A review of scanning probe microscopy investigations of liposome-DNA complexes

Liposome-DNA complexes are one of the most promising systems for the protection and delivery of nucleic acids to combat neoplastic, viral, and genetic diseases. In addition, they are being used as models in the elucidation of many biological phenomena such as viral infection and transduction. In order to understand these phenomena and to realize the mechanism of nucleic acid transfer by liposome-DNA complexes, studies at the molecular level are required. To this end, scanning probe microscopy (SPM) is increasingly being used in the characterization of lipid layers, lipid aggregates, liposomes, and their complexes with nucleic acid molecules. The most attractive attributes of SPM are the potential to image samples with subnanometer spatial resolution under physiological conditions and provide information on their physical and mechanical properties. This review describes the application of scanning tunneling microscopy and atomic force microscopy, the two most commonly applied SPM techniques, in the characterisation of liposome-DNA complexes.     

New insights into the nature of the wool fibre surface

Wool fibres have been treated to remove the covalently bound lipid and characterised using lipid analysis, wettability and scanning probe microscopy. A model substrate consisting of alternating stripes of hydrophobic (predominantly CH₃ terminated molecules) and hydrophilic (COOH terminated molecules) surfaces, micro-printed onto a gold-coated mica surface was assessed using the SPM techniques of adhesion, friction and phase imaging and showed that SPM can easily distinguish these surfaces. When KOH/methanol treated wool fibres were examined, SPM showed an increase in coefficient of friction and a decrease in adhesion as the lipid is removed. The increased friction is consistent with studies on the model surface and confirms the hypothesis that the lipid layer decreases the surface friction of fibres. The decreased adhesion is consistent with results in the literature on hair, but is at odds with the results on the model surface. The strong contrast shown between the methyl and carboxylic acid surfaces in the friction image of the micro-patterned surface, and the complete absence of any such contrast developing with time of treatment of the wool fibres strongly suggests that the surface lipid is not present as a discrete outer layer on the fibre. A new model is proposed in which the lipid is intimately associated with the surface proteins and allows for changes in lipid concentration at the surface in response to changes in environmental conditions.     

Single molecule imaging of supported planar lipid bilayer--reconstituted human insulin receptors by in situ scanning probe microscopy

A 480-kDa disulfide-linked heterodimer single-pass transmembrane protein, the insulin receptor, is autophosphorylated upon insulin binding to its extracellular domain. Remarkably, the structural basis for this activation process remained largely unknown until the recent cryoelectron microscopy studies of the insulin-insulin receptor complex by Luo et al. [Science 285 (1999) 1077]. We report here the results of an in situ study by high-resolution scanning probe microscopy of the full-length insulin receptor reconstituted within supported planar lipid bilayers. Our preliminary studies confirm that (1) the intact receptor can be reconstituted constitutively within a lipid vesicle and (2) fusion of the receptor-containing vesicles to mica resulted in the formation of molecular flat 5.5-nm-thick supported planar bilayers populated by two populations of protrusions, the shape and size of which are consistent with those of the insulin receptor's intra- and extracellular domains as modeled by the cryo-EM data of Ottensmeyer et al. [Biochemistry 39 (2000) 12103]. These results establish a framework for real-time studies of insulin-insulin receptor binding by in situ SPM and single molecule force spectroscopy.     

扫描探针显微镜及其在生物医学研究中的应用

本文介绍了一类可以从原子水平到微米尺寸观察物质结构的三维成像工具——扫描探针显微镜(SPM),重点介绍了扫描隧道显微镜(STM)和原子力显微镜(AFM)的基本原理,以及SPM在细胞生物学、核酸和小分子成像等生物医学研究领域的一些应用。SPM不久将可能成为大多数生命科学实验室的一项重要技术。     

