Microscopy Conference 2023

Session chairs

Prof. Dr. phil. nat.

Prof. Dr.

naRNA is a canonical neutrophil extracellular trap component

Abstract text (incl. figure legends and references) Neutrophil extracellular traps (NETs) have been established as a key feature of cellular innate immunity that is mediated by polymorphonuclear neutrophils (PMNs), the primary leukocyte population in humans. NETs form web-like structures that are composed of DNA, histones, and antimicrobial proteins. They trap and kill microbial invaders thereby enhancing host defense. Whilst DNA has been in focus as a primary structural component of NETs, we here characterize naRNA (NET-associated RNA), as a new canonical, abundant, and largely unexplored NET component. In order to contribute to the understanding of the localization of these RNAs and the composition of the NET structures we have analysed PMNs on the ultrastructural level both by scanning and transmission electron microscopy. Immunolabeling using anti-rRNA antibodies confirmed the presence of RNA in NET structures on ultrathin sections of plastic embedded samples corroborating fluorescence microscopy date. To show the localization of RNA to the NET structures also in SEM we established an immunolabeling protocol for this kind of samples.

LS 6: Pathology, pathogens and diagnostics

LS 7: Advances in sample preparation

Poster

Electron microscopic analysis of wrapping glial differentiation in adult Drosophila

Abstract text (incl. figure legends and references) Axonal wrapping starts in the developing Drosophila larva and is similar to the non-myelinating Schwann cell in the vertebrate PNS. Myelin is a more sophisticated form of axonal wrapping in vertebrates but has not been reported for Drosophila so far. Nevertheless, myelin-like structures have been described in several invertebrate species, e.g., in earthworms, shrimps, prawns, and cockroaches. Here we demonstrate that myelin-like structures also exist in the Drosophila nervous system. Abutting the myelinated axon segments, we found a prominent lacunar system formed by glial cell processes. The glial lacunar system comprises an exceptionally wide, liquid-filled extracellular space that possibly constitutes an ion reservoir meeting the high demand for ions needed for sustained firing of action potentials. We therefore analysed the localization of the only voltage-gated sodium channel encoded by the Drosophila genome termed Para. Antibody labelling indicates that Para predominantly localizes in motor axons at a position that coincides with the localization of the glial lacunae. To further test this we labelled the endogenous Para protein by inserting an Apex2 encoding exon, which allows to detect Para protein localization in the electron microscope. Our findings will be discussed in the light of the evolution of fast and precise signal transduction in the peripheral nervous system.

Constructing artificial membrane structures on graphene to resolve membrane protein conformational dynamics

Abstract text (incl. figure legends and references) Mechanistic understanding of membrane-based processes requires detailed insights of the membrane environment and its impact on protein structure and function. Membrane properties such as fluidity, curvature, and lipid phase segregation have tremendous influence to membrane protein structure. To explore the conformational spectra of membrane protein on variable membrane structures, we tether liposomes as membrane curvature models and fuse them into planar lipid bilayer on graphene-coated substrate. For this purpose, ultra small unilamellar vesicles (USUVs) are prepared with a mean hydrodynamic diameter of 40±15nm. Using biotin-streptavidin interaction, stable immobilization of the USUVs on graphene-supported lipid monolayer (GSLM) is confirmed by negative staining electron microscopy (EM). The USUVs can thus be used to integrate individual ABC transporter MsbA per liposome on graphene-coated EM grid to determine the correlation of protein structure with membrane curvature. By fusing the USUVs on GSLM on glass substrate, fluorescence recovery after photobleaching reveals a fluidic membrane structure. Based on these artificial membrane structures on graphene, we aim to combine single molecule fluorescence microscopy and cryo-EM to correlatively investigate the time-resolved conformational dynamics of membrane protein in controlled membrane environment.

