Toxic HypF-N Oligomers Selectively Bind the Plasma Membrane to Impair Cell Adhesion Capability

The deposition of fibrillar protein aggregates in human organs is the hallmark of several pathological states, including highly debilitating neurodegenerative disorders and systemic amyloidoses. It is widely accepted that small oligomers arising as intermediates in the aggregation process, released by fibrils, or growing in secondary nucleation steps are the cytotoxic entities in protein-misfolding diseases, notably neurodegenerative conditions. Increasing evidence indicates that cytotoxicity is triggered by the interaction between nanosized protein aggregates and cell membranes, even though little information on the molecular details of such interaction is presently available. In this work, we propose what is, to our knowledge, a new approach, based on the use of single-cell force spectroscopy applied to multifunctional substrates, to study the interaction between protein oligomers, cell membranes, and/or the extracellular matrix. We compared the interaction of single Chinese hamster ovary cells with two types of oligomers (toxic and nontoxic) grown from the N-terminal domain of the Escherichia coli protein HypF. We were able to quantify the affinity between both oligomer type and the cell membrane by measuring the mechanical work needed to detach the cells from the aggregates, and we could discriminate the contributions of the membrane lipid and protein fractions to such affinity. The fundamental role of the ganglioside GM1 in the membrane-oligomers interaction was also highlighted. Finally, we observed that the binding of toxic oligomers to the cell membrane significantly affects the functionality of adhesion molecules such as Arg-Gly-Asp binding integrins, and that this effect requires the presence of the negatively charged sialic acid moiety of GM1.     

Plasma Membrane Turnover in Plant Cells

Steer, M. W. 1988. Plasma membrane turnover in plant cells.—J.exp. Bot. 39: 987–996. Plasma membrane turnover in plant cells occurs as a consequenceof secretion, which incorporates new membrane into the cellsurface and endocytosis, which internalizes surface membrane.Development of methods that provide estimates of the rate ofnew membrane flow to the cell surface has allowed the estimationof turnover times for the plasma membrane. These times rangefrom 10 min for a non-expanding secretory cell to 3 h for anelongating epidermal cell. At least part, if not all, of thereturn route into the cell is via endocytotic vesicles. Quantitativestudies are required to establish the precise level of flowthrough this route. However, turnover times estimated from theabundance of coated patches on the plasma membrane are comparableto those estimated from secretion studies. The effect of thesehigh turnover rates on a number of plasma membrane functionsare discussed and assessed. Key words: Plasma membrane, endocytosis, secretion, plant cells     

Cell Membrane Disruption Stimulates NO/PKG Signaling and Potentiates Cell Membrane Repair in Neighboring Cells

Resealing of a disrupted plasma membrane at the micron-diameter range requires Ca(2+)-regulated exocytosis. Repeated membrane disruptions reseal more quickly than the initial wound, and this potentiation of membrane resealing persists for at least 24 hours after the initial wound. Long-term potentiation of membrane resealing requires CREB-dependent gene expression, which is activated by the PKC- and p38 MAPK-dependent pathway in a wounded cell. The present study demonstrates that membrane resealing is potentiated in both wounded and neighboring cells in MDCK cells. Wounding of cells expressing CREB133, a mutant variant of CREB, does not show the potentiated response of cell membrane resealing in either wounded or neighboring cells. Furthermore, wounding of cells induces CREB phosphorylation, not only in wounded cells, but also in neighboring cells. Inhibition of the nitric oxide/PKG signaling pathway suppresses CREB phosphorylation in neighboring cells, but not in wounded cells. The potentiation of membrane resealing in neighboring cells is suppressed if the nitric oxide/PKG pathway is inhibited during the initial wound. Together, these results suggest that the nitric oxide/PKG pathway stimulates CREB phosphorylation in neighboring cells so that subsequent cell membrane disruptions of the neighboring cells reseal more quickly.     