Nanotechnologies in proteomics

Progress in proteomic researches is largely determined by development and implementation of new methods for the revelation and identification of proteins in biological material in a wide concentration range (from 10(-3) M to single molecules). The most perspective approaches to address this problem involve (i) nanotechnological physicochemical procedures for the separation of multicomponent protein mixtures; among these of particular interest are biospecific nanotechnological procedures for selection of proteins from multicomponent protein mixtures with their subsequent concentration on solid support; (ii) identification and counting of single molecules by use of molecular detectors. The prototypes of biospecific nanotechnological procedures, based on the capture of ligand biomolecules by biomolecules of immobilized ligate and the concentration of the captured ligands on appropriate surfaces, are well known; these are affinity chromatography, magnetic biobeads technology, different biosensor methods, etc. Here, we review the most promising nanotechnological approaches for selection of proteins and kinetic characterization of their complexes based on these biospecific methods with subsequent MS/MS identification of proteins and protein complexes. Two major groups of methods for the analysis and identification of individual molecules and their complexes by use of molecular detectors will be reviewed: scanning probe microscopy (SPM) (including atomic-force microscopy) and cryomassdetector technology.     

Three-dimensional development of luminal projections and junctional complexes in the developing central nervous system blood vessels of the rat

Summary The luminal surface features and Junctional complexes from developing blood vessels in the rat central nervous system have been studied by high-voltage electron microscopy and scanning electron microscopy. Developing blood vessels exhibit three types of luminal projections; marginal folds or ridges at Junctional complexes, ridges not at Junctional complexes and microvilli. Both types of ridges are associated with troughs or depressions in the luminal surface of the endothelial cell. Those ridges not associated with Junctional complexes take part in the production of enclosed tunnels in the endothelial cell cytoplasm. Fusion of the external leaflets of Junctional complexes between adjacent endothelial cells occurred, initially, near the luminal surface of the blood vessel with other small fusion sites forming in the direction of the basal lamina secondarily. Further fusion activity to produce the zonula occludens type junction appeared to spread outwards from the smaller fusion sites.Supported in part by a NIH HVEM Travel Grant and the Medical College of Georgia     

Gene delivery mediated by cationic liposomes: from biophysical aspects to enhancement of transfection

Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo. However, the relationship between the features of the lipid-DNA complexes (`lipoplexes') and their mode of interaction with cells, the efficiency of gene transfer and gene expression remain to be clarified. To gain insights into these aspects, the size and zeta potential of cationic liposomes (composed of 1,2-dioleoyl-3- (trimethylammonium) propane (DOTAP) and its mixture with phosphatidylethanolamine (PE)), and their complexes with DNA at different (+/-) charge ratios were determined. A lipid mixing assay was used to assess the interaction of liposomes and lipoplexes with monocytic leukaemia cells. The use of inhibitors of endocytosis indicated that fusion of the cationic liposomes with cells occurred mainly at the plasma membrane level. However, very limited transfection of these cells was achieved using the above complexes. It is possible that the topology of the cationic liposome-DNA complexes does not allow the entry of DNA into cells through a fusion process at the plasma membrane. In an attempt to enhance transfection mediated by lipoplexes composed of DOTAP and its equimolar mixture with dioleoylphosphatidylethanolamine (DOPE) two different strategies were explored: (i) association of a targeting ligand (transferrin) to the complexes to promote their internalization, presumably by receptor-mediated endocytosis; and (ii) association of synthetic fusogenic peptides (GALA or the influenza haemagglutinin Nterminal peptide HA-2) to the complexes to promote endosomal destabilization and release of the genetic material into the cytoplasm. These strategies were effective in enhancing transfection in a large variety of cells, including epithelial and lymphoid cell lines, as well as human macrophages, especially with the use of optimized lipid/ DNA (+/-) charge ratios. Besides leading to high levels of transfection, the ternary complexes of cationic liposomes, DNA, and protein or peptide, have the advantages of being active in the presence of serum and being non-toxic. Moreover, such ternary complexes present a net negative charge and, thus, are likely to alleviate the problems associated with the use of highly positively charged complexes in vivo, such as avid complexation with serum proteins. Overall, the results indicate that these complexes, and their future derivatives, may constitute viable alternatives to viral vectors for gene delivery in vivo.     