LS 5: Correlative and multimodal microscopy

Theoretical framework and experimental solution for the air-water interface adsorption problem in cryoEM

Abstract text (incl. figure legends and references) Introduction: As cryogenic electron microscopy (cryoEM) gains traction in the structural biology community as a method of choice for determining atomic structures of biological complexes, it has been increasingly recognized that many complexes that behave well under conventional negative-stain electron microscopy tend to have preferential orientation, aggregate or simply mysteriously "disappear" on cryoEM grids, but the reasons for such misbehavior are not well understood, limiting systematic approaches to solving the problem. Objective: This study aims to develop a theoretical formulation that explains the phenomenon of particles preferential orientation and aggregation during cryoEM sample preparation. Materials &amp; methods: We first develop a mathematical model to calculate the surface energy of particles that migrate to the air-water interface (AWI). Then we employed GroEL as a model protein to confirm our formulations using cryogenic electron tomography (cryoET) technique. Results: By conducting cryogenic electron tomography (cryoET) with the widely-tested sample, GroEL, we demonstrate that, in standard buffer solution, nearly all particles migrate to the AWI. Gradual reduction of the surface tension by introducing surfactants decreased the percentage of particles exposed to the surface. By conducting single-particle cryoEM, we confirm that the surfactants do not damage the biological complex, thus suggesting that they might offer a practical, simple and general solution to the problem for high-resolution cryoEM. Application of this solution to a real-world AWI adsorption problem with a more challenging membrane protein, namely, ClC-1 channel, has led to its first atomic structure using cryoEM. Conclusion: Our formulation predicts that all particles migrate to the air-water interface (AWI) to lower the total potential surface energy —rationalizing the use of surfactant, which is a direct solution to lowering the surface tension of the aqueous solution. Figure 1. Side-view schematics of a hole of cryoEM grid, highlighting particle behavior and distribution, in relation to AWI. Figure 2. GroEL particle distribution in vitrified samples with different concentrations of FFC8. Figure 3. Near-atomic cryoEM reconstructions of GroEL, demonstrating no structural damage introduced by the surfactants at the indicated concentrations—the proposed solution to the AWI problem. Figure 4. Example of the AWI adsorption problem in cryoEM and application to real-world AWI adsorption problem.

LS 2: Cryo-tomography

A fast and uranium-free method for platinum replica preparation of membrane cortices

Abstract text (incl. figure legends and references) Conventional room-temperature preparation of biological samples for electron microscopy (EM) is often time-consuming and involves treatment of the sample with different fixation agents to stabilize the structure and enable its later observation in a vacuum under a high-energy electron beam. The fixation agents in general are extremely hazardous chemicals, some of which are subject to strict regulations, with uranium salts being the most complicated due to radioactivity. Some alternatives to uranium were proposed, but their use is mostly limited to post-staining of sections or negative staining. Here we propose to expand the range of their use by substituting uranyl acetate (UA) in the production of the platinum replicas from cell cortices. Our sample preparation procedure is based on previously published methods (1, 2). Cells, cultured on the glass coverslips, washed off culture medium with Ca2+-free Ringer solution, additionally &quot;glued&quot; using PLL 0.1 mg/ml, swollen in hypotonic buffer (1/3 of normal PHEM concentration), and subsequently unroofed by sonication in PHEM buffer of normal concentration. Cell cortices were immediately fixed in a mixture of glutaraldehyde and tannic acid in PHEM. After extensive wash with deionized water the samples were post-fixed with different metal salts in water, then dehydrated in ethanol series followed by hexamethyldisilazane, and air dried instead of critical-point drying to spare time. Platinum replicas were made by coating samples with 2 nm Pt/C; no baking out with carbon was used. Replicas were released with the help of HF acid and after extensive wash transferred to Formvar-coated 75 mesh TEM grids. Images were then acquired with a high-resolution SEM Hitachi S5000 or TEM Philips CM10 with bottom-mounted camera TemCam F-416 (in the latter case the images were digitally inverted to resemble SEM). The quality of all samples was compared to UA-treated sample taken as standard. Experiments revealed that UA could be substituted by gadolinium, hafnium, or iron chloride, as well as acetate salts of gadolinium, neodymium, samarium, or ammonium molybdate or osmium tetroxide without losing sample quality. Negligible sample thickness after unroofing allowed to shorten the time down to two hours from starting the preparation to image acquisition. With little modifications, it could be adapted for CLEM or gold-immuno EM by altering the primary fixation and introducing immunolabelling prior EM preparation procedure, and thus is a good alternative to existing routines. 1. Heuser, J. Traffic, 2000, 1, 545-52 2. Sochacki, K. A. &amp; Taraska, J. W. 2017, 1663, 219-230

PS 24
Poster session LS 7: Advances in sample preparation

Appointment

Session chairs

Programme

PS 24Poster session LS 7: Advances in sample preparation

Appointment

Session chairs

Programme

PS 24
Poster session LS 7: Advances in sample preparation