A diffusion barrier maintains distribution of membrane proteins in polarized neurons

The asymmetric distribution of proteins to distinct domains in the plasma membrane is crucial to the function of many polarized cells. In epithelia, distinct apical and basolateral surfaces are maintained by tight junctions that prevent diffusion of proteins and lipids between the two domains. Polarized neurons maintain axonal and somatodendritic plasma membrane domains without an obvious physical barrier. Indeed, the artificial lipid Dil encounters no diffusion barrier at the presumptive domain boundary, the axon hillock. By measuring the lateral mobility of membrane proteins using optical tweezers, we show here that some membrane proteins exhibit markedly reduced mobility in the initial segment of the axon. Disruption of F-actin and low levels of dimethyl sulphoxide (DMSO) abolish this diffusion barrier and lead to redistribution of membrane markers that had previously been polarized. Immobilization in the initial segment may reflect, at least in part, differential tethering to cytoskeletal components. Therefore, the ability to maintain a polarized distribution of membrane proteins depends on a specialized domain at the initial segment of the axon, which restricts lateral mobility and serves as a new type of diffusion barrier that acts in the absence of cell-cell contact.     

Pericytes regulate the blood-brain barrier

The blood-brain barrier (BBB) consists of specific physical barriers, enzymes and transporters, which together maintain the necessary extracellular environment of the central nervous system (CNS). The main physical barrier is found in the CNS endothelial cell, and depends on continuous complexes of tight junctions combined with reduced vesicular transport. Other possible constituents of the BBB include extracellular matrix, astrocytes and pericytes, but the relative contribution of these different components to the BBB remains largely unknown. Here we demonstrate a direct role of pericytes at the BBB in vivo. Using a set of adult viable pericyte-deficient mouse mutants we show that pericyte deficiency increases the permeability of the BBB to water and a range of low-molecular-mass and high-molecular-mass tracers. The increased permeability occurs by endothelial transcytosis, a process that is rapidly arrested by the drug imatinib. Furthermore, we show that pericytes function at the BBB in at least two ways: by regulating BBB-specific gene expression patterns in endothelial cells, and by inducing polarization of astrocyte end-feet surrounding CNS blood vessels. Our results indicate a novel and critical role for pericytes in the integration of endothelial and astrocyte functions at the neurovascular unit, and in the regulation of the BBB.     

Targeting of transmembrane and GPI-anchored forms of N-CAM to opposite domains of a polarized epithelial cell.

The calcium-independent neural cell adhesion molecule N-CAM is expressed transiently during development in many tissues, including epithelia. The three naturally occurring principal isoforms of N-CAM differ in the way in which they associate with the membrane and in their cytoplasmic domains. These isoforms are generated by developmentally regulated alternative splicing of a single gene: the large cytoplasmic domain (ld) form (relative molecular mass 180,000 (Mr 180K] is specific for post-mitotic neurons; the 120K small cytoplasmic domain (ssd) and 140K small surface domain (sd) forms also occur on other cell types. One function of the different isoforms could be to specify cellular localization; for example, glycosyl phosphatidyl inositol (GPI)-membrane anchoring acts as a targeting signal for expression on the apical surface of polarized epithelial cells. Neurons and epithelial cells may use similar mechanisms for polarizing their plasma membrane proteins. We have therefore investigated the targeting of GPI-anchored (ssd N-CAM, 120K) and transmembrane forms of N-CAM (sd N-CAM, 140K; ld N-CAM, 180K) by comparing the expression of each after transfection of the appropriate complementary DNAs into polarized epithelial cells. We find that isoforms with alternative modes of membrane association are targeted to different surfaces of polarized epithelial cells: ssd N-CAM is expressed on the apical surface, whereas sd and ld N-CAM are expressed on the basolateral surface. These results suggest that the different isoforms of N-CAM determine their own diverse cellular destinations. They also support the hypothesis that the GPI anchor acts as an apical targeting signal in epithelia.     

