Modulation of spectrin-actin assembly by erythrocyte adducin

The spectrin-based membrane skeleton, an assembly of proteins tightly associated with the plasma membrane, determines the shape and mechanical properties of erythrocytes. Spectrin, the most abundant component of this assembly, is an elongated and flexible molecule that, with potentiation by protein 4.1, is cross-linked at its ends by short actin filaments to form a lattice beneath the membrane. These and other proteins stabilize the plasma membrane, organize integral membrane proteins and maintain specialized regions of the cell surface. A membrane-skeleton-associated calmodulin-binding protein of erythrocytes is a major substrate for Ca2+- and phospholipid-dependent protein kinase C (ref. 5), and thus is a target for Ca2+ by two regulatory pathways. Here we demonstrate that this protein, called adducin: (1) binds tightly in vitro to spectrin-actin complexes but with much less affinity either to spectrin or to actin alone; (2) promotes assembly of additional spectrin molecules onto actin filaments; and (3) is inhibited in its ability to induce the binding of additional spectrin molecules to actin by micromolar concentrations of calmodulin and Ca2+. Adducin may be involved in the action of Ca2+ on erythrocyte membrane skeleton and in the assembly of spectrin-actin complexes.     

Appearance of new variants of membrane skeletal protein 4.1 during terminal differentiation of avian erythroid and lenticular cells

The erythrocyte plasma membrane is lined with a network of extrinsic proteins, mainly spectrin and actin, which constitute a reticulum tethered to the intrinsic anion transport protein of the lipid bilayer through a linker protein, ankyrin. Protein 4.1 forms a stable ternary complex with spectrin and actin, thereby strengthening the reticulum and anchoring it directly to the lipid bilayer or to another intrinsic protein, glycophorin. It has been found recently that spectrin, ankyrin and protein 4.1 are not erythrocyte-specific; this has elucidated further the mechanisms of plasma membrane assembly and modelling during the differentiation of diverse tissues. We have shown previously that protein 4.1 in chickens is most abundant in erythrocytes and lens cells, but is scarce or absent from other spectrin-rich cell types. In addition, it exists as a family of related polypeptides showing differential expression in these two tissues, suggesting variant-specific functions. Here we show that the pattern of protein 4.1 variants changes during the terminal differentiation of erythroid and lenticular cells, with novel variants appearing in postmitotic cells. The accumulation of these variants may lead to the final stabilization of the plasma membrane skeletons of these cells.     

Fission yeast mod5p regulates polarized growth through anchoring of tea1p at cell tips

Microtubules have a central role in eukaryotic cell polarity, in part through interactions between microtubule end-binding proteins and the cell cortex. In the fission yeast Schizosaccharomyces pombe, microtubules and the polarity modulator tea1p maintain cylindrical cell shape and strictly antipodal cell growth. The tea1p protein is transported to cell tips by association with growing microtubule plus ends; once at cell tips, tea1p releases from microtubule ends and associates with the cell cortex, where it coordinates polarized growth. Here we describe a cortical protein, mod5p, that regulates the dynamic behaviour of tea1p. In mod5Delta cells, tea1p is efficiently transported on microtubules to cell tips but fails to anchor properly at the cortex and thus fails to accumulate to normal levels. mod5p contains a signal for carboxy-terminal prenylation and in wild-type cells is associated with the plasma membrane at cell tips. However, in tea1Delta cells, although mod5p remains localized to the plasma membrane, mod5p is no longer restricted to the cell tips. We propose that tea1p and mod5p act in a positive-feedback loop in the microtubule-mediated regulation of cell polarity.     

