Monitoring biomolecular interactions by time-lapse atomic force microscopy

The atomic force microscope (AFM) is a unique imaging tool that enables the tracking of single macromolecule events in response to physiological effectors and pharmacological stimuli. Direct correlation can therefore be made between structural and functional states of individual biomolecules in an aqueous environment. This review explores how time-lapse AFM has been used to learn more about normal and disease-associated biological processes. Three specific examples have been chosen to illustrate the capabilities of this technique. In the cell, actin polymerizes into filaments, depolymerizes, and undergoes interactions with numerous effector molecules (i.e., severing, capping, depolymerizing, bundling, and cross-linking proteins) in response to many different stimuli. Such events are critical for the function and maintenance of the molecular machinery of muscle contraction and the dynamic organization of the cytoskeleton. One goal is to use time-lapse AFM to examine and manipulate some of these events in vitro, in order to learn more about how these processes occur in the cell. Aberrant protein polymerization into amyloid fibrils occurs in a multitude of diseases, including Alzheimer's and type 2 diabetes. Local amyloid deposits may cause organ dysfunction and cell death; hence, it is of interest to learn how to interfere with fibril formation. One application of time-lapse AFM in this area has been the direct visualization of amyloid fibril growth in vitro. This experimental approach holds promise for the future testing of potential therapeutic drugs, for example, by directly visualizing at which level of fibril assembly (i.e., nucleation, elongation, branching, or lateral association of protofibrils) a given active compound will interfere. Nuclear pore complexes (NPCs) are large supramolecular assemblies embedded in the nuclear envelope. Transport of ions, small molecules, proteins, RNAs, and RNP particles in and out of the nucleus occurs via NPCs. Time-lapse AFM has been used to structurally visualize the response of individual NPC particles to various chemical and physical effectors known to interfere with nucleocytoplasmic transport. Taken together, such time-lapse AFM studies could provide novel insights into the molecular mechanisms of fundamental biological processes under both normal and pathological conditions at the single molecule level.     

纤维结合素分子中Asp1961～Glu1978序列对三结构域多肽在大肠杆菌中表达的影响

构建了人纤维结合素（ＦＮ）的三功能结构域重组多肽的两个表达质粒,分别编码两个重组多肽：ＣＨ６２（ＦＮ的Ｐｒｏ１２３９～Ｓｅｒ１５１５经Ｍｅｔ和Ａｌａ１６９０～Ｖａ１２０４９相连）和ＣＨ６３（从ＣＨ６２中删除了Ｉｌｅ１８５０～Ｇｌｕ１９７８）．ＣＨ６２在大肠杆菌中的表达效率很低,而ＣＨ６３的表达效率则很高,结果提示ＦＮ分子中的Ａｓｐ１９６１～Ｇｌｕ１９７８序列是影响三结构域多肽在大肠杆菌中表达的关键结构．ＣＨ６３经过溶解和复性后,可通过肝素－琼脂糖亲和层析得到纯品,所得纯品具有结合肝素和结合细胞的功能,且结合细胞的能力比双结构域ＦＮ多肽更强,表明两个结合细胞的功能结构域均有活性．ＣＨ６３的制备为进一步研究具有更强的抑制肿瘤转移作用的基因工程制品奠定了基础．     

In vitro conversion of full-length mammalian prion protein produces amyloid form with physical properties of PrP(Sc)

The "protein only" hypothesis postulates that the infectious agent of prion diseases, PrP(Sc), is composed of the prion protein (PrP) converted into an amyloid-specific conformation. However, cell-free conversion of the full-length PrP into the amyloid conformation has not been achieved. In an effort to understand the mechanism of PrP(Sc) formation, we developed a cell-free conversion system using recombinant mouse full-length PrP with an intact disulfide bond (rPrP). We demonstrate that rPrP will convert into the beta-sheet-rich oligomeric form at highly acidic pH (<5.5) and at high concentrations, while at slightly acidic or neutral pH (>5.5) it assembles into the amyloid form. As judged from electron microscopy, the amyloid form had a ribbon-like assembly composed of two non-twisted filaments. In contrast to the formation of the beta-oligomer, the conversion to the amyloid occurred at concentrations close to physiological and displayed key features of an autocatalytic process. Moreover, using a shortened rPrP consisting of 106 residues (rPrP 106, deletions: Delta23-88 and Delta141-176), we showed that the in vitro conversion mimicked a transmission barrier observed in vivo. Furthermore, the amyloid form displayed a remarkable resistance to proteinase K (PK) and produced a PK-resistant core identical with that of PrP(Sc). Fourier transform infrared spectroscopy analyses showed that the beta-sheet-rich core of the amyloid form remained intact upon PK-digestion and accounted for the extremely high thermal stability. Electron and real-time fluorescent microscopy revealed that proteolytic digestion induces either aggregation of the amyloid ribbons into large clumps or further assembly into fibrils composed of several ribbons. Fibrils composed of ribbons were very fragile and had a tendency to fragment into short pieces. Remarkably, the amyloid form treated with PK preserved high seeding activity. Our work supports the protein only hypothesis of prion propagation and demonstrates that formation of the amyloid form that recapitulates key physical properties of PrP(Sc) can be achieved in vitro in the absence of cellular factors or a PrP(Sc) template.     

