Conical tomography of freeze-fracture replicas: a method for the study of integral membrane proteins inserted in phospholipid bilayers

We have used conical tomography to study the structure of integral proteins in their phospholipid bilayer environments. Complete conical series were collected from replicas of the water channel aquaporin-0 (AQP0), a 6.6 nm side tetramer with a molecular weight of approximately 120 kDa that was purified and reconstituted in liposomes. The replicas were tilted at 38 degrees , 50 degrees or 55 degrees and rotated by 2.5 degrees , 4 degrees , or 5 degrees increments until completing 360 degrees turns. The elliptical paths of between 6 and 12 freeze-fracture particles aligned the images to a common coordinate system. Using the weighted back projection algorithm, small volumes of the replicas were independently reconstructed to reconstitute the field. Using the Fourier Shell Correlation computed from reconstructions of even and odd projections of the series, we estimated a resolution of 2-3 nm, a value that was close to the thickness of the replica (approximately 1.5 nm). The 3D reconstructions exhibited isotropic resolution along the x-y plane, which simplified the analysis of particles oriented randomly in the membrane plane. In contrast to reconstructions from single particles imaged using random conical tilt [J. Mol. Biol. 325 (2003) 210], the reconstructions using conical tomography allowed the size and shape of individual particles representing the AQP0 channel to be identified without averaging or imposing symmetry. In conclusion, the reconstruction of freeze-fracture replicas with electron tomography has provided a novel experimental approach for the study of integral proteins inserted in phospholipid bilayers.     

Human cataract lens membrane at subnanometer resolution

Human pathologies often originate from molecular disorders. Therefore, imaging technology as one of the bases for the identification and understanding of pathologies must provide views of single molecules at subnanometer resolution. Membrane proteins mediate many of life's most important processes, and their malfunction is often lethal or leads to severe disease. The membrane proteins aquaporin-0 (AQP0) and connexons form junctional microdomains between healthy lens core cells in which AQP0 form square arrays surrounded by connexons. Malfunction of both proteins results in the formation of cataract. We have used high-resolution atomic force microscopy (AFM) to image junctional microdomains in membranes from an individual human eye lens with senile cataract. Images at subnanometer resolution report individual helix-connecting loops of four amino acid residues on the AQP0 surface. We describe the supramolecular assembly and the conformational state of AQP0 in junctional microdomains, where a mixture of truncated junctional and full-length water channel AQP0 form square arrays. Imaging of microdomain borders revealed individual AQP0 tetramers and no associated connexon, indicating a lack of metabolite transport, waste accumulation, and enlarged regions of non-adhering membranes, causing cataract in this individual. This first high-resolution view of the membrane of this pathological human tissue provides insights into cataract pathology at the single membrane protein level, and indicates the power of the AFM as a future tool in medical imaging at subnanometer resolution.     

The Kohonen self-organizing map: a tool for the clustering and alignment of single particles imaged using random conical tilt

An important step in determining the three-dimensional structure of single macromolecules is to bring common features in the images into register through alignment and classification. Here, we took advantage of the striking computational properties of the Kohonen self-organizing map (SOM) to align and classify images of channels obtained by random conical geometry into more homogeneous subsets. First, we used simulations with artificially created images to deduce simple geometrical rules governing the mapping of bounded (differing in size and shape) and unbounded (differing in in-plane orientation) variations in the output plane. Second, we measured the effect of noise on the accuracy of the algorithm to separate homogeneous subsets. Finally, we applied the rules ascertained in the previous steps to separate freeze-fracture images of the cytoplasmic and external domains of the small (approximately 118 kDa) aquaporin-0 water channel. Comparison with the results obtained from a similar input set using alignment-through-classification showed that both methods converged to stable classes exhibiting the same overall shapes (tetragonal and octagonal) for the cytoplasmic and external views of the channel. Processing with the SOM, however, was simplified by the utilization of the geometric rules governing the mapping of bounded and unbounded variations as well as the lack of subjectivity in selecting the reference images during alignment.     