Synapsin I is a spectrin-binding protein immunologically related to erythrocyte protein 4.1

The membrane-associated cytoskeleton is considered to be the apparatus by which cells regulate the properties of their plasma membranes, although recent evidence has indicated additional roles for the proteins of this structure, including an involvement in intracellular transport and exocytosis (see refs 1-3 for review). Of the membrane skeletal proteins, to date only spectrin (fodrin) and ankyrin have been purified and characterized from non-erythroid sources. Protein 4.1 in the red cell is a spectrin-binding protein that enhances the binding of spectrin to actin and can apparently bind to at least one transmembrane protein Immunoreactive forms of 4.1 have been detected in several cell types, including brain. Here we report the purification of brain 4.1 on the basis of its cross-reactivity with erythrocyte 4.1 and spectrin-binding activity. We further show that brain 4.1 is identical to the synaptic vesicle protein, synapsin I, one of the brain's major substrates for cyclic AMP and Ca2+-calmodulin-dependent kinases. Spectrin and synapsin are present in brain homogenates in an approximately 1:1 molar ratio. Although synapsin I has been implicated in synaptic transmission, no activity has been previously ascribed to it.     

Targeted recycling of PECAM from endothelial surface-connected compartments during diapedesis

Leukocytes enter sites of inflammation by squeezing through the borders between endothelial cells that line postcapillary venules at that site. This rapid process, called transendothelial migration (TEM) or diapedesis, is completed within 90 s after a leukocyte arrests on the endothelial surface. In this time, the leukocyte moves in ameboid fashion across the endothelial borders, which remain tightly apposed to it during transit. It is not known how the endothelial cell changes its borders rapidly and reversibly to accommodate the migrating leukocyte. Here we show that there is a membrane network just below the plasmalemma at the cell borders that is connected at intervals to the junctional surface. PECAM-1, an integral membrane protein with an essential role in TEM, is found in this compartment and constitutively recycles evenly along endothelial cell borders. During TEM, however, recycling PECAM is targeted to segments of the junction across which monocytes are in the act of migration. In addition, blockade of TEM with antibodies against PECAM specifically blocks the recruitment of this membrane to the zones of leukocyte migration, without affecting the constitutive membrane trafficking.     

植物在渗透胁迫下信号转导的级联机制

研究植物在渗透胁迫下信号转导的级联机制,对于有效调控植物在逆境中的生长发育,提高其抗逆性和生存能力具有重要的指导意义．根据近年来国内外的研究成果和报道,综述了植物细胞在渗透胁迫下感受到信号后,相继产生许多信号分子,这些信号分子通过细胞壁-质膜-细胞骨架连续体,引起细胞骨架蛋白变构而传递信息,并与细胞膜蛋白、第二信使系统以及调节因子构成了信号传递网,最终有条不紊地引起特定的基因表达和应答．     

Hereditary spherocytosis associated with deletion of human erythrocyte ankyrin gene on chromosome 8

Hereditary spherocytosis (HS) is one of the most common hereditary haemolytic anaemias. HS red cells from both autosound dominant and recessive variants are spectrin-deficient, which correlates with the severity of the disease. Some patients with recessive HS have a mutation in the spectrin alpha-2 domain (S.L.M. et al., unpublished observations), and a few dominant HS patients have an unstable beta-spectrin that is easily oxidized, which damages the protein 4.1 binding site and weakens spectrin-actin interactions. In most patients, however, the cause of spectrin deficiency is unknown. The alpha- and beta-spectrin loci are on chromosomes 1 and 14 respectively. The only other genetic locus for HS is SPH2, on the short arm of chromosome 8 (8p11). This does not correspond to any of the known loci of genes for red cell membrane proteins including protein 4.1 (1p36.2-p34), the anion exchange protein (AE1, band 3; 17q21-qter), glycophorin C (2q14-q21), and beta-actin (7pter-q22). Human erythrocyte ankyrin, which links beta-spectrin to the anion exchange protein, has recently been cloned. We now show that the ankyrin gene maps to chromosome 8p11.2, and that one copy is missing from DNA of two unrelated children with severe HS and heterozygous deletions of chromosome 8 (del(8)(p11-p21.1)). Affected red cells are also ankyrin-deficient. The data suggest that defects or deficiency or ankyrin are responsible for HS at the SPH2 locus.     

