植物细胞中的RGD模体及受体

Arg-Gly-Asp(RGD)模体是动物细胞底物黏附分子的基本识别结构,许多胞外黏附蛋白是通过RGD模体与质膜受体整合素结合的,它参与细胞的跨膜信号转导,介导多种生物学过程。越来越多的实验表明植物细胞中也存在RGD结合模体,现就近年来植物细胞在这方面的研究进展进行综述。     

壁膜连接中断影响植物细胞响应机械应力的实验研究

目的:研究植物细胞响应机械应力的形态学变化;初步探讨壁膜连接对于植物细胞响应机械应力的意义,及其是否参与机械应力响应过程。方法:将植物单细胞包埋后进行精确机械力加载并培养数日,每天抽样进行显微观测和图像分析监测其形态变化;在此基础上进行质壁分离,并采用RGD多肽处理中断细胞壁膜连接后进行应力加载,监测其形态变化。结果:植物细胞经机械应力加载后,其分裂方向趋于垂直应力加载方向。经壁膜连接中断处理后进行加载的植物细胞失去沿主应力线垂直方向分裂的能力,细胞分裂方向呈无序状态。结论:壁膜连接特别是以RGD序列识别为基础的细胞连接参与了植物细胞对机械应力的响应过程。     

KDEL-Containing Auxin-Binding Protein Is Secreted to the Plasma Membrane and Cell Wall

The auxin-binding protein ABP1 has been postulated to mediate auxin-induced cellular changes associated with cell expansion. This protein contains the endoplasmic reticulum (ER) retention signal, the tetrapeptide lysine-aspartic acid-glutamic acid-leucine (KDEL), at its carboxy terminus, consistent with previous subcellular fractionation data that indicated an ER location for ABP1. We used electron microscopic immunocytochemistry to identify the subcellular localization of ABP1. Using maize (Zea mays) coleoptile tissue and a black Mexican sweet (BMS) maize cell line, we found that ABP1 is located in the ER as expected, but is also on or closely associated with the plasma membrane and within the cell wall. Labeling of the Golgi apparatus suggests that the transport of ABP1 to the cell wall occurs via the secretory system. Inhibition of secretion of an ABP homolog into the medium of BMS cell cultures by brefeldin A, a drug that specifically blocks secretion, is consistent with this secretion pathway. The secreted protein was recognized by an anti-KDEL peptide antibody, strongly supporting the interpretation that movement of this protein out of the ER does not involve loss of the carboxy-terminal signal. Cells starved for 2,4-dichlorophenoxyacetic acid for 72 h retained less ABP in the cell and secreted more of it into the medium. The significance of our observations is 2-fold. We have identified a KDEL-containing protein that specifically escapes the ER retention system, and we provide an explanation for the apparent discrepancy that most of the ABP is located in the ER, whereas ABP and auxin act at the plasma membrane.     

茉莉素的信号转导与植物的防御反应

文章从茉莉素信号途径及其与其他信号途径交叉作用的角度 ,概括了茉莉素在植物防御反应中的作用     

Localized Patch Clamping of Plasma Membrane of a Polarized Plant Cell : Laser Microsurgery of the Fucus spiralis Rhizoid Cell Wall

We used an ultraviolet laser to rupture a small region of cell wall of a polarized Fucus spiralis rhizoid cell and gained localized access to the plasma membrane at the growing apex. Careful control of cell turgor enabled a small portion of plasma membrane-bound cytoplasm to be exposed. Gigaohm seals allowing single-channel recordings were obtained with a high success rate using this method with conventional patch clamp techniques.     

壳梭孢素对质膜H~ -ATPase活力影响机制的研究进展

壳梭孢素 (FC)作为一种重要的研究工具广泛用于研究酸介导的生长反应和依赖于质子推动力的膜运输系统 ,FC刺激质膜H _ATPase的活性是通过FC结合蛋白 (FCBP)与H _ATPase发生作用。FCBP是 1 4_3_3蛋白家族成员之一     

Rapid Changes in Plasma Membrane Protein Phosphorylation during Initiation of Cell Wall Digestion