Immobilization of biotinylated DNA on 2-D streptavidin crystals

The structural study of transient nucleoprotein complexes by electron microscopy is hampered by the coexistence of multiple interaction states leading to an heterogeneous image population. To tackle this problem, we have investigated the controlled immobilization of double stranded DNA molecules and of nucleoprotein complexes onto a support suitable for cryo-electron microscopy observation. The DNA was end-labeled with a biotin moiety in order to decorate, or to be incorporated into, two-dimensional streptavidin crystals formed in contact of a biotinylated lipid layer. The binding specificity and efficiency were examined by radioactively labeled oligonucleotides and by direct visualization of unstained and hydrated nucleic acid molecules in cryo-electron microscopy. By using RNA polymerase we further show that, once immobilized, femtomolar amounts of DNA template are suitable to interact with the enzyme. The image analysis of the RNA polymerase-DNA complexes showed that a three-dimensional model can be retrieved from such samples.     

Gene delivery mediated by cationic liposomes: from biophysical aspects to enhancement of transfection.

Cationic liposomes complexed with DNA have been used extensively as non-viral vectors for the intracellular delivery of reporter or therapeutic genes in culture and in vivo. However, the relationship between the features of the lipid-DNA complexes ('lipoplexes') and their mode of interaction with cells, the efficiency of gene transfer and gene expression remain to be clarified. To gain insights into these aspects, the size and zeta potential of cationic liposomes (composed of 1,2-dioleoyl-3- (trimethylammonium) propane (DOTAP) and its mixture with phosphatidylethanolamine (PE)), and their complexes with DNA at different (+/-) charge ratios were determined. A lipid mixing assay was used to assess the interaction of liposomes and lipoplexes with monocytic leukaemia cells. The use of inhibitors of endocytosis indicated that fusion of the cationic liposomes with cells occurred mainly at the plasma membrane level. However, very limited transfection of these cells was achieved using the above complexes. It is possible that the topology of the cationic liposome-DNA complexes does not allow the entry of DNA into cells through a fusion process at the plasma membrane. In an attempt to enhance transfection mediated by lipoplexes composed of DOTAP and its equimolar mixture with dioleoylphosphatidylethanolamine (DOPE) two different strategies were explored: (i) association of a targeting ligand (transferrin) to the complexes to promote their internalization, presumably by receptor-mediated endocytosis; and (ii) association of synthetic fusogenic peptides (GALA or the influenza haemagglutinin N-terminal peptide HA-2) to the complexes to promote endosomal destabilization and release of the genetic material into the cytoplasm. These strategies were effective in enhancing transfection in a large variety of cells, including epithelial and lymphoid cell lines, as well as human macrophages, especially with the use of optimized lipid/DNA (+/-) charge ratios. Besides leading to high levels of transfection, the ternary complexes of cationic liposomes, DNA, and protein or peptide, have the advantages of being active in the presence of serum and being non-toxic. Moreover, such ternary complexes present a net negative charge and, thus, are likely to alleviate the problems associated with the use of highly positively charged complexes in vivo, such as avid complexation with serum proteins. Overall, the results indicate that these complexes, and their future derivatives, may constitute viable alternatives to viral vectors for gene delivery in vivo.     

