Microscopy Conference 2023

Session chairs

Prof. Dr.

Cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET) provide us a high-resolution gateway to the molecular processes that underlie life. These techniques allow us to view biological molecules in their native state and at high resolution, to image and determine their dynamics and, moreover, their socio-molecular interactions. Still, improvements and developments in sample preparation, workflows that integrate different tools and co-localization strategies, along with new image processing approaches are needed to exploit the full potential of these methods. This session will discuss the latest technological advances and present tangible examples that demonstrate the strength of both cryoEM and cryoET.

On the way to objective tomogram analysis

Abstract text (incl. figure legends and references) Cryo-electron tomography (Cryo-ET) has become an essential tool for in situ structural biology. Recent developments both in hardware and software lead to (relatively) high throughput in tilt-series acquisition as well as more automated tilt-series alignment and tomogram reconstruction. For subsequent tomogram analysis, most of the method development has been focused on automated particle picking and subtomogram averaging (STA). While these techniques are essential for high-resolution structure determination, they are often "blind" to the rich cellular environment from which the particles come and leave a large portion of the tomograms unexplored. In my talk I will discuss advantages and ways of incorporating contextual information into the particle picking and STA workflows and show how using meta data from STA can be used to analyze the remaining parts of cellular tomograms.

LS 1: High-resolution cryo-EM

LS 2: Cryo-tomography

Invited talk

Elucidating molecular machineries essential in cell migration and virus assembly using cryo-ET

Assistant Professor

Invited speaker

Structural basis of proteasome impairment by protein aggregates in ALS/FTD

Abstract text (incl. figure legends and references) Protein aggregation is a hallmark of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, the mechanisms linking aggregation to neurotoxicity remain poorly understood, partly because only limited information is available on the native structure of protein aggregates inside cells. We address this pressing issue utilizing the latest developments in cryo-electron tomography (cryo-ET). We use cryo-focused ion beam to prepare thin lamellas of vitrified cells containing protein aggregates, and subsequently image them in three dimensions by cryo-ET. This allows us to analyse aggregate structure within pristinely preserved cellular environments and at molecular resolution. Using this approach, we analysed neuronal poly-GA and TDP-43 aggregates. The expansion of a GGGGCC repeat in the C9orf72 gene is the most common cause of familial ALS/FTD. This mutation leads to the translation of poly-GA and other dipeptide repeat proteins, which aggregate in neurons. On the other hand, most ALS/FTD cases are sporadic and display aggregation of C-terminal fragments of TDP-43. Our in situ cryo-ET studies show that both poly-GA [ref #1; Fig.1] and TDP-43 [ref #2, Fig.2] aggregates recruit large amounts of proteasomes in neurons. Subtomogram averaging revealed that association with aggregates alters the conformational landscape of the proteasome. This leads to defects in proteasomal degradation and accumulation of proteasomal substrates in the cells. To understand these processes with mechanistic detail, we reconstituted the poly-GA/proteasome interaction in vitro [ref #3]. Combined single-particle, cryo-ET and biochemical analyses suggest a structural basis of proteasome impairment by poly-GA. Altogether, our studies highlight proteasome inhibition as an important factor of neurotoxicity in ALS/FTD, pointing to possible therapeutic avenues. References [1] Guo et al., Cell (2018) 172 (4), 696-705, DOI: 10.1016/j.cell.2017.12.030 [2] Riemenschneider et al., EMBO Rep (2022) 23 (6), e53890, DOI: 10.15252/embr.202153890 [3] Qu et al., manuscript in preparation Legends Fig 1. C9orf72 poly-GA aggregates consist of polymorphic twisted ribbons (red) that recruit high numbers of proteasomes (light blue). Other large macromolecules, such as ribosomes (gold) or TRiC/CCT (purple) are mostly excluded from the aggregate and accumulate at its periphery. Further analysis of proteasome structure by subtomogram averaging suggests that poly-GA-associated proteasomes are functionally impaired. Guo et al., Cell (2018). Fig 2. Aggregates formed by C-terminal fragments of TDP-43 ("TDP-25") appear amorphous (red) and recruit high numbers of proteasomes (light blue). Other large macromolecules, such as ribosomes (gold) or TRiC/CCT (green) are mostly excluded from the aggregate and accumulate at its periphery. Further analysis of proteasome structure by subtomogram averaging suggests that poly-GA-associated proteasomes are functionally impaired. Riemenschneider et al., EMBO Rep (2022).

LS 5: Correlative and multimodal microscopy

Abstract talk

Prof Dr Dr h.c.

