Contribution of cryoelectron microscopy of vitreous sections to the understanding of biological membrane structure

Abstract

Using cryoelectron microscopy of vitreous sections, we investigated in situ the ultrastructure of biological membranes, selected from several cell types for their diverse biological functions. Here we describe how to visualize the two membrane leaflets and tightly apposed membranes, lying as close as 1.1 nm apart, by tuning the imaging conditions. We show how defects in membrane stacks may be clues to resolving their structure. Details of membrane proteins are also resolved, as well as protein lattices with correlations between stacked membranes. Imaging the cell in its native hydrated state can now be done in the nanometer resolution range, which should open unique routes for investigating structure-function relationships.thylakoid | transmembrane proteins | multilamellar body | Golgi | cortical alveoli B iological membranes are highly specialized dynamic entities that limit structural and functional compartments and control the exchange of matter and energy. They may exhibit a wide diversity of shapes with variable curvature, including spherical, tubular, planar, sponge, or cubic bicontinuous organizations (1). They are composed of hundreds of lipid and protein species, in some cases modified by sugars. Their structure and composition vary among species, cell types, and organelles and, even within a given membrane type, in time and space. This structural and chemical variability is directly related to their functional properties (2). Lipids (mostly phospholipids) represent more than 50% of membrane components. Their amphiphilic nature leads to the formation of bilayers. The hydrophobic chains are buried inside the membrane, and the polar heads are exposed to the outer, hydrated environment. Proteins may be bound to the lipid bilayer through electrostatic interactions or partially embedded within the bilayer.Transmission electron microscopy provides various techniques for exploring cell ultrastructure. Among these, cryoelectron microscopy of vitreous sections (CEMOVIS) provides snapshots of unstained frozen-hydrated cells and tissues in their native state. The analysis of ordered fibrillar objects like DNA in sperm cells (3), bacteria (4), and dinoflagellates (5) has benefited from this approach. Nevertheless, cryoelectron tomography (CET) is often preferred over CEMOVIS to overcome limitations arising from superimpositions on 2D projections and to obtain 3D views. CET has been used to successfully image entire small cells, organelles, and thin cellular extensions (6, 7), as well as thicker objects sliced into cryosections (8-11). Spectacular results have been obtained with CET in combination with image analysis; for example, the Ccadherin atomic structure has been fit into averaged subtomograms of epidermal desmosomes (8). However, the relatively low resolution of CET (generally between 5 and 8 nm; 3 nm in the optimum cases) remains insufficient to address many biological questions, especially those dealing with membrane structure.Fine structural details, particularly the bilayer nature of membranes, have been...

Cryo FIB-SEM: Volume imaging of cellular ultrastructure in native frozen specimens

“…In contrast to conventional transmission electron microscopy (TEM), which relies on the preferential binding of contrasting agents to proteins and lipids, in cryo-electron microscopy the image is generated by the density of the biological material itself. Therefore, these data contain highly valuable direct biological information suitable for modelling of macromolecules or membrane assemblies in their native context (Al-Amoudi et al, 2007;Leforestier et al, 2012). By cryo-electron tomography (CET) (Bouchet-Marquis and Hoenger, 2011) three dimensional (3D) views of organelles and cells are generated.…”

In situ structural analysis of Golgi intracisternal protein arrays

“…However, these tomographic studies were restricted to descriptions of membrane architecture and, in the best cases, the classification of membrane coats, due to the resolution limitations imposed by conventional sample preparation, involving dehydration, plastic embedding, and staining with heavy-metal contrasting agents. To date, cryo-ET studies of the Golgi have been extremely limited (2,(21)(22)(23).In this study, we used cryo-FIB of vitreous Chlamydomonas cells followed by cryo-ET to image the native molecular landscape of the Golgi with unprecedented resolution and sample integrity. Our tomograms revealed new structures within the Golgi cisternae, including ordered membrane-associated protein arrays, dark granular aggregates, and bundles of filaments near the trans-Golgi coated buds.…”

“…However, these tomographic studies were restricted to descriptions of membrane architecture and, in the best cases, the classification of membrane coats, due to the resolution limitations imposed by conventional sample preparation, involving dehydration, plastic embedding, and staining with heavy-metal contrasting agents. To date, cryo-ET studies of the Golgi have been extremely limited (2,(21)(22)(23).…”

“…focused ion beam | cryo-electron tomography | Chlamydomonas | Golgi | glycosyltransferase C ryo-electron tomography (cryo-ET) provides the unique ability to visualize macromolecules and supramolecular structures within frozen hydrated cells (1)(2)(3)(4). Biological material is immobilized in vitreous ice, preserving cellular structures in a near-native state.…”

Observer la cellule et ses structures membranaires à l’échelle du nanomètre

“…On obtient ainsi des instantanés de la cellule dans son état natif, sans coloration ni marquage [6]. Nous allons montrer ici ce que peut apporter cette méthode à l'étude des systèmes membranaires à l'aide de quelques exemples puisés dans différents types cellulaires [7].…”