Andrew Leis

Disclosure of the mycobacterial outer membrane: Cryo-electron tomography and vitreous sections reveal the lipid bilayer structure

The cell walls of mycobacteria form an exceptional permeability barrier, and they are essential for virulence. They contain extractable lipids and long-chain mycolic acids that are covalently linked to peptidoglycan via an arabinogalactan network. The lipids were thought to form an asymmetrical bilayer of considerable thickness, but this could never be proven directly by microscopy or other means. Cryo-electron tomography of unperturbed or detergent-treated cells of Mycobacterium smegmatis embedded in vitreous ice now reveals the native organization of the cell envelope and its delineation into several distinct layers. The 3D data and the investigation of ultrathin frozen-hydrated cryosections of M. smegmatis, Myobacterium bovis bacillus Calmette–Guérin, and Corynebacterium glutamicum identified the outermost layer as a morphologically symmetrical lipid bilayer. The structure of the mycobacterial outer membrane necessitates considerable revision of the current view of its architecture. Conceivable models are proposed and discussed. These results are crucial for the investigation and understanding of transport processes across the mycobacterial cell wall, and they are of particular medical relevance in the case of pathogenic mycobacteria.

Visualizing cells at the nanoscale

Cryogenic electron tomography (cryo- ET) enables the 3D visualization of biological material at a previously unseeable scale. Carefully controlled cryogenic specimen preparation avoids the artefacts that are notorious to conventional electron microscopy specimen preparation. To date, studies employing cryo- ET have mostly been restricted to isolated macromolecular assemblies, small prokaryotic cells or thin regions of eukaryotic cells owing to the limited penetration depth of electrons through ice-embedded preparations. Recent progress in cryosectioning makes it possible to acquire tomograms from many kinds of vitrified cells and tissues. The systematic and comprehensive interpretation of such tomograms will provide unprecedented insight into the molecular organization of cellular landscapes.

Micromachining tools and correlative approaches for cellular cryo-electron tomography.

A principal limitation of cryo-transmission electron microscopy performed on cells or tissues is the accessible specimen thickness. This is exacerbated in tomography applications, where the aspect ratio (and thus the apparent specimen thickness) changes considerably during specimen tilting. Cryo-ultramicrotomy is the most obvious way of dealing with this problem; however, frozen-hydrated sections suffer from potentially inconsistent compression that cannot be corrected with certainty, and furthermore, yields of sections that satisfy all of the conditions necessary for tomographic imaging are poor. An alternative approach that avoids mechanical deformations is the use of focused ion beam (FIB) instrumentation, where thinning of the frozen-hydrated specimen occurs through the process of sputtering with heavy ions, typically gallium. Here, we use correlative cryo-fluorescence microscopy to navigate large cellular volumes and to localize specific cellular targets. We show that the selected targets in frozen-hydrated specimens can be accessed directly by focused ion beam milling. We also introduce a novel cryo-planing procedure as a method that could facilitate thinning of large areas of vitreous ice prior to cryo-fluorescence, FIB thinning, and cryo-electron tomography.

Correlative cryo-light microscopy and cryo-electron tomography: from cellular territories to molecular landscapes

In cell biology, visual techniques such as light and electron microscopy provide the most intuitive means by which to study structure and function; however, no single microscopy technique is capable of providing all of the desired information. As a consequence, many separate techniques have evolved, each with unique capabilities. The most informative approaches are global in the sense that they take advantage of multiple imaging modalities spanning a range of spatial scales and frequencies, preferably encompassing preservation of the hydrated nature of the cell. Correlative microscopy utilizes complementary visual techniques that allow the experimenter to capture significant proportions of a population of cells, to identify features of interest, and to then capture high-resolution snapshots that represent bona fide cellular events.

Cryo-electron tomography of cells: connecting structure and function.

Cryo-electron tomography (cryo-ET) allows the visualization of cellular structures under close-to-life conditions and at molecular resolution. While it is inherently a static approach, yielding structural information about supramolecular organization at a certain time point, it can nevertheless provide insights into function of the structures imaged, in particular, when supplemented by other approaches. Here, we review the use of experimental methods that supplement cryo-ET imaging of whole cells. These include genetic and pharmacological manipulations, as well as correlative light microscopy and cryo-ET. While these methods have mostly been used to detect and identify structures visualized in cryo-ET or to assist the search for a feature of interest, we expect that in the future they will play a more important role in the functional interpretation of cryo-tomograms.

