Uncoupling of a nerve cell membrane junction by calcium-ion removal

Calcium ion participates in maintaining electrical connections between the nerve cells of Retzius (Hirudo medicinalis). The conductance across the junction between these cells decreases with decreasing concentration of free, extracellular Ca(++) At a certain level of Ca(++) withdrawal from the cell system, junctional conductance reaches a critical low point at which the cells become functionally disconnected: the nerve impulses which are normally discharged in synchrony by the cells become asynchronous. These effects of Ca(++) on junctional connection are irreversible, in contrast to those on nonjunctional surface membrane permeability.     

Plasma membrane compartmentalization in yeast by messenger RNA transport and a septin diffusion barrier

Asymmetric localization of proteins plays a key role in many cellular processes, including cell polarity and cell fate determination. Using DNA microarray analysis, we identified a plasma membrane protein-encoding mRNA (IST2) that is transported to the bud tip by an actomyosin-based process. mRNA localization created a higher concentration of IST2 protein in the bud compared with that of the mother cell, and this asymmetry was maintained by a septin-mediated membrane diffusion barrier at the mother-bud neck. These results indicate that yeast creates distinct plasma membrane compartments, as has been described in neurons and epithelial cells.     

Cell corpse engulfment mediated by C. elegans phosphatidylserine receptor through CED-5 and CED-12

During apoptosis, phosphatidylserine, which is normally restricted to the inner leaflet of the plasma membrane, is exposed on the surface of apoptotic cells and has been suggested to act as an "eat-me" signal to trigger phagocytosis. It is unclear how phagocytes recognize phosphatidylserine. Recently, a putative phosphatidylserine receptor (PSR) was identified and proposed to mediate recognition of phosphatidylserine and phagocytosis. We report that psr-1, the Caenorhabditis elegans homolog of PSR, is important for cell corpse engulfment. In vitro PSR-1 binds preferentially phosphatidylserine or cells with exposed phosphatidylserine. In C. elegans, PSR-1 acts in the same cell corpse engulfment pathway mediated by intracellular signaling molecules CED-2 (homologous to the human CrkII protein), CED-5 (DOCK180), CED-10 (Rac GTPase), and CED-12 (ELMO), possibly through direct interaction with CED-5 and CED-12. Our findings suggest that PSR-1 is likely an upstream receptor for the signaling pathway containing CED-2, CED-5, CED-10, and CED-12 proteins and plays an important role in recognizing phosphatidylserine during phagocytosis.     

Bidirectional transmembrane signaling by cytoplasmic domain separation in integrins

Although critical for development, immunity, wound healing, and metastasis, integrins represent one of the few classes of plasma membrane receptors for which the basic signaling mechanism remains a mystery. We investigated cytoplasmic conformational changes in the integrin LFA-1 (alphaLbeta2) in living cells by measuring fluorescence resonance energy transfer between cyan fluorescent protein-fused and yellow fluorescent protein-fused alphaL and beta2 cytoplasmic domains. In the resting state these domains were close to each other, but underwent significant spatial separation upon either intracellular activation of integrin adhesiveness (inside-out signaling) or ligand binding (outside-in signaling). Thus, bidirectional integrin signaling is accomplished by coupling extracellular conformational changes to an unclasping and separation of the alpha and beta cytoplasmic domains, a distinctive mechanism for transmitting information across the plasma membrane.     

水稻和昆虫细胞自噬期间自噬体发生的细胞学机理

【目的】从细胞学角度研究细胞自噬过程中膜结构细胞器的变化及其与自噬体发生的关系。【方法】以基因互作引起细胞调亡现象发生的水稻爪粳杂种F1和天然化合物印楝素、喜树碱、苦参碱诱导凋亡的昆虫细胞Sf9为材料,通过透射电子显微镜和荧光显微镜技术观察细胞自噬凋亡期间自噬体的特征。【结果】水稻杂种F1花粉母细胞在不同发育时期存在不同程度的退化,部分细胞内线粒体形态发生显著变化,形成长哑铃型或线管状,最后两端弯曲形成双层膜结构的细胞器;绒毡层细胞均出现内质网膨大,且逐渐包裹周围其它细胞器,如线粒体,形成双层膜结构的自噬体,最终引发细胞的自噬性凋亡。喜树碱和苦参碱诱发凋亡的细胞胞内线粒体形态发生拉长、弯曲,最后闭合形成包裹其它细胞器的双层膜结构自噬体;印楝素和喜树碱诱发凋亡的细胞胞内核通过分页和出芽的方式形成双层膜结构自噬体。【结论】动植物细胞自噬凋亡过程中,内质网、线粒体和细胞核3种膜结构细胞器均是自噬体膜结构的直接来源,然而3种膜结构细胞器参与自噬体形成的程度和方式因物种、细胞种类以及自噬诱发因素的变化而不同。     