The ultrastructure of the plasmodesmata of the salt glands ofTamarix as revealed by transmission and freeze-fracture electron microscopy

Summary Numerous plasmodesmata occur in the walls between the secretory cells ofTamarix salt glands. The plasmalemma bounds the plasmodesmata and is continuous from cell to cell. In freeze-fracture, the e-face of the plasmalemma within the plasmodesmata is virtually devoid of intramembranous particles while, in contrast, the p-face is decidedly enriched with particles. The axial components appear to be a tightly curved membrane bilayer, as judged from measurements and their appearance in freeze-fracture, and the e-face of this membrane is also devoid of particles. Observations from both thin sections and freeze-fracture replicas indicate the presence of a circular cluster of six particles around the axial component near the cytoplasmic termini of the plasmodesmata. These particles extend from the p-face of the axial component to the p-face of the plasmalemma. These observations are summarized in a model.     

The condensing effect of glucagon on phospholipid bilayers

Glucagon forms discoidal particles with dimyristoylphosphatidylcholine at temperatures below the phase transition. Under these conditions and at a lipid to protein molar ratio of 20 : 1, glucagon is observed to induce a closer packing of the phospholipid bilayer. Similar effects are observed upon the interaction of glucagon with dilauroylphosphatidylcholine. In the region of the phase transition the discoidal particles are observed by freeze-fracture electron microscopy to undergo end-to-end association leading to the formation of multilamellar structures containing only a few layers and having a large internal volume. Above the phase transition temperature the properties of the lipid appear to be unperturbed by glucagon according to either freeze-fracture or densitometer studies. These results further support the importance of phospholipid phase transitions in peptide-lipid interactions.     

Freeze-fracture studies of annulate lamellae in zebrafish oocytes

Summary The zebrafish oocyte contains prominent stacks of annulate lamellae (AL) located primarily in a subcortical position of the ooplasm. Many lamellae comprising a stack eventually exhibit continuity with the rough-surfaced endoplasmic reticulum which is present in abundance in larger oocytes. Pore structure of both AL and nuclear envelope (NE) was studied and compared by use of freeze-fracture electron microscopy. In freeze-fracture replicas, the NE and AL pores were easily distinguished, and a variety of fracture planes with respect to the stacked AL were generated. The pore diameter of NE and AL is similar (100nm). The number of nuclear pores varied from an average of 40 pores/m² in early stage oocytes to nearly double this number in later stage oocytes. For AL, the center-to-center spacing (120–130 nm) and the number of pores per square micrometer (56–67) did not change markedly regardless of oocyte developmental stage examined. Hexagonal packing of AL pores is a common feature. The AL pores have an angular margin with octagonal symmetry suggested in some cases. The AL pore interior contains fibrillar and particulate components and, depending upon the fracture plane, may appear to be filled with a plug of material. Both P- and E-membrane fracture faces of AL have a relative scarcity of intramembranous particles. The non-porous membranes that extend from the AL, however, have a higher concentration of intramembranous particles.     

Supramolecular organization of lysophosphatidylcholine-packaged Gramicidin A

Heat derived gramicidin A'/L-alpha-lysophosphatidylcholine complexes were separated on a sucrose gradient to form two fractions: Fraction A which had an approximately constant Gramicidin A' to phospholipid ratio of 8 to 10 lipid molecules per Gramicidin A' molecule and Fraction B which had a larger but variable ratio. Fluorescence and circular dichroism studies confirmed Fraction A to be a lipid-incorporated channel state. Electron microscopic studies, using uranyl acetate negative staining, showed fraction A to be a membranous state with the formation of bilayer vesicles, that is, the interaction of peptide and phospholipid micelles causes the lipid to reorganize into a bilayer structure. Freeze-fracture replicas of the channel incorporated state demonstrated the presence of a supramolecular organization of particles exhibiting a tendency to form rows with a 50-60 A periodicity along the row and with 70-80 A distance between rows. An idealized working model for the incorporated state is presented.     

Supramolecular organization of lysophosphatidylcholine-packaged gramicidin A′

Heat derived gramicidin A′/l-α-lysophosphatidylcholine complexes were separated on a sucrose gradient to form two fractions: Fraction A which had an approximately constant Gramicidin A′ to phospholipid ratio of 8 to 10 lipid molecules per Gramicidin A′ molecule and Fraction B which had a larger but variable ratio. Fluorescence and circular dichroism studies confirmed Fraction A to be a lipid-incorporated channel state. Electron microscopic studies, using uranyl acetate negative staining, showed fraction A to be a membranous state with the formation of bilayer vesicles, that is, the interaction of peptide and phospholipid micelles causes the lipid to reorganize into a bilayer structure. Freeze-fracture replicas of the channel incorporated state demonstrated the presence of a supramolecular organization of particles exhibiting a tendency to form rows with a 50–60 Å periodicity along the row and with 70–80 Å distance between rows. An idealized working model for the incorporated state is presented.     

