NON-FOULING AFFINITY PLATFORMS FOR PROTEIN IMMOBILIZATION IN ELECTRON MICROSCOPY

Single-particle reconstruction has grown significantly with the improvements in various data collection and computational strategies including CTF fitting, the use of vitrified samples and the utilization of ultra-sensitive direct electron detectors. Although these improvements have contributed significantly to the recent evolution of 3D reconstruction analysis, the way samples are prepared for electron microscopy has remained largely unchanged. We report the development of TEM grids that are modified with non-fouling coatings bearing surface grafted nitrilotriacetic acid substituents that promotes specific capture of protein targets for high resolution TEM analysis. The utilization of these grids for specific adsorption of the targeted protein onto the grid surface results in well-controlled surface concentration enhancements and a days-to-minutes reduction in time required for the preparation of a purified sample for cryoEM analysis from an E. coli expression system. The selective and reversible capture of his-tag T7 bacteriophage, RplL, and GroEL from crude lysates, as well as purified nanodisc-solubilized his-malFGK2, on these NTA-modified grids with an exceptionally low level of adsorption by non-target proteins has been observed. Our data illustrates the utility of these grids for selective capture from complex mixtures, detergent-solubilized membrane protein isolates, and expression systems yielding low copy numbers of the desired target in a manner that is well-suited for single particle reconstruction analysis

New science exploration from XFEL: a new paradigm for structural visualisation of macromolecules

X-rays have a long-standing history as an investigative probe in the sciences, and in particular their application to the biological and biomedical sciences has provided an enormous contribution to these fields. Indeed structural biology, the study of the molecules of life at an atomic scale via macromolecular crystallography, has been a major benefactor of advances in x-ray radiation sources. Currently two major bottlenecks exist within this field, the need for well diffracting crystals and radiation damage limitations. The advent of fourth generation x-ray sources, X-ray Free-electron Lasers (XFEL) heralds a shift in the way such experiments are performed. XFELs, due to their high brilliance and ultra short (fs) pulses, hope to decouple radiation dose limitations from spatial resolution by outrunning this radiation damage in short exposures, ‘diffraction before destruction’. This thesis is concerned with exploring experimental methodologies made possible by XFELs, including establishing the experimental infrastructure required at the worlds second XFEL, SACLA, and performing initial experiments. Firstly the potential of performing gas-phase small angle x-ray scattering experiments (gSAXS) is investigated. The current need for gas-phase structural information will be presented and the experimental parameters and projected signal requirements will then be explored. The results of experiments at a synchrotron radiation source with various biomolecules will be presented. It is shown that with the current experimental set-up experiments are fundamentally limited by the signal to noise ratio (SNR) pointing to the necessity of XFEL. Secondly the application of coherent diffractive imaging (CDI) to biological systems at synchrotron and XFEL sources is explored, and the development of experimental systems at both sources is outlined. A method for combining complimentary scattering experiments at both sources is demonstrated and the results of its application to the assembly mechanism of the self-assembling, non-crystalline, macromolecule, the RNAi microsponge, are presented. The microsponge is found to have a nucleating origin leading to a core-shell like nanostructure in the fully formed molecule