Human nectin-like 1, a novel membrane protein interacting with protein 4.1 R

Human nectin-like 1 (NECL1) full-length cDNA was cloned by bioinformatics method when searching for candidate membrane proteins interacting with members of protein 4.1 family. The cytoplasmic and extracellular regions of NECL1 were expressed in and purified from E. coli, and the polyclonal antibody was produced. Interaction between the cytoplasmic region of NECL1 and the 30 kD membrane binding domain of protein 4.1 on red blood cell (4. 1R) was demonstrated by IAsys-biosensor system and GST pull-down experiment. Results of biotin-labeled peptide ELISA further demonstrated the key amino acids for the binding. The interaction research of NECL1's cytoplasmic domain provides basis for further study of the functions of NECL1 in nervous system.     

显式拓扑优化的拉压杆模型自动提取方法

为了实现从显式拓扑优化到拉压杆模型的自然过渡,同时保持拓扑优化结构与拉压杆模型的拓扑一致性,以可移动变形组件拓扑优化为例,建立了显式拓扑优化拉压杆模型自动提取方法。该方法是计算机图形学和结构优化的结合,采用Voronoi骨架提取和形状优化,由骨架提取、框架提取和形状优化3部分构成。结果表明,该方法自动构建了受力合理且几何规则的拉压杆模型;Voronoi骨架提取从显式拓扑优化结构提取了光滑的中轴骨架;以类桁架指标为约束的形状优化实现了拉压杆模型从框架结构到桁架结构的质变。     

Cell surface clustering of Cadherin adhesion complex induced by antibody coated beads

Cadherin receptors mediate cell-cell adhesion, signal transduction and assembly of cytoskeletons. How a single transmembrane molecule Cadherin can be involved in multiple functions through modulating its binding activities with many membrane adhesion molecules and cytoskeletal components is an unanswered question which can be elucidated by clues from bead experiments. Human lung cells expressing N-Cadherin were examined. After co-incubation with anti-N-Cadherin monoclonal antibody coated beads, cell surface clustering of N-Cadherin was induced. Immunofluorescent detection demonstrated that in addition to Cadherin, β-Catenin, α-Catenin, α-Actinin and Actin fluorescence also aggregated respectively at the membrane site of bead attachment. Myosin heavy chain (MHC), another major component of Actin cytoskeleton, did not aggregate at the membrane site of bead attachment. Adhesion unrelated protein Con A and polylysine conjugated beads did not induce the clustering of adhesion molecules. It is indicated that the Cadherin/Catenins/α-Actinin/Actin complex is formed at Cadherin mediated cell adherens junction; occupancy and cell surface clustering of Cadherin is crucial for the formation of Cadherin adhesion protein complexes.     

Inhibition of exocytosis by intracellularly applied antibodies against a chromaffin granule-binding protein

Exocytotic secretion requires the interaction and fusion of secretory vesicles with the plasma membrane. This process could be mediated by specific recognition molecules acting as intracellular, membrane-bound receptors and ligands. One possible component of such a recognition site on the plasma membrane is a protein of relative molecular mass (Mr) 51,000 (51K) that has been isolated from bovine adrenal chromaffin cells. This protein binds strongly to chromaffin granules, the secretory vesicles of these cells. To determine the function of this membrane-anchored chromaffin granule-binding protein in exocytosis, we tested the effect of intracellularly injected antibodies on secretion. Here we show, by two independent techniques in two different cell types, that antibodies against this protein inhibit exocytosis. In rat pheochromocytoma cell cultures, monospecific antibodies, applied by erythrocyte ghost fusion, impair the release of 3H-noradrenaline. The same antibodies, introduced into individual chromaffin cells through a patch pipette, block exocytosis, as revealed by the measurement of membrane capacitance. These results demonstrate the functional involvement in exocytosis of a plasma membrane protein with high affinity for secretory vesicles.     