Water transport in AQP0 aquaporin: molecular dynamics studies

A double lipid bilayer structure containing opposing tetramers of AQP0 aquaporin, in contact through extracellular face loop regions, was recently modeled using an intermediate-resolution map obtained by electron crystallographic methods. The pores of these water channels were found to be critically narrow in three regions and subsequently interpreted to be those of a closed state of the channel. The subsequent determination of a high-resolution AQP0 tetramer structure by X-ray crystallographic methods yielded a pore model featuring two of the three constrictions as noted in the EM work and water molecules within the channel pore. The extracellular-side constriction region of this AQP0 structure was significantly larger than that of the EM-based model and similar to that of the highly water permeable AQP1. The X-ray-based study of AQP0 however could not ascertain if the water molecules found in the pore were the result of water entering from one or both ends of the channel, nor whether water could freely pass through all constriction points. Additionally, this X-ray-based structure could not provide an answer to the question of whether the double lipid bilayer configuration of AQP0 could functionally maintain a water impermeable state of the channel. To address these questions we conducted molecular dynamics simulations to compare the time-dependent behavior of the AQP0 and AQP1 channels within lipid bilayers. The simulations demonstrate that AQP0, in single or double lipid bilayers, is not closed to water transport and that thermal motions of critical side-chains are sufficient to facilitate the movement of water past any of its constriction regions. These motional requirements do however lead to significant free energy barriers and help explain physiological observations that found water permeability in AQP0 to be substantially lower than in the AQP1 pore.     

Mechanism for maturation-related reorganization of flavivirus glycoproteins

Flaviviruses, such as dengue, West Nile, and yellow fever viruses, assemble as fusion-incompetent particles and subsequently undergo a large reorganization of their glycoprotein envelope resulting in formation of mature infectious virions. Here we used a combination of three-dimensional cryo-electron tomography and two-dimensional image analysis to study pleomorphic maturation intermediates of dengue virus 2. Icosahedral symmetries of immature and mature regions within one particle were mismatched relative to each other. Furthermore, the orientation of the two regions relative to each other differed among particles. Therefore, there cannot be a specific pathway determining the maturation of all particles. Instead, the region with mature structure expands when glycoproteins on its boundary acquire suitable orientation and conformation to allow them to become a stable part of the mature region. This type of maturation is possible because the envelope glycoproteins are anchored to the phospholipid bilayer that is a part of flavivirus virions and are thus restricted to movement on the two-dimensional surface of the particle. Therefore, compounds that limit movement of the glycoproteins within the virus membrane might be used as inhibitors of flavivirus maturation.     

Structural Information, Resolution, and Noise in High-Resolution Atomic Force Microscopy Topographs

AFM has developed into a powerful tool in structural biology, providing topographs of proteins under close-to-native conditions and featuring an outstanding signal/noise ratio. However, the imaging mechanism exhibits particularities: fast and slow scan axis represent two independent image acquisition axes. Additionally, unknown tip geometry and tip-sample interaction render the contrast transfer function nondefinable. Hence, the interpretation of AFM topographs remained difficult. How can noise and distortions present in AFM images be quantified? How does the number of molecule topographs merged influence the structural information provided by averages? What is the resolution of topographs? Here, we find that in high-resolution AFM topographs, many molecule images are only slightly disturbed by noise, distortions, and tip-sample interactions. To identify these high-quality particles, we propose a selection criterion based on the internal symmetry of the imaged protein. We introduce a novel feature-based resolution analysis and show that AFM topographs of different proteins contain structural information beginning at different resolution thresholds: 10 Å (AqpZ), 12 Å (AQP0), 13 Å (AQP2), and 20 Å (light-harvesting-complex-2). Importantly, we highlight that the best single-molecule images are more accurate molecular representations than ensemble averages, because averaging downsizes the z-dimension and “blurs” structural details.Abbreviations: 2D, two-dimensional; 3D, three-dimensional; ACV, auto-correlation value; AFM, atomic force microscopy; AQP0, aquaporin-0; AQP2, aquaporin-2; AqpZ, aquaporin-Z; bR, bacteriorhodopsin; CCV, cross-correlation value; CTF, contrast transfer function; DPR, differential phase residual; EM, electron microscopy; FRC, Fourier ring correlation; FSC, Fourier shell correlation; IS, internal symmetry; LH2, light-harvesting-complex 2; RMSD, root mean-square deviation; SD, standard deviation; SNR, signal/noise ratio; SSNR, spectral signal/noise ratio     

Water channel structures analysed by electron crystallography

Background

The mechanisms underlying water transport through aquaporin (AQP) have been debated for two decades. The water permeation phenomenon of AQP seems inexplicable because the Grotthuss mechanism does not allow for simultaneous fast water permeability and inhibition of proton transfer through the hydrogen bonds of water molecules.