Localization of apical epithelial determinants by the basolateral PDZ protein Scribble

The generation of membrane domains with distinct protein constituents is a hallmark of cell polarization. In epithelia, segregation of membrane proteins into apical and basolateral compartments is critical for cell morphology, tissue physiology and cell signalling. Drosophila proteins that confer apical membrane identity have been found, but the mechanisms that restrict these determinants to the apical cell surface are unknown. Here we show that a laterally localized protein is required for the apical confinement of polarity determinants. Mutations in Drosophila scribble (scrib), which encodes a multi-PDZ (PSD-95, Discs-large and ZO-1) and leucine-rich-repeat protein, cause aberrant cell shapes and loss of the monolayer organization of embryonic epithelia. Scrib is localized to the epithelial septate junction, the analogue of the vertebrate tight junction, at the boundary of the apical and basolateral cell surfaces. Loss of scrib function results in the misdistribution of apical proteins and adherens junctions to the basolateral cell surface, but basolateral protein localization remains intact. These phenotypes can be accounted for by mislocalization of the apical determinant Crumbs. Our results show that the lateral domain of epithelia, particularly the septate junction, functions in restricting apical membrane identity and correctly placing adherens junctions.     

安徽省风轮菜属CIinopodium植物营养器官的比较解剖研究

本文研究了安徽省风轮菜属断血流、风轮菜、剪刀菜和光风轮四种植物营养器官的解剖构造。发现断血流、剪刀菜和光风轮的茎都有内皮层,凯氏点或凯氏带也非常明显。并根据茎和根的构造特点编制了四种植物的检索表。     

门极换向晶闸管(GCT)扩散阻挡层的研究

通过对多种阻挡层特性的分析,选定了熔点高、化学稳定性好、电阻率低的TiN薄膜作为门极换向晶闸管阳极的阻挡层.采用磁控溅射技术生长的TiN,经俄歇电子谱仪分析表明,TiN阻挡层系统能有效地阻挡硅和铝的相互扩散,可作为门极换向晶闸管阳极理想的阻挡层材料.     

X-ray structures of LeuT in substrate-free outward-open and apo inward-open states

Neurotransmitter sodium symporters are integral membrane proteins that remove chemical transmitters from the synapse and terminate neurotransmission mediated by serotonin, dopamine, noradrenaline, glycine and GABA (γ-aminobutyric acid). Crystal structures of the bacterial homologue, LeuT, in substrate-bound outward-occluded and competitive inhibitor-bound outward-facing states have advanced our mechanistic understanding of neurotransmitter sodium symporters but have left fundamental questions unanswered. Here we report crystal structures of LeuT mutants in complexes with conformation-specific antibody fragments in the outward-open and inward-open states. In the absence of substrate but in the presence of sodium the transporter is outward-open, illustrating how the binding of substrate closes the extracellular gate through local conformational changes: hinge-bending movements of the extracellular halves of transmembrane domains 1, 2 and 6, together with translation of extracellular loop 4. The inward-open conformation, by contrast, involves large-scale conformational changes, including a reorientation of transmembrane domains 1, 2, 5, 6 and 7, a marked hinge bending of transmembrane domain 1a and occlusion of the extracellular vestibule by extracellular loop 4. These changes close the extracellular gate, open an intracellular vestibule, and largely disrupt the two sodium sites, thus providing a mechanism by which ions and substrate are released to the cytoplasm. The new structures establish a structural framework for the mechanism of neurotransmitter sodium symporters and their modulation by therapeutic and illicit substances.     

