间隙连接课件

Simplified drawing of a cross-section through part of the wall of the intestine. This long, tubelike organ is constructed from epithelial tissues (red), connective tissues (green), and muscle tissues (yellow). Each tissue is an organized assembly of cells held together by cell -cell adhesions, extracellular matrix, or both. 第一节 细胞连接 cell junction 是指在细胞质膜的特化区域，通过膜蛋白、细胞骨 架或胞外基质形成的细胞与细胞间或细胞与细胞外 基质间的联结结构。 分为三大类，即： 封闭连接（occluding junction） 锚定连接（anchoring junction） 通讯连接（communicating junction） (1) Occluding junctions: Tight junction (2) Anchoring junctions: Desmosomes and Adhesion belts; Focal contacts and hemidesmosomes (3)Communicating junctions: Gap junction； Chemical synapse； Plasmodesmata. Tight junctions create an impermeable seal Gap junctions provide direct chemical and electrical communication. 一、封闭连接 紧密连接 存在于脊椎动物的上皮细胞间。 连接区域CAM构成焊接线，也称嵴线。相 邻质膜紧密结合，没有缝隙。 主要作用：封闭相邻细胞间的接缝，防止 溶液渗入，构成脑血屏障和睾血屏障。 A current model of a tight junction 连接区域由跨膜整合蛋白质形成的 嵴线，嵴线: Claudin， Occludin Tight Junction between two type I pneumocytes vExperiment evidence demonstrating that tight junctions create a permeability barrier vAnother role of tight junctions is to block the lateral movement of membrane proteins vTight junctions form the blood-brain barrier. The role of tight junctions in transcellular transport Construction of an anchoring junction. Highly schematized drawing showing the two classes of proteins that constitute such a junction: intracellular attachment proteins and transmembrane linker proteins. 细胞内锚蛋白 跨膜粘结蛋白 二、锚定连接 锚定连接（anchoring junctions） 中间纤维有关的：桥粒（desmosome） 半桥粒（hemidesmosome） 肌动蛋白有关的：黏合带（adhesion belt） 黏合斑（focal adhesion） 类型 锚定连接将相邻细胞的骨架系统或将细胞与基质相连 形成一个坚挺、有序的细胞群体。根据细胞骨架纤维 不同： 桥粒（desmosome）是相邻细胞间形成的纽扣式结构。通过质 膜下的致密斑连接中间纤维，跨膜蛋白为钙粘素。主要分布在承 受拉力的组织中，如皮肤、口腔、血管和心肌中。 半桥粒（hemidesmosome）位于上皮细胞基面与基膜之间，它 与桥粒的不同之处在于：只在质膜内侧形成桥粒斑结构，其另 一侧为基膜；穿膜连接蛋白为整合素而不是钙粘素；细胞内 的附着蛋白为角蛋白。 2.1 、桥粒与半桥粒 The distribution of desmosomes and hemidesmosomes in epithelial cells of the small intestine. The keratin filament networks of adjacent cells are indirectly connected to one another through desmosomes and to the basal lamina through hemidesmosomes. DesmosomeDesmosome 半桥粒（hemidesmosome）：位于上皮细胞基面 与基膜之间，连接蛋白为整合素。连接的细胞内 骨架成分为角蛋白。 锚定连接（anchoring junctions） 中间纤维有关的：桥粒（desmosome） 半桥粒（hemidesmosome） 肌动蛋白有关的：黏合带（adhesion belt） 黏合斑（focal adhesion） 类型 锚定连接将相邻细胞的骨架系统或将细胞与基质相连 形成一个坚挺、有序的细胞群体。