CDC42——新生上皮细胞的舵手

关于上皮细胞极性的形成过程近年来已有很多研究，人们发现与内质网和高尔基体转运有关的一些蛋白质如果丢失，上皮细胞的极性将被取消，黏膜面和细胞面的蛋白排列混乱。然而近日Jaffe等人发现，CDC42也是一种与内质网和高尔基体转运有关的蛋白质，其在上皮细胞的极性形成中起着很独特的作用。     

受体介导式入胞的分子机理(2)

早期内体除了接受来自细胞膜的囊泡之外,还接受来自高尔基体和晚期内体的囊泡,并且将内体中的物质以囊泡的形式重新分配到细胞膜、高尔基体和晚期内体(如图1,见本刊第11期第13页),这种重新分配也称分选。例如,细胞膜上的脂质和膜蛋白能够被早期内体分选,一部分回到胞膜重新利用,另一部分被转运到溶酶体降解。     

肝细胞极化的结构功能特征及在肝脏疾病时的病理表现

肝细胞极化的形成和维持是肝细胞发挥正常功能的保证。与简单极化上皮细胞不同,肝细胞在肝脏血管与胆小管间形成多个极化膜面,并由紧密连接分隔。极化肝细胞膜及细胞内骨架结构与功能复杂并有序,其分子组成及物质转运机制近年来已被逐渐认识。由于肝细胞极化与肝脏生理功能及多种肝脏疾病时的病理改变有着密切关系,该文就目前肝细胞极化分子和细胞水平研究现状进行综述,并探讨此领域研究发展方向。     

消化腺电镜图象

图1.肝小叶局部扫描电镜(SEM)图象:图为兔肝小叶局部断面,有的肝细胞被断开暴露出细胞质及核(N);可见肝细胞彼此相连,形成许多肝细胞索(板)。在相邻细胞索之间,为宽窄及形态不一的窦状隙(*)。×1,350 图2.肝小叶间胆管超薄切片透射电镜图象:L为其管腔,其管壁由单层立方或柱状上皮组成。在上皮细胞的游离面(腔面),有散在的微绒毛(mv)和少量纤毛(↑)。 N为上皮细胞核。×6,000 图3.双核肝细胞SEM图象:肝细胞呈多边形,     

蛋白的高尔基体定位

高尔基体既是蛋白质修饰、分选、水解加工的场所,又是分泌物质的转运站,每时每刻都有大量的蛋白进出高尔基体。在这种情况下,高尔基体仍能保持完整且高度有序的结构,表明高尔基体驻留蛋白有精确的定位信号,以保证它们定位于正确的区隔,而不会沿着分泌途径被运输出去。高尔基体内有几种不同类别的膜蛋白,包括糖基转移酶、周缘膜蛋白、病毒蛋白和受体等。研究显示,有多种定位信号和定位机制参与了蛋白的高尔基体定位。     

内皮屏障与极性运输

极化细胞的极性分布和功能行使,需要不同机制相互协作,改变胞内蛋白的运输和分布,并对环境变化做出极性应激。内皮细胞(endothelial cells,ECs)是一类具有极性特征的单层特化上皮细胞,在结构和功能上形成面向血液的顶端区域(apical membrane domain)和面向下方平滑肌细胞的基底侧区域(basolateral membrane domain)。内皮功能障碍和细胞极性丢失,与心血管疾病及癌症的发生紧密相关。在炎症和免疫应答中,内皮细胞通过胞内蛋白的持续分选维持极性,协助血液中大分子跨过内皮屏障完成生理功能,同时,对血液或组织中的生理变化做出极性应答。     

囊泡转运复合物Retromer和SNX蛋白家族在发育和疾病中的作用

逆向囊泡转运复合物Retromer主要负责介导货物蛋白从内体向反式高尔基体或细胞表面逆向转运,是细胞内囊泡转运分选系统的重要成员.Retromer复合物主要含有两个亚复合体:货物选择复合体VPS26-VPS29-VPS35和膜结合复合体SNX-BAR.本文着重综述了Retromer复合物和SNX蛋白家族参与囊泡转运过程的分子机制以及它们在发育中对Wnt信号的调控作用;并讨论了Retromer复合物在细胞极性形成、细胞凋亡、神经元信号传递中的重要作用;以及该复合物与帕金森和阿尔茨海默病等退行性疾病之间的关系.     