Elektron kryo-mikroskopické techniky v biologickém výzkumu a nanotechnologiích

Příprava biologických vzorků pro transmisní elektronovou mikroskopii není triviální úkol. Vzorky musí odolat vakuu přítomném v mikroskopu, a proto je často nutné uplatnit nefyziologické postupy při jejich zpracování. Tyto postupy obvykle zahrnují fixaci na bázi aldehydů, nahrazení vody alkoholem (t.j. dehydrataci/substituci), a zalití do pryskyřice, která vytváří podporu pro následnou přípravu tenkých řezů, které pak mohou být vloženy do mikroskopu. V posledním desetiletí získala dominantní postavení v oblasti výzkumu buněčné biologie metoda kryo-fixace (vitrifikace) za pomoci ultrarychlého vysokotlakého zmrazování a následná kryo-substituce a zalití vzorků do pryskyřice při nízkých teplotách. Tímto způsobem byli úspěšně vitrifikovány různé biologické vzorky s tloušťkou až několik stovek mikrometrů do stavu, který byl srovnatelný s jejich in vivo strukturou. Kryo-fixace izolovaných biologických objektů (s omezenou tloušťkou do několika mikrometrů) je možná i v tenké vrstvě vitrifikované vody za pomoci imerzní kryo-fixace při normálním tlaku. V kombinaci s kryo-elektronovou mikroskopií se tato metoda stala nejefektivnejším a základním principem pro tvorbu elektron kryo-mikroskopických obrázků plně hydratovaných vzorků s velmi vysokým rozlišením na úrovni několika desetin nanometrů. Obě tyto metody...Preparation of biological samples for transmission electron microscopy is not a trivial task. The samples must withstand a vacuum environment present inside a microscope, and it is often necessary to use non-physiological procedures for their processing. These procedures usually involve aldehyde-based fixation, replacing water with alcohol (i.e. dehydration/substitution), and embedding into a resin, which creates support for the subsequent preparation of thin sections that can be placed into the microscope. In the last decade, the method of cryo-fixation (vitrification) using ultra-fast high-pressure freezing followed by freeze substitution and low-temperature resin embedding gained a dominant position in the cell biology research. In this way, a range of biological samples with a thicknesses up to several hundreds of micrometers was successfully vitrified to a state that was closely related to their in vivo structures. The cryo-fixation of isolated biological objects (with a limited thickness up to several micrometers) is possible in a thin layer of vitrified water by plunge freezing at ambient pressure. In combination with electron cryo-microscopy, this method has become the most effective and fundamental principle for the high-resolution studies and image analysis of fully hydrated samples...Ústav buněčné biologie a patologie 1. LF UKInstitute of Cell Biology and Pathology First Faculty of Medicine Charles UniversityFirst Faculty of Medicine1. lékařská fakult

Characterising the elastic and viscoelastic interaction between the cell and its matrix in 3D: because it takes two to salsa dance

The extracellular matrix (ECM) is a three-dimensional, acellular component of all organs and tissues. The ECM has elastic and viscoelastic properties, quantified through the elastic modulus (i.e. stiffness) and stress relaxation, respectively, that guide cell fate. Stiffness and stress relaxation drive cellular plasticity in homeostasis and disease. Therefore, to represent the mechanics of the ECM in vitro it is necessary to employ models that recapitulate these properties. Among said models are hydrogels: polymeric networks whose mass mainly consists of water. Importantly, hydrogel viscoelasticity remains an understudied property, notably in cell-loaded materials. Hence, this thesis investigated the elastic and viscoelastic properties of diverse cell-free and cell-loaded hydrogels. The hydrogels evaluated in this thesis include organ-derived ECM, gelatine methacryloyl, agarose, human-derived platelet-poor plasma, alginate and pluronic. These hydrogels have tissue engineering and regenerative medicine (TERM) potential. Particular emphasis was placed on investigating and mathematically modelling the elastic and viscoelastic fate of cell-loaded hydrogels. Our data show that increasing polymer concentration tailored hydrogel elasticity and viscoelasticity. Hydrogel architecture, composition and the bonds forming the polymer network dictated hydrogel elasticity and viscoelasticity. Also, cells altered hydrogel stiffness and stress relaxation in a polymer type, hydrogel concentration and time-dependent manner. A generalised Maxwell model of viscoelasticity further revealed cell-induced changes in hydrogel time-dependent mechanics. Overall, this thesis furthers our understanding of cell-matrix biology in vitro. The data presented here also have implications for the TERM field and areas of hydrogel-based research for cellular applications

Analysing the lattice transition of thin filaments in striated muscle

Thin filaments, through interaction with thick filaments, form the contractile apparatus of striated muscle. Therefore, the length and arrangement of the thin filaments are of key importance to the function of the muscle. The thin filaments from adjacent sarcomeres are anchored at the Z-disc. In 1968 Pringle predicted that thin filament are organised in the Z-disc in a rhomboid lattice rather than a square lattice. Previous experimental evidence has been insufficient to verify Pringle’s suggestion. In the A-band the thin filaments interdigitate with the thick filaments on a hexagonal lattice, hence from the Z-disc to the A-band, there is a transition of the lattice from square to hexagonal. In this project, I have firstly used Fourier analysis and electron tomography to investigate the thin filament lattice in the Z-disc. I have used electron tomography to determine how the lattice transition occurs between the Z-disc and the A-band. Electron tomography of these samples also allowed me to determine the lengths of thin filaments, showing unequivocally that they are of variable lengths in cardiac muscle