Plasma Membrane Localization of Gαz Requires Two Signals

Three covalent attachments anchor heterotrimeric G proteins to cellular membranes: the α subunits are myristoylated and/or palmitoylated, whereas the γ chain is prenylated. Despite the essential role of these modifications in membrane attachment, it is not clear how they cooperate to specify G protein localization at the plasma membrane, where the G protein relays signals from cell surface receptors to intracellular effector molecules. To explore this question, we studied the effects of mutations that prevent myristoylation and/or palmitoylation of an epitope-labeled α subunit, α_z. Wild-type α_z (α_z-WT) localizes specifically at the plasma membrane. A mutant that incorporates only myristate is mistargeted to intracellular membranes, in addition to the plasma membrane, but transduces hormonal signals as well as does α_z-WT. Removal of the myristoylation site produced a mutant α_z that is located in the cytosol, is not efficiently palmitoylated, and does not relay the hormonal signal. Coexpression of βγ with this myristoylation defective mutant transfers it to the plasma membrane, promotes its palmitoylation, and enables it to transmit hormonal signals. Pulse-chase experiments show that the palmitate attached to this myristoylation-defective mutant turns over much more rapidly than does palmitate on α_z-WT, and that the rate of turnover is further accelerated by receptor activation. In contrast, receptor activation does not increase the slow rate of palmitate turnover on α_z-WT. Together these results suggest that myristate and βγ promote stable association with membranes not only by providing hydrophobicity, but also by stabilizing attachment of palmitate. Moreover, palmitoylation confers on α_z specific localization at the plasma membrane.     

Human Cells Display Reduced Apoptotic Function Relative to Chimpanzee Cells

Previously published gene expression analyses suggested that apoptotic function may be reduced in humans relative to chimpanzees and led to the hypothesis that this difference may contribute to the relatively larger size of the human brain and the increased propensity of humans to develop cancer. In this study, we sought to further test the hypothesis that humans maintain a reduced apoptotic function relative to chimpanzees by conducting a series of apoptotic function assays on human, chimpanzee and macaque primary fibroblastic cells. Human cells consistently displayed significantly reduced apoptotic function relative to the chimpanzee and macaque cells. These results are consistent with earlier findings indicating that apoptotic function is reduced in humans relative to chimpanzees.     

Heterogeneity of Arabinogalactan-Proteins on the Plasma Membrane of Rose Cells

Arabinogalactan-proteins (AGPs) have been purified from the plasma membrane of suspension-cultured Paul's Scarlet rose (Rosa sp.) cells. The two most abundant and homogeneous plasma membrane AGP fractions were named plasma membrane AGP1 (PM-AGP1) and plasma membrane AGP2 (PM-AGP2) and had apparent molecular masses of 140 and 217 kD, respectively. Both PM-AGP1 and PM-AGP2 had [beta]-(1-3)-, [beta]-(1,6)-, and [beta]-(1,3,6)-galactopyranosyl residues, predominantly terminal [alpha]-arabinofuranosyl residues, and (1,4)- and terminal glucuronopyranosyl residues. The protein moieties of PM-AGP1 and PM-AGP2 were both rich in hydroxyproline, alanine, and serine, but differed in the abundance of hydroxyproline, which was 1.6 times higher in PM-AGP2 than in PM-AGP1. Another difference was the overall protein content, which was 3.7% (w/w) in PM-AGP1 and 15% in PM-AGP2. As judged by their behavior on reverse-phase chromatography, PM-AGP1 and PM-AGP2 were not more hydrophobic than AGPs from the cell wall or culture medium. In contrast, a minor plasma membrane AGP fraction eluted later on reverse-phase chromatography and was more negatively charged at pH 5 than either PM-AGP1 or PM-AGP2. The more negatively charged fraction contained molecules with a glycosyl composition characteristic of AGPs and included at least two different macromolecules. The results of this investigation indicate that Rosa plasma membrane contains at least four distinct AGPs or AGP-like molecules. These molecules differed from each other in size, charge, hydrophobicity, amino-acyl composition, and/or protein content.     

Formation of Hybrid Cells and Heterokaryons by Fusion of Enucleated and Nucleated Cells

OBSERVATIONS on the synchronous behaviour of nuclei in naturally occurring binucleate and multinucleate cells have indicated that certain nuclear events are regulated by cytoplasmic factors^1,2. And the importance of cytoplasmic factors in regulating gene expression has been demonstrated in hybrid cells and heterokaryons formed by fusion of cells through the use of inactivated Sendai virus^3,4. We wish to describe a modification of virus-induced cell fusion, whereby hybrids and heterokaryons can be created by fusing nucleated cells of one species with enucleated cells from another. Using this technique the relative contribution of nucleus and cytoplasm in controlling the expression of specific functions can be better assessed.     