Cryo-EM Studies of Virus-Antibody Immune Complexes

Antibodies play critical roles in neutralizing viral infections and are increasingly used as therapeutic drugs and diagnostic tools. Structural studies on virus-antibody immune complexes are important for better understanding the molecular mechanisms of antibody-mediated neutralization and also provide valuable information for structure-based vaccine design.Cryo-electron microscopy(cryo-EM) has recently matured as a powerful structural technique for studying bio-macromolecular complexes. When combined with X-ray crystallography, cryo-EM provides a routine approach for structurally characterizing the immune complexes formed between icosahedral viruses and their antibodies. In this review, recent advances in the structural understanding of virus-antibody interactions are outlined for whole virions with icosahedral T = pseudo 3(picornaviruses) and T = 3(flaviviruses) architectures, focusing on the dynamic nature of viral shells in different functional states. Glycoprotein complexes from pleomorphic enveloped viruses are also discussed as immune complex antigens. Improving our understanding of viral epitope structures using virus-based platforms would provide a fundamental road map for future vaccine development.     

Atomic-resolution STM structure of DNA and localization of the retinoic acid binding site

Single-molecule imaging by scanning tunnelling microscopy (STM) yields the atomic-resolution (0.6A) structure of individual B-type DNA molecules. The strong correlation between these STM structures and those predicted from the known base sequence indicates that sequencing of single DNA molecules using STM may be feasible. There is excellent agreement between the STM and X-ray structures, but subtle differences exist due to radial distortions. We show that the interactions of other molecules with DNA, their binding configurations, and the structure of these complexes can be studied at the single-molecule level. The anti-cancer drug retinoic acid (RA) binds selectively to the minor groove of DNA with up to 6 RA molecules per DNA turn and with the plane of the RA molecule approximately parallel to the DNA symmetry axis. Similar studies for other drug molecules will be valuable in the a priori evaluation of the effectiveness of anti-cancer drugs.     

The potential use of atomic force microscopy for studying corrosion of metals in the presence of bacterial biofilms — an overview

This communication outlines the principles of application of scanning probe microscopy (SPM) as a tool for studying physico-chemical and biological phenomena and discusses the potential use of atomic force microscopy (AFM) , a form of SPM, for investigation of bacterial biofilms formed on metal surfaces and for studying corrosion of these surfaces in the presence of such biofilms. AFM images showing biofilms developed in pure cultures of either Pseudomonas species on copper, or by a marine isolate of sulphate-reducing bacterium on 304 stainless steel are presented to demonstrate usefulness of the SPM technique for both quantitative and qualitative determination of biocorrosion.     

Structure of single-stranded nucleic acids in the presence of ribosomal protein S1.

We have developed a new method for mounting nucleic acids and nucleic acidprotein complexes for high-resolution electron microscopy, and have used it to characterize the interaction between ribosomal protein S1 and single-stranded nucleic acids. We find that SI unwinds most, but not all of the secondary structure present in MS2 RNA and øX174 viral DNA. The binding of S1 to DNA and RNA is not highly co-operative, and has a stoichiometry of one S1 per 10 to 15 nucleotides. We have not observed any tendency for S1 nucleic acid complexes to form aggregates in either 0·01 m-Na⁺ or 0·1 m-Na⁺. An analogous protein isolated from the 30 S ribosomal subunit of Caulobacter crescentus is indistinguishable from Escherichia coli S1 in these studies. The mono-N-ethylmaleimide derivative of E. coli S1 will bind to both MS2 RNA and øX174 viral DNA with a stoichiometry of one N-ethylmaleimide-S1 per 10 to 15 nucleotides, but will not unwind the secondary structure of either of them.     

Cholesterol's decoupling effect on membrane partitioning and permeability revisited: Is there anything beyond Fick's law of diffusion?

In general, Fick's law of diffusion describes membrane permeation of hydrophobic or amphiphilic molecules. In contrast to this, Thomae et al. recently identified the volume ratio between barrier and aqueous compartments as important additional determinants of membrane permeability (P_m) [A.V. Thomae, T. Koch, C. Panse, H. Wunderli-Allenspach, and S.D. Kramer, Comparing the lipid membrane affinity and permeation of drug-like acids: the intriguing effects of cholesterol and charged lipids, Pharm. Res. 24 (2007) 1457-1472.]. This new theory was supported by the striking observation that low concentrations of cholesterol increased P_m of salicylic acid. As Fick's law is of fundamental importance to all membrane transport processes, we reinvestigated this phenomenon. We measured the electrophoretic mobility of vesicles and used electrochemical scanning microscopy to study the adsorption of the SA anion to lipid vesicular bilayers and SA transport through planar lipid bilayers, respectively. As predicted by Fick's law, P_m of SA decreased continuously with increasing cholesterol content. Thomae et al. made the contrasting artifactual observation because their kinetic approach lacked the required time resolution and led to an underestimation of P_m by five orders of magnitude. We conclude that there is nothing beyond Fick's law of diffusion. It is still valid.     