水稻抗白叶枯病相关基因的亚细胞定位

利用亚细胞定位技术对水稻抗白叶枯病相关基因的表达产物进行了分析,为基因功能分析提供了重要信息。通过前期基因芯片筛选、半定量/定量PCR的验证及基因功能注释,从高抗白叶枯病水稻品种Y73中筛选了18个潜在的与抗白叶枯病相关的基因,并在烟草叶片细胞中进行亚细胞定位分析。通过观察融合有绿色荧光标记(sGFP)的表达产物,初步了解这些基因在烟草细胞中的表达位置。经过激光共聚焦显微镜观察后发现: 其中4个基因的 sGFP 融合蛋白定位于细胞核上,可能发挥了转录因子的功能;4个基因的 sGFP 融合蛋白定位于细胞质膜上,推测可能与细胞质膜的稳定或控制蛋白转运相关;4个基因的 sGFP 融合蛋白同时定位于细胞核和细胞质膜上;另有6个基因的 sGFP 融合表达后没有观察到明显的定位信号。     

Cytoprotective role of Ca2+- activated K+ channels in the cardiac inner mitochondrial membrane

Ion channels on the mitochondrial inner membrane influence cell function in specific ways that can be detrimental or beneficial to cell survival. At least one type of potassium (K+) channel, the mitochondrial adenosine triphosphate-sensitive K+ channel (mitoKATP), is an important effector of protection against necrotic and apoptotic cell injury after ischemia. Here another channel with properties similar to the surface membrane calcium-activated K+ channel was found on the mitochondrial inner membrane (mitoKCa) of guinea pig ventricular cells. MitoKCa significantly contributed to mitochondrial K+ uptake of the myocyte, and an opener of mitoKCa protected hearts against infarction.     

Integrins regulate Rac targeting by internalization of membrane domains

Translocation of the small GTP-binding protein Rac1 to the cell plasma membrane is essential for activating downstream effectors and requires integrin-mediated adhesion of cells to extracellular matrix. We report that active Rac1 binds preferentially to low-density, cholesterol-rich membranes, and specificity is determined at least in part by membrane lipids. Cell detachment triggered internalization of plasma membrane cholesterol and lipid raft markers. Preventing internalization maintained Rac1 membrane targeting and effector activation in nonadherent cells. Regulation of lipid rafts by integrin signals may regulate the location of membrane domains such as lipid rafts and thereby control domain-specific signaling events in anchorage-dependent cells.     

The direction of membrane lipid flow in locomoting polymorphonuclear leukocytes

The objective of this study was to determine the direction of membrane lipid flow in locomoting cells. The plasma membrane of human polymorphonuclear leukocytes was stained with a fluorescent lipid analog dihexadecanoyl indocarbocyanine. A line was photobleached on the cell surface perpendicular to the direction of cell motion. Low-light-level fluorescence microscopy and digital image-processing techniques were used to analyze a series of images taken at short intervals after photobleaching. The bleached line remained visible for about 5 seconds before being erased by diffusional recovery. Examination of fluorescence intensity profiles allowed a comparison to be made between the velocities of line and cell movement. Results indicate that the bleached line moves forward with the same velocity as the cell during locomotion, refuting the retrograde lipid flow model of locomotion. Instead, the plasma membrane lipid appears to move forward according to either the unit movement of membrane or the tank track model of locomotion.     