Structural characterization of Ebola virus uncoating

Viruses initiate infection of host cells by entering through a variety of different pathways. Their entry is concluded by the release of the viral genome into the cytoplasm, where the cellular machinery gets repurposed for virus replication. Prerequisite for genome release is the uncoating of the viral particles, a process which requires the destabilization of interactions established during virus assembly. Ebola viruses (EBOVs) are highly pathogenic, enveloped RNA viruses of remarkable filamentous morphology. Their shape is dictated by the viral matrix protein VP40, which forms a tubular scaffold underneath the viral envelope and confers stability to the particles during EBOV transmission. EBOVs enter host cells via the endocytic pathway and release their genome into the cytoplasm after fusion of their envelope with the endosomal membrane. The first line of defence against a viral infection is blocking viral entry, and EBOV entry has accordingly been well investigated with respect to receptor engagement and potential membrane fusion triggers. However, key mechanisms governing the final step of virus entry are still unknown, including the central question of how these unusually shaped virions undergo uncoating. Whether and how the VP40 matrix disassembles to enable membrane fusion; whether uncoating involves additional triggers; and finally, how and where the viral genome gets released from the viral particles and nucleocapsids remains to be elucidated. In this thesis, I investigate EBOV uncoating during entry into host cells and shed light on the fate of the most abundant and versatile viral protein, VP40. As a main tool, I use in situ cryo-electron tomography and provide structural insights into EBOV uncoating both in vitro and in infected host cells at molecular resolution. I discover that at low endosomal pH, the VP40 matrix detaches from the viral envelope and disassembles. This is caused by the disruption of electrostatic interactions between membrane lipids and anionic amino acids exposed on the surface of VP40 dimers, which I show are the structural units of the VP40 matrix. The strong effect of low pH on the integrity of the VP40 matrix is a consequence of acidification of the viral lumen, which I further investigate to uncover its mechanism. I show that protons diffuse passively across the viral envelope independently of a dedicated ion channel, which might be relevant for other late-penetrating viruses lacking viroporins. Finally, I provide the first high-resolution images of Ebola virions in endolysosomal compartments of infected cells. These images confirm the disassembly of the VP40 matrix in virions located in acidified compartments while clearly showing that their nucleocapsids remain intact. Together, these findings reveal that VP40 matrix disassembly is an essential step during EBOV uncoating, which precedes membrane fusion and genome release from the nucleocapsids. Overall, this thesis extends the current understanding of virus uncoating and indicates that pH-driven structural remodeling of viral matrix proteins may act as a switch coupling matrix uncoating to membrane fusion during host cell entry of enveloped viruses

The drying of foods using supercritical carbon dioxide

Food drying techniques such as air and freeze drying are not ideal: high temperatures used during air drying result in degradation of nutrients and sensorial properties, while freeze drying is expensive and therefore only applicable to high value foods. As an alternative to such drying techniques, drying with supercritical carbon dioxide was investigated here. Initially, carrot was dried using this technique. Addition of a co-solvent (ethanol) to the supercritical fluid was used as a method to increase the water solubility in the supercritical fluid and therefore aid drying. Analysis of the dried and rehydrated product structure, rehydration properties and mechanical properties was carried out which gave an indication of product quality. Drying of agar, containing varying concentrations of sugar was carried out on a laboratory and pilot plant scale. Gel structure and gel properties were studied. Addition of sugar to agar gel pieces improved structural retention considerably during drying. Fourier transform infrared analysis was used to investigate interactions that may be responsible for structural differences seen during supercritical drying. Changes in experimental parameters such as flow rate and depressurisation rate did not appear to have a significant effect on the dried gel structure. The supercritical drying technique investigated allowed food products to be dried and unique structures to be created with different rehydration and textural properties to the equivalent food products dried by air or freeze drying