Luminal particles within cellular microtubules

The regulation of microtubule dynamics is attributed to microtubule-associated proteins that bind to the microtubule outer surface, but little is known about cellular components that may associate with the internal side of microtubules. We used cryoelectron tomography to investigate in a quantitative manner the three dimensional structure of microtubules in intact mammalian cells. We show that the lumen of microtubules in this native state is filled with discrete, globular particles with a diameter of 7 nm and spacings between 8 and 20 nm in neuronal cells. Cross-sectional views of microtubules confirm the presence of luminal material in vitreous sections of brain tissue. Most of the luminal particles had connections to the microtubule wall, as revealed in tomograms. A higher accumulation of particles was seen near the retracting plus ends of microtubules. The luminal particles were abundant in neurons, but were also observed in other cells, such as astrocytes and stem cells.

Chromatin Organization and Radio Resistance in the Bacterium Gemmata obscuriglobus

The organization of chromatin has a major impact on cellular activities, such as gene expression. For bacteria, it was suggested that the spatial organization of the genetic material correlates with transcriptional levels, implying a specific architecture of the chromosome within the cytoplasm. Accordingly, recent technological advances have emphasized the organization of the genetic material within nucleoid structures. Gemmata obscuriglobus, a member of the phylum Planctomycetes, exhibits a distinctive nucleoid structure in which chromatin is encapsulated within a discrete membrane-bound compartment. Here, we show that this soil and freshwater bacterium tolerates high doses of UV and ionizing radiation. Cryoelectron tomography of frozen hydrated sections and electron microscopy of freeze-substituted cells have indicated a more highly ordered condensed-chromatin organization in actively dividing and stationary-phase G. obscuriglobus cells. These three-dimensional analyses revealed a complex network of double membranes that engulf the condensed DNA. Bioinformatics analysis has revealed the existence of a putative component involved in nonhomologous DNA end joining that presumably plays a role in maintaining chromatin integrity within the bacterium. Thus, our observations further support the notion that packed chromatin organization enhances radiation tolerance.

Cryo-electron tomography of biological specimens

This paper describes the important considerations in cryo-electron tomography (CET) of biological specimens and identifies the areas where digital signal processing can make a decisive contribution.

Correlation Microscopy: Bridging the Gap between Light- and Cryo-Electron Microscopy

Cryo-electron tomography (cryo-ET) of frozen, hydrated, biological samples on carbon-coated copper grids is a powerful technique for quasi in vivo studies of cellular structures with nanometerscale resolution [1]. A major inconvenience of this method exists in the difficulty of locating and unequivocally identifying the structures of interest within a ice embedded sample. Additionally, kinetic experiments are almost impossible, due to the fact that the information about the dynamic state in vitrified samples has been lost. By establishing a direct correlation between cryofluorescence microscopy and cryo-electron microscopy (cryo-EM), we propose an effective way to overcome these limitations, opening the way for a wide spectrum of novel applications. Our concept of correlation microscopy is based on the possibility of identifying and determining the position of fluorescently-labelled structures in vitrified samples directly on the TEM grid by means of cryofluorescence microscopy, and the recovery of these positions during subsequent investigations with cryo-EM. The entire vitrified grid can be imaged and mapped with epifluorescence under cryo-conditions thanks to a new designed and homemade cryo-holder for TEM grids, adapted to a fully automated, inverted light microscope (Zeiss Axiovert 200) with long working-distance objectives (20x and 40x magnification). The cryo-holder is devised to keep the sample at liquid nitrogen temperature (lN2) and it is efficiently isolated from the external environment to ensure a thermal equilibrium and to prevent undesirable contamination during investigations. The absolute coordinates of the area of interest on the grid can be determined and recorded by cryo-light microscopy and directly recovered in the electron microscope with a MatLab-based program, integrated within the TOM toolbox [2]. Fluorescently labelled neurons grown on carbon-coated TEM grids have been embedded in vitreous ice and successfully used for first studies. In the case of thin specimens suitable for cryo-electron tomography (where the thickness of the amorphous ice does not exceed 200-400 nm), the resolution is comparable to investigations in buffer solutions. The ‘blurring’ of the fluorescence signal and the decrease in resolution is directly related to the thickness of the ice, which can be estimated from focusing and transmitted light measurements. Our current applications involve the localisation of synapses by immunolabelling of glutamate receptors with fluorescent dyes and the distribution of fluorescent markers in vitreous cryo-sections. Future applications will involve labelling of extra-cellular molecules, structures and receptors with Quantum Dots [3], which are ideal candidates for correlation microscopy due to their fluorescence and electron-dense characteristics, which allow visualisation and targeting of the label both with cryo-light microscopy and cryo-EM, especially cryo-ET.

Correlative cryo-fluorescence and electron microscopy

Cryo-electron tomography (CET) can provide 3-dimensional information about the structural basis of complex cellular processes on the scale of a few nanometres. However, a main drawback in locating features of interest at the level of magnification offered by electron microscopy results from the fact that biological material embedded in amorphous ice has intrinsically low contrast and is sensitive to irradiation by the electron beam. Due to these circumstances, it is complicated to retrieve areas of interest without inducing a priori damage by the search process.