Cryo-scanning transmission electron tomography of vitrified cells

Cryo-electron tomography (cryo-ET) is a formidable technique to observe the inner workings of vitrified cells at a nanometric resolution in near-native conditions and in three-dimensions. One consequent drawback of this technique is the sample thickness, for two reasons: i) achieving proper vitrification of the sample gets increasingly difficult with sample thickness, and ii) cryo-ET relies on transmission electron microscopy (TEM), requiring thin samples for proper electron transmittance (<500 nm). For samples exceeding this thickness limit, thinning methods can be used to render the sample amenable for cryo-ET. Cryo-focused ion beam (cryo-FIB) milling is one of them and despite having hugely benefitted the fields of animal cell biology, virology, microbiology, and even crystallography, plant cells are still virtually unexplored by cryo-ET, in particular because they are generally orders of magnitude bigger than bacteria, viruses, or animal cells (at least 10 μm thick) and difficult to process by cryo-FIB milling. Here, we detail a preparation method where abaxial epidermal onion cell wall peels are separated from the epidermal cells and subsequently plunge frozen, cryo-FIB milled, and screened by cryo-ET in order to acquire high resolution tomographic data for analyzing the organization of the cell wall. This protocol was validated in: Curr Biol (2022), DOI: 10.1016/j.cub.2022.04.024.

Superhelical simian virus 40 DNA migrates more rapidly during electrophoresis in agarose gels than covalently closed DNA free of superhelical turns (relaxed DNA). The difference in electrophoretic mobility between superhelical and relaxed DNA was used to monitor the activity of a protein from human tissue culture cells which converts superhelical DNA into relaxed DNA. Purified DNA-relaxing protein removes both negative and positive superhelical turns and acts in a catalytic manner. The relaxation of DNA proceeds in a stepwise fashion and DNA intermediates with decreasing numbers of superhelical turns are seen during the course of the reaction.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) catalyzes the formation of intracellular cholesterol esters in various tissues. We recently reported the cloning and expression of human macrophage ACAT cDNA. In the current study, we report the production of specific polyclonal antibodies against ACAT by immunizing rabbits with the recombinant fusion protein composed of glutathione S-transferase and the first 131 amino acids of ACAT protein. Immunoblot analysis showed that the antibodies cross-reacted with a 50-kDa protein band from a variety of human cell lines. These antibodies immunodepleted more than 90% of detergent-solubilized ACAT activities from six different human cell types, demonstrating that the 50-kDa protein is the major ACAT catalytic component in these cells. In multiple human tissues examined, the antibodies recognized protein bands with various molecular weights. These antibodies also cross-reacted with the ACAT protein in Chinese hamster ovary cells. Immunoblot analysis showed that the ACAT protein contents in human fibroblast cells, HepG2 cells, or Chinese hamster ovary cells were not affected by sterol in the medium, demonstrating that the main mechanism for sterol-dependent regulation of ACAT activity in these cells is not change in ACAT protein content. As revealed by indirect immunofluorescent microscopy, the ACAT protein in tissue culture cells was located in the endoplasmic reticulum. This finding, along with earlier studies, suggests that cholesterol concentration in the endoplasmic reticulum may be the major determinant for regulating ACAT activity in the intact cells.

Transmission electron microscopy can be used for the characterisation of a wide range of thin specimens, but soft matter and aqueous samples such as gels, nanoparticle dispersions, and emulsions will dry out and collapse under the microscope vacuum, therefore losing information on their native state and ultimately limiting the understanding of the sample. This study examines commonly used techniques in transmission electron microscopy when applied to the characterisation of cryogenically frozen Pickering emulsion samples. Oil-in-water Pickering emulsions stabilised by 3 to 5nm platinum nanoparticles were cryogenically frozen by plunge-freezing into liquid ethane to retain the native structure of the system without inducing crystallisation of the droplet oil cores. A comparison between the droplet morphology following different sample preparation methods has confirmed the effectiveness of using plunge-freezing to prepare these samples. Scanning transmission electron microscopy imaging showed that dry droplets collapse under the microscope vacuum, changing their shape and size (average apparent diameter: ∼342nm) compared to frozen samples (average diameter: ∼183nm). Cryogenic electron tomography was used to collect additional information of the 3D shape and size of the emulsion droplets, and the position of the stabilising nanoparticles relative to the droplet surface. Cryogenic energy dispersive X-ray and electron energy loss spectroscopy were used to successfully obtain elemental data and generate elemental maps to identify the stabilising nanoparticles and the oil phase. Elemental maps generated from spectral data were used in conjunction with electron tomography to obtain 3D information of the oil phase in the emulsion droplets. Beam-induced damage to the ice was the largest limiting factor to the sample characterisation, limiting the effective imaging resolution and signal-to-noise ratio, though careful consideration of the imaging parameters used allowed for the characterisation of the samples presented in this study. Ultimately this study shows that cryo-methods are effective for the representative characterisation of Pickering emulsions.