Fhit Delocalizes Annexin A4 from Plasma Membrane to Cytosol and Sensitizes Lung Cancer Cells to Paclitaxel

Fhit protein is lost or reduced in a large fraction of human tumors, and its restoration triggers apoptosis and suppresses tumor formation or progression in preclinical models. Here, we describe the identification of candidate Fhit-interacting proteins with cytosolic and plasma membrane localization. Among these, Annexin 4 (ANXA4) was validated by co-immunoprecipitation and confocal microscopy as a partner of this novel Fhit protein complex. Here we report that overexpression of Fhit prevents Annexin A4 translocation from cytosol to plasma membrane in A549 lung cancer cells treated with paclitaxel. Moreover, paclitaxel administration in combination with AdFHIT acts synergistically to increase the apoptotic rate of tumor cells both in vitro and in vivo experiments.     

NK Cells Respond to Haptens by the Activation of Calcium Permeable Plasma Membrane Channels

Natural Killer (NK) cells mediate innate immunity to infected and transformed cells. Yet, NK cells can also mount hapten-specific recall responses thereby contributing to contact hypersensitivity (CHS). However, since NK cells lack antigen receptors that are used by the adaptive immune system to recognize haptens, it is not clear if NK cells respond directly to haptens and, if so, what mediates these responses. Here we show that among four haptens the two that are known to induce NK cell-dependent CHS trigger the rapid influx of extracellular Ca²⁺ into NK cells and lymphocyte cell lines. Thus lymphocytes can respond to haptens independent of antigen presentation and antigen receptors. We identify the Ca²⁺-permeable cation channel TRPC3 as a component of the lymphocyte response to one of these haptens. These data suggest that the response to the second hapten is based on a distinct mechanism, consistent with the capacity of NK cells to discriminate haptens. These findings raise the possibility that antigen-receptor independent activation of immune cells contributes to CHS.     

Virus Movements on the Plasma Membrane Support Infection and Transmission between Cells

How viruses are transmitted across the mucosal epithelia of the respiratory, digestive, or excretory tracts, and how they spread from cell to cell and cause systemic infections, is incompletely understood. Recent advances from single virus tracking experiments have revealed conserved patterns of virus movements on the plasma membrane, including diffusive motions, drifting motions depending on retrograde flow of actin filaments or actin tail formation by polymerization, and confinement to submicrometer areas. Here, we discuss how viruses take advantage of cellular mechanisms that normally drive the movements of proteins and lipids on the cell surface. A concept emerges where short periods of fast diffusive motions allow viruses to rapidly move over several micrometers. Coupling to actin flow supports directional transport of virus particles during entry and cell-cell transmission, and local confinement coincides with either nonproductive stalling or infectious endocytic uptake. These conserved features of virus–host interactions upstream of infectious entry offer new perspectives for anti-viral interference.     

The Superoxide Synthases of Plasma Membrane Preparations from Cultured Rose Cells

Preparations of plasma membranes isolated from cultured rose (Rosa damascena Mill. cv Gloire de Guilan) cells synthesized O2- when incubated with either NADH or NADPH, as measured by an O2--specific assay based on the chemiluminescence of lucigenin. The activities were strongly dependent on the presence of Triton X-100. The Km for NADH was 159 [mu]M; that for NADPH was 19 [mu]M. Neither NADH- nor NADPH-dependent activity was inhibited by azide, an inhibitor of peroxidase, nor by antimycin A, an inhibitor of mitochondrial electron transport; both activities were inhibited by 30 to 100 nM diphenylene iodonium, an inhibitor of the mammalian NADPH oxidase. The NADH- and NADPH-dependent activities could be distinguished by detergent solubilization and ultracentrifugation: the NADH-dependent activity sedimented more easily, whereas the NADPH-dependent activity remained in suspension. One or both of these enzymes may provide the O2- seen when plant cells are exposed to pathogens or pathogen-associated elicitors; however, plasma membranes from rose cells treated with a Phytophthora elicitor had the same activity as control cells.     

凋亡细胞被识别和吞噬机制

凋亡细胞能被吞噬细胞吞噬,这对于正常组织的动态平衡和免疫反应是非常重要的。在凋亡细胞被吞噬（engulfment）的过程中,吞噬细胞表面存在大量的受体来识别凋亡细胞发出的信号,如：“吃我（eat-me）”信号、缺少存在于健康细胞上的“不吃我（don’t-eat-me）”信号以及由凋亡细胞分泌的可溶性“来吃我（come-get-me）”信号。至少有7种线虫（Caenorhabditis elegans）吞噬基因（它们在哺乳动物中存在同系物）组成了两条平行但部分重叠的吞噬信号通路,并且通过一个类似于巨胞CL（macropinocytosis）的“栓系-激活（tether and tickle）”保守机制吞噬凋亡细胞,这个机制因吞噬细胞和凋亡细胞的种类以及细胞凋亡后的时间差异而不同。     

