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。     

Occludin 蛋白与细胞间紧密连接关系及其临床意义

细胞间紧密连接(tight junctions)广泛存在于上皮细胞及内皮细胞之间,其作用是保持细胞间结构的完整性,确保其功能的正常发挥,紧密连接上有很多种蛋白,occludin蛋白是其中主要蛋白之一,occludin蛋白的结构发生变化会导致紧密连接结构及功能的改变,而紧密连接结构与功能的紊乱是很多临床疾病共同的病理生理学特点,如肿瘤、中风及炎症性肺疾病。Occludin蛋白的结构及功能的改变受很多机制的调控,本文主要对occludin蛋白的结构、功能、调控机制及其与紧密连接之间的关系进行叙述。     

上皮细胞间紧密连接功能的研究进展

紧密连接(tight junction,TJ)广泛存在于所有上皮或内皮细胞间连接的最顶端,是物质经旁细胞途径转运的结构和功能基础。TJ是由跨膜蛋白和胞浆蛋白两大类构成的大分子复合物,主要行使"屏障"和"栅栏"功能,前者可对物质的大小和电荷进行选择,进而调控旁细胞途径的物质转运;后者则通过调控顶膜和基底侧膜两个功能区之间的脂质和蛋白等物质的自由弥散形成高度极性化的细胞。近年来,关于TJ在各种上皮细胞中的作用及调控机制的研究日益增多。本文重点综述了上皮细胞间TJ研究的最新进展,包括TJ的构成、结构和功能检测以及调控机制,并以几类研究比较集中的上皮类型为例介绍TJ研究的现状,这将为防治与TJ改变相关的上皮屏障功能障碍性疾病提供新的思路。     

MCT在精子发生中的表达规律及调控机制的研究进展

单羧酸转运蛋白(monocarboxylate transporters,MCTs)是哺乳动物细胞膜上一类重要的跨膜转运蛋白,主要负责乳酸盐、丙酮酸盐、酮体等单羧酸类化合物的跨膜转运。MCT基因在睾丸生精上皮细胞发育、分化过程中具有不同程度表达,并通过多种途径调节精子发生过程。开展对MCT基因在精子发生过程的作用研究有助于人们从能量代谢角度进一步阐明生精细胞发育和精子发生的调控机制。本研究着重从MCT在精子发生过程中的表达定位、功能及调节机制进行综述。     

PAR3在上皮细胞紧密连接形成中作用和分子机制研究

细胞极性的建立是组织发育和器官形成的重要环节。而其中紧密连接是上皮细胞极性建立和维持的重要结构,也是极性破坏的靶点。因此,紧密连接对上皮细胞极性来说十分重要。保守的PAR3-PAR6-aPKC极性复合体在紧密连接的形成过程中发挥中枢作用。PAR3可与JAMs、TIAM1及LIMK2等分子相互作用,在多个信号通路中发挥调节作用,其相互作用机制复杂。PAR3还可受到来自胞外信号作用于EGFR等受体型酪氨酸磷酸化蛋白激酶的调控。由于PAR3在紧密连接形成的过程中至关重要,有关PAR3的蛋白磷酸化和EGFR等信号转导通路影响PAR3,从而调控紧密连接形成的机制成为了新的研究热点。     

KPNB1和Ran蛋白共同介导新城疫病毒基质蛋白的入核转运

【目的】鉴定与新城疫病毒(Newcastle disease virus,NDV)基质蛋白(matrix protein,M)入核相关的细胞蛋白,以阐明NDV M蛋白细胞核定位的分子机制。【方法】从鸡胚成纤维细胞中分别克隆核转运受体蛋白KPNA1–KPNA6和KPNB1基因,将其构建到真核表达载体,并与表达NDV M蛋白的重组真核表达载体分别共转染HEK-293T细胞,通过免疫共沉淀方法鉴定与NDV M蛋白相互作用的核转运受体蛋白。另外,将M蛋白与Ran蛋白突变体或与M蛋白互作的核转运受体蛋白缺失体分别共表达,通过荧光共定位确定M蛋白入核转运相关的细胞蛋白。【结果】构建的重组真核表达载体在HEK-293T细胞中能够正确表达;通过间接免疫荧光观察发现,重组蛋白中除Myc-KPNA2蛋白定位在细胞质外,其它核转运受体蛋白均与M蛋白表现出相同的细胞核定位。免疫共沉淀试验结果表明,M蛋白与KPNA1蛋白和KPNB1蛋白均存在相互作用。进一步通过荧光共定位观察发现,M蛋白与KPNA1蛋白缺失体(DN-KPNA1)共表达不改变M蛋白的细胞核定位,而与KPNB1蛋白缺失体(DN-KPNB1)共表达后导致M蛋白变为细胞质定位,说明M蛋白入核转运需要KPNB1蛋白的参与。另外,将M蛋白与Ran蛋白突变体Ran-Q69L共表达,荧光观察发现M蛋白同样由细胞核定位变为细胞质定位,说明M蛋白入核转运还需要Ran蛋白的辅助。【结论】KPNB1和Ran蛋白共同介导NDV M蛋白的入核转运,其过程是KPNB1蛋白首先和M蛋白发生相互作用并形成复合物,然后通过Ran蛋白的辅助作用完成入核转运。     

