Imaging and 3D reconstruction of membrane protein complexes by cryo-electron microscopy and single particle analysis

Cryo-electron microscopy (cryo-EM) in combination with single particle image processing and volume reconstruction is a powerful technology to obtain medium-resolution structures of large protein complexes, which are extremely difficult to crystallize and not amenable to NMR studies due to size limitation. Depending on the stability and stiffness as well as on the symmetry of the complex, three-dimensional reconstructions at a resolution of 10-30 ˚ can be achieved. In this range of resolution, we may not be able to answer A chemical questions at the level of atomic interactions, but we can gain detailed insight into the macromolecular architecture of large multi-subunit complexes and their mechanisms of action. In this thesis, several prevalently large membrane protein complexes of great physiological importance were examined by various electron microscopy techniques and single particle image analysis. The core part of my work consists in the imaging of a mammalian V-ATPase, frozen-hydrated in amorphous ice and of the completion of the first volume reconstruction of this type of enzyme, derived from cryo-EM images. This ubiquitous rotary motor is essential in every eukaryotic cell and is of high medical importance due to its implication in various diseases such as osteoporosis, skeletal cancer and kidney disorders. My contribution to the second and third paper concerns the volume reconstruction of two bacterial outer membrane pore complexes from cryo-EM images recorded by my colleague Mohamed Chami. PulD from Klebsiella oxytoca constitutes a massive translocating pore capable of transporting a fully folded cell surface protein PulA through the membrane. It is part of the Type II secretion system, which is common for Gram-negative bacteria. The second volume regards ClyA, a pore-forming heamolytic toxin of virulent Escherichia coli and Salmonella enterica strains that kill target cells by inserting pores into their membranes. To the last two papers, I contributed with cryo-negative stain imaging of the cell division protein DivIVA from Bacillus subtilis and with image processing of the micrographs displaying the siderophore receptor FrpB from Neisseria meningitidis

Chloroplast Division Protein ARC3: Effects on FtsZ2 Assembly and GTPase Activity

Chloroplasts evolved from cyanobacterial endosymbiotic ancestors and their division is a complex process initiated by assembly of cytoskeletal FtsZ proteins into a ring structure at the division site (Z-ring). The cyanobacterial Z-ring positioning system (MinCDE proteins) is also conserved in chloroplasts except that MinC was lost and replaced by the eukaryotic ARC3. Both MinC and ARC3 act as negative regulators of FtsZ assembly, but ARC3 bears little sequence similarity with MinC. Here, light scattering assays, co-sedimentation, light microscopy, GTPase assay and transmission electron microscopy in conjunction with single particle analysis have been used to elucidate the structure of ARC3 and its effect on its main target in chloroplast division: FtsZ2. Analysis of FtsZ2 in vitro assembly reactions in the presence and absence of GMPCPP showed that ARC3 promotes FtsZ2 debundling and disassembly of existing filaments in a concentration-dependent manner and requires GTP hydrolysis. 3D reconstruction of ARC3 revealed an almost circular molecule in which the FtsZ-binding N-terminus and the C-terminal PARC6-binding MORN domain are in close proximity and suggests a model for PARC6-enabled binding of ARC3 to FtsZ2. The latter is corroborated by in vivo data

Unraveling the Thousand Word Picture: An Introduction to Super-Resolution Data Analysis

Super-resolution microscopy provides direct insight into fundamental biological processes occurring at length scales smaller than light’s diffraction limit. The analysis of data at such scales has brought statistical and machine learning methods into the mainstream. Here we provide a survey of data analysis methods starting from an overview of basic statistical techniques underlying the analysis of super-resolution and, more broadly, imaging data. We subsequently break down the analysis of super-resolution data into four problems: the localization problem, the counting problem, the linking problem, and what we’ve termed the interpretation problem

Nuclear pores as versatile reference standards for quantitative superresolution microscopy

Quantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions. Here, we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ reference structures to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag, HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use (1) as three-dimensional resolution standards for calibration and quality control, (2) to quantify absolute labeling efficiencies and (3) as precise reference standards for molecular counting. These cell lines will enable the broader community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements

Structure determination of membrane-located complexes: Aquaporin 8 and YscC secretin