嗜中性粒细胞的高尔基体极性研究

用四氧化锇浸染技术研究了嗜中性粒细胞发育过程中高尔基体的极性变化。结果表明,在大鼠嗜中性粒细胞发育过程中,高尔基体的超微结构与嗜锇反应有着一系列的变化。从原粒细胞到分叶核粒细胞,不仅高尔基体的形状与大小有变化,而且高尔基体的极性也在不断变化。呈嗜锇反应的高尔基体生成面并不总是位于凸面,因此不能单凭高尔基体的形状(凹面与凸面)来鉴别高尔基体的极性。嗜中性粒细胞高尔基体的主要功能是产生两种不同的细胞质颗粒,即嗜天青颗粒与特殊颗粒。嗜天青颗粒产生于早幼粒细胞阶段,从高尔基体的成熟面形成,这一阶段的高尔基体成熟面是凹面。特殊颗粒产生于中幼粒细胞阶段,也是从高尔基体的成熟面形成,但这一阶段的高尔基体成熟面是凹面。尽管早幼粒细胞高尔基体的凹凸面与中幼粒细胞相反,但两种颗粒都是从高尔基体的成熟面分泌出来的,与其他分泌细胞形成分泌颗粒的方式相同。     

胆汁淤积性肝损伤治疗靶点与药物治疗进展

胆汁淤积(cholestasis)是由于胆汁合成、分泌、转运、排泄等代谢功能紊乱引起的肝内胆汁淤留,肠中缺乏胆汁,血中胆汁成分过多的一种病理状态。胆汁酸信号传递过程以及胆汁淤积过程中炎症的发展和延续这些与胆汁淤积相关的肝损伤方面的分子机制已经取得重大进展。核受体作为肝脏疾病的靶标被广泛讨论,了解核受体在病理生理条件下的调节,是肝脏疾病的潜在治疗方法。本文基于中药治疗和分子免疫机制受体水平方面对胆汁淤积性肝病进行综述。     

内质网-高尔基体中间体功能研究进展

内质网-高尔基体中间体(ERGIC)的发现来自于对病毒蛋白胞内转运的研究.最初认为ERGIC是内质网和高尔基体之间的膜泡运输分选站,主要调控早期分泌途径中的货物分选及双向运输.随着研究的深入,发现ER-GIC在细胞应激条件下发挥其他重要细胞学功能,包括在自噬过程中调控早期自噬体膜的形成,以及在非经典蛋白分泌途径中扮演蛋...     

Epithelial polarity requires septin coupling of vesicle transport to polyglutamylated microtubules

In epithelial cells, polarized growth and maintenance of apical and basolateral plasma membrane domains depend on protein sorting from the trans-Golgi network (TGN) and vesicle delivery to the plasma membrane. Septins are filamentous GTPases required for polarized membrane growth in budding yeast, but whether they function in epithelial polarity is unknown. Here, we show that in epithelial cells septin 2 (SEPT2) fibers colocalize with a subset of microtubule tracks composed of polyglutamylated (polyGlu) tubulin, and that vesicles containing apical or basolateral proteins exit the TGN along these SEPT2/polyGlu microtubule tracks. Tubulin-associated SEPT2 facilitates vesicle transport by maintaining polyGlu microtubule tracks and impeding tubulin binding of microtubule-associated protein 4 (MAP4). Significantly, this regulatory step is required for polarized, columnar-shaped epithelia biogenesis; upon SEPT2 depletion, cells become short and fibroblast-shaped due to intracellular accumulation of apical and basolateral membrane proteins, and loss of vertically oriented polyGlu microtubules. We suggest that septin coupling of the microtubule cytoskeleton to post-Golgi vesicle transport is required for the morphogenesis of polarized epithelia.     

Modulation of transcytotic and direct targeting pathways in a polarized thyroid cell line.

Two biosynthetic pathways exist for delivery of membrane proteins to the apical surface of epithelial cells, direct transport from the trans-Golgi network (TGN) and transcytosis from the basolateral membrane. Different epithelial cells vary in the expression of these mechanisms. Two extremes are MDCK cells, that use predominantly the direct route and hepatocytes, which deliver all apical proteins via the basolateral membrane. To determine how epithelial cells establish a particular targeting phenotype, we studied the apical delivery of endogenous dipeptidyl peptidase IV (DPPIV) at early and late stages in the development of monolayers of a highly polarized epithelial cell line derived from Fischer rat thyroid (FRT). In 1 day old monolayers, surface delivery of DPPIV from the TGN was unpolarized (50%/50%) but a large basal to apical transcytotic component resulted in a polarized apical distribution. In contrast, after 7 days of culture, delivery of DPPIV was mainly direct (85%) with no transcytosis of the missorted component. A basolateral marker, Ag 35/40 kD, on the other hand, was directly targeted (90-98%) at all times. These results indicate that the sorting machinery for apical proteins develops independently from the sorting machinery for basolateral proteins and that the sorting site relocates progressively from the basal membrane to the TGN during development of the epithelium. The transient expression of the transcytotic pathway may serve as a salvage pathway for missorted apical proteins when the polarized phenotype is being established.     