囊泡转运蛋白SM蛋白家族的研究进展

真核细胞中含有多种不同功能的转运囊泡。虽然转运途径和携带物质各异,但细胞转运的基本分子机制却呈现出高度相似性和保守性。大多数转运途径都需要一种SNARE（Soluble NSF Attachment Protein Receptor）蛋白质复合体介导转运膜泡与靶膜的融合。同时,另一个蛋白家族,Secl／Muncl8蛋白（SM蛋白）也在囊泡运输中发挥重要作用。但是相比于对SNARE蛋白的认识的一致性,在不同的研究中SM蛋白的功能及其与SNARE复合体的相互作用方式却不尽相同。以下综述近年来有关SM蛋白结构和功能的研究进展,并归纳SM蛋白分子的作用机制、功能以及应用。     

肝细胞极性与膜蛋白分选的分子机制

肝细胞是高度特化的极性上皮细胞,细胞质膜蛋白的分选和极性转运对于肝细胞极性的建立与维持至关重要.首先,膜蛋白在内质网中合成,随后经高尔基体加工修饰,再由反面高尔基体进一步分选,最后通过膜泡运输等不同的机制分别转运到胆汁腔面或窦状隙面,行使其特殊的功能.近些年来,细胞内负责转运的细胞器和主要的分选信号已逐步被揭示.特别是循环内体也被证明参与了胆汁腔面和窦状隙面膜蛋白的极性分选和转运.肝细胞的极性一旦遭到破坏,将会引起胆汁分泌障碍以及其他肝脏功能的损伤,从而可能导致肝脏糖脂代谢紊乱,甚至丧失正常的生理功能.因此,深入研究肝脏细胞极性的形成与维持机制,将为多种肝脏疾病的预防和治疗寻找到新的方向和靶点,具有重要的理论和临床实践意义.     

脂筏、紧密连接和血脑屏障的关系

血脑屏障破坏是缺血性脑卒中急性期发生脑水肿及神经元毒性损害的核心病理过程之一,目前尚无特效保护方法。血脑屏障通透性调节的中心环节是内皮细胞的紧密连接,而紧密连接结构蛋白表达水平和位置分布的变化与脑微血管通透性的改变及脑水肿的程度密切相关。脂筏是高流动性的细胞膜脂质双层内富含胆固醇的特殊脂质和蛋白质的动态微区,它参与细胞蛋白转运。血脑屏障上有大量的脂筏存在,紧密连接结构蛋白分布于脂筏中,其功能受胆固醇调节,且脂筏上紧密结合的脂质有利于蛋白质的寡聚化。因此,基于脂筏调节血脑屏障紧密连接可能为脑保护研究提供新的药物靶点。     

肾小管髓襻升支粗段claudin-10缺失减少Na^+通透性并引起高镁血症和肾钙化北大核心CSCD

在肾小管髓襻升支粗段（thick ascending limb,TAL）,Cl-只能通过跨细胞途径进行重吸收,而约50%的Na＋以及Ca2＋和Mg2＋通过旁细胞途径重吸收。位于上皮细胞间的紧密连接蛋白复合体可以调控旁细胞途径对离子的选择性和通透性,对肾小管的重吸收起重要作用。Claudin-10是紧密连接蛋白claudin家族的成员,可在TAL表达,但其功能尚不了解。     

Basolateral expression of a chimeric protein in which the transmembrane and cytoplasmic domains of vesicular stomatitis virus G protein have been replaced by those of the influenza virus hemagglutinin

Two integral membrane proteins, influenza virus hemagglutinin (HA) and vesicular stomatitis virus G protein, are transported to and accumulated on the apical and basolateral surfaces, respectively, of the plasma membrane of polarized epithelial cells. We have used chimeric constructions to identify the domains of HA and G proteins which contain the signals for polarized transport. Previously, we have shown that a chimeric protein containing the cleavable leader and the ectodomain of HA fused to the anchoring and cytoplasmic domains of G is transported to the apical surface of polarized MDCK cells (McQueen, N.L., Nayak, D.P., Stephens, E.B., and Compans, R.W. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9318-9322). In this report we show that a chimera containing the cleavable leader and ectodomain of G fused to the anchoring and cytoplasmic domains of HA is transported to the basolateral surface of polarized cells. Another chimera which contains the leader sequence of G fused to leader minus HA is transported to the apical surface of polarized cells. These results taken together suggest that the signals for the polarized transport of HA and G proteins may reside in their ectodomains.     