柄海鞘生殖系统的组织学研究

１９９８年５月至１９９９年４月每月１次 研究了柄海鞘的生殖系统的组织学、发育及季节变化。柄海鞘为雌雄同体的尾索动物,其卵巢和精巢相间排列,但不同期发育。精巢由外腊和无数生精小管组成。外膜由单层扁平上皮和薄层肌肉构成并向腺体内延伸参与形成生殖小管的基膜,生殖上皮位于基膜上,它不断的向腔内分裂增殖出精原细胞、初级精母细胞次级精母细胞、精细胞、精细胞和精子。精原细胞较大,圆形或卵圆形,核较大,与细胞同型     

中华宽体金线蛭AP神经元对压力刺激部位信息的传出调制效应

水蛭前宝塔神经元(AP)是神经节前侧囊中胞体最大的神经元,它接受多种感觉传入,但传出功能未知。通过应用细胞内、外电生理学记录结合方法,揭示了AP神经元的输出效应可能是调制水蛭体壁肌肉细胞膜的兴奋水平。     

超薄MoN扩散阻挡层的扩散阻挡性能分析

采用磁控溅射法,制备了超薄MoN扩散阻挡层,并对Cu/MoN/Si体系进行真空退火。用四点探针(FPP)、X射线衍射(XRD)、场发射扫描电子显微镜(FESEM)进行薄膜电性能和微结构表征。分析结果表明,MoN作为Cu扩散阻挡层结构具有良好的热稳定性,失效温度达到600℃,明显优于Mo扩散阻挡层。     

Vasopressin stimulates formation of coated pits in rat kidney collecting ducts

The water permeability of collecting ducts is greatly increased by the antidiuretic hormone, vasopressin. This permeability increase is associated with the appearance of intramembrane particle (IMP) clusters on the luminal plasma membranes of principal cells of the collecting duct epithelium. IMP aggregates have also been related to an increase in water permeability of two other vasopressin-sensitive epithelia, the amphibian urinary bladder and the amphibian epidermis, and it has been proposed that these specialized membrane domains might represent specific water-permeable membrane patches, induced by the hormone in their respective epithelia. Using a cytochemical probe for membrane cholesterol, filipin, we show here that the membrane patches in rat kidney are selectively devoid of filipin-sterol complexes and that when identified in thin sections, they have a cytoplasmic bristle coat: both of these features are characteristics of coated pits which, in other systems, are involved in receptor-mediated endocytosis. We also show that vasopressin induces the appearance of coated pits on collecting duct luminal membranes in Brattleboro homozygous rats, which have hereditary diabetes insipidus.     

A mechanism for asymmetric segregation of age during yeast budding

Ageing and the mortality that ensues are sustainable for the species only if age is reset in newborns. In budding yeast, buds are made young whereas ageing factors, such as carbonylated proteins and DNA circles, remain confined to the ageing mother cell. The mechanisms of this confinement and their relevance are poorly understood. Here we show that a septin-dependent, lateral diffusion barrier forms in the nuclear envelope and limits the translocation of pre-existing nuclear pores into the bud. The retention of DNA circles within the mother cell depends on the presence of the diffusion barrier and on the anchorage of the circles to pores mediated by the nuclear basket. In accordance with the diffusion barrier ensuring the asymmetric segregation of nuclear age-determinants, the barrier mutant bud6Delta fails to properly reset age in buds. Our data involve septin-dependent diffusion barriers in the confinement of ageing factors to one daughter cell during asymmetric cell division.     

Template-directed synthesis of a genetic polymer in a model protocell

Contemporary phospholipid-based cell membranes are formidable barriers to the uptake of polar and charged molecules ranging from metal ions to complex nutrients. Modern cells therefore require sophisticated protein channels and pumps to mediate the exchange of molecules with their environment. The strong barrier function of membranes has made it difficult to understand the origin of cellular life and has been thought to preclude a heterotrophic lifestyle for primitive cells. Although nucleotides can cross dimyristoyl phosphatidylcholine membranes through defects formed at the gel-to-liquid transition temperature, phospholipid membranes lack the dynamic properties required for membrane growth. Fatty acids and their corresponding alcohols and glycerol monoesters are attractive candidates for the components of protocell membranes because they are simple amphiphiles that form bilayer membrane vesicles that retain encapsulated oligonucleotides and are capable of growth and division. Here we show that such membranes allow the passage of charged molecules such as nucleotides, so that activated nucleotides added to the outside of a model protocell spontaneously cross the membrane and take part in efficient template copying in the protocell interior. The permeability properties of prebiotically plausible membranes suggest that primitive protocells could have acquired complex nutrients from their environment in the absence of any macromolecular transport machinery; that is, they could have been obligate heterotrophs.     

