Freeze-fracture of lipids and model membrane systems

Model membrane systems are used extensively in all aspects of membrane research, and freeze-fracture is the preeminent procedure for directly visualizing local structure in these lipid dispersions. Here we describe in detail the formation of liposomes and how freeze-fracture is routinely employed as a complementary technique to biophysical and biochemical procedures in the characterization of multilamellar vesicles (most commonly known as liposomes) and unilamellar vesicles. Many preparative procedures exist for the formation of multi- and unilamellar vesicles. Examples of each system are given and their properties as well as freeze-fracture morphology are discussed. The detection of lipid-phase transitions is considered, in particular, with emphasis on the application of freeze-fracture to the study of lipid polymorphism. We briefly discuss the fracturing of apolar lipids which do not adopt bilayer structures but which can be stabilized into microemulsions by a phospholipid monolayer. Finally, a critical assessment is made of filipin as a morphological marker for cholesterol domains in the plane of the bilayer.     

Atomic force microscopy of nanometric liposome adsorption and nanoscopic membrane domain formation

Scanning probe microscopy studies of membrane fusion and nanoscopic structures were performed using hydrated single lipids and lipid mixtures. Extruded vesicles of DMPC and mixtures at various concentrations of DLPC, DPPC and cholesterol were deposited on freshly cleaved mica and studied in a fluid environment by AFM. The nanostructures formed by these extruded liposomes ranged from isolated unilamellar vesicles to flat sheet membranes and were marked influenced by thermodynamic phase behavior. For DMPC membrane, intact bilayers exhibited a phase transition process in agreement with large bilayer patches. In the DLPC, DPPC and cholesterol mixtures, nanoscopic domain diameters ranged from approximately 25 to 48nm with height differences of approximately 1.4nm; all values were lipid composition-dependent. Our data support and extend previous studies of microscopic domains and phase boundaries of the same mixtures in giant unilamellar vesicles determined by confocal light microscopy. Our approach for preparing and utilizing supported membrane structures is potentially relevant to studies of native cell membranes.     

Synaptic membrane proteins form stable microdomains in early endosomes

In the plasma membrane, membrane proteins are frequently organized in microdomains that are stabilized both by protein‐protein and protein‐lipid interactions, with the membrane lipid cholesterol being instrumental for microdomain stability. However, it is unclear whether such microdomains persist during endocytotic membrane trafficking. We used stimulated emission‐depletion microscopy to investigate the domain structure of the endosomes. We developed a semiautomatic method for counting the individual domains, an approach that we have validated by immunoelectron microscopy. We found that in endosomes derived from neuroendocrine PC12 cells synaptophysin and several SNARE proteins are organized in microdomains. Cholesterol depletion by methyl‐β‐cyclodextrin disintegrates most of the domains. Interestingly, no change in the frequency of microdomains was observed when endosomes were fused with protein‐free liposomes of similar size (in what constitutes a novel approach in modifying acutely the lipid composition of organelles), regardless of whether the membrane lipid composition of the liposomes was similar or very different from that of the endosomes. Similarly, Rab depletion from the endosome membranes left the domain structure unaffected. Furthermore, labeled exogenous protein, introduced into endosomes by liposome fusion, equilibrated with the corresponding microdomains. We conclude that synaptic membrane proteins are organized in stable but dynamic clusters within endosomes, which are likely to persist during membrane recycling. Microsc. Res. Tech. 2010. © 2009 Wiley‐Liss, Inc.     

Time influence on the interaction between Cyt2Aa2 and lipid/cholesterol bilayers

Protein–membrane interactions are still an important topic of investigation. One of the suitable experimental techniques used by the scientific community to address such question is atomic force microscopy. In a previous work, we have reported that the binding mechanism between the cytolytic and antimicrobial protein (Cyt2Aa2) and lipid/cholesterol bilayers was concentration‐dependent, leading to either the formation of holes in the bilayer or aggregates. Here we study such binding mechanism as a function of time at low protein concentrations (10 µg/mL). We demonstrate that although holes are formed during the first stages of the protein–lipid interaction, a reparation process due to molecular mobility in the bilayer leads to a homogenous and isotropic protein–lipid/cholesterol layer within 3 hr. The combination of imaging, force spectroscopy, and phase contrast delivered information about topography dynamics (molecular mobility), layer thickness, and mechanical properties of the protein–lipid/cholesterol system. These results highlight the importance of the observation time in (such type of) protein–lipid interactions (at low protein concentrations).     