根据细胞骨架纤维 不同： Adhesion belts and focal adhesion Adhesion belts between epithelial cells in the small intestine. This beltlike anchoring junction encircles each of the interacting cells. Its most obvious feature is a contractile bundle of actin filaments running along the cytoplasmic surface of the junctional plasma membrane. The actin filaments are joined from cell to cell by transmembrane linker proteins (cadherins), whose extracellular domain binds to the extracellular domain of an identical cadherin molecule on the adjacent cell. 2.2、 粘合带与粘合斑 Adhesion belt or Adherens junction 2.3 功能 Determining the size of a gap-junction channel. When fluorescent molecules of various sizes are injected into one of two cells coupled by gap junctions, molecules smaller than about 1000 daltons can pass into the other cell but larger molecules cannot. 三、通讯连接 （一）间隙连接 gap junction 连接处有24nm的缝隙。 基本单位称连接子，由6个相同或相似的跨 膜蛋白亚单位（connexin）环绕而成。 允许小于1.5kD的分子通过，通透性可调。 A model of a gap junction. The drawing shows the interacting plasma membranes of two adjacent cells. The apposed lipid bilayers (red) are penetrated by protein assemblies called connexons (green), each of which is thought to be formed by six identical protein subunits (called connexins). Two connexons join across the intercellular gap to form a continuous aqueous channel connecting the two cells. Gap junctions as seen in the electron microscope. Thin-section (A) and freeze-fracture (B) electron micrographs of a large and a small gap junction between fibroblasts in culture. In (B) each gap junction is seen as a cluster of homogeneous intramembrane particles associated exclusively with the cytoplasmic fracture face (P face) of the plasma membrane. A proposed model for how gap-junction channels may close in response to a rise in Ca2+ or a fall in pH in the cytosol. A small rotation of each subunit closes the channel. The model is based on an image analysis of electron micrographs of rapidly frozen tissue in which the structure of gap junction channels in their presumed open state was compared with their structure in a Ca2+-induced closed state. It is possible that a similar mechanism operates in the opening and closing of the gated ion channels discussed later Chapter . AFM image of connexon A. Open state in Ca2+ free buffer; B. Close 间隙连接的功能及其调节机制 间隙连接在代谢偶联中的作用 间隙连接允许小分子代谢物和信号分子通过, 是细胞间代谢 偶联的基础 代谢偶联现象在体外培养细胞中的证实 代谢偶联作用在协调细胞群体的生物学功能方面起重要作用. 间隙连接在神经冲动信息传递过程中的作用 电突触(electronic junction) 快速实现细胞间信号通讯 间隙连接调节和修饰相互独立的神经元群的行为 间隙连接在早期胚胎发育和细胞分化过程中的作用 胚胎发育中细胞间的偶联提供信号物质的通路, 从而为某一 特定细胞提供它的“位置信息”，并根据其位置影响其分化。 肿瘤细胞之间间隙的连接明显减少或消失，间隙联接类似 “肿瘤抑制因子”。 （二）胞间连丝 plasmodesmata 由穿过细胞壁的原生质构成，直径约 2040nm。中央有SER形成的连丝小管。 功能上与动物细胞间的间隙连接类似。 通透性可调节。某些植物病毒能制造特殊 的蛋白质，使胞间连丝的有效孔径扩大。 Plasmodesmata of cultured plant cells Plasmodesmata 三、化学突触 synapse 存在于可兴奋细胞间，通过释放神经递质传 导兴奋。 由突触前膜、突触后膜、突触间隙组成。 突触前神经元突起末梢膨大，称突触小体。 突触小体内有突触小泡，内含神经递质。 (1) Occluding junctions: Tight junction (2) Anchoring junctions: Desmosomes and Adhesion belts; Focal contacts and hemidesmosomes (3)Communicating junctions: Gap junction； Chemical synapse； Plasmodesmata. Tight junctions create an impermeable seal Gap junctions provide direct chemical and electrical communication. Various Cell Junctional ComplexesVarious Cell Junctional Complexes 封闭连接紧密连接上皮组织 锚定连接 肌动蛋白 (微丝) 粘合带 粘合斑 中间纤维 桥粒 半桥粒 通讯连接 间隙连接 化学突触 胞间连丝 Claudin Occludin E-cadherin Integrin Cadherin Integrin Connexin 上皮组织 上皮组织基部 上皮、心肌、表皮 上皮组织基部 动物组织 植物细胞 神经与神经或肌细胞 各类连接的比较 第二节 细胞粘附分子 Cell Adhesion Molecule，CAM Organ-specific adhesion of dissociated vertebrate embryo cells determined by a radioactive cell-binding assay. The rate of cell adhesion can be measured by determining the number of radioactively labeled cells bound to the cell aggregates after various periods of time. The rate of adhesion is greater between cells of the same kind. In a commonly used modification of this assay, cells labeled with a fluorescent or radioactive marker are allowed to bind to a monolayer of unlabeled cells in culture. 可大致分为四类： I. 钙粘素 (Cadherin) II. 选择素 (Selectin) III. 免疫球蛋白超家族 (lgSF) IV. 整合素 (Integrin) Cell adhesion molecules 细胞粘着分子（cell adhesion molecule，CAM）: 是参与细胞与细胞之间及细胞与细胞外基质之间相互作用的分子。 胞外区，肽链的N端部分，带有糖链，负责与配体的识别； 跨膜区，多为一次跨膜； 胞质区，肽链的C端部分，一般较小，或与质膜下的骨架成分 直接相连，或与胞内的化学信号分子相连，以活化信号转导途 径。 细胞黏着分子都是跨膜糖蛋白， 分子结构由三部分组成： 属同亲性CAM，其作用 依赖于Ca2。至今已鉴定 出30种以上钙粘素，分布 于不同的组织。 I. 钙粘素（cadherin） 钙粘素结构模型 哺乳动物细胞表面的主要钙粘素分子 1介导细胞连接，在成年脊椎动物，E-钙粘素是保持上皮细 胞相互粘合的主要CAM，是粘合带的主要构成成分。 2参与细胞分化，钙粘素对于胚胎细胞的早期分化及成体组 织的构筑有重要作用。在发育过程中通过调控钙粘素表达的种 类与数量可决定胚胎细胞间的相互作用，从而通过细胞的微环 境，影响细胞的分化，参与器官形成过程。 3抑制细胞迁移，很多种癌组织中细胞表面的E钙粘素减少 或消失，以致癌细胞易从瘤块脱落，成为侵袭与转移的前提。 因而有人将E钙粘素视为转移抑制分子。 钙粘素的作用： II. 选择素（Selectin） 选择素（selectin）属异亲性CAM，其作用依赖于Ca2。主要参与白细 胞与脉管内皮细胞之间的识别与粘合。 P选择素：贮存于血小板及内 皮细胞； E选择素：存在于活化的血管 内皮细胞表面； L选择素：广泛存在于各种白 细胞的表面，参与炎症部位 白细胞的出脉管过程。外周 淋巴细胞归巢. The protein-carbohydrate interaction that initiates the transient adhesion of neutrophils to endothelial cells at sites of inflammation. (A) The lectin domain of P-selectin binds to the specific oligosaccharide shown in (B), which is present on both cell-surface glycoprotein and glycolipid molecules. The lectin domain of the selectins is homologous to lectin domains found on many other carbohydrate-binding proteins in animals; because the binding to their specific sugar ligand requires extracellular Ca2+, they are called C-type lectins. A three-dimensional structure of one of these lectin domains, determined by x-ray crystallography, is shown in (C); its bound sugar is colored blue. Gal = galactose; GlcNAc = N-acetylglucosamine; Fuc = fucose; NANA = sialic acid. III、免疫球蛋白超家族 Ig-superfamily 含免疫球蛋白（Ig）样结构域，即二硫键维 系的两组反向平行的折叠。 