A two-tiered mechanism for stabilization and immobilization of E-cadherin

Epithelial tissues maintain a robust architecture which is important for their barrier function, but they are also remodelled through the reorganization of cell-cell contacts. Tissue stability requires intercellular adhesion mediated by E-cadherin, in particular its trans-association in homophilic complexes supported by actin filaments through beta- and alpha-catenin. How alpha-catenin dynamic interactions between E-cadherin/beta-catenin and cortical actin control both stability and remodelling of adhesion is unclear. Here we focus on Drosophila homophilic E-cadherin complexes rather than total E-cadherin, including diffusing 'free' E-cadherin, because these complexes are a better proxy for adhesion. We find that E-cadherin complexes partition in very stable microdomains (that is, bona fide adhesive foci which are more stable than remodelling contacts). Furthermore, we find that stability and mobility of these microdomains depend on two actin populations: small, stable actin patches concentrate at homophilic E-cadherin clusters, whereas a rapidly turning over, contractile network constrains their lateral movement by a tethering mechanism. alpha-Catenin controls epithelial architecture mainly through regulation of the mobility of homophilic clusters and it is largely dispensable for their stability. Uncoupling stability and mobility of E-cadherin complexes suggests that stable epithelia may remodel through the regulated mobility of very stable adhesive foci.     

Distinct antigen receptor repertoires of two classes of murine epithelium-associated T cells

T lymphocytes are found not only as recirculating cells in the lymphoid system, but also as immobile cells in certain epithelia. T-cell antigen receptors (TCR) of both alpha/beta and gamma/delta-heterodimer subtypes can exhibit an extremely high degree of diversity. The diversity of alpha/beta TCRs derives from the use of a large number of variable (V) gene segments, as well as junctional diversity generated during rearrangement of these segments, whereas the diversity of gamma/delta TCRs derives largely from junctional elements, with a smaller contribution from a limited number of V gene segments. Many T cells in the epidermal and intestinal epithelia of mice express TCR composed of gamma/delta heterodimers. We demonstrate here that gamma/delta TCRs of T cells in both these tissues are restricted in V gene usage, with different elements predominating. The TCR junctional diversity of epidermal T cells, however, is extremely limited, whereas that of intestinal T cells is extremely diverse. The distinctive features of these two populations suggest that they develop or are selected differently for particular tissue-specific functions.     

Structural basis for selective recognition of ESCRT-III by the AAA ATPase Vps4

The AAA+ ATPases are essential for various activities such as membrane trafficking, organelle biogenesis, DNA replication, intracellular locomotion, cytoskeletal remodelling, protein folding and proteolysis. The AAA ATPase Vps4, which is central to endosomal traffic to lysosomes, retroviral budding and cytokinesis, dissociates ESCRT complexes (the endosomal sorting complexes required for transport) from membranes. Here we show that, of the six ESCRT--related subunits in yeast, only Vps2 and Did2 bind the MIT (microtubule interacting and transport) domain of Vps4, and that the carboxy-terminal 30 residues of the subunits are both necessary and sufficient for interaction. We determined the crystal structure of the Vps2 C terminus in a complex with the Vps4 MIT domain, explaining the basis for selective ESCRT-III recognition. MIT helices alpha2 and alpha3 recognize a (D/E)xxLxxRLxxL(K/R) motif, and mutations within this motif cause sorting defects in yeast. Our crystal structure of the amino-terminal domain of an archaeal AAA ATPase of unknown function shows that it is closely related to the MIT domain of Vps4. The archaeal ATPase interacts with an archaeal ESCRT-III-like protein even though these organisms have no endomembrane system, suggesting that the Vps4/ESCRT-III partnership is a relic of a function that pre-dates the divergence of eukaryotes and Archaea.     