Direct Selective Laser Sintering of Hard Metal Powders: Experimental Study and Simulation

Direct selective laser sintering (SLS) technology can be used to produce 3D hard metal functional parts from commercial available powders. Unlike conventional sintering, it does not require dedicated tools, such as dies. Hence, total production time and cost can be reduced. The large shape freedom offered by such a process makes the use of, for example, sintered carbides components viable in domains where they were not applied before. Successful results have been obtained in the production of sintered carbide or hard metal parts through SLS. The investigation focuses on tungsten carbide–cobalt (WC-Co) powder mixture. This material is characterised by its high mechanical properties and its high wear resistance and is widely used in the field of cutting tools. This paper is devoted to the experimental study and the simulation of direct selective laser sintering of WC-Co hard metal powders.     

Correlative fluorescence and transmission electron microscopy: an elegant tool to study the actin cytoskeleton of whole-mount (breast) cancer cells

Elucidating the structure and dynamics of lamellipodia and filopodia in response to different stimuli is a topic of continuing interest in cancer cells as these structures may be attractive targets for therapeutic purposes. Interestingly, a close functional relationship between these actin-rich protrusions and specialized membrane domains has been recently demonstrated. The aim of this study was therefore to investigate the fine organization of these actin-rich structures and examine how they structurally may relate to detergent-resistant membrane (DRM) domains in the MTLn3 EGF/serum starvation model. For this reason, we designed a straightforward and alternative method to study cytoskeleton arrays and their associated structures by means of correlative fluorescence (/laser)- and electron microscopy (CFEM).
  CFEM on whole mounted breast cancer cells revealed that a lamellipodium is composed of an intricate filamentous actin web organized in various patterns after different treatments. Both actin dots and DRM's were resolved, and were closely interconnected with the surrounding cytoskeleton. Long actin filaments were repeatedly observed extending beyond the leading edge and their density and length varied after different treatments. Furthermore, CFEM also allowed us to demonstrate the close structural association of DRMs with the cytoskeleton in general and the filamentous/dot-like structural complexes in particular, suggesting that they are all functionally linked and consequently may regulate the cell's fingertip dynamics. Finally, electron tomographic modelling on the same CFEM samples confirmed that these extensions are clearly embedded within the cytoskeletal matrix of the lamellipodium.     

Nidogens-Extracellular matrix linker molecules

Nidogens/entactins are a family of highly conserved, sulfated glycoproteins. Biochemical studies have implicated them as having a major structural role in the basement membrane. However despite being ubiquitous components of this specialized extracellular matrix and having a wide spectrum of binding partners, genetic analysis has shown that they are not required for the overall architecture of the basement membrane. Rather in development they play an important role in its stabilization especially in tissues undergoing rapid growth or turnover. Nidogen breakdown has been implicated as a key event in the basement membrane degradation occurring in mammary gland involution. A number of studies, most compellingly those in C. elegans, demonstrated that nidogens may have other nonstructural roles and be involved in axonal pathfinding and synaptic transmission.     

Energy-filtered cryotransmission electron microscopy of liposomes prepared from human stratum corneum lipids

We used cryo-TEM to examine the morphology of vesicles formed from lipids of the human stratum corneum (hSC). Human stratum corneum lipid liposomes (hSCLLs) were prepared in buffer at various pH values, using different preparation methods (film method, extrusion, ultrasonication, detergent dialysis). The morphology of hSCLLs at pH 7.4 differed markedly from that of liposomes formed by phospholipids, showing folds, stacks and membrane thickening. At pH 5.0, corresponding to natural conditions at the skin surface, membrane structures are essentially the same as those prepared at pH 7.4. Sharp edges in hSCLLs, branching membranes and stable membrane stacks were explained by the presence of ceramides, the major components and structural elements of human stratum corneum lipids (hSCLs). Thickened areas in the membranes may be caused by the local accumulation of triacylglycerols and cholesterol esters in the hydrophobic interior of the bilayer.     

Structural diversity of sphingomyelin microdomains

In cells plasma membrane, sphingomyelin (SM) plays a key role in the formation of a category of lipid microdomains enriched in cholesterol (Chl) often referred to as rafts. Atomic force microscopy (AFM) was used to analyze the mesoscopic topography of enriched SM microdomains in supported bilayers made of SM/dioleoylphosphatidylcholine (SM/DOPC) and SM/palmitoyl-oleoyl-phosphatidylcholine (SM/POPC) equimolar mixtures, in buffer, at room temperature. Gel–fluid phase separation occurs in both SM/DOPC and SM/POPC bilayers. The gel phase SM-enriched microdomains adopt a variety of size, shape and mesoscopic structure, from homogeneous flat domains of a few hundreds of nanometer in diameter to domains of several micrometers made of closely packed globular structures. Gel–gel phase separation in SM domains is also observed which gives rise to different structures for the diunsaturated and the mixed-saturated PC species. These differences could also extend to the interactions with Chl. This suggests that studies on rafts formation commonly performed using SM/DOPC mixture as a model should also include the physiologically more relevant POPC species.     