Expression ofChlamydomonas actin-gfp fusion gene in tobacco suspension cell and polymerization of the actin-gfp proteinin vitro

The fusion gene of actin (cDNA ofChlamydomonas reinhardtii) and green fluorescence protein (gfp) had been constructed into two expression vectors which could be expressed inE. coli and tobacco suspension cells BY2. The correct expression was observed inE. coli and BY2 with a fluorescence microscopy. The fusion protein, which took part in the membrane skeleton, was mainly located peripherally along the membrane, specially the fusion protein was distributed around nucleus and cell plate, while the fusion protein also forms F-actin in the cell. The fusion protein was purified from Bl21plus by ammonium sulfate fractionation, ion exchange chromatography and hydrophobic interaction chromatography. The purified production could polymerize into F-actin when the actin polymerizing buffer was added. It was demonstrated that the characteristics and function of actin inChlamydomonas was similar with those of animals and higher plants.     

基于直方图的形状描述及骨架图匹配算法

考虑物体形状的多变性,给出了一种基于二维直方图的物体形状描述及骨架图匹配算法.该算法利用骨架长度和骨架上点的圆盘半径作为特征量来描述形状.首先基于二维直方图给出一种形状及骨架端点的特征描述方法,其次结合改进的最优子序列匹配算法实现骨架图的匹配.算法中的形状及骨架端点的特征描述在一些常见非刚性变换下具有不变性.实验结果表明,该算法能够实现一些非刚性变换下的物体形状的骨架图匹配和较高精度的形状检索,且复杂度较低.     

Crystal structure of bacterial multidrug efflux transporter AcrB

AcrB is a major multidrug exporter in Escherichia coli. It cooperates with a membrane fusion protein, AcrA, and an outer membrane channel, TolC. We have determined the crystal structure of AcrB at 3.5 A resolution. Three AcrB protomers are organized as a homotrimer in the shape of a jellyfish. Each protomer is composed of a transmembrane region 50 A thick and a 70 A protruding headpiece. The top of the headpiece opens like a funnel, where TolC might directly dock into AcrB. A pore formed by three alpha-helices connects the funnel with a central cavity located at the bottom of the headpiece. The cavity has three vestibules at the side of the headpiece which lead into the periplasm. In the transmembrane region, each protomer has twelve transmembrane alpha-helices. The structure implies that substrates translocated from the cell interior through the transmembrane region and from the periplasm through the vestibules are collected in the central cavity and then actively transported through the pore into the TolC tunnel.     

Abolition of actin-bundling by phosphorylation of human erythrocyte protein 4.9

Protein 4.9, first identified as a component of the human erythrocyte membrane skeleton, binds to and bundles actin filaments. Protein 4.9 is a substrate for various kinases, including a cyclic AMP(cAMP)-dependent one, in vivo and in vitro. We show here that phosphorylation of protein 4.9 by the catalytic subunit of cAMP-dependent protein kinase reversibly abolishes its actin-bundling activity, but phosphorylation by protein kinase C has no such effect. A quantitative immunoassay showed that human erythrocytes contain 43,000 trimers of protein 4.9 per cell, which is equivalent to one trimer for each actin oligomer in these red blood cells. As analogues of protein 4.9 have been identified together with analogues of other erythroid skeletal proteins in non-erythroid tissues of numerous vertebrates, phosphorylation and dephosphorylation of protein 4.9 may be the basis for a mechanism that regulates actin bundling in many cells.     

