TY - JOUR AB - Poxviruses are among the largest double-stranded DNA viruses, with members such as variola virus, monkeypox virus and the vaccination strain vaccinia virus (VACV). Knowledge about the structural proteins that form the viral core has remained sparse. While major core proteins have been annotated via indirect experimental evidence, their structures have remained elusive and they could not be assigned to individual core features. Hence, which proteins constitute which layers of the core, such as the palisade layer and the inner core wall, has remained enigmatic. Here we show, using a multi-modal cryo-electron microscopy (cryo-EM) approach in combination with AlphaFold molecular modeling, that trimers formed by the cleavage product of VACV protein A10 are the key component of the palisade layer. This allows us to place previously obtained descriptions of protein interactions within the core wall into perspective and to provide a detailed model of poxvirus core architecture. Importantly, we show that interactions within A10 trimers are likely generalizable over members of orthopox- and parapoxviruses. AU - Datler, Julia AU - Hansen, Jesse AU - Thader, Andreas AU - Schlögl, Alois AU - Bauer, Lukas W AU - Hodirnau, Victor-Valentin AU - Schur, Florian KM ID - 14979 JF - Nature Structural & Molecular Biology KW - Molecular Biology KW - Structural Biology SN - 1545-9993 TI - Multi-modal cryo-EM reveals trimers of protein A10 to form the palisade layer in poxvirus cores ER - TY - JOUR AB - The extracellular matrix (ECM) serves as a scaffold for cells and plays an essential role in regulating numerous cellular processes, including cell migration and proliferation. Due to limitations in specimen preparation for conventional room-temperature electron microscopy, we lack structural knowledge on how ECM components are secreted, remodeled, and interact with surrounding cells. We have developed a 3D-ECM platform compatible with sample thinning by cryo-focused ion beam milling, the lift-out extraction procedure, and cryo-electron tomography. Our workflow implements cell-derived matrices (CDMs) grown on EM grids, resulting in a versatile tool closely mimicking ECM environments. This allows us to visualize ECM for the first time in its hydrated, native context. Our data reveal an intricate network of extracellular fibers, their positioning relative to matrix-secreting cells, and previously unresolved structural entities. Our workflow and results add to the structural atlas of the ECM, providing novel insights into its secretion and assembly. AU - Zens, Bettina AU - Fäßler, Florian AU - Hansen, Jesse AU - Hauschild, Robert AU - Datler, Julia AU - Hodirnau, Victor-Valentin AU - Zheden, Vanessa AU - Alanko, Jonna H AU - Sixt, Michael K AU - Schur, Florian KM ID - 15146 IS - 6 JF - Journal of Cell Biology SN - 0021-9525 TI - Lift-out cryo-FIBSEM and cryo-ET reveal the ultrastructural landscape of extracellular matrix VL - 223 ER - TY - JOUR AB - The actin cytoskeleton plays a key role in cell migration and cellular morphodynamics in most eukaryotes. The ability of the actin cytoskeleton to assemble and disassemble in a spatiotemporally controlled manner allows it to form higher-order structures, which can generate forces required for a cell to explore and navigate through its environment. It is regulated not only via a complex synergistic and competitive interplay between actin-binding proteins (ABP), but also by filament biochemistry and filament geometry. The lack of structural insights into how geometry and ABPs regulate the actin cytoskeleton limits our understanding of the molecular mechanisms that define actin cytoskeleton remodeling and, in turn, impact emerging cell migration characteristics. With the advent of cryo-electron microscopy (cryo-EM) and advanced computational methods, it is now possible to define these molecular mechanisms involving actin and its interactors at both atomic and ultra-structural levels in vitro and in cellulo. In this review, we will provide an overview of the available cryo-EM methods, applicable to further our understanding of the actin cytoskeleton, specifically in the context of cell migration. We will discuss how these methods have been employed to elucidate ABP- and geometry-defined regulatory mechanisms in initiating, maintaining, and disassembling cellular actin networks in migratory protrusions. AU - Fäßler, Florian AU - Javoor, Manjunath AU - Schur, Florian KM ID - 12421 IS - 1 JF - Biochemical Society Transactions KW - Biochemistry SN - 0300-5127 TI - Deciphering the molecular mechanisms of actin cytoskeleton regulation in cell migration using cryo-EM VL - 51 ER - TY - JOUR AB - Regulation of the Arp2/3 complex is required for productive nucleation of branched actin networks. An emerging aspect of regulation is the incorporation of subunit isoforms into the Arp2/3 complex. Specifically, both ArpC5 subunit isoforms, ArpC5 and ArpC5L, have been reported to fine-tune nucleation activity and branch junction stability. We have combined reverse genetics and cellular structural biology to describe how ArpC5 and ArpC5L differentially affect cell migration. Both define the structural stability of ArpC1 in branch junctions and, in turn, by determining protrusion characteristics, affect protein dynamics and actin network ultrastructure. ArpC5 isoforms also affect the positioning of members of the Ena/Vasodilator-stimulated phosphoprotein (VASP) family of actin filament elongators, which mediate ArpC5 isoform–specific effects on the actin assembly level. Our results suggest that ArpC5 and Ena/VASP proteins are part of a signaling pathway enhancing cell migration. AU - Fäßler, Florian AU - Javoor, Manjunath AU - Datler, Julia AU - Döring, Hermann AU - Hofer, Florian AU - Dimchev, Georgi A AU - Hodirnau, Victor-Valentin AU - Faix, Jan AU - Rottner, Klemens AU - Schur, Florian KM ID - 12334 IS - 3 JF - Science Advances KW - Multidisciplinary SN - 2375-2548 TI - ArpC5 isoforms regulate Arp2/3 complex–dependent protrusion through differential Ena/VASP positioning VL - 9 ER - TY - DATA AB - Regulation of the Arp2/3 complex is required for productive nucleation of branched actin networks. An emerging aspect of regulation is the incorporation of subunit isoforms into the Arp2/3 complex. Specifically, both ArpC5 subunit isoforms, ArpC5 and ArpC5L, have been reported to fine-tune nucleation activity and branch junction stability. We have combined reverse genetics and cellular structural biology to describe how ArpC5 and ArpC5L differentially affect cell migration. Both define the structural stability of ArpC1 in branch junctions and, in turn, by determining protrusion characteristics, affect protein dynamics and actin network ultrastructure. ArpC5 isoforms also affect the positioning of members of the Ena/Vasodilator-stimulated phosphoprotein (VASP) family of actin filament elongators, which mediate ArpC5 isoform–specific effects on the actin assembly level. Our results suggest that ArpC5 and Ena/VASP proteins are part of a signaling pathway enhancing cell migration. AU - Schur, Florian KM ID - 14562 TI - Research data of the publication "ArpC5 isoforms regulate Arp2/3 complex-dependent protrusion through differential Ena/VASP positioning" ER - TY - COMP AB - A precise quantitative description of the ultrastructural characteristics underlying biological mechanisms is often key to their understanding. This is particularly true for dynamic extra- and intracellular filamentous assemblies, playing a role in cell motility, cell integrity, cytokinesis, tissue formation and maintenance. For example, genetic manipulation or modulation of actin