effects characterized by syncytia formation and cell death. Recent studies have demonstrated that binding of HIV to its cellular receptor CD4 is necessary for viral entry. We find that binding of HIV to CD4 induces rapid and sustained phosphorylation of CD4 which could involve protein kinase C. HIV-induced CD4 phosphorylation can be blocked by antibody against CD4 and monoclonal antibody against the HIV envelope glycoprotein gp120, indicating that a specific interaction between CD4 and gp120 is required for phosphorylation. Electron microscopy shows that a protein kinase C inhibitor does not impair binding of HIV to CD4+ cells, but causes an apparent accumulation of virus particles at the cell surface, at the same time inhibiting viral infectivity. These results indicate a possible role for HIV-induced CD4 phosphorylation in viral entry and identify a potential target for antiviral therapy.     

Ecto-protein kinase activity on the external surface of neural cells

ATP is secreted in association with neurotransmitters at certain synapses and neuromuscular junctions. Extracellular ATP is known to exert potent effects on the activity of cells in the nervous system, where it can act as a neurotransmitter or as a modulator regulating the activity of other neurohormones. We have suggested that such modulation may involve the activity of extracellular protein phosphorylation systems. It is well known that intracellular protein kinases are important in the regulation of various neuronal functions, but protein kinases which use extracellular ATP to phosphorylate proteins localized at the external surface of the plasma membrane (ecto-protein kinases) have not been demonstrated in neuronal cells. Here we present direct evidence for the existence of an ecto-protein kinase and demonstrate endogenous substrates for its activity at the surface of intact neural cells. The phosphorylation of one of these surface proteins is selectively stimulated during cell depolarization. In addition, neuronal cell adhesion molecules (N-CAMs) appear to be among the substrates of ecto-protein kinase activity. These results suggest a role for surface protein phosphorylation in regulating specific functions of developing and mature neurones.     

带电粒子输运双群模型中的直向前近似及其可能的应用

在粒子输运双群模型中，引入直向前近似，计算了双群模型中的直向前粒子输运．通过计算，检验了该近似的可用性，进而讨论了将带电粒子输运双群模型推广到三维非均匀介质的可能性     

H-2-restricted cytolytic T cells specific for HLA can recognize a synthetic HLA peptide

It is generally accepted that T lymphocytes recognize antigens in the context of molecules encoded by genes in the major histocompatibility complex (MHC). MHC class II-restricted T cells usually recognize degraded or denatured rather than native forms of antigen on the surface of class II-bearing antigen presenting cells. It has recently been shown that short synthetic peptides corresponding to mapped antigenic sites of the influenza nucleoprotein (NP) can render uninfected target cells susceptible to lysis by NP-specific class I-restricted cytolytic T cells (CTL). These and earlier experiments that showed specific recognition of NP deletion mutant transfectants suggest that class I-restricted recognition might also involve processed antigenic fragments. One important issue arising from these studies is whether the model applies not only to viral proteins that are expressed internally (such as NP) but also to antigens normally expressed as integral membrane proteins at the cell surface. We have recently isolated class I-restricted mouse CTL clones that recognize class I gene products of the human MHC (HLA) as antigens in mouse cell HLA-transfectants. Here we show that these anti-HLA CTL can lyse HLA-negative syngeneic mouse cells in the presence of a synthetic HLA peptide. These results suggest that the model applies generally.     

Transferrin receptor on endothelium of brain capillaries

The blood/brain barrier prevents the passive diffusion of proteins and metabolites from cerebral blood vessels into tissue spaces around neuronal and glial cells. To provide nutrients for these cells, transport mechanisms must exist and indeed have been demonstrated for metabolites. We now show that monoclonal antibodies against rat and human transferrin receptors label blood capillaries in the brain but not in other tissues. In the rat this labelling occurs after injection of antibody into the blood, thus the receptors seem to be accessible at the endothelial surface. It is possible that transferrin receptors are expressed on these cells to allow transport of transferrin (and thus iron) into brain tissues.