Effect of Thrombin on Human Amnion Mesenchymal Cells,Mouse Fetal Membranes,and Preterm Birth

Here, we investigated the effects of thrombin on matrix metalloproteinases (MMPs) and prostaglandin (PG) synthesis in fetal membranes. Thrombin activity was increased in human amnion from preterm deliveries. Treatment of mesenchymal, but not epithelial, cells with thrombin resulted in increased MMP-1 and MMP-9 mRNA and enzymatic activity. Thrombin also increased COX2 mRNA and PGE₂ in these cells. Protease-activated receptor-1 (PAR-1) was localized to amnion mesenchymal and decidual cells. PAR-1-specific inhibitors and activating peptides indicated that thrombin-induced up-regulation of MMP-9 was mediated via PAR-1. In contrast, thrombin-induced up-regulation of MMP-1 and COX-2 was mediated through Toll-like receptor-4, possibly through thrombin-induced release of soluble fetal fibronectin. In vivo, thrombin-injected pregnant mice delivered preterm. Mmp8, Mmp9, and Mmp13, and PGE₂ content was increased significantly in fetal membranes from thrombin-injected animals. These results indicate that thrombin acts through multiple mechanisms to activate MMPs and PGE2 synthesis in amnion.     

大鼠肾脏纤维结合素的分布以及慢性缺氧对其影响的免疫组化研究

本文采用冰冻切片过氧化酶免疫组织化学染色技术观察正常大鼠肾脏中纤维结合素(FN)的分布及慢性缺氧对其影响。56只成年雄性Wistar大鼠随机分为7组:正常对照0天、5天、15天和30天组以及缺氧5天、15天和30天组。缺氧组采用10％氧浓度常压缺氧箱持续缺氧,对照组则呼吸空气。结果基线组及各对照组肾动脉壁、肾小管外周、肾小球囊及球内毛细血管间均可见FN阳性染色;缺氧5天及15天组FN阳性染色无明显改变;缺氧30天组FN阳性染色明显增多。该研究对于深入探讨慢性呼吸衰竭病人水钠潴留和肾功能异常的发生机理具有一定意义。     

人5～6月胎儿眼球壁组织中纤维粘连蛋白的免疫细胞化学

本文对4例(8眼)5～6月人胎儿眼球壁组织中纤维粘连蛋白(fibronectin,FN)进行了光镜和电镜免疫细胞化学定位观察。结果显示:Descemet膜及虹膜、瞳孔膜、脉络膜和视网膜的血管壁为FN强阳性反应;小梁组织和Schlemm管为FN阴性反应。视网膜血管电镜免疫细胞化学显示:FN阳性反应产物主要分布于血管周细胞的外侧,而与内皮细胞相邻的内侧很少,提示:血管的周细胞可能是FN合成和分泌的主要细胞。     

Expression of fibronectin,laminin and ribosomes in normal and nocodazole-treated neonatal heart cells in culture: a study by laser scanning confocal microscopy and immunocytochemistry

Polyclonal and monoclonal antibodies were used to examine the effects of the synthetic microtubule disruptive drug nocodazole on the subcellular expression of fibronectin, laminin, and ribosomes in primary cultures of neonatal cardiac ventricular cells. Non-invasive serial optical sectioning was carried out by immunolaser scanning confocal microscopy. In addition, fibronectin and laminin were immunolabelled with peroxidase or gold conjugates for electron-microscopic examination. Immunolabelling for the large 60S ribosome subunit in fibroblast-like non-myocytes showed that punctate ribosome structures with a multi-subunit composition were present in perinuclear region. Double immunostaining with antibodies directed against ribosomes and cellular fibronectin indicated that the punctate structures were cisternae of the rough endoplasmic reticulum. No clear effects of nocodazole treatment were detected on the distribution of cytoskeleton-bound ribosomes. Following immunolabelling for both glycoproteins and double immunolabelling for cellular fibronectin and the 60 S ribosome subunit, fibronectin and laminin were found in the perinuclear cisternae of the rough endoplasmic reticulum and in pleomorphic secretory vesicles. The cisternal stacks of the Golgi complex appeared either unstained or were only weakly labelled. When these cells were exposed to nocodazole, fibronectin and laminin accumulated in peripheral parts of the cytoplasm, including cellular processes. These peripheral accumulations of immunostaining for fibronectin and laminin did not reflect Golgi staining, as shown by double labelling experiments versus wheat-germ-agglutinin staining, and, by exposing cultures to a high dose of brefeldin A.     