Specific dephosphorylation of membrane proteins in Rous sarcoma virus-transformed chick embryo fibroblasts

Chick embryo fibroblasts (CEF) infected with avian sarcoma virus become rapidly transformed as a result of expression of the viral src gene in the form of a single polypeptide of molecular weight 60,000 (pp60src) with protein kinase activity and suggested preferential association with the plasma membrane. Studies with normal avian and mammalian cells have revealed the presence of an antigenically related protein which seems to have similar kinase activity, but which is present at less than 1% of the levels of virally induced src protein found in transformed cells. As dynamic phosphorylation is important in numerous regulatory processes, the phenotypic expression of transformation may arise from an imbalance in one or more regulatory mechanisms that are controlled by protein phosphorylation. The cell membrane is affected during transformation, including its phosphotransferase activity. The latter has been shown using isolated membrane fractions whose properties may be changed during preparation. Therefore, we have compared the phosphorylation state of individual membrane proteins found in intact normal and RSV-transformed cells and report here the identification of two heavily phosphorylated, acidic membrane proteins in normal CEF which are specifically dephosphorylated on transformation by wild-type and temperature-sensitive Rous sarcoma viruses.     

Substrate twinning activates the signal recognition particle and its receptor

Signal sequences target proteins for secretion from cells or for integration into cell membranes. As nascent proteins emerge from the ribosome, signal sequences are recognized by the signal recognition particle (SRP), which subsequently associates with its receptor (SR). In this complex, the SRP and SR stimulate each other's GTPase activity, and GTP hydrolysis ensures unidirectional targeting of cargo through a translocation pore in the membrane. To define the mechanism of reciprocal activation, we determined the 1.9 A structure of the complex formed between these two GTPases. The two partners form a quasi-two-fold symmetrical heterodimer. Biochemical analysis supports the importance of the extensive interaction surface. Complex formation aligns the two GTP molecules in a symmetrical, composite active site, and the 3'OH groups are essential for association, reciprocal activation and catalysis. This unique circle of twinned interactions is severed twice on hydrolysis, leading to complex dissociation after cargo delivery.     

Spectrin assembly in avian erythroid development is determined by competing reactions of subunit homo- and hetero-oligomerization

Erythroid differentiation entails the biogenesis of a membrane skeleton, a network of proteins underlying and interacting with the plasma membrane, whose major constituent is the heterodimeric protein spectrin, composed of two structurally similar but distinct subunits, alpha (relative molecular mass (Mr) 240,000) and beta (Mr 220,000), which interact side-on with each other to form a long rod-like molecule. Interaction of this network with the membrane is mediated by the binding of the beta subunit to ankyrin, which in turn binds to the cytoplasmic domain of the transmembrane anion transporter (also referred to as band 3). Purified alpha and beta subunits of spectrin from the membrane of mature red blood cells will spontaneously heterodimerize, suggesting that assembly of the spectrin-actin skeleton is a simple self-assembly process, but in vivo studies with developing chicken embryo erythroid cells have indicated that assembly in vivo is more complex. We now present evidence that newly synthesized spectrin subunits in vivo or in vitro rapidly adopt one of two competing conformations, a heterodimer or a homo-oligomer. These competing reactions seem to determine the overall extent of spectrin assembled during erythroid development by determining which conformation will assemble onto the membrane-skeleton (the heterodimer) and which conformations are targeted for degradation (the homo-oligomers).     

统计骨架信息的形状描述新方法

针对形状描述与识别问题,提出了骨架结构特征直方图,利用提取的形状骨架信息,解决了形状轮廓的起点选择、旋转、平移以及缩放等不变性。首先,计算每对骨架点关于骨架中心的测地路径长度比例特征和骨架点圆盘半径比例特征,然后结合上述2个特征统计构造直方图矩阵来描述图像形状。实验结果表明,该形状描述不仅具有刚性变化不变性,在一些非刚性变化下也能够实现高效率和高精度的形状检索。     

细胞挤入狭缝的润滑理论分析

血细胞的入口阻力构成血液循环外周阻力的重要组成部分。本文首次提出一个简化的力学模型,试图探讨细胞挤入二维狭缝的运动特性。本模型的细胞外形根据显微录相设为已知,细胞膜沿其表面滑移。对膜与缝壁间的薄血浆层应用润滑理论,算出压力和剪应力分布,给出了细胞所受阻力。本文得到的细胞运动规律,与显微录相定性一致地模拟了细胞的入口过程。