Jason Pierson

M. tuberculosis and M. leprae Translocate from the Phagolysosome to the Cytosol in Myeloid Cells

M. tuberculosis and M. leprae are considered to be prototypical intracellular pathogens that have evolved strategies to enable growth in the intracellular phagosomes. In contrast, we show that lysosomes rapidly fuse with the virulent M. tuberculosis- and M. leprae-containing phagosomes of human monocyte-derived dendritic cells and macrophages. After 2 days, M. tuberculosis progressively translocates from phagolysosomes into the cytosol in nonapoptotic cells. Cytosolic entry is also observed for M. leprae but not for vaccine strains such as M. bovis BCG or in heat-killed mycobacteria and is dependent upon secretion of the mycobacterial gene products CFP-10 and ESAT-6. The cytosolic bacterial localization and replication are pathogenic features of virulent mycobacteria, causing significant cell death within a week. This may also reveal a mechanism for MHC-based antigen presentation that is lacking in current vaccine strains.

Direct Visualization by Cryo-EM of the Mycobacterial Capsular Layer: A Labile Structure Containing ESX-1-Secreted Proteins

The cell envelope of mycobacteria, a group of Gram positive bacteria, is composed of a plasma membrane and a Gram-negative-like outer membrane containing mycolic acids. In addition, the surface of the mycobacteria is coated with an ill-characterized layer of extractable, non-covalently linked glycans, lipids and proteins, collectively known as the capsule, whose occurrence is a matter of debate. By using plunge freezing cryo-electron microscopy technique, we were able to show that pathogenic mycobacteria produce a thick capsule, only present when the cells were grown under unperturbed conditions and easily removed by mild detergents. This detergent-labile capsule layer contains arabinomannan, α-glucan and oligomannosyl-capped glycolipids. Further immunogenic and proteomic analyses revealed that Mycobacterium marinum capsule contains high amounts of proteins that are secreted via the ESX-1 pathway. Finally, cell infection experiments demonstrated the importance of the capsule for binding to cells and dampening of pro-inflammatory cytokine response. Together, these results show a direct visualization of the mycobacterial capsular layer as a labile structure that contains ESX-1-secreted proteins.

Structural mechanism of glutamate receptor activation and desensitization

Ionotropic glutamate receptors are ligand-gated ion channels that mediate excitatory synaptic transmission in the vertebrate brain. To gain a better understanding of how structural changes gate ion flux across the membrane, we trapped rat AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) and kainate receptor subtypes in their major functional states and analysed the resulting structures using cryo-electron microscopy. We show that transition to the active state involves a ‘corkscrew’ motion of the receptor assembly, driven by closure of the ligand-binding domain. Desensitization is accompanied by disruption of the amino-terminal domain tetramer in AMPA, but not kainate, receptors with a two-fold to four-fold symmetry transition in the ligand-binding domains in both subtypes. The 7.6 Å structure of a desensitized kainate receptor shows how these changes accommodate channel closing. These findings integrate previous physiological, biochemical and structural analyses of glutamate receptors and provide a molecular explanation for key steps in receptor gating.

Cryo-electron microscopy: A primer for the non-microscopist

Cryo‐electron microscopy (cryo‐EM) is increasingly becoming a mainstream technology for studying the architecture of cells, viruses and protein assemblies at molecular resolution. Recent developments in microscope design and imaging hardware, paired with enhanced image processing and automation capabilities, are poised to further advance the effectiveness of cryo‐EM methods. These developments promise to increase the speed and extent of automation, and to improve the resolutions that may be achieved, making this technology useful to determine a wide variety of biological structures. Additionally, established modalities for structure determination, such as X‐ray crystallography and nuclear magnetic resonance spectroscopy, are being routinely integrated with cryo‐EM density maps to achieve atomic‐resolution models of complex, dynamic molecular assemblies. In this review, which is directed towards readers who are not experts in cryo‐EM methodology, we provide an overview of emerging themes in the application of this technology to investigate diverse questions in biology and medicine. We discuss the ways in which these methods are being used to study structures of macromolecular assemblies that range in size from whole cells to small proteins. Finally, we include a description of how the structural information obtained by cryo‐EM is deposited and archived in a publicly accessible database.

Three-dimensional ultrastructure of the septin filament network in Saccharomyces cerevisiae

Septins are essential for membrane compartmentalization and remodeling. Electron tomography of yeast bud necks shows filaments perpendicular and parallel to the mother-bud axis that resemble in vitro septin arrays. Filaments are still present, although disordered, in mutants lacking a single septin, underscoring the importance of septin assembly.

