A new method to measure bilayer thickness: Cryo-electron microscopy of frozen hydrated liposomes and image simulation

Tahara, Yoshikazu; Fujiyoshi, Yoshinori

doi:10.1016/0968-4328(94)90039-6

Cited by 71 publications

(56 citation statements)

References 11 publications

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“…This observation is consistent with the presence of mycolyl residues in the structure and the shorter chain lengths of corynomycolic acids (32 to 36 carbons) compared to those of mycobacteria (70 to 90 carbons). The separation of the density peaks of the PM bilayer (3.9 Ϯ 0.4 nm in M. bovis BCG) corresponds to the separation measured by cryoEM and X-ray scattering in liposomes made of phosphatidylcholine with acyl chain lengths of 16 to 18 carbons (25,52). This is consistent with the lengths of the main fatty acid constituents of the PM (11).…”

Section: Discussionsupporting

confidence: 71%

Direct Visualization of the Outer Membrane of Mycobacteria and Corynebacteria in Their Native State

et al. 2008

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The cell envelope of mycobacteria, which include the causative agents of tuberculosis and leprosy, is crucial for their success as pathogens. Despite a continued strong emphasis on identifying the multiple chemical components of this envelope, it has proven difficult to combine its components into a comprehensive structural model, primarily because the available ultrastructural data rely on conventional electron microscopy embedding and sectioning, which are known to induce artifacts. The existence of an outer membrane bilayer has long been postulated but has never been directly observed by electron microscopy of ultrathin sections. Here we have used cryo-electron microscopy of vitreous sections (CEMOVIS) to perform a detailed ultrastructural analysis of three species belonging to the Corynebacterineae suborder, namely, Mycobacterium bovis BCG, Mycobacterium smegmatis, and Corynebacterium glutamicum, in their native state. We provide new information that accurately describes the different layers of the mycobacterial cell envelope and challenges current models of the organization of its components. We show a direct visualization of an outer membrane, analogous to that found in gram-negative bacteria, in the three bacterial species examined. Furthermore, we demonstrate that mycolic acids, the hallmark of mycobacteria and related genera, are essential for the formation of this outer membrane. In addition, a granular layer and a low-density zone typifying the periplasmic space of gram-positive bacteria are apparent in CEMOVIS images of mycobacteria and corynebacteria. Based on our observations, a model of the organization of the lipids in the outer membrane is proposed. The architecture we describe should serve as a reference for future studies to relate the structure of the mycobacterial cell envelope to its function.

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Section: Discussionsupporting

confidence: 71%

Direct Visualization of the Outer Membrane of Mycobacteria and Corynebacteria in Their Native State

et al. 2008

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“…By this technique, a single electron-dense line representing a row of phosphorus atoms was observed in the surface of isolated lipid droplets. It contrasts with two parallel lines seen in the liposomal membrane (27), which is a phospholipid bilayer, and thus proves for the first time that the lipid droplet surface is a phospholipid monolayer.…”

Section: Discussionmentioning

confidence: 70%

“…To directly visualize the lipid droplet surface structure, we adopted cryoelectron microscopy that can visualize ultrastructures at high resolution. Liposomal membrane, or a phospholipid bilayer, was observed as two parallel lines by this method without any fixation or staining (27).…”

Section: Cryoelectron Microscopy Of Lipid Droplets-conventionalmentioning

confidence: 99%

The Surface of Lipid Droplets Is a Phospholipid Monolayer with a Unique Fatty Acid Composition

Tauchi-Sato

Ozeki

Houjou

et al. 2002

Journal of Biological Chemistry

640

523

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We found that caveolin-2 is targeted to the surface of lipid droplets (Fujimoto, T., Kogo, H., Ishiguro, K., Tauchi, K., and Nomura, R. (2001) J. Cell Biol. 152, 1079 -1085) and hypothesized that the lipid droplet surface is a kind of membrane. To elucidate the characteristics of the lipid droplet surface, we isolated lipid droplets from HepG2 cells and analyzed them by cryoelectron microscopy and by mass spectrometry. By use of cryoelectron microscopy at the stage temperature of 4.2 K, the lipid droplet surface was observed as a single line without any fixation or staining, indicating the presence of a single layer of phospholipids. This result appeared consistent with the hypothesis that the lipid droplet surface is derived from the cytoplasmic leaflet of the endoplasmic reticulum membrane and may be continuous to it. However, mass spectrometry revealed that the fatty acid composition of phosphatidylcholine and lysophosphatidylcholine in lipid droplets is different from that of the rough endoplasmic reticulum. The ample presence of free cholesterol in lipid droplets also suggests that their surface is differentiated from the bulk endoplasmic reticulum membrane. On the other hand, although caveolin-2␤ and adipose differentiation-related protein, both localizing in lipid droplets, were enriched in the low density floating fraction, the fatty acid composition of the fraction was distinct from lipid droplets. Collectively, the result indicates that the lipid droplet surface is a hemi-membrane or a phospholipid monolayer containing cholesterol but is compositionally different from the endoplasmic reticulum membrane or the sphingolipid/cholesterol-rich microdomain.Lipid droplets have been regarded as a depot of neutral lipids. They exist most abundantly in adipose cells and steroidproducing cells but can be found in virtually any kind of cell. The core of lipid droplets is occupied by triacylglycerol and cholesterol ester in various ratios depending on the cell type (1), but information on the lipid droplet surface has been scarce. Recently we as well as others showed that caveolins can exist in the lipid droplet surface (2-4). Caveolins, i.e. caveolin-1, 2, 3, are membrane proteins that are incorporated to the sphingolipid/cholesterol-enriched membrane microdomain and form the framework of caveolae (5). Furthermore, lipid droplets were reported to contain other microdomain proteins, i.e. Lyn and mitogen-activated protein kinase, as well as abundant free cholesterol (6 -8). These results suggest that the lipid droplet surface is a kind of membrane and that it might have some similarity to the microdomain.However, electron microscopy of conventional resin-embedded ultrathin sections cannot visualize any membranous structure around the lipid droplet. In the ultrathin section of specimens fixed by aldehydes and then by osmium tetroxide, the lipid droplet content appears vacant, and its periphery is usually seen as a thin intermittent line. In many diagrams, the lipid droplet surface has been depicted as a phospholipid ...

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“…As a comparison, the lipid bilayer of isolated lipid vesicles (arrow in Figures 1) and of membranes in non-junctional areas (arrows in Figure 2a) was resolved into two dark stripes, corresponding to the lipid polar head groups of both lipid lea¯ets, which have higher electron scattering properties than the alkyl chains (Lepault et al, 1985;Tahara & Fujiyoshi, 1994). The two outer stripes on both sides of a junction correspond therefore to the lipid lea¯ets of the associated vesicles.…”

Section: Resultsmentioning

confidence: 99%

Structural analysis of junctions formed between lipid membranes and several annexins by cryo-electron microscopy 1 1Edited by M. F. Moody

Lambert

Gerke

Bader

et al. 1997

Journal of Molecular Biology

108

103

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A new method to measure bilayer thickness: Cryo-electron microscopy of frozen hydrated liposomes and image simulation

Cited by 71 publications

References 11 publications

Direct Visualization of the Outer Membrane of Mycobacteria and Corynebacteria in Their Native State

Direct Visualization of the Outer Membrane of Mycobacteria and Corynebacteria in Their Native State

The Surface of Lipid Droplets Is a Phospholipid Monolayer with a Unique Fatty Acid Composition

Structural analysis of junctions formed between lipid membranes and several annexins by cryo-electron microscopy 1 1Edited by M. F. Moody

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