Cryo-electron tomography of SARS-CoV-2 replication and assembly sites reconstituted in cells

Abstract text (incl. figure legends and references) Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is an enveloped virus with a positive single-stranded RNA (ssRNA) genome. Upon infection, endoplasmic reticulum (ER)-derived double-membrane vesicles (DMVs) serve as replication organelles. Recently a structure spanning the DMVs was identified serving as a putative pore dedicated to the translocation of the newly synthesized viral genome and mRNAs from the DMV lumen into the cytoplasm. The viral membrane (M) and envelope (E) proteins drive virion budding and are responsible for the recruitment of the viral genome packaged by the nucleocapsid (N) protein at the ER-Golgi Intermediate Compartment (ERGIC). However, the exact roles of structural proteins in virus budding are poorly understood and the proteins involved in DMV biogenesis remain to be identified. Here we apply in situ cryo-electron tomography (cryo-ET) to reconstitute the formation of DMVs and virus assembly. To elucidate the DMV pore structure and the role of each structural protein in SARS-CoV-2 budding, we use a transfection-based system allowing us to express different combinations of viral proteins together with a fluorescent marker. Cryo-light microscopy is applied to target transfected cells for cryo-focused ion beam milling and produced lamellae are subjected to cryo-electron tomography. Our data provide information on VLP assembly and the composition and structure of the SARS-CoV-2 DMV pore. In summary, in situ cryo-ET of here established transfection-based system provides an invaluable tool to study SARS-CoV-2 replication and virus assembly at biosafety level 1 condition.

LS 6: Pathology, pathogens and diagnostics

Prof.

Characterization of vRNP clustering in influenza A virus-infected cells using in-situ cryo-electron tomography

Abstract text (incl. figure legends and references) Influenza A virus has an eight-segment single-stranded RNA genome. Each genome segment is packaged into a rod-shaped vRNP complex, eight of which form a bundle containing one copy of each genome segment. During viral assembly, newly produced vRNPs subvert the recycling endosome to travel from the ER exit sites, where they phase-separate in the presence of Rab11, to the plasma membrane where budding takes place. On this route, vRNP selection and bundle formation occur, yet neither the site of clustering nor the mechanism behind are fully understood on a molecular level.To shed light on this important step in the viral replication cycle, we employed in-situ cryo-electron tomography. To analyze the spatial distribution of vRNPs inside cryoFIB-milled cells, we established a segmentation and cluster analysis workflow. Our data show that vRNPs localize to so far uncharacterized compartments that comprise double membranes zippered together by head-to-head interactions of viral hemagglutinin, which is the main mediator of viral entry. vRNPs cluster around these membranes, with numerous points of vRNP-vRNP and vRNP-membrane interactions. Taken together, our data show a previously unknown function of HA during viral assembly in intracellular membrane remodeling and vRNP clustering.

SavvyScan2: enhancements to a multichannel flexible scanning TEM system

Abstract text (incl. figure legends and references) SavvyScan is a system that incorporates flexible pattern scanning and simultaneous acquisition from multiple diffraction detector segments in a Scanning Transmission Electron Microscope (STEM). The microscope operations are fully manageable by SerialEM while the extended hardware is based on multipurpose PC cards from Spectrum Instrumentation GmbH. Interface software is released as open source. In the first publication of the system we presented spiral scans of integrated differential phase contrast (iDPC) images and introduced the useful idea of splitting iDPC to separate depth contrast and phase contrast based on image shift corrections (1). Here we report on recent enhancements to the system. The range of image sizes has been extended based on first-in-first-out acquisition that is in principle limitless. The rate of digital averaging has been extended to maximum to improve signal to noise ratio and the channels are processed and recorded simultaneously to shorten acquisition time. The system now supports automated tilt series acquisition at multiple sites based on the navigation features in SerialEM. SavvyScan software recognizes the Record scans and save them in a multi-slice mrc format per detector channel. The newly supported remote Python scripting in SerialEM is utilized for special tasks such as automated alignment of the diffraction disk at high precision. Additional scan patterns have been introduced, including Lissajous curve and continuous 4X4 raster scans that may potentially reduce instability artifacts in single particle analysis. We learn to exploit the multiple signals from concentric annular segments on the Opal diode (El-Mul Technologies) and HAADF (Fischione) detectors for discrimination of materials on the basis of atomic stoichiometry. (1) Seifer, S.; Houben, L.; Elbaum, M. Flexible STEM with Simultaneous Phase and Depth Contrast. Microscopy and Microanalysis 2021, 1–12. https://doi.org/10.1017/S1431927621012861.

LS 2
Cryo-electron microscopy and cryo-electron tomography

Appointment

Session chairs

Description

Programme

LS 2Cryo-electron microscopy and cryo-electron tomography

Appointment

Session chairs

Description

Programme

LS 2
Cryo-electron microscopy and cryo-electron tomography