Catch the KIF5B train to the apical surface

As epithelial cells become polarized, they develop new pathways to send proteins to the apical or basolateral domains of their plasma membrane. In this issue of Developmental Cell, Jaulin et al. (2007) show that as polarity develops, there is a shift in the kinesin motor protein used to transport an apical protein to the cell surface.     

Myosin VI is required for sorting of AP-1B-dependent cargo to the basolateral domain in polarized MDCK cells

In polarized epithelial cells, newly synthesized membrane proteins are delivered on specific pathways to either the apical or basolateral domains, depending on the sorting motifs present in these proteins. Because myosin VI has been shown to facilitate secretory traffic in nonpolarized cells, we investigated its role in biosynthetic trafficking pathways in polarized MDCK cells. We observed that a specific splice isoform of myosin VI with no insert in the tail domain is required for the polarized transport of tyrosine motif containing basolateral membrane proteins. Sorting of other basolateral or apical cargo, however, does not involve myosin VI. Site-directed mutagenesis indicates that a functional complex consisting of myosin VI, optineurin, and probably the GTPase Rab8 plays a role in the basolateral delivery of membrane proteins, whose sorting is mediated by the clathrin adaptor protein complex (AP) AP-1B. Our results suggest that myosin VI is a crucial component in the AP-1B-dependent biosynthetic sorting pathway to the basolateral surface in polarized epithelial cells.     

Recycling endosomes of polarized epithelial cells actively sort apical and basolateral cargos into separate subdomains

The plasma membranes of epithelial cells plasma membranes contain distinct apical and basolateral domains that are critical for their polarized functions. However, both domains are continuously internalized, with proteins and lipids from each intermixing in supranuclear recycling endosomes (REs). To maintain polarity, REs must faithfully recycle membrane proteins back to the correct plasma membrane domains. We examined sorting within REs and found that apical and basolateral proteins were laterally segregated into subdomains of individual REs. Subdomains were absent in unpolarized cells and developed along with polarization. Subdomains were formed by an active sorting process within REs, which precedes the formation of AP-1B-dependent basolateral transport vesicles. Both the formation of subdomains and the fidelity of basolateral trafficking were dependent on PI3 kinase activity. This suggests that subdomain and transport vesicle formation occur as separate sorting steps and that both processes may contribute to sorting fidelity.     

PDZ proteins retain and regulate membrane transporters in polarized epithelial cell membranes

PDZ proteins retain and regulate membrane transporters in polarized epithelial cell membranes. Am J Physiol Cell Physiol 288: C20–C29, 2005; doi:10.1152/ajpcell.00368.2004.—The plasma membrane of epithelial cells is subdivided into two physically separated compartments known as the apical and basolateral membranes. To obtain directional transepithelial solute transport, membrane transporters (i.e., ion channels, cotransporters, exchangers, and ion pumps) need to be targeted selectively to either of these membrane domains. In addition, the transport properties of an epithelial cell will be maintained only if these membrane transporters are retained and properly regulated in their specific membrane compartments. Recent reports have indicated that PDZ domain-containing proteins play a dual role in these processes and, in addition, that different apical and basolateral PDZ proteins perform similar tasks in their respective membrane domains. First, although PDZ-based interactions are dispensable for the biosynthetic targeting to the proper membrane domain, the PDZ network ensures that the membrane proteins are efficiently retained at the cell surface. Second, the close spatial positioning of functionally related proteins (e.g., receptors, kinases, channels) into a signal transduction complex (transducisome) allows fast and efficient control of membrane transport processes. retention of apical and basolateral membrane proteins; transducisomes; protein complex formation     

Vectorial insertion of apical and basolateral membrane proteins in polarized epithelial cells revealed by quantitative 3D live cell imaging