Plasma membrane vesicles from wild carrot cells grown in suspension culture were isolated by aqueous two-phase partitioning, and ATP-dependent phosphorylation was measured with [γ-³²P]ATP in the presence and absence of calcium. Treatment of the carrot cells with the cell wall digestion enzymes, driselase, in a sorbitol osmoticum for 1.5 min altered the protein phosphorylation pattern compared to that of cells treated with sorbitol alone. Driselase treatment resulted in decreased phosphorylation of a band of M_r 80,000 which showed almost complete calcium dependence in the osmoticum treated cells; decreased phosphorylation of a band of M_r 15,000 which showed little calcium activation, and appearance of a new band of calcium-dependent phosphorylation at M_r 22,000. These effects appeared not to be due to nonspecific protease activity and neither in vivo nor in vitro exposure to driselase caused a significant loss of Coomassie blue-staining bands on the gels of the isolated plasma membranes. However, protein phosphorylation was decreased. Adding driselase to the in vitro reaction mixture caused a general decrease in the membrane protein phosphorylation either in the presence or absence of calcium which did not mimic the in vivo response. Cells labeled in vivo with inorganic ³²P also showed a response to the Driselase treatment. An enzymically active driselase preparation was required for the observed responses.     

MAPK和活性氧参与植物抗病防卫反应的信号转导

促分裂原活化蛋白激酶（MAPK）级联途径和活性氧参与调控植物过敏性细胞死亡。本文介绍促分裂原活化蛋白激酶级联途径在植物抗病防卫反应信号转导中的作用研究进展,并对活性氧积累与MAPK之间的关系作了分析。     

Functional Interaction between the Fanconi Anemia D2 Protein and Proliferating Cell Nuclear Antigen (PCNA) via a Conserved Putative PCNA Interaction Motif

Fanconi Anemia (FA) is a rare recessive disease characterized by congenital abnormalities, bone marrow failure, and cancer susceptibility. The FA proteins and the familial breast cancer susceptibility gene products, BRCA1 and FANCD1/BRCA2, function cooperatively in the FA-BRCA pathway to repair damaged DNA and to prevent cellular transformation. Activation of this pathway occurs via the mono-ubiquitination of the FANCD2 protein, targeting it to nuclear foci where it co-localizes with FANCD1/BRCA2, RAD51, and PCNA. The regulation of the mono-ubiquitination of FANCD2, as well as its function in DNA repair remain poorly understood. In this study, we have further characterized the interaction between the FANCD2 and PCNA proteins. We have identified a highly conserved, putative FANCD2 PCNA interaction motif (PIP-box), and demonstrate that mutation of this motif disrupts FANCD2-PCNA binding and precludes the mono-ubiquitination of FANCD2. Consequently, the FANCD2 PIP-box mutant protein fails to correct the mitomycin C hypersensitivity of FA-D2 patient cells. Our results suggest that PCNA may function as a molecular platform to facilitate the mono-ubiquitination of FANCD2 and activation of the FA-BRCA pathway.Fanconi anemia (FA)² is a rare recessive disorder characterized by developmental abnormalities, progressive bone marrow failure, and pronounced cancer susceptibility (1). FA patients are particularly susceptible to early-onset acute myelogenous leukemia and squamous cell carcinoma of the head, neck, and gynecologic regions (2). FA patient cells are hypersensitive to the clastogenic effects of DNA cross-linking agents, e.g. mitomycin C (MMC), and agents that inhibit DNA replication, e.g. aphidicolin (APH) (3, 4). There are currently thirteen genetically defined FA complementation groups (A, B, C, D1, D2, E, F, G, I, J, L, M, and N), and all thirteen genes have been identified (5).A central step in the activation of the FA-BRCA pathway is the mono-ubiquitination of the FANCD2 and FANCI proteins, catalyzed by the core FA E2/E3 holoenzyme complex (5, 6). The mono-ubiquitination of FANCD2 and FANCI signals their translocation to discrete nuclear foci, where they co-localize with the BRCA1 and RAD51 DNA repair proteins, as well as the major cellular DNA polymerase processivity factor PCNA (3, 4, 7–9). Several studies have suggested an important role for the FA-BRCA pathway in a DNA replication-associated DNA repair process, e.g. homologous recombination (HR), and/or translesion DNA synthesis (TLS) (3, 4, 10–12). Accordingly, additional proteins with established roles in the DNA replication stress response, including ATR, CHK1, HCLK2, and RPA, modulate DNA damage-inducible FANCD2 mono-ubiquitination (13–15). Our understanding of the regulation of this critical post-translational modification, however, is incomplete.We, and others (4, 7) have previously reported an association between FANCD2 and PCNA. FANCD2 and PCNA co-localize in nuclear foci following treatment with agents that inhibit DNA replication. Like FANCD2, PCNA is mono-ubiquitinated following exposure to DNA-damaging agents (16, 17). While FANCD2 and PCNA are mono-ubiquitinated by different E3 ubiquitin ligases, FANCL and RAD18 (16–19), respectively, both proteins are de-ubiquitinated by the USP1 enzyme (20, 21). The functional significance of the FANCD2-PCNA interaction, however, has not been determined.In addition to its role as a DNA polymerase processivity factor, PCNA interacts with many DNA repair proteins, e.g. MSH3, XPG, and p21^Cip1/Waf1 (22). These interactions typically occur in a hydrophobic pocket of the PCNA homotrimer, termed the interdomain connecting loop (ICL). Proteins that interact with the PCNA ICL harbor a highly conserved PCNA-binding motif called the PIP-box, defined by the amino acid sequence QXXhXXaa, where h represents amino acids with moderately hydrophobic side chains, e.g. leucine, isoleucine, or methionine (L, I, M), a represents amino acids with highly hydrophobic, aromatic side chains, e.g. phenylalanine and tyrosine (F, Y), and X is any amino acid (23).Here, we describe an important functional interaction between FANCD2 and PCNA. We have identified a highly conserved putative PIP-box in FANCD2, and demonstrate that mutation of this motif disrupts the FANCD2-PCNA interaction, and precludes both the spontaneous and DNA damage-inducible mono-ubiquitination of FANCD2. Consequently, the FANCD2 PIP-box mutant fails to correct the MMC hypersensitivity of FA-D2 patient-derived cells. However, the mutant protein retains the ability to localize to chromatin, interact with FANCE, and undergo DNA damage-inducible phosphorylation. Our results suggest that PCNA may act as a molecular platform for the mono-ubiquitination of FANCD2 and for the activation of the FA-BRCA pathway.     