一般不依赖Ca2，亲同性或亲异性CAM。 N-CAM存在于神经细胞。 Pe-CAM存在于血小板及大多数免疫细胞。 I-CAM及V-CAM在活化的血管内皮细胞表达。 免疫球蛋白结构域是指借二硫键维系的反向平行折叠结构。 免疫球蛋白超家族的功能 1. 介导淋巴细胞和免疫应答所需要的细胞之间粘着 2. 神经系统的发育. IV、整联蛋白 整合素（integrin）大多为异亲性细胞黏着分子，其作用依赖于Ca2。介导细 胞与细胞间的相互作用及细胞与细胞外基质间的相互作用。几乎所有动植物细胞均 表达整合素。 subunit binds RGD domain on fibronectin a subunit binds calcium Ca2+ necessary for substrate binding RGD 精氨酸，甘氨酸，天冬氨酸 纤维蛋白原 RGD（可溶性血液蛋白） 血小板 整联蛋白 血凝块形成机制 1.The mechanical- structural function of focal adhesion, which is carried out by actin filaments and associated proteins. 2. Signaling function from extracellular surfacenucleus (where they stimulate the transcription of genes involved in cell growth and proliferation), which is carried out by tyrosine kinase (Src and FAK) Extracellular Cell Matrix (ECM) 第三节、细胞外基质 MACROMOLECULAR ORGANIZATION OF ECM Scanning electron micrograph of fibroblasts in connective tissue. The tissue is from the cornea of a rat. The extracellular matrix surrounding the fibroblasts is composed largely of collagen fibrils. The glycoproteins, glycosaminoglycans, and proteoglycans, which normally form a hydrated gel filling the interstices of the fibrous network, have been removed by enzyme and acid treatment. 内容提要 第一节 ECM的成分 第二节 ECM的生物学作用 概念：细胞外基质（extracellular matrix，ECM）是指分部 于细胞外空间，由细胞分泌的蛋白和多糖所构成的网络 结构。 类型：结构蛋白：胶原、弹性蛋白。强度和韧性 蛋白聚糖：糖胺聚糖与蛋白聚糖。抗压。 粘联糖蛋白：层粘连蛋白和纤连蛋白。 功能：粘连；支持；改变细胞微环境；信号功能。 影响细胞的存活、死亡、增殖和分化。 一、 ECM的成分 一、胶原Collagen 是人体最丰富的蛋白，占蛋白总量的30以上。 参与形成结缔组织，如骨、韧带、基膜、皮肤。 组成：胶原纤维的基本结构单位是原胶原，由原 胶原交联而成。 类型：已发现20多种，由不同基因编码。 型了解较多。I、II、III、V、XI型为有横纹的纤维 结构。 原胶原：3条多肽链形成的螺 旋，含三种结构：螺旋区、非 螺旋区及球形结构域。 原胶原每条链由重复的Gly -X-Y序列构成。X=pro， Y=hypro, hylys，重复序列 使链卷曲为左手螺旋。 三股链绕成右手超螺旋。 合成：由成纤维细胞、软骨细胞、成骨细胞、上皮细胞分泌。在RER上合成，形成三股螺 旋之前于Pro及Lys残基上进行羟基化修饰。 抗坏血酸的作用 前原胶原 前胶原 原胶原 胶原纤维 The covalent intramolecular and intermolecular cross-links formed between modified lysine side chains within a collagen fibril. The cross- links are formed in several steps. First, certain lysine and hydroxylysine residues are deaminated by the extracellular enzyme lysyl oxidase to yield highly reactive aldehyde groups. The aldehydes then react spontaneously to form covalent bonds with each other or with other lysine or hydroxylysine residues. Most of the cross-links form between the short nonhelical segments at each end of the collagen molecules. How the staggered arrangement of collagen molecules gives rise to the striated appearance of a negatively stained fibril. (A) Since the negative stain fills only the space between the molecules, the stain in the gaps between the individual