The three-dimensional cytoarchitecture of the interstitial tissue in the rat kidney

Summary The cytoarchitecture of the interstitial tissue of the rat kidney was studied by combined scanning and transmission electron microscopy. The renal interstitium is composed of an elaborate network of stellate sustentacular cells. In the cortex, sustentacular cells radiate thin branching processes to form a fine reticulum, which supports intertubular spaces. In the medulla, these cells extend thick processes horizontally along the basal surfaces of the thin limbs or vasa recta, reinforcing their attenuate walls. The horizontal processes connect with each other at their terminals, compartmentalizing the interstitial space into thin layers. The medullary sustentacular cells contain abundant small lipid droplets. The network of sustentacular cells houses vasa recta, keeping them in parallel position to each other and to the tubules. The arterial vasa recta are accompanied by pericytes, which frequently contain lipid droplets larger in size than those in the sustentacular cells. Venous vasa recta extend numerous basal microvilli, which anchor the venous wall to adjacent tubules or vessels. Numerous free cells, round in shape, are found in the sustentacular cell network, especially in the cortex. They consist of macrophages and occasional lymphocytes. Some macrophages extend long pseudopodia, while others make intimate contact with lymphocytes, suggesting their high level of activity.     

Antioxidant-mediated protective effect of potato peel extract in erythrocytes against oxidative damage

Potato peels are waste by-product of the potato processing industry. They are reportedly rich in polyphenols. Our earlier studies have shown that extracts derived from potato peel (PPE) possess strong antioxidant activity in chemical and biological model systems in vitro, attributable to its polyphenolic content. The main objective of this study was to investigate the ability of PPE to protect erythrocytes against oxidative damage, in vitro. The protection rendered by PPE in erythrocytes was studied in terms of resistance to oxidative damage, morphological alterations as well as membrane structural alterations. The total polyphenolic content in PPE was found to be 3.93 mg/g powder. The major phenolic acids present in PPE were predominantly: gallic acid, caffeic acid, chlorogenic acid and protocatechuic acid. We chose the experimental prooxidant system: FeSO₄ and ascorbic acid to induce lipid peroxidation in rat RBCs and human RBC membranes. PPE was found to inhibit lipid peroxidation with similar effectiveness in both the systems (about 80–85% inhibition by PPE at 2.5 mg/ml). While PPE per se did not cause any morphological alteration in the erythrocytes, under the experimental conditions, PPE significantly inhibited the H₂O₂-induced morphological alterations in rat RBCs as revealed by scanning electron microscopy. Further, PPE was found to offer significant protection to human erythrocyte membrane proteins from oxidative damage induced by ferrous–ascorbate. In conclusion, our results indicate that PPE is capable of protecting erythrocytes against oxidative damage probably by acting as a strong antioxidant.     