Deregulation of Apoptotic Factors Bcl-xL and Bax Confers Apoptotic Resistance to Myeloid-derived Suppressor Cells and Contributes to Their Persistence in Cancer

Myeloid-derived suppressor cells (MDSCs) are heterogeneous immature myeloid cells that accumulate in response to tumor progression. Compelling data from mouse models and human cancer patients showed that tumor-induced inflammatory mediators induce MDSC differentiation. However, the mechanisms underlying MDSC persistence is largely unknown. Here, we demonstrated that tumor-induced MDSCs exhibit significantly decreased spontaneous apoptosis as compared with myeloid cells with the same phenotypes from tumor-free mice. Consistent with the decreased apoptosis, cell surface Fas receptor decreased significantly in tumor-induced MDSCs. Screening for changes of key apoptosis mediators downstream the Fas receptor revealed that expression levels of IRF8 and Bax are diminished, whereas expression of Bcl-xL is increased in tumor-induced MDSCs. We further determined that IRF8 binds directly to Bax and Bcl-x promoter in primary myeloid cells in vivo, and IRF8-deficient MDSC-like cells also exhibit increased Bcl-xL and decreased Bax expression. Analysis of CD69 and CD25 levels revealed that cytotoxic T lymphocytes (CTLs) are partially activated in tumor-bearing hosts. Strikingly, FasL but not perforin and granzymes were selectively activated in CTLs in the tumor-bearing host. ABT-737 significantly increased the sensitivity of MDSCs to Fas-mediated apoptosis in vitro. More importantly, ABT-737 therapy increased MDSC spontaneous apoptosis and decreased MDSC accumulation in tumor-bearing mice. Our data thus determined that MDSCs use down-regulation of IRF8 to alter Bax and Bcl-xL expression to deregulate the Fas-mediated apoptosis pathway to evade elimination by host CTLs. Therefore, targeting Bcl-xL is potentially effective in suppression of MDSC persistence in cancer therapy.     

Arabidopsis Synaptotagmin SYT1, a Type I Signal-anchor Protein,Requires Tandem C2 Domains for Delivery to the Plasma Membrane

The correct localization of integral membrane proteins to subcellular compartments is important for their functions. Synaptotagmin contains a single transmembrane domain that functions as a type I signal-anchor sequence in its N terminus and two calcium-binding domains (C₂A and C₂B) in its C terminus. Here, we demonstrate that the localization of an Arabidopsis synaptotagmin homolog, SYT1, to the plasma membrane (PM) is modulated by tandem C2 domains. An analysis of the roots of a transformant-expressing green fluorescent protein-tagged SYT1 driven by native SYT1 promoter suggested that SYT1 is synthesized in the endoplasmic reticulum, and then delivered to the PM via the exocytotic pathway. We transiently expressed a series of truncated proteins in protoplasts, and determined that tandem C₂A-C₂B domains were necessary for the localization of SYT1 to the PM. The PM localization of SYT1 was greatly reduced following mutation of the calcium-binding motifs of the C₂B domain, based on sequence comparisons with other homologs, such as endomembrane-localized SYT5. The localization of SYT1 to the PM may have been required for the functional divergence that occurred in the molecular evolution of plant synaptotagmins.     

Phospholipase C-activating Plasma Membrane Receptors and Calcium Signaling in Immortalized Human Airway Epithelial Cells

Mechanical clearance of inhaled dust particles and microorganisms is an important part of the innate defense mechanisms of mammalian airways. Airway epithelia are composed of various cell types with different degrees of cell polarity. Serous cells regulate composition and volume of luminal periciliary fluid and mucus. Autocrine, paracrine, or neuronal messengers determine the secretory and reabsorptive rates of electrolytes and water via cAMP-or inositol triphosphate/calcium-mediated intracellular signals. Comparison of the expression of calcium-mobilizing receptor types (G protein-coupled-, growth factor-, and cytokine receptors) in two types of human immortalized airway epithelial cells (S9, 16HBE14o-) revealed that receptor populations were qualitatively and quantitatively different in the two cell types. Sustained calcium signals were elicited by activation of purinergic receptors in 16HBE14o-cells or muscarinic acetylcholine or histamine receptors in S9 cells. These G protein-coupled receptors mobilized calcium from intracellular stores and activated capacitative calcium influx. The experimental cells may represent different types of original airway epithelial cells and seem to be suited as model cells to study cell signaling and protein expression during interaction with pathogens or their secretory products (e.g., virulence factors).     