Scope of review

The AQP1 structure determined by electron crystallography provided the first insights into the proton exclusion mechanism despite fast water permeation. Although several studies have provided clues about the mechanism based on the AQP structure, each proposed mechanism remains incomplete. The present review is focused on AQP function and structure solved by electron crystallography in an attempt to fill the gaps between the findings in the absence and presence of lipids.

Major conclusions

Many AQP structures can be superimposed regardless of the determination method. The AQP fold is preserved even under conditions lacking lipids, but the water arrangement in the channel pore differs. The differences might be explained by dipole moments formed by the two short helices in the lipid bilayer. In addition, structure analyses of double-layered two-dimensional crystals of AQP suggest an array formation and cell adhesive function.

General significance

Electron crystallography findings not only have contributed to resolve some of the water permeation mechanisms, but have also elucidated the multiple functions of AQPs in the membrane. The roles of AQPs in the brain remain obscure, but their multiple activities might be important in the regulation of brain and other biological functions. This article is part of a Special Issue entitled Aquaporins.     

Structural analysis of the isolated chloroplast coupling factor and the N,N'-dicyclohexylcarbodiimide binding proteolipid

Negative staining of purified spinach dicyclohexylcarbodiimide (DCCD) sensitive ATPase revealed a population of 110 Å subunits attached by stalks to short string-like aggregates. The interpretation of these data is that 110 Å CF₁ are attached by stalks to an aggregate of CF₀.The CF₁-CF₀ complex was incorporated into phospholipid vesicles; freezefracture analysis of this preparation revealed a homogeneous population of particles spanning the lipid bilayer; these averaged 96 Å in diameter. The DCCD binding proteolipid (apparent molecular weight 7500), an integral component of CF₀, was isolated from membranes by butanol extraction and was incorporated rated into phospholipid vesicles. Freeze-fracture analysis of the DCCD-binding proteolipid/vesicle preparation revealed a population of particles averaging 83 Å in diameter suggesting that the DCCD-binding proteolipid self-associates in lipid to form a stable complex. This complex may be required for proton transport across chloroplast membranes in vivo. The size difference between CF₀ and DCCD-proteolipid freeze-fracture particles may be related to differences in polypeptide composition of the two complexes.     

Arrays of plasma-membrane “rosettes” involved in cellulose microfibril formation of Spirogyra

The cell-wall structure and plasma-membrane particle arrangement during cell wall formation of the filamentous chlorophycean alga Spirogyra sp. was investigated with the freeze-fracture technique. The cell wall consists of a thick outer slime layer and a multilayered inner wall with ribbon-like microfibrils. This inner wall shows three differing orientations of microfibrils: random orientation on its outside, followed by axial bundles of parallel microfibrils, and several internal layers of bands of mostly five to six parallel associated microfibrils with transverse to oblique orientation. The extraplasmatic fracture face of the plasma membrane shows microfibril imprints, relatively few particles, and “terminal complexes” arranged in a hexagonal package at the end of the imprint of a microfibril band. The plasmatic fracture face of the plasma membrane is rich in particles. In places, it reveals hexagonal arrays of “rosettes”. These rosettes are best demonstrable with the double-replica technique. These findings on rosette arrays of the zygnematacean alga Spirogyra are compared in detail with the published data on the desmidiacean algae Micrasterias and Closterium.     