Relationship between Cell Wall, Cytoplasmic Membrane, and Bacterial Motility

High-resolution electron microscopy of polarly flagellated bacteria revealed that their flagella originate at a circular, differentiated portion of the cytoplasmic membrane approximately 25 nm in diameter. The flagella also have discs attaching them to the cell wall. These attachment discs are extremely resistant to lytic damage and are firmly bound to the flagella. The cytoplasm beneath the flagellum contains a granulated basal body about 60 nm in diameter, and a specialized polar membrane. The existence of membrane-bound basal bodies is shown to be an artifact arising from adherence of cell wall and cytoplasmic membrane fragments to flagella in lysed preparations. Based on structures observed, a mechanism to explain bacterial flagellar movement is proposed. Flagella are considered to be anchored to the cell wall and activated by displacement of underlying cytoplasmic membrane to which they are also firmly attached. An explanation for the membrane displacement is given.     

壳梭孢素对质膜H^＋—ATPase活力影响机制的研究进展

壳梭孢素（FC）作为一种重要的研究工具广泛用于研究酸介导的生长反应和依赖于质子推动力的膜运输系统,FC刺激质膜H^ -ATPase的活性是通过FC结合蛋白（FCBP）与H^ -ATPase发生作用,FCBP是14－3－3蛋白家族成员之一。     

壳梭孢素对质膜H+-ATPase活力影响机制的研究进展

壳梭孢素（FC）作为一种重要的研究工具广泛用于研究酸介导的生长反应和依赖于质子推动力的膜运输系统,FC刺激质膜H+_ATPase的活性是通过FC结合蛋白（FCBP）与H+_ATPase 发生作用。FCBP是14-3-3蛋白家族成员之一。     

Plasma Membrane Turnover in Plant Cells

Steer, M. W. 1988. Plasma membrane turnover in plant cells.—J.exp. Bot. 39: 987–996. Plasma membrane turnover in plant cells occurs as a consequenceof secretion, which incorporates new membrane into the cellsurface and endocytosis, which internalizes surface membrane.Development of methods that provide estimates of the rate ofnew membrane flow to the cell surface has allowed the estimationof turnover times for the plasma membrane. These times rangefrom 10 min for a non-expanding secretory cell to 3 h for anelongating epidermal cell. At least part, if not all, of thereturn route into the cell is via endocytotic vesicles. Quantitativestudies are required to establish the precise level of flowthrough this route. However, turnover times estimated from theabundance of coated patches on the plasma membrane are comparableto those estimated from secretion studies. The effect of thesehigh turnover rates on a number of plasma membrane functionsare discussed and assessed. Key words: Plasma membrane, endocytosis, secretion, plant cells     