Preferential Transfer of Certain Plasma Membrane Proteins onto T and B Cells by Trogocytosis

T and B cells capture antigens via membrane fragments of antigen presenting cells (APC) in a process termed trogocytosis. Whether (and how) a preferential transfer of some APC components occurs during trogocytosis is still largely unknown. We analyzed the transfer onto murine T and B cells of a large panel of fluorescent proteins with different intra-cellular localizations in the APC or various types of anchors in the plasma membrane (PM). Only the latter were transferred by trogocytosis, albeit with different efficiencies. Unexpectedly, proteins anchored to the PM''s cytoplasmic face, or recruited to it via interaction with phosphinositides, were more efficiently transferred than those facing the outside of the cell. For proteins spanning the PM''s whole width, transfer efficiency was found to vary quite substantially, with tetraspanins, CD4 and FcRγ found among the most efficiently transferred proteins. We exploited our findings to set immunodiagnostic assays based on the capture of preferentially transferred components onto T or B cells. The preferential transfer documented here should prove useful in deciphering the cellular structures involved in trogocytosis.     

Circulating monoclonal IgM proteins in B cell chronic lymphocytic leukemia: their identification, characterization and relationship to membrane IgM

Accumulated evidence indicates that there is a circulating monoclonal Ig protein related to the leukemic cell-associated Ig in the majority of patients with B cell chronic lymphocytic leukemia (CLL) despite the failure to demonstrate such a protein by conventional serum electrophoresis. Methodology has been developed to reveal these hidden monoclonal bands and to show that they are related to the leukemia-associated membrane Ig (mIg). Of nine CLL cases with stainable mIgM and without discernable plasma Ig bands, marked hypogammaglobulinemia was evident in six. In the other three, a significant amount of protein was present in the gamma region. IgM was isolated from the plasma of these patients by affinity chromatography with Sepharose-4B, conjugated with affinity purified anti-human IgM antibodies. One to 3 mg were isolated from 20 to 40 ml of plasma. Agarose electrophoresis revealed a monoclonal Ig band in the isolated IgM in all cases. Eight of these IgM proteins were analyzed by high-pressure liquid chromatography. Five were found to be pentameric IgM. In the remaining three, various amounts of monomeric IgM were detected. Attempts to make anti-idiotypic antibodies to the isolated proteins have been successful. Thus far, a rabbit anti-idiotypic antiserum was obtained in one case and two mouse monoclonal anti-idiotypic antibodies in two additional cases. Immunofluorescence analysis revealed that plasma IgM and mIgM shared similar idiotypic determinants. One other monoclonal antibody was shown to be specific for a V region marker of a minor Ig population. These findings indicate that B leukemic lymphocytes do secrete a small amount of IgM and lend further support to the thesis that the maturation defect in CLL is incomplete. It is also feasible to isolate the secreted IgM and to produce anti-idiotypic antibodies to them. In view of the potential therapeutic effect of anti-idiotypic antibodies, this may offer an alternative and efficient approach to generate a large panel of anti-idiotypic antibodies for clinical trials. The possibility also exists that this approach is applicable to other B cell proliferative disorders such as the non-Hodgkin B cell lymphomas.     

Rapid Proliferation and Differentiation of a Subset of Circulating IgM Memory B Cells to a CpG/Cytokine Stimulus In Vitro

Circulating human IgM expressing memory B cells have been incompletely characterized. Here, we compared the phenotype and in vitro functional response (capacity to proliferate and differentiate to antibody secreting cells) in response to CpG and a cytokine cocktail (IL-2, IL-6, and IL-10) of sorted naïve B cells, IgM memory B cells and isotype-switched circulating memory B cells. Compared to naïve B cells, IgM memory B cells had lower integrated mean fluorescence intensity (iMFI) of BAFF-R, CD38, CD73, and IL-21R, but higher iMFI of CD95, CD11c, TLR9, PD-1, and CD122. Compared to switched memory B cells, IgM memory B cells had higher iMFI of BAFF-R, PD-1, IL-21R, TLR9, and CD122, but lower iMFI of CD38, CD95, and CD73. Four days after receiving the CpG/cytokine cocktail, higher frequencies of IgM than switched memory B cells—and these in turn greater than naïve cells—proliferated and differentiated to antibody secreting cells. At this time point, a small percentage (median of 7.6%) of stimulated IgM memory B cells changed isotype to IgG. Thus, among the heterogeneous population of human circulating IgM memory B cells a subset is capable of a rapid functional response to a CpG/cytokine stimulus in vitro.