黄缘盒龟肝脏与肾脏的超微结构

在透射电子显微镜下观察黄缘盒龟的肝脏和肾脏的超微结构.结果显示肝实质内结缔组织少,肝小叶分界不清楚;肝细胞具两种形态,即L细胞和D细胞.肾小球由毛细血管构成;肾小囊外壁由扁平细胞构成,内壁可见足细胞附于其上;颈段由单层纤毛立方上皮细胞构成,细胞核较大,细胞游离面有很多纤毛伸向管腔,细胞核周围排列密集的线粒体;近曲小管由单层柱状上皮细胞构成,细胞核圆形,位于细胞中部,胞质内含有丰富的线粒体;中间段由单层纤毛立方上皮构成;远曲小管由单层柱状上皮细胞构成,细胞核位于细胞中部,其周围分布有许多线粒体,细胞底部可见丰富的质膜内褶;收集管由单层柱状上皮细胞组成,胞质内可见较丰富的膜迷路.黄缘盒龟肝脏的超微结构与其他爬行动物相似.肾脏结构与其生态习性有关.     

Structural plasticity and dynamic selectivity of acid-sensing ion channel-spider toxin complexes

Acid-sensing ion channels (ASICs) are voltage-independent, amiloride-sensitive channels involved in diverse physiological processes ranging from nociception to taste. Despite the importance of ASICs in physiology, we know little about the mechanism of channel activation. Here we show that psalmotoxin activates non-selective and Na(+)-selective currents in chicken ASIC1a at pH?7.25 and 5.5, respectively. Crystal structures of ASIC1a-psalmotoxin complexes map the toxin binding site to the extracellular domain and show how toxin binding triggers an expansion of the extracellular vestibule and stabilization of the open channel pore. At pH?7.25 the pore is approximately 10?? in diameter, whereas at pH?5.5 the pore is largely hydrophobic and elliptical in cross-section with dimensions of approximately 5 by 7??, consistent with a barrier mechanism for ion selectivity. These studies define mechanisms for activation of ASICs, illuminate the basis for dynamic ion selectivity and provide the blueprints for new therapeutic agents.     

Cingulin, a new peripheral component of tight junctions

The tight junction (Zonula occludens), a belt-like region of contact between cells of polarized epithelia, serves as a selective barrier to small molecules and as a total barrier to large molecules, and is involved in the separation between lumenal and basolateral compartments of the epithelium. In the electron microscope, tight junctions show focal regions of apparent fusion between the adjoining cell membranes, and freeze-fractured membranes display an elaborate network of branching and anastomosing strands. Very little is known about the molecular composition and architecture of tight junctions. The first specific zonula occludens-associated protein, designated ZO-1, has recently been identified in mammalian epithelial and endothelial cells. Here we describe the identification and purification of a new component of this junctional complex in avian brush-border cells, which we name cingulin. Cingulin is an acidic, heat-stable protein, with a highly elongated shape. Immunofluorescence and immunoelectron microscopy of brush-border cells with anti-cingulin antibodies show that cingulin is localized in the apical zone of the terminal web, at the endofacial surfaces of the zonula occludens.     

纳米材料在植物细胞生物学研究中的应用

纳米材料已广泛应用于药物载体、生物传感器、成像技术以及基因治疗等研究,相对于动物细胞而言,纳米材料在植物细胞生物学中的应用相对滞后,目前主要集中在量子点探针标记技术和纳米基因载体介导外源基因遗传转化两方面。据此,笔者主要介绍了近年来的量子点合成及功能化等方面的进展,特别对于在植物细胞成像中应用进行了评述。另外还介绍了纳米基因载体的种类和特征,以及在植物完整细胞或原生质体中介导外源基因遗传转化等方面的研究进展。笔者认为已有的纳米材料存在粒径过大或自身的细胞毒性过大,限制了其在植物细胞生物学中的进一步应用,所以针对植物细胞自身特征,设计合成新型的纳米材料将是未来研究的焦点。