Effect of cholesterol on the physical properties of pulmonary surfactant films: atomic force measurements study

Atomic force measurements were performed on supported pulmonary surfactant (PS) films to address the effect of cholesterol on the physical properties of lung surfactant films. We recently found that cholesterol in excess of a physiological proportion abolishes surfactant function, and is the reason that surfactant fails to lower the surface tension upon compression. In this study, we investigated how the loss of mechanical stability observed earlier is related to the local mechanical properties of the film by local force measurements. The presence of 20% of cholesterol in bovine lipid extract surfactant (BLES) resulted in a decrease of the observed adhesive interaction, and an increase in rigidity of the film. We discuss the implication the increased rigidity might have on the functional failure of PS.     

Presynaptic proteins of ribbon synapses in the retina

The synapses of photoreceptors and bipolar cells in the retina are characterized ultrastructurally by the presence of an electron-dense bar, the synaptic ribbon, lying perpendicular to the plasma membrane at the active zone and extending about 0.5 microm into the cytoplasm. Hence, these synapses are known as ribbon synapses. All neurons that make ribbon synapses release neurotransmitter tonically. That is, neurotransmitter is released continuously from these neurons and the rate of release is modulated in response to graded changes in the membrane potential. This contrasts with action potential-driven, phasic release from other neurons. Similar to other synapses, neurotransmitter is released at ribbon synapses by the calcium-dependent exocytosis of synaptic vesicles. Most components of the molecular machinery governing transmitter release are conserved between ribbon and conventional synapses, but several differences that may be important determinants of tonic transmitter release have been identified in the retina by immunohistochemistry. For example, the presynaptic calcium channels of bipolar cells and photoreceptors are different from those elsewhere in the brain. Differences have also been found in the proteins involved in synaptic vesicle recruitment to the active zone and in synaptic vesicle fusion. These differences and others are discussed in terms of their implications for neurotransmitter release from photoreceptors and bipolar cells in the retina.     

Structure and function of endothelial caveolae

Caveolae are spherical invaginations of the plasma membrane and associated vesicles that are found at high surface densities in most cells, endothelia included. Their structural framework has been shown to consist of oligomerized caveolin molecules interacting with cholesterol and sphingolipids. Caveolae have been involved in many cellular functions such as endocytosis, signal transduction, mechano-transduction, potocytosis, and cholesterol trafficking. Some confusion still persists in the field with respect to the relationship between caveolae and the lipid rafts, which have been involved in many of the above functions. In addition to all these, endothelial caveolae have been involved in capillary permeability by their participation in the process of transcytosis. This short review will focus on their structure and components, methods used to determine these components, and the role of caveolae in the transendothelial exchanges between blood plasma and the interstitial fluid.     

基于LVQ工况识别的混合动力汽车自适应能量管理控制策略

为提高混合动力汽车的燃油经济性,选取6种典型行驶工况代表“市区”、“郊区”和“高速公路”3类主要工况,采用基于规则的模糊能量管理控制策略,以整车燃油经济性为目标,在3类主要工况下用改进型粒子群优化算法优化发动机联合工作曲线与发动机关闭曲线系数,得到相应的优化后的隶属度函数的参数;运用学习向量量化(LVQ)算法识别车辆运行工况,动态选择相应的模糊控制策略,使混合动力汽车控制策略对选定的几种代表性工况具有自适应性,从而提高整车的燃油经济性。仿真对比结果表明,相比于传统混合动力汽车,燃油经济性提高了3.4%。     

S形检测试件五轴联动数控加工方法研究

作为国际标准试件中的新成员,S形检测试件由于其造型复杂,一直是加工领域中的一个难题。在UG NX环境下,通过三维建模、工艺规划、数控编程得到了S形件加工后置指令。基于虚拟机床技术理论,搭建了后置指令加工仿真通用平台,并验证了S形件数控程序的准确性。提出并对比分析了多种编程加工方法下S形件的理论误差。选取最优编程方法进行切削实验,实现了S形件的五轴联动加工。     

Freeze-Fracture of physiologically identified neuromuscular junctions from single frog muscle fibers

We developed a technique for freeze-fracturing single physiologically identified neuromuscular junctions. This technique permits direct comparison of quantal content with morphological variables such as active zone length per unit terminal length for the same cell. The technique was developed to elucidate the structural basis for variability in transmitter release at the neuromuscular junction. The procedures were as follows: (1) record quantal content by conventional intracellular recordings; (2) mark cells for identification by fluorescent dye injection; (3) fix and stain endplate cholinesterase; (4) glycerinate and remove single fibers from the muscle; (5) draw endplate morphology; (6) freeze-fracture single muscle fibers; (7) examine in a transmission electron microscope; and (8) photograph and measure nerve terminal membrane ultrastructure. We found that approximately 15% of freeze-fractured single muscle fibers exhibited nerve terminal active zones. To demonstrate the usefulness of this technique, physiological and morphological information from an identified junction is presented. Freeze-fracture of identified cells has several advantages over thin sections, which cannot accurately show such things as active zone length, spacing, or intramembrane particles. This technique also has applications to the study of active zone ultrastructure in situations where neurotransmitter release is known to differ from normal levels. In addition, direct correlations between membrane structure and function can be studied in other preparations by this method.     