Systemic lupus erythematosus autoantibodies were used to identify and to characterize new human chromosome-associated proteins. Previous immunolocalization studies in human and murine tissue culture cells showed that some of these monoclonal antibodies recognize nuclear antigens that associate with condensed chromosomes during mitosis. One antibody was selected for screening a human HeLa S3 cDNA expression library, and cDNAs that code for an antigen of 31-33 kDa were isolated. Immunological, biochemical and cell fractionation data indicate that the 31- to 33-kDa antigen corresponds to the chromosome-associated protein recognized by the original monoclonal antibody. Sequence analysis shows that we isolated a novel human gene. Immunolocalization to human tissue culture cells shows that during interphase the antigen is dispersed in the nucleus and that during mitosis it associates exclusively with condensed chromosomes. A similar pattern of localization was also observed in mouse fibroblasts, suggesting that the antigen is conserved among different species. Finally, we show that part of the antigen remains bound to the scaffold/matrix component, even after high salt extraction.

RNA and DNA metabolism in human tissue culture cells studied with tritiated cytidine

The morphology of the vitreoretinal border region, also termed the inner limiting membrane, was studied in spontaneously hypertensive rats (SHR rats), in Wistar Kyoto rats (WKY rats), and in Buffalo rats (BUF rats). The region was examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) in order to visualize a possible increase in thickness with raised arterial pressure. The median thickness of the lamina densa in TEM in the three groups varied from 34 to 68 nm. In hypertensive SHR rats the lamina densa showed thickening up to the 10th month and was unchanged thereafter. The same pattern was shown in WKY rats, although the thickness increased also after the tenth month in these controls. In BUF rats, however, the lamina densa decreased in thickness with age. The median thickness of the whole vitreoretinal border region varied between: SHR rats: 42 and 84 nm (SEM) and 51 and 127 nm (TEM); WKY rats: 84 nm (SEM) and 51 and 68 nm (TEM); BUF rats: 42 nm (SEM) and 34 and 68 nm (TEM). SHR rats showed in SEM an increase in thickness with age. In TEM, however, only one very old hypertensive rat had a thickened vitreoretinal border region. In WKY rats both SEM and TEM showed an increase in thickness with age, whereas BUF rats showed a decrease. Fundus photographs disclosed narrow, straight and irregular retinal arterioles in hypertensive SHR rats. Light microscopy of older SHR rats demonstrated retinal degeneration from the outer photoreceptor layer to the inner nuclear layer. This was also found in some of the older WKY rats.

The effects of electron-beam damage on the Fe 3+ /ΣFe (total iron) ratio were measured by electron energy-loss spectroscopy (EELS) with a transmission electron microscope (TEM). Spectra were acquired from crushed and ion-beam-thinned cronstedtite. For fluences below 1 × 10 4 e/A 2 , the Fe 3+ /ΣFe values from crushed grains range between 0.43 and 0.49, consistent with undamaged material. These measurements were acquired from flakes 180 to 1000 A thick. With increase in fluence, samples 3+ /ΣFe values >0.5. The critical fluence for radiation damage by 100 kV electrons as defined by Fe 3+ /ΣFe 4 e/A 2 . The absorbed dose to the speciman during acquisition of a typical EELS spectrum is large, with values around 2.2 × 10 10 Gy (J/kg), equivalent to the deposition of 620 eV/A 3 . Cooling to liquid N 2 temperature did not significantly slow the damage process. Ion-beam thinning produces an amorphous layer on crystal surfaces. Spectra from the thinnest regions, which are amorphous, exhibit Fe 3+ /ΣFe >0.7. With increase in sample thickness, the Fe 3+ /ΣFe values decrease to a minimum, consistent with data from the undamaged material. The increase of Fe 3+ /ΣFe with respect to electron-beam irradiation is likely caused by loss of H. At low fluences, the loss of H is negligible, thus allowing consistent Fe 3+ /ΣFe values to be measured. The cronstedtite study illustrates the care required when using EELS to measure Fe 3+ /ΣFe values. Similar damage effects occur for a range of high-valence and mixed-oxidation state metals in minerals. EELS is the only spectroscopic method that can be used routinely to determine mixed-valence ratios at the nanometer scale, but care is required when measuring these data. Consideration needs to be given to the incident beam current, fluence, fluence rate, and sample thickness.