Improving the technique of vitreous cryo-sectioning for cryo-electron tomography: Electrostatic charging for section attachment and implementation of an anti-contamination glove box

Cryo-electron tomography of vitreous cryo-sections is the most suitable method for exploring the 3D organization of biological samples that are too large to be imaged in an intact state. Producing good quality vitreous cryo-sections, however, is challenging. Here, we focused on the major obstacles to success: contamination in and around the microtome, and attachment of the ribbon of sections to an electron microscopic grid support film. The conventional method for attaching sections to the grid has involved mechanical force generated by a crude stamping or pressing device, but this disrupts the integrity of vitreous cryo-sections. Furthermore, attachment is poor, and parts of the ribbon of sections are often far from the support film. This results in specimen instability during image acquisition and subsequent difficulty with aligning projection images. Here, we have implemented a protective glove box surrounding the cryo-ultramicrotome that reduces the humidity around and within the microtome during sectioning. We also introduce a novel way to attach vitreous cryo-sections to an EM grid support film using electrostatic charging. The ribbon of vitreous cryo-sections remains in place during transfer and storage and is devoid of stamping related artefacts. We illustrate these improvements by exploring the structure of putative cellular 80S ribosomes within 50nm, vitreous cryo-sections of Saccharomyces cerevisiae.

Toward visualization of nanomachines in their native cellular environment

The cellular nanocosm is made up of numerous types of macromolecular complexes or biological nanomachines. These form functional modules that are organized into complex subcellular networks. Information on the ultra-structure of these nanomachines has mainly been obtained by analyzing isolated structures, using imaging techniques such as X-ray crystallography, NMR, or single particle electron microscopy (EM). Yet there is a strong need to image biological complexes in a native state and within a cellular environment, in order to gain a better understanding of their functions. Emerging methods in EM are now making this goal reachable. Cryo-electron tomography bypasses the need for conventional fixatives, dehydration and stains, so that a close-to-native environment is retained. As this technique is approaching macromolecular resolution, it is possible to create maps of individual macromolecular complexes. X-ray and NMR data can be ‘docked’ or fitted into the lower resolution particle density maps to create a macromolecular atlas of the cell under normal and pathological conditions. The majority of cells, however, are too thick to be imaged in an intact state and therefore methods such as ‘high pressure freezing’ with ‘freeze-substitution followed by room temperature plastic sectioning’ or ‘cryo-sectioning of unperturbed vitreous fully hydrated samples’ have been introduced for electron tomography. Here, we review methodological considerations for visualizing nanomachines in a close-to-physiological, cellular context. EM is in a renaissance, and further innovations and training in this field should be fully supported.

Immunogold Labeling of Thawed Cryosections

Publisher Summary Immunogold labeling is an established approach to localize gene products in their cellular environment. This chapter focuses on the current protocols for cryoimmunogold labeling in order to localize subcellular proteins of interest. The chapter describes the types of grids that are most often used and how they can be prepared for labeling. Electron microscopic (EM) grids are made from metals to conduct heat and charge away from the sample. Carbon, Formvar, or a combination of the two films can be applied to the metal to provide support for the cryosection. The specimen preparation that is a prerequisite for efficient labeling is presented in the chapter. The chapter outlines a method for ultrathin sectioning. Another method for immunolabeling, which enables the localization of specific proteins and cellular components, reagents and solutions are discussed. The chapter explores the cryosectioning and the application of this technique for tomography. The chapter focuses on basic principles and variations in order to maximize labeling effciency.

Self-assembled monolayers improve protein distribution on holey carbon cryo-EM supports

Poor partitioning of macromolecules into the holes of holey carbon support grids frequently limits structural determination by single particle cryo-electron microscopy (cryo-EM). Here, we present a method to deposit, on gold-coated carbon grids, a self-assembled monolayer whose surface properties can be controlled by chemical modification. We demonstrate the utility of this approach to drive partitioning of ionotropic glutamate receptors into the holes, thereby enabling 3D structural analysis using cryo-EM methods.

Exploring vitreous cryo-section-induced compression at the macromolecular level using electron cryo-tomography; 80S yeast ribosomes appear unaffected.

Vitreous cryo-section-induced compression influences the interpretation and the reliability of electron microscopy images and tomographic reconstructions. Previous studies of this deformation have been focused at the cellular level where considerable compression occurs, yet the degree of possible intracellular macromolecular deformation has remained unclear. Here, electron cryo-tomographic reconstructions of vitreous cryo-sections show that 80S ribosomes, both intracellular and in an isolated state, appear able to resist section-induced compression. Our observations indicate that vitreous section-induced compression is non-uniform between whole cells that have been sectioned and their intracellular macromolecular complexes. We conclude that electron cryo-tomography of vitreous cryo-sections, in spite of section-induced compression, is a suitable technique for charting the structural organization of cellular nanomachines, such as ribosomes, in a cellular environment.