molecules in each row accounts for the dark staining bands. An electron micrograph of a portion of a negatively stained fibril is shown below (B). The staggered arrangement of the collagen molecules maximizes the tensile strength of the aggregate. TEM image of collagen Electron micrograph of a cross-section of tadpole skin. Note the plywoodlike arrangement of collagen fibrils, in which successive layers of fibrils are laid down nearly at right angles to each other. This arrangement is also found in mature bone and in the cornea. 胶原组织 作用 1、细胞外基质的骨架结构 2、刚性及抗张力 二、弹性蛋白 elastin 构成弹性纤维 弹性蛋白是高度疏水的非糖基化蛋白，具有两个明显的特征: 构象呈无规则卷曲状态，疏水短肽，赋予分子以弹性； 通过Lys残基相互交连成网状结构。 老年组织中弹性蛋白的生成减少，降解增强，以致组 织失去弹性。 Stretching a network of elastin molecules. The molecules are joined together by covalent bonds (indicated in red) to generate a cross-linked network. In the model shown each elastin molecule in the network can expand and contract as a random coil, so that the entire assembly can stretch and recoil like a rubber band. A network of elastic fibers. These scanning electron micrographs show a low-power view of a segment of a dogs aorta (A) and a high-power view of the dense network of longitudinally oriented elastic fibers in the outer layer of the same blood vessel (B). All of the other components have been digested away with enzymes and formic acid. (K.S. Haas et al., Anat. Rec. 230:86-96.) Elastin 三、糖胺聚糖及蛋白聚糖 GAG是重复二糖单位构成的无分枝长链多糖。 二糖单位通常由氨基已糖和糖醛酸组成。 1糖胺聚糖(Glycosaminoglycan) 分为：透明质酸(HA)、硫酸软骨素、硫酸皮肤素 、硫酸乙酰肝素、肝素、硫酸角质素。 除HA及肝素外，其他GAG不游离存在。 HA是唯一不硫酸化的GAG，含多达10万个糖基。 可结合大量水分子，赋予组织一定的抗压性。 糖氨聚糖的分子特性及分布 2蛋白聚糖 是GAG(除HA)与核心蛋白的共价结合物。 核心蛋白在高尔基体中装配GAG链。 先合成4糖连接桥（Xyl-Gal-Gal-GlcUA）连 接到Ser上，然后延长糖链。 HA能够以非共价键连接许多蛋白聚糖，形 成巨分子。 A Proteoglycan Complex. Galactose N-Acetyl-D-glucosamine D-glucuronic acid N-Acetyl-D-galactosamine 显著特点是多态性：不同的核心蛋白, 不同的氨基聚糖； 软骨中的蛋白聚糖是最大巨分子之一, 赋予软骨以凝 胶样特性和抗变形能力； 蛋白聚糖可视为细胞外的激素富集与储存库，可与多 种生长因子结合，完成信号的传导。 蛋白聚糖的特性与功能 Scanning electron micrograph of a basal lamina in the cornea of a chick embryo. Some of the epithelial cells (E) have been removed to expose the upper surface of the matlike basal lamina (BL). A network of collagen fibrils (C) in the underlying connective tissue interacts with the lower face of the lamina. 四、纤粘连蛋白 Fibronectin（FN） 含糖4.59.5，有57个有特定功能的 结构域。类型： FN有20多种亚单位。同一基因编码，转录后拼接不同 ，形成异型分子。 血浆FN： V字形二聚体，可溶，存在于血浆、体液。 细胞FN：多聚体，不溶，存在于ECM及细胞表面。 Fibronectin helps connect the inside of the cell to the outside 纤连蛋白的主要功能： 介导细胞粘着，进而调节细胞的形状和细胞骨 架的组织，促进细胞铺展； 在胚胎发生过程中，纤连蛋白对于许多类型细 胞的迁移和分化是必需的； 在创伤修复中，纤连蛋白促进巨噬细胞和其它 免疫细胞迁移到受损部位； 在血凝块形成中，纤连蛋白促进血小板附着于 血管受损部位。 层粘连蛋白是高分子糖蛋白（820KD）,动物胚胎 及成体组织的基膜的主要结构组分之一； 层粘连蛋白的由一条重链和两条轻链构成