Mitochondrial DNA from human tissue culture cells contains heterogeneous sequences located within a previously identified, evolutionarily conserved region termed CSB-2. CSB-2 is located near the origin of heavy-strand mitochondrial DNA synthesis and the major transcriptional promoters for each strand of human mitochondrial DNA. Nucleotide sequence analysis of cloned mitochondrial DNA and electrophoretic analysis of appropriate small fragments from cellular mitochondrial DNA show that the variability is limited to a homopolymer sequence which can range in length from 6 to 12 residues. In vitro transcriptional analyses, using several of these cloned length polymorphs as templates and partially purified human mitochondrial RNA polymerase, demonstrate that the most common polymorphs will support accurate transcriptional initiation.

Strength, elasticity, and biocompatibility make spider silk an attractive resource for the production of artificial biomaterials. Spider silk proteins, spidroins, contain hundreds of repeated poly alanine/glycine-rich blocks and are difficult to produce recombinantly in soluble form. Most previous attempts to produce artificial spider silk fibers have included solubilization steps in nonphysiological solvents. It is here demonstrated that a miniature spidroin from a protein in dragline silk of Euprosthenops australis can be produced in a soluble form in Escherichia coli when fused to a highly soluble protein partner. Although this miniature spidroin contains only four poly alanine/glycine-rich blocks followed by a C-terminal non-repetitive domain, meter-long fibers are spontaneously formed after proteolytic release of the fusion partner. The structure of the fibers is similar to that of dragline silks, and although self-assembled from recombinant proteins they are as strong as fibers spun from redissolved silk. Moreover, the fibers appear to be biocompatible because human tissue culture cells can grow on and attach to the fibers. These findings enable controlled production of high-performance biofibers at large scale under physiological conditions.

Chapter 6 - The Use of Targeted Proteomics to Determine the Stoichiometry of Large Macromolecular Assemblies

Transposons are highly abundant in eukaryotic genomes, but their mobilization must be finely tuned to maintain host organism fitness and allow for transposon propagation. Forty percent of the human genome is comprised of transposable element sequences, and the most abundant cut-and-paste transposons are from the hAT superfamily. We found that the hAT transposase TcBuster from Tribolium castaneum formed filamentous structures, or rodlets, in human tissue culture cells, after gene transfer to adult mice, and ex vivo in cell-free conditions, indicating that host co-factors or cellular structures were not required for rodlet formation. Time-lapsed imaging of GFP-laced rodlets in human cells revealed that they formed quickly in a dynamic process involving fusion and fission. We delayed the availability of the transposon DNA and found that transposition declined after transposase concentrations became high enough for visible transposase rodlets to appear. In combination with earlier findings for maize Ac elements, these results give insight into transposase overproduction inhibition by demonstrating that the appearance of transposase protein structures and the end of active transposition are simultaneous, an effect with implications for genetic engineering and horizontal gene transfer.

ABSTRACTInterferon-gamma is a cytokine secreted by natural killer cells, CD4 TH1 cells and CD8 cytotoxic lymphocytes in response to mitogenic and antigenic stimuli. It is endowed with multiple biological activities and because of that is one of the first targets of recombinant DNA technology. Human interferon-gamma (hIFNγ) is expressed so far in Escherichia coli and various eukaryotic hosts. The disadvantages of the bacterial hIFNγ and unsatisfactory results from the eukaryotic expression were the reason to employ transient expression of the hIFNγ gene in CAP-T and HEKF cells, widely used for industrial production of human proteins. The obtained results demonstrated that (unlike with other human genes): i) cell viability decreased very fast and reached the critical level of 50% after five days only; ii) cell density did not exceed 0.7x105 cells/mL and sharply decreased after four days of cultivation; iii) the yield of hIFNy secreted in the supernatant did not exceed 5.8 mg/L.

Advanced Design of Dumbbell-shaped Genetic Minimal Vectors Improves Non-coding and Coding RNA Expression

The diverse populations of microtubule polymers in cells are functionally distinguished by different posttranslational modifications, including polyglutamylation. Polyglutamylation is enriched on subsets of microtubules including those found in the centrioles, mitotic spindle, and cilia. However, whether this modification alters intrinsic microtubule dynamics or affects extrinsic associations with specific interacting partners remains to be determined. Here we identify the microtubule-binding protein centriole and spindle-associated protein (CSAP), which colocalizes with polyglutamylated tubulin to centrioles, spindle microtubules, and cilia in human tissue culture cells. Reducing tubulin polyglutamylation prevents CSAP localization to both spindle and cilia microtubules. In zebrafish, CSAP is required for normal brain development and proper left-right asymmetry, defects that are qualitatively similar to those reported previously for depletion of polyglutamylation-conjugating enzymes. We also find that CSAP is required for proper cilia beating. Our work supports a model in which polyglutamylation can target selected microtubule-associated proteins, such as CSAP, to microtubule subpopulations, providing specific functional capabilities to these populations.