Outer Hair Cell Lateral Wall Structure Constrains the Mobility of Plasma Membrane Proteins

Nature’s fastest motors are the cochlear outer hair cells (OHCs). These sensory cells use a membrane protein, Slc26a5 (prestin), to generate mechanical force at high frequencies, which is essential for explaining the exquisite hearing sensitivity of mammalian ears. Previous studies suggest that Slc26a5 continuously diffuses within the membrane, but how can a freely moving motor protein effectively convey forces critical for hearing? To provide direct evidence in OHCs for freely moving Slc26a5 molecules, we created a knockin mouse where Slc26a5 is fused with YFP. These mice and four other strains expressing fluorescently labeled membrane proteins were used to examine their lateral diffusion in the OHC lateral wall. All five proteins showed minimal diffusion, but did move after pharmacological disruption of membrane-associated structures with a cholesterol-depleting agent and salicylate. Thus, our results demonstrate that OHC lateral wall structure constrains the mobility of plasma membrane proteins and that the integrity of such membrane-associated structures are critical for Slc26a5’s active and structural roles. The structural constraint of membrane proteins may exemplify convergent evolution of cellular motors across species. Our findings also suggest a possible mechanism for disorders of cholesterol metabolism with hearing loss such as Niemann-Pick Type C diseases.     

Interaction of Partially Purified Phytotoxins from Phytophthora cactorum on Apple Cell Plasma Membrane

The ‘crude’ filtrate (CF) of Phytophthora cactorum containing phytotoxin(s), having some properties similar to the toxins isolated from other Phytophthora species, was processed by three steps (acetone precipitation, dialysis and gel filtration chromatography). The CF fractions corresponding to the progressive steps of purification were tested for phytotoxicity on tomato seedlings and for activity on cell trans-membrane electrical potential (Em) of susceptible and resistant apple rootstocks (Malus domestica). The fractions (F4), obtained from chromatography on Sephadex G50 fine and eluted in the zone corresponding to a molecular weight of 15 ± 2 kD, induced a specific alteration on susceptible apple cell membranes. These metabolites, even though incompletely purified, are able to induce a high and specific activity on susceptible apple cell Em only, not on resistant ones. As a consequence, they may be of potential use in screening for insensitive cells.     

植物细胞壁蛋白质组学研究进展

植物细胞壁蛋白质在细胞代谢和发育调控、细胞壁组分修饰、信号转导及胁迫响应等生物学事件中具有重要功能.最近,国内外学者开展了大量植物细胞壁蛋白质组学的研究工作,并取得了巨大进展.本文详述了细胞壁蛋白质的分类、提取、鉴定及生物信息学分析的最新进展,总结了植物细胞壁蛋白质组学的应用和面临的挑战,提出了植物细胞壁蛋白质组学研究的框架图,以期为植物细胞壁蛋白质组学的广泛研究提供借鉴.     

Vesicle-associated Membrane Protein 4 is Implicated in Trans-Golgi Network Vesicle Trafficking

The trans-Golgi network (TGN) plays a pivotal role in directing proteins in the secretory pathway to the appropriate cellular destination. VAMP4, a recently discovered member of the vesicle-associated membrane protein (VAMP) family of trafficking proteins, has been suggested to play a role in mediating TGN trafficking. To better understand the function of VAMP4, we examined its precise subcellular distribution. Indirect immunofluorescence and electron microscopy revealed that the majority of VAMP4 localized to tubular and vesicular membranes of the TGN, which were in part coated with clathrin. In these compartments, VAMP4 was found to colocalize with the putative TGN-trafficking protein syntaxin 6. Additional labeling was also present on clathrin-coated and noncoated vesicles, on endosomes and the medial and trans side of the Golgi complex, as well as on immature secretory granules in PC12 cells. Immunoprecipitation of VAMP4 from rat brain detergent extracts revealed that VAMP4 exists in a complex containing syntaxin 6. Converging lines of evidence implicate a role for VAMP4 in TGN-to-endosome transport.