Basal lamina and other extracellular matrix produced by bovine granulosa cells in anchorage-independent culture

Bovine granulosa cells from 3–7 mm follicles were cultured without anchorage in soft agar/methylcellulose solution for 14 days, with or without 50 ng/ml basic fibroblast growth factor. The granulosa cells divided to form colonies of cells. These were analysed by light and electron microscopy, immunohistochemistry and Western immunoblotting. In approximately 20% of the colonies extracellular matrix was clearly visible at the light-microscope level. Ultrastructurally the matrix resembled a basal lamina 30–100 nm thick and was composed of tangled fibres or cords. Unidentified spherical structures of less than 50 nm diameter were sometimes present and attached to this basal lamina. The basal lamina of follicles had similar features, except that the basal lamina produced in vitro was a large aggregate of many convoluted layers. The cells produced collagen type IV and the cellular form of fibronectin. Intercellular areas not associated with basal lamina were identified. Ruthenium red staining revealed these areas to be rich in proteoglycan granules. Free granules were clustered near the cell surface, and the lumina of these areas were rich in fibres decorated with ruthenium red. This material did not resemble follicular fluid of antral follicles. Thus, granulosa cells in anchorage-independent cultures have a follicular cell morphology and secrete two distinct extracellular matrices, one similar to the follicular basal lamina.This study was funded by the Flinders Medical Centre Research Foundation, Flinders University, and the National Health and Medical Research Council of Australia     

The immunoglobulin superfamily: An insight on its tissular, species, and functional diversity

The immunoglobulin superfamily (IgSF) is a heterogenic group of proteins built on a common fold, called the Ig fold, which is a sandwich of two β sheets. Although members of the IgSF share a similar Ig fold, they differ in their tissue distribution, amino acid composition, and biological role. In this paper we report an up-to-date compilation of the IgSF where all known members of the IgSF are classified on the basis of their common functional role (immune system, antibiotic proteins, enzymes, cytokine receptors, etc.) and their distribution in tissue (neural system, extracellular matrix, tumor marker, muscular proteins, etc.), or in species (vertebrates, invertebrates, bacteria, viruses, fungi, and plants). The members of the family can contain one or many Ig domains, comprising two basic types: the constant domain (C), with seven strands, and the variable domain (V), with eight, nine, or ten strands. The different overviews of the IgSF led to the definition of new domain subtypes, mainly concerning the C type, based on the distribution of strands within the two sheets. The wide occurrence of the Ig fold and the much less conserved sequences could have developed from a common ancestral gene and/or from a convergent evolutionary process. Cell adhesion and pattern recognition seem to be the common feature running through the entire family. Received: 4 June 1997 / Accepted: 15 September 1997     

Covisualization by computation optical-sectioning microscopy of integrin and associated proteins at the cell membrane of living onion protoplasts

Summary Using higher-resolution wide-field computational optical-sectioning fluorescence microscopy, the distribution of antigens recognized by antibodies against animal ₁ integrin, fibronectin, and vitronectin has been visualized at the outer surface of enzymatically protoplasted onion epidermis cells and in depectinated cell wall fragments. On the protplast all three antigens are colocalized in an array of small spots, as seen in raw images, in Gaussian filtered images, and in images restored by two different algorithms. Fibronectin and vitronectin but not ₁ integrin antigenicities colocalize as puncta in comparably prepared and processed images of the wall fragments. Several control visualizations suggest considerable specificity of antibody recognition. Affinity purification of onion cell extract with the same anti-integrin used for visualization has yielded protein that separates in SDS-PAGE into two bands of about 105–110 and 115–125 kDa. These bands are again recognized by the visualizationi antibody, which was raised against the extracellular domain of chicken ₁ integrin, and are also reconized by an antibody against the intracellular domain of chicken ₁ integrin. Because ₁ integrin is a key protein in numerous animal adhesion sites, it appears that the punctate distribution of this protein in the cell membranes of onion epidermis represents the adhesion sites long known to occur in cells of this tissue. Because vitronectin and fibronectin are matrix proteins that bind to integrin in animals, the punctate occurrence of antigenically similar proteins both in the wall (matrix) and on enzymatically prepared protoplasts reinforces the concept that onion cells have adhesion sites with some similarity to certain kinds of adhesioni sites in animals.