Characterization of ultrastructure and collagen composition of the teratoma membrane: Comparison to the amniotic membrane

The structural and morphological properties of the teratoma membrane were investigated to better understand the pathogenesis of ovarian teratomas. A mature cystic teratoma and amnion were obtained from patients who underwent laparoscopic cystectomy and uncomplicated delivery, respectively. The teratoma membrane was divided into three layers according to the results of the histological analysis. Each layer showed distinct morphological properties, including an outer layer that was uniformly arranged, a middle layer with an irregular pattern of fibers, and an inner layer that was structurally dense with a wavy pattern of fibers. The morphology of the layers of the amniotic membrane was the reverse that of the teratoma membrane. In the teratoma membrane, the outer layer was primarily composed of type III collagen and the inner layer had a large amount of type III and IV collagen. The amniotic membrane showed a small amount of type III collagen in the outer layer, whereas the inner layer had large amounts of type I, III, and IV collagen. In the teratoma membrane, the collagen fibrils were arranged regularly in the outer layer, but irregularly in the inner layer. In the amniotic membrane, the arrangement of collagen fibrils was the reverse that of the teratoma membrane. Additionally, the collagen fibrils in the teratoma membrane were thinner than those of the amniotic membrane and had slightly shorter d‐spacing. Two membranes showed the differences in collagen fibril arrangement, which may caused by the different functional roles. Microsc. Res. Tech. 76:432–441, 2013. © 2013 Wiley Periodicals, Inc.     

液压阀口典型激振流动及其可视化实验方法

液压阀口通常伴随剧烈的介质流动，诱发空化相变、流体自激、结构振动等复杂气液固耦合激振问题。通过流动可视化实验，获得流动现象的直观捕捉，有助于深入理解现象的物理本质，揭示问题的内在机制，获得合理的建模依据，进而提出解决问题的有效办法。在分析液压阀口流动可视化实验研究进展基础上，综合实验内容要素，阐述了 “看”、“听”、“演”的可视化实验研究方法与技术手段。     

Odor discrimination by G protein-coupled olfactory receptors

The vertebrate olfactory system possesses a remarkable capacity to recognize and discriminate a variety of odorants by sending the coding information from peripheral olfactory sensory neurons in the olfactory epithelium to the olfactory bulb of the brain. The recognition of odorants appear to be mediated by a G protein-coupled receptor superfamily that consists of approximately 1% of total genes in vertebrates. Since the first discovery of the olfactory receptor gene superfamily in the rat, similar chemosensory receptors have been found in various species across different phyla. The functions of these receptors, however, had been uncharacterized until the recently successful functional expression and ligand screening of some olfactory receptors in various cell expression systems. The functional cloning of odorant receptors from single olfactory neurons allowed for the identification of multiple receptors that recognized a particular odorant of interest. Reconstitution of the odorant responses demonstrated that odorant receptors recognized various structurally-related odorant molecules with a specific molecular receptive range, and that odor discrimination is established based on a combinatorial receptor code model in which the identities of different odorants are encoded by a combination of odorant receptors. The receptor code for an odorant changes at different odorant concentrations, consistent with our experience that perceived quality of an odorant changes at different concentrations. The molecular bases of odor discrimination at the level of olfactory receptors appear to correlate well with the receptive field in the olfactory bulb where the input signal is further processed to create the specific odor maps.     

Preparation of cell membranes for high resolution imaging by AFM

Studies of cell membrane structure by atomic force microscopy (AFM) have been limited because of the softness of cell membranes. Here, we utilize a new technique of sample preparation to lay red blood cell membranes on the top of a mica surface to obtain high resolution images by in-situ AFM on both sides of cell membranes. Our results indicate that the location of oligosaccharides and proteins in red blood cell membranes might be different from the current membrane model. The inner membrane leaflet is covered by dense proteins with fewer free lipids than expected. In contrast, the outer membrane leaflet is quite smooth; oligosaccharides and peptides supposed to protrude out of the outer membrane leaflet surface might be actually hidden in the middle of hydrophilic lipid heads; transmembrane proteins might form domains in the membranes revealed by PNGase F and trypsin digestion. Our result could be significant to interpret some functions about red blood cell membranes and guide to heal the blood diseases related to cell membranes.