Scanning electron-microscopic study of sperm retention and migration in the vagino-cervical region of the rabbit

Summary The pattern of sperm retention and migration in the vagino-cervical region of rabbit was studied by means of scanning electron microscopy. The following phenomena were observed: 1) Spermatozoa located in the vagina and at the orifice of the ectocervix are usually distributed diffusely. They appear to be resting on the epithelial surface; many are structurally abnormal or decapitated. 2) The great majority of spermatozoa, however, seems to be anchored or retained in narrow epithelial channels with their heads in close file formations. This phenomenon was observed particularly in the fornix vaginae as late as 24 h post coitum. 3) A great number of spermatozoa invading the cervix evidently migrates in groups along the mucosal surface. Their heads are oriented toward the uterus and contact the epithelial cells. Spermatozoa that migrate beyond the cervico-uterine junction are distributed in the same manner. 4) Spermatozoa colonizing the cervical crypts appear to be attached via the anterior margins of their heads to the epithelial cells or to the tips of kinocilia. Their tails project into the crypt lumen. It is suggested that mainly three factors may be responsible for these phenomena: (i) the fact that only motile spermatozoa overcome the vagino-cervical barrier; (ii) the tendency of spermatozoa to move along the mucosa in close vicinity to the epithelial cells; and (iii) the inability to recognize mechanical barriers on the migration route (e.g., cervical crypts) and to overcome them quickly. This may be one of many possible causes leading to sperm retention in the vagino-cervical region.Supported by a grant from the Deutsche Forschungsgemeinschaft     

Zernike phase contrast electron microscopy of ice-embedded influenza A virus

The ultrastructure of the frozen-hydrated influenza A virus was examined by Zernike phase contrast electron microscopy. Using this new microscopy, not only lipid bilayers but also individual glycoprotein spikes on viral envelopes were clearly resolved with high contrast in micrographs taken in focus. In addition to spherical and elongated virions, three other classes of virions were distinguished on the basis of the features of their viral envelope: virions with a complete matrix layer, which were the most predominant, virions with a partial matrix layer, and virions with no matrix layer under the lipid bilayer. About 450 glycoprotein spikes were present in an average-sized spherical virion. Eight ribonucleoprotein complexes, that is, a central one surrounded by seven others, were distinguished in one viral particle. Thus, Zernike phase contrast electron microscopy is a powerful tool for resolving the ultrastructure of viruses, because it enables high-contrast images of ice-embedded particles free of contrast transfer function artifacts that can be a problem in conventional cryo-electron microscopy.     

高水分、富含淀粉植物组织的扫描电镜制备技术优化

应用常规高真空扫描电子显微镜观察生物样品必须经过脱水和干燥处理,但无论采用临界点干燥还是冷冻干燥方法,都存在样品表面不同程度失真的问题。植物高水分、富含淀粉组织样品经处理后,容易出现淀粉流失、细胞壁变形等现象,从而造成扫描图像粗糙,无法获得真实的细胞内部结构。本文通过对CO_2临界点干燥、化学固定样品冷冻干燥和新鲜样品冷冻干燥3种扫描电镜样品制备技术中后期制样进行机械断裂和液氮脆断改进,优化出两种植物高水分、富含淀粉组织的扫描电镜样品制备方法:(1)样品首先进行FAA化学固定,经冷冻干燥后用液氮脆断,对断面喷金镀膜和扫描电镜观察。利用该方法所得细胞结构完整,细胞壁整齐,淀粉粒和蛋白轮廓明确,可用于分析淀粉粒和蛋白颗粒在细胞内的分布。(2)新鲜样品直接进行冷冻干燥,经液氮脆断后对断面喷金镀膜和扫描电镜观察。利用该方法所得细胞壁整齐,淀粉粒轮廓更清晰,并且无蛋白颗粒干扰,用于分析淀粉粒在细胞内的分布更加理想。     

鳋科（寄生桡足类）胸板的扫描电镜比较研究

本文利用扫描电镜观察了我国鳋科的5个属的种类。首次发现在它们的胸部腹面游泳足连接板之下均有一板状结构,其形状与栉齿的着生情况在不同属之间存在着明显的差异,同属不同种之间也不尽相同,这是一个过去未曾被注意过的结构,权且称之为胸板。它是一个独立的结构,不与连接板相连。胸板上的栉齿从形态-功能-适应三位一体分析,它与附着寄主有夭,以免被水流冲走,与无栉齿的相比,应是一个进步性状。