Although epithelial cells are known to exhibit a polarized distribution of membrane components, the pathways responsible for delivering membrane proteins to their appropriate domains remain unclear. Using an optimized approach to three-dimensional live cell imaging, we have visualized the transport of newly synthesized apical and basolateral membrane proteins in fully polarized filter-grown Madin-Darby canine kidney cells. We performed a detailed quantitative kinetic analysis of trans-Golgi network (TGN) exit, passage through transport intermediates, and arrival at the plasma membrane using cyan/yellow fluorescent protein-tagged glycosylphosphatidylinositol-anchored protein and vesicular stomatitis virus glycoprotein as apical and basolateral reporters, respectively. For both pathways, exit from the TGN was rate limiting. Furthermore, apical and basolateral proteins were targeted directly to their respective membranes, resolving current confusion as to whether sorting occurs on the secretory pathway or only after endocytosis. However, a transcytotic protein did reach the apical surface after a prior appearance basolaterally. Finally, newly synthesized proteins appeared to be delivered to the entire lateral or apical surface, suggesting-contrary to expectations-that there is not a restricted site for vesicle docking or fusion adjacent to the junctional complex.     

The transmembrane domain of the respiratory syncytial virus F protein is an orientation-independent apical plasma membrane sorting sequence

The processes that facilitate transport of integral membrane proteins though the secretory pathway and subsequently target them to particular cellular membranes are relevant to almost every field of biology. These transport processes involve integration of proteins into the membrane of the endoplasmic reticulum (ER), passage from the ER to the Golgi, and post-Golgi trafficking. The respiratory syncytial virus (RSV) fusion (F) protein is a type I integral membrane protein that is uniformly distributed on the surface of infected nonpolarized cells and localizes to the apical plasma membrane of polarized epithelial cells. We expressed wild-type or altered RSV F proteins to gain a better understanding of secretory transport and plasma membrane targeting of type I membrane proteins in polarized and nonpolarized epithelial cells. Our findings reveal a novel, orientation-independent apical plasma membrane targeting function for the transmembrane domain of the RSV F protein in polarized epithelial cells. This work provides a basis for a more complete understanding of the role of the transmembrane domain and cytoplasmic tail of viral type I integral membrane proteins in secretory transport and plasma membrane targeting in polarized and nonpolarized cells.     

TGN38 recycles basolaterally in polarized Madin-Darby canine kidney cells.

Sorting of newly synthesized plasma membrane proteins to the apical or basolateral surface domains of polarized cells is currently thought to take place within the trans-Golgi network (TGN). To explore the relationship between protein localization to the TGN and sorting to the plasma membrane in polarized epithelial cells, we have expressed constructs encoding the TGN marker, TGN38, in Madin-Darby canine kidney (MDCK) cells. We report that TGN38 is predominantly localized to the TGN of these cells and recycles via the basolateral membrane. Analyses of the distribution of Tac-TGN38 chimeric proteins in MDCK cells suggest that the cytoplasmic domain of TGN38 has information leading to both TGN localization and cycling through the basolateral surface. Mutations of the cytoplasmic domain that disrupt TGN localization also lead to nonpolarized delivery of the chimeric proteins to both surface domains. These results demonstrate an apparent equivalence of basolateral and TGN localization determinants and support an evolutionary relationship between TGN and plasma membrane sorting processes.     

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.     

Identification of a basolateral membrane targeting signal within the cytoplasmic domain of myelin/oligodendrocyte glycoprotein

Oligodendrocytes possess two distinct membrane compartments--uncompacted plasma membrane (cell body, processes) and compact myelin. Specific targeting mechanisms must exist to establish and maintain these membrane domains. Polarized epithelial cells have the best characterized system for targeting components to apical and basolateral compartments. Since oligodendrocytes arise from neuroepithelial cells, we investigated whether they might utilize targeting paradigms similar to polarized epithelial cells. Myelin/oligodendrocyte glycoprotein (MOG) is a transmembrane Ig-like molecule restricted to uncompacted oligodendroglial plasma membrane. We stably expressed MOG in Madin-Darby canine kidney (MDCK) Type II epithelial cells, which have been extensively used in protein-targeting studies. Data from surface biotinylation assays and confocal microscopy revealed that MOG sorts exclusively to the basolateral membrane of MDCK cells. Expression vectors containing progressive truncations of MOG from the cytoplasmic C-terminus were expressed in MDCK cells to localize basolateral sorting signals. A loss of only four C-terminal residues results in some MOG expression at the apical surface. More strikingly, removal of the C-terminal membrane associated hydrophobic domain from MOG results in complete loss of basolateral sorting and specific targeting to the apical membrane. These data suggest that myelinating oligodendrocytes may utilize a sorting mechanism similar to that of polarized epithelia.