Low Rho activity in hepatocytes prevents apical from basolateral cargo separation during trans‐Golgi network to surface transport

Hepatocytes, the main epithelial cells of the liver, organize their polarized membrane domains differently from ductal epithelia. They also differ in their biosynthetic delivery of single‐membrane‐spanning and glycophosphatidylinositol‐anchored proteins to the apical domain. While ductal epithelia target apical proteins to varying degrees from the trans‐Golgi network (TGN) to the apical surface directly, hepatocytes target them first to the basolateral domain, from where they undergo basolateral‐to‐apical transcytosis. How TGN‐to‐surface transport differs in both scenarios is unknown. Here, we report that the basolateral detour of a hepatocyte apical protein is due, in part, to low RhoA activity at the TGN, which prevents its segregation from basolateral transport carriers. Activating Rho in hepatocytic cells, which switches their polarity from hepatocytic to ductal, also led to apical‐basolateral cargo segregation at the TGN as is typical for ductal cells, affirming a central role for Rho‐signaling in different aspects of the hepatocytic polarity phenotype. Nevertheless, Rho‐induced cargo segregation was not sufficient to target the apical protein directly; thus, failure to recruit apical targeting machinery also contributes to its indirect itinerary.     

Oncostatin M regulates membrane traffic and stimulates bile canalicular membrane biogenesis in HepG2 cells

Hepatocytes are the major epithelial cells of the liver and they display membrane polarity: the sinusoidal membrane representing the basolateral surface, while the bile canalicular membrane is typical of the apical membrane. In polarized HepG2 cells an endosomal organelle, SAC, fulfills a prominent role in the biogenesis of the canalicular membrane, reflected by its ability to sort and redistribute apical and basolateral sphingolipids. Here we show that SAC appears to be a crucial target for a cytokine-induced signal transduction pathway, which stimulates membrane transport exiting from this compartment promoting apical membrane biogenesis. Thus, oncostatin M, an IL-6-type cytokine, stimulates membrane polarity development in HepG2 cells via the gp130 receptor unit, which activates a protein kinase A-dependent and sphingomyelin-marked membrane transport pathway from SAC to the apical membrane. To exert its signal transducing function, gp130 is recruited into detergent-resistant membrane microdomains at the basolateral membrane. These data provide a clue for a molecular mechanism that couples the biogenesis of an apical plasma membrane domain to the regulation of intracellular transport in response to an extracellular, basolaterally localized stimulus.     

Intracellular redirection of plasma membrane trafficking after loss of epithelial cell polarity

In polarized Madin-Darby canine kidney epithelial cells, components of the plasma membrane fusion machinery, the t-SNAREs syntaxin 2, 3, and 4 and SNAP-23, are differentially localized at the apical and/or basolateral plasma membrane domains. Here we identify syntaxin 11 as a novel apical and basolateral plasma membrane t-SNARE. Surprisingly, all of these t-SNAREs redistribute to intracellular locations when Madin-Darby canine kidney cells lose their cellular polarity. Apical SNAREs relocalize to the previously characterized vacuolar apical compartment, whereas basolateral SNAREs redistribute to a novel organelle that appears to be the basolateral equivalent of the vacuolar apical compartment. Both intracellular plasma membrane compartments have an associated prominent actin cytoskeleton and receive membrane traffic from cognate apical or basolateral pathways, respectively. These findings demonstrate a fundamental shift in plasma membrane traffic toward intracellular compartments while protein sorting is preserved when epithelial cells lose their cell polarity.     

N-聚糖和O-聚糖在极性化细胞蛋白质分拣中的作用

极性化上皮细胞的质膜因其所含蛋白质、脂质等组分不同,可以分为细胞膜顶端和细胞膜基底侧端两个区域,而新合成的蛋白质向这两个区域的有效分拣是上皮细胞维持其自身极性及正常功能所必需的。细胞膜基底侧端蛋白质的分拣主要由位于该蛋白质胞质区的信号肽所介导,关于这方面的研究是比较深入的;而细胞膜顶端蛋白质的分拣机制目前尚未阐明,因而显得比较复杂。近年来,糖类分子作为生物体内细胞识别和调控过程的信息分子日益受到关注,人们通过干扰聚糖合成、基因突变以及构建糖基化缺陷细胞株等实验方法,逐渐地认识到糖类分子在极性化上皮细胞的蛋白质分拣调节中起重要作用。由于糖分子本身结构非常复杂,而且目前缺乏研究糖类分子的有效手段,使得糖生物学的研究远远落后于蛋白质和核酸的研究。从而导致探讨糖类分子在蛋白质分拣过程的具体机制相对来说比较困难。本综述拟简要概括糖类分子中N-聚糖和O-聚糖在极性化上皮细胞的蛋白质分拣过程中的作用,以及两种聚糖在此过程中行使分拣信号功能的可能机制。     