A biological membrane is key to the life of a cell. In all living cells a biological membrane separates a inner life and an outer environment. Plasma membrane has many functions such as responding to external signals and transmitting them into the cell, providing a barrier to the water soluble molecules, transporting molecules via channels, cell-to-cell communication, separating cellular reactions by compartments and creating ion gradients between them, which are used for converting an energy and signal transduction. Most of the membrane functions are performed by embedded proteins, which are constantly in motion. The homeostasis of water, the most abundant molecule in living organisms, is crucial for physiology of all cells. The main interest in structure determination of the water channel aquaporin 8 (AQP8) is connected with its unique position within the aquaporin family. The human AQP8 cDNA has been cloned in 1998 and since then the intensive studies concerning its intravesicular localization and activity have been preformed. Expressed in the inner mitochondrial membrane of several mammalian tissues, found in liver, gastrointestinal tract, testis, airways and kidney cells, the ammonia-permeable AQP8 could be essential for the organism metabolism. By sequence alignment it is evident that AQP8 creates a separate subfamily, which is apart from all the other mammalian aquaporins. The special constriction region of the pore, which determines the solute permeability, is unique in AQP8 and makes it permeable to both ammonia and hydrogen peroxide in addition to water. For mammalian aquaporins, the structures solved up to now, all belong to the water-permeable subfamily. To better understand AQP8 selectivity and gating mechanism the high-resolution structure is necessary. To assess the structure, human AQP8 was overexpressed in methylotrophic yeast Pichia pastoris as a His-tagged protein. A wide screen of different detergents and detergent-lipid combinations for optimal protein purification and 2D crystallization was essential to obtain well-ordered AQP8 crystal arrays. Removal of amino acids constituting affinity tags was necessary to achieve highly ordered crystals diffracting up to 3 Å. Atomic force microscopy, electron microscopy and gold labeling experiments revealed the double-layered nature of 2D crystals, with tetrameric organization of AQP8, which had termini exposed outwards of the 2D crystal. In parallel, alignments to AQP4 revealed a similar, extraordinary long N-terminal of AQP8. In analogy to AQP4, where only the short isoform is able to crystallize, 2D crystallization of the shorter AQP8 construct, with removed N-terminus, was initiated. The translocation of proteins across the biological membrane is an essential part of cellular life. The type III secretion system (T3SS) is a major factor for the virulence or symbiosis of many Gram-negative bacteria that infect plants and animals. Bacterial effector proteins are delivered via T3SS injectisome from the pathogen cytoplasm into the eukaryotic host cells, in which they modulate the host innate immune response. The outer membrane-localized YscC oligomer belongs to the secretin family and is one of the main components of the injectisome. In this study, the YscC complex was expressed in the avirulent strain of Yesinia enterocolitica and purified in order to construct a 3D model from cryo Electron Microscopy single particle images. The 12 Å-resolution 3D structure of the closed YscC complex was calculated from 30000 projections of vitrified YscC. Various approaches like rotary metal shadowing of trypsin digested oligomers, mass spectrometry and Scanning Transmission Electron Microscopy were used for oligomer symmetry evaluation. The generated 3D structure of the YscC complex disclosed the N-terminal flexible domain, which forms the part of a large chamber between the bacterial outer and inner membranes, the conical shape periplasmic domain, and two differently sized ring-shaped domains linked by fine density connectors corresponding to the outer membrane spanning regions. The sample trypsinization revealed the protease-resistant core of the protein. Comparison of the sequence and structure of YscC to other, close or more distant related secretins made it possible to define homology regions located both on the N- and C-termini of the protein

Structure of the cell envelope of Halobacterium halobium

The structure of the isolated cell envelope of Halobacterium halobium is studied by X-ray diffraction, electron microscopy, and biochemical analysis. The envelope consists of the cell membrane and two layers of protein outside. The outer layer of protein shows a regular arrangement of the protein or glycoprotein particles and is therefore identified as the cell wall. Just outside the cell membrane is a 20 A-thick layer of protein. It is a third structure in the envelope, the function of which may be distinct from that of the cell membrane and the cell wall. This inner layer of protein is separated from the outer protein layer by a 65 Å-wide space which has an electron density very close to that of the suspending medium, and which can be etched after freeze-fracture. The space is tentatively identified as the periplasmic space. At NaCl concentrations below 2.0 M, both protein layers of the envelope disintegrate. Gel filtration and analytical ultracentrifugation of the soluble components from the two protein layers reveal two major bands of protein with apparent mol wt of ~16,000 and 21,000. At the same time, the cell membrane stays essentially intact as long as the Mg++ concentration is kept at ≥ 20 mM. The cell membrane breaks into small fragments when treated with 0.1 M NaCl and EDTA, or with distilled water, and some soluble proteins, including flavins and cytochromes, are released. The cell membrane apparently has an asymmetric core of the lipid bilayer

Anomalous transport in the crowded world of biological cells

A ubiquitous observation in cell biology is that diffusion of macromolecules and organelles is anomalous, and a description simply based on the conventional diffusion equation with diffusion constants measured in dilute solution fails. This is commonly attributed to macromolecular crowding in the interior of cells and in cellular membranes, summarising their densely packed and heterogeneous structures. The most familiar phenomenon is a power-law increase of the MSD, but there are other manifestations like strongly reduced and time-dependent diffusion coefficients, persistent correlations, non-gaussian distributions of the displacements, heterogeneous diffusion, and immobile particles. After a general introduction to the statistical description of slow, anomalous transport, we summarise some widely used theoretical models: gaussian models like FBM and Langevin equations for visco-elastic media, the CTRW model, and the Lorentz model describing obstructed transport in a heterogeneous environment. Emphasis is put on the spatio-temporal properties of the transport in terms of 2-point correlation functions, dynamic scaling behaviour, and how the models are distinguished by their propagators even for identical MSDs. Then, we review the theory underlying common experimental techniques in the presence of anomalous transport: single-particle tracking, FCS, and FRAP. We report on the large body of recent experimental evidence for anomalous transport in crowded biological media: in cyto- and nucleoplasm as well as in cellular membranes, complemented by in vitro experiments where model systems mimic physiological crowding conditions. Finally, computer simulations play an important role in testing the theoretical models and corroborating the experimental findings. The review is completed by a synthesis of the theoretical and experimental progress identifying open questions for future investigation.Comment: review article, to appear in Rep. Prog. Phy

Experimental approaches for addressing fundamental biological questions in living, functioning cells with single molecule precision

In recent years, single molecule experimentation has allowed researchers to observe biological processes at the sensitivity level of single molecules in actual functioning, living cells, thereby allowing us to observe the molecular basis of the key mechanistic processes in question in a very direct way, rather than inferring these from ensemble average data gained from traditional molecular and biochemical techniques. In this short review, we demonstrate the impact that the application of single molecule bioscience experimentation has had on our understanding of various cellular systems and processes, and the potential that this approach has for the future to really address very challenging and fundamental questions in the life sciences