Cooperativity and allostery in aquaporin 0 regulation by Ca2+

Aquaporin 0 (AQP0) is essential for eye lens homeostasis as is regulation of its water permeability by Ca²⁺, which occurs through interactions with calmodulin (CaM), but the underlying molecular mechanisms are not well understood. Here, we use molecular dynamics (MD) simulations on the microsecond timescale under an osmotic gradient to explicitly model water permeation through the AQP0 channel. To identify any structural features that are specific to water permeation through AQP0, we also performed simulations of aquaporin 1 (AQP1) and a pure mixed lipid bilayer under the same conditions. The relative single-channel water osmotic permeability coefficients (p_f) calculated from all of our simulations are in reasonable agreement with experiment. Our simulations allowed us to characterize the dynamics of the key structural elements that modulate the diffusion of water single-files through the AQP0 and AQP1 pores. We find that CaM binding influences the collective dynamics of the whole AQP0 tetramer, promoting the closing of both the extracellular and intracellular gates by inducing cooperativity between neighboring subunits.     

Interaction of cytochrome c and its precursor apocytochrome c with various phospholipids

The effects of cytochrome c and apocytochrome c on the structural properties of various membrane phospholipids in model systems were compared by binding, calorimetric, permeability, ³¹P n.m.r. and freeze-fracture experiments. Both cytochrome c and apocytochrome c experience strong interactions only with negatively charged phospholipids; apocytochrome c interacted more strongly than cytochrome c. These interactions are primarily electrostatic but also have a hydrophobic character. Cytochrome c as well as apocytochrome c induces changes in the structure of cardiolipin liposomes as is shown by ³¹P n.m.r. and freeze-fracture electron microscopy. Cytochrome c does not affect the bilayer structure of phosphatidylserine. In contrast, interaction of apocytochrome c with this phospholipid results in changes of the ³¹P n.m.r. bilayer spectrum of the liposomes and also particles are observed at the fracture faces. The results are discussed in relation to the import of the protein into the mitochondrion.     

水通道蛋白1-C末端肽段/GST融合蛋白的原核表达(简报)

水通道蛋白(Aquaporin,AQP)是一类选择性高效转运水分子的细胞膜通道蛋白,广泛存在于原核和真核生物细胞的细胞膜上,主要介导自由水分子的被动跨膜转运,对保持细胞内外液环境的稳态平衡起着重要的作用.     

Freeze-fracture observations of the lactating rat mammary gland. Membrane events during milk fat secretion

Membrane events during milk fat secretion were analyzed by freeze-fracture of the rat mammary gland. Two modes of milk fat secretion were observed: extrusion of fat droplets surrounded by a portion of the apical plasma membrane of the alveolar epithelial cells and, less frequently, release into the alveolar lumen of fat droplets contained in intracytoplasmic vacuoles. The extrusion process consists of two asynchronous events: clearing of membrane particles (probably including integral membrane proteins) and bulging of the apical plasma membrane. Most fat droplets are extruded with a bilayer membrane envelope (milk fat globule membrane) partially devoid of particles. The segregation of membrane particles may represent the onset of a process of structural degradation of the milk fat globule membrane.     

水通道 AQP1 敲除小鼠肿瘤血管生成障碍及肿瘤生长减缓

血管生成是肿瘤生长、浸润和转移的必要步骤. 肿瘤血管生成涉及瘤旁组织血管内皮细胞增殖、向肿瘤细胞团内迁移以及管腔形成,目前机理尚不完全清楚. 水通道 AQP1 在多种肿瘤血管内皮高表达,提示其可能参与肿瘤血管的生成过程. 应用 AQP1 敲除小鼠荷瘤实验证实了 AQP1 在黑色素瘤生长和血管新生中的作用. 结果表明,皮下接种的黑色素瘤在 AQP1 敲除小鼠的生长较之在野生型小鼠延迟近 30％ (P<0.01). 免疫组化与肿 瘤病理形态学分析显示, AQP1 在野生型小鼠黑色素瘤血管内皮细胞上高表达,而在 AQP1 敲除小鼠黑色素瘤血管内皮细胞呈阴性表达. 在病理结构上,黑色素瘤细胞围绕血管分支呈岛状分布. 野生型小鼠黑色素瘤内血管管腔较细小,而 AQP1(－/－)小鼠黑色素瘤内血管床显著膨大. AQP1(－/－)小鼠肿瘤内平均微血管密度 (47/mm²) 较之 AQP1(＋/＋) 肿瘤 (142/mm²) 减少 67％ (P<0.01). 围绕 AQP1(－/－) 肿瘤血管的肿瘤细胞岛周边坏死区域明显大于 AQP1(＋/＋)肿瘤. 上述结果提出确切证据表明, AQP1 缺失使肿瘤血管生成发生障碍,从而影响了肿瘤血液供应和肿瘤生长. AQP1参与肿瘤血管生成的机理值得深入研究.