Trafficking of immunoglobulin receptors in epithelial cells:signals and cellular factors

A typical feature of epithelial cells is the polarized distribution of their respective plasma membrane proteins. Apical and basolateral proteins can be sorted both in the trans-Golgi network and endosomes, or in both locations. Inclusion into basolateral carriers in the TGN requires the presence of distinct cytoplasmic determinants, which also appear to be recognized in endosomes. Inactivation of the basolateral sorting information leads to the efficient apical delivery, probably due to the unmasking of a recessive apical signal. Factors associated with the cytosolic face of organelles probably not only recognize these signals to mediate the inclusion of the proteins into the correct transport vesicles, but also target the carriers to the corresponding plasma membrane domain. Our interest has focused on analyzing at the molecular level how epithelial MDCK cells generate and maintain a polarized phenotype, taking advantage of immunoglobulin receptors to study the biosynthetic and endocytic pathways and the corresponding sorting events.     

GPI-anchored proteins are directly targeted to the apical surface in fully polarized MDCK cells

The polarity of epithelial cells is dependent on their ability to target proteins and lipids in a directional fashion. The trans-Golgi network, the endosomal compartment, and the plasma membrane act as sorting stations for proteins and lipids. The site of intracellular sorting and pathways used for the apical delivery of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are largely unclear. Using biochemical assays and confocal and video microscopy in living cells, we show that newly synthesized GPI-APs are directly delivered to the apical surface of fully polarized Madin-Darby canine kidney cells. Impairment of basolateral membrane fusion by treatment with tannic acid does not affect the direct apical delivery of GPI-APs, but it does affect the organization of tight junctions and the integrity of the monolayer. Our data clearly demonstrate that GPI-APs are directly sorted to the apical surface without passing through the basolateral membrane. They also reinforce the hypothesis that apical sorting of GPI-APs occurs intracellularly before arrival at the plasma membrane.     

Polarized sorting and trafficking in epithelial cells

The polarized distribution of proteins and lipids at the surface membrane of epithelial cells results in the formation of an apical and a basolateral domain, which are separated by tight junctions. The generation and maintenance of epithelial polarity require elaborate mechanisms that guarantee correct sorting and vectorial delivery of cargo molecules. This dynamic process involves the interaction of sorting signals with sorting machineries and the formation of transport carriers. Here we review the recent advances in the field of polarized sorting in epithelial cells. We especially highlight the role of lipid rafts in apical sorting.     

Transport of vesicular stomatitis virus G protein to the cell surface is signal mediated in polarized and nonpolarized cells

Current model propose that in nonpolarized cells, transport of plasma membrane proteins to the surface occurs by default. In contrast, compelling evidence indicates that in polarized epithelial cells, plasma membrane proteins are sorted in the TGN into at least two vectorial routes to apical and basolateral surface domains. Since both apical and basolateral proteins are also normally expressed by both polarized and nonpolarized cells, we explored here whether recently described basolateral sorting signals in the cytoplasmic domain of basolateral proteins are recognized and used for post TGN transport by nonpolarized cells. To this end, we compared the inhibitory effect of basolateral signal peptides on the cytosol-stimulated release of two basolateral and one apical marker in semi-intact fibroblasts (3T3), pituitary (GH3), and epithelial (MDCK) cells. A basolateral signal peptide (VSVGp) corresponding to the 29-amino acid cytoplasmic tail of vesicular stomatitis virus G protein (VSVG) inhibited with identical potency the vesicular release of VSVG from the TGN of all three cell lines. On the other hand, the VSVG peptide did not inhibit the vesicular release of HA in MDCK cells not of two polypeptide hormones (growth hormone and prolactin) in GH3 cells, whereas in 3T3 cells (influenza) hemagglutinin was inhibited, albeit with a 3x lower potency than VSVG. The results support the existence of a basolateral-like, signal-mediated constitutive pathway from TGN to plasma membrane in all three cell types, and suggest that an apical-like pathway may be present in fibroblast. The data support cargo protein involvement, not bulk flow, in the formation of post-TGN vesicles and predict the involvement of distinct cytosolic factors in the assembly of apical and basolateral transport vesicles.