Effect of Drought Stress on Growth and Water Physiological Characteristics of Poa sibirica

In order to develop the resources of native turfgrass,the morphological traits and drought resistance of native Siberian bluegrass (Poa sibirica,abbreviated as PS) was evaluated using the introduced Kentucky bluegrass 'Midnight' (Poa pratensis,abbreviated as PP) as a control.Two water schemes were imposed to plants in this pot culture study in greenhouse.One was with drought stress persistent limiting water supply for 20 days,the other was re-hydrated until 14 days after drought.The leaf shape,turf color,water status and cell plasma membrane permeability were evaluated.Similar changing trends with these parameters were shown for both species,and there were not significant differences with most evaluations during drought and re-water periods.The values leaf width and length of PS were higher while leaf color intensity was slightly lower than that of PP,but the greenness of PS leaf was still visually acceptable.There were not significant differences with cell membrane stability between the two species.In comparison,the native wild species PS possessed the potential for to be domesticated into a new cultivar for turf industry.     

Vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells

Viruses employ many different strategies to enter host cells. Vaccinia virus, a prototype poxvirus, enters cells in a pH-dependent fashion. Live cell imaging showed that fluorescent virus particles associated with and moved along filopodia to the cell body, where they were internalized after inducing the extrusion of large transient membrane blebs. p21-activated kinase 1 (PAK1) was activated by the virus, and the endocytic process had the general characteristics of macropinocytosis. The induction of blebs, the endocytic event, and infection were all critically dependent on the presence of exposed phosphatidylserine in the viral membrane, which suggests that vaccinia virus uses apoptotic mimicry to enter cells.     

Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells

Many proteins associated with the plasma membrane are known to partition into submicroscopic sphingolipid- and cholesterol-rich domains called lipid rafts, but the determinants dictating this segregation of proteins in the membrane are poorly understood. We suppressed the tendency of Aequorea fluorescent proteins to dimerize and targeted these variants to the plasma membrane using several different types of lipid anchors. Fluorescence resonance energy transfer measurements in living cells revealed that acyl but not prenyl modifications promote clustering in lipid rafts. Thus the nature of the lipid anchor on a protein is sufficient to determine submicroscopic localization within the plasma membrane.     

Tunicamycin enhances the antiviral and anticellular activity of interferon

The inhibitory effects of interferon on virus multiplication and cell growth are significantly enhanced by treatment with tunicamycin. Potentiation of antiviral activity was found only with enveloped viruses and not with nonbudding viruses. Changes in the plasma membrane of treated cells may account for this effect, since enveloped viruses bud from the cell surface as a terminal step.     

iRhom2 regulation of TACE controls TNF-mediated protection against Listeria and responses to LPS

Innate immune responses are vital for pathogen defense but can result in septic shock when excessive. A key mediator of septic shock is tumor necrosis factor-α (TNFα), which is shed from the plasma membrane after cleavage by the TNFα convertase (TACE). We report that the rhomboid family member iRhom2 interacted with TACE and regulated TNFα shedding. iRhom2 was critical for TACE maturation and trafficking to the cell surface in hematopoietic cells. Gene-targeted iRhom2-deficient mice showed reduced serum TNFα in response to lipopolysaccharide (LPS) and could survive a lethal LPS dose. Furthermore, iRhom2-deficient mice failed to control the replication of Listeria monocytogenes. Our study has identified iRhom2 as a regulator of innate immunity that may be an important target for modulating sepsis and pathogen defense.     

Activation of the HIV-1 LTR by T cell mitogens and the trans-activator protein of HTLV-I

To investigate the mechanism by which immune activation augments replication of the human immunodeficiency virus type 1 (HIV-1) in infected T cells, four different classes of T cell mitogens were evaluated for their effects on the HIV-1 long terminal repeat (LTR). Phytohemagglutinin (PHA), a mitogenic lectin; phorbol 12-myristic 13-acetate, a tumor promoter; ionomycin, a calcium ionophore; and tat-1, the trans-activator protein from the human T cell leukemia/lymphoma virus type I (HTLV-I) each stimulated the HIV-1 LTR. Studies of deleted forms of the LTR supported a central role in these responses for the HIV-1 enhancer, which alone was sufficient for mitogen inducibility, but also suggested that other 5' positive and negative regulatory elements contribute to the overall magnitude of the response. Synergistic activation of the HIV-1 LTR (up to several thousandfold) was observed with combinations of these mitogens and the HIV-1--derived tat-III protein. Cyclosporin A, an immunosuppressive agent, inhibited PHA-mediated activation of the HIV-1 LTR but was without effect in the presence of other mitogens. Thus, HIV-1 gene expression and replication appear to be regulated, via the HIV-1 LTR, by the same mitogenic signals that induce T cell activation.     

Uncoupling of an epithelial cell membrane junction by calcium-ion removal

Calcium takes part in maintaining ion communication between salivary gland cells (Chironomus thummi). Its withdrawal from the cell systems results in virtual disconnection of ion communication, at Ca(++) concentrations which do not noticeably affect cell adhesion. The junctional membrane surfaces. which are normally quite freely permeable to ions, become as impermeable as the nonjunctional membrane surfaces; each cell seals itself off irreversibly as a unit. In maintaining ion communication Mg(++) substitutes for Ca(++)     

甘蔗叶绿体荧光参数、MDA含量及膜透性与耐旱性的关系

水分胁迫加速蔗叶活性氧产生并削弱蔗叶活性氧清除能力;随水分胁迫强度的加剧,膜脂过氧化产物丙二醛（ Ｍ Ｄ Ａ）含量迅速提高,细胞质膜透性增大,蔗叶叶绿体２,６ 二氯酚靛酚光还原活性下降,蔗叶叶绿体光系统Ⅱ原初光能转换效率、 Ｐ ＳⅡ潜在活性、叶绿体光合量子产额降低．上述荧光参数的受抑程度与品种的相对耐旱性强弱、膜脂过氧化作用产物 Ｍ Ｄ Ａ 含量增加及质膜相对透性提高程度密切相关     

Insulin-stimulated release of lipoprotein lipase by metabolism of its phosphatidylinositol anchor

Lipoprotein lipase (LPL) plays a critical role in the metabolism of plasma lipoproteins. In 3T3-L1 adipocytes, insulin elicits the rapid release of LPL through mechanisms that are independent of energy metabolism and protein synthesis. Some of the metabolic actions of insulin may be mediated by the activation of a specific phospholipase that hydrolyzes a glycosyl phosphatidylinositol (PI) molecule. The insulin-sensitive glycosyl-PI is structurally similar to the glycolipid membrane anchor of a number of proteins. LPL appears to be anchored to the 3T3-L1 cell surface by glycosyl-PI, and its rapid release by insulin may be due to activation of a glycosyl-PI-specific phospholipase C.     

Direct evidence that endogenous GM1 ganglioside can mediate thymocyte proliferation

The B subunit of cholera toxin, which is multivalent and binds exclusively to a specific ganglioside, GM1, was mitogenic for rat thymocytes. When exposed to the B subunit, the cells proliferated, as measured by 3H-labeled thymidine incorporation. Mitogenesis depended on the direct interaction of the B subunit with GM1 on the surface of the cells. This demonstrates that endogenous plasma membrane gangliosides can mediate proliferation in lymphocytes.     

Complex I binding by a virally encoded RNA regulates mitochondria-induced cell death

Human cytomegalovirus infection perturbs multiple cellular processes that could promote the release of proapoptotic stimuli. Consequently, it encodes mechanisms to prevent cell death during infection. Using rotenone, a potent inhibitor of the mitochondrial enzyme complex I (reduced nicotinamide adenine dinucleotide-ubiquinone oxido-reductase), we found that human cytomegalovirus infection protected cells from rotenone-induced apoptosis, a protection mediated by a 2.7-kilobase virally encoded RNA (beta2.7). During infection, beta2.7 RNA interacted with complex I and prevented the relocalization of the essential subunit genes associated with retinoid/interferon-induced mortality-19, in response to apoptotic stimuli. This interaction, which is important for stabilizing the mitochondrial membrane potential, resulted in continued adenosine triphosphate production, which is critical for the successful completion of the virus' life cycle. Complex I targeting by a viral RNA represents a refined strategy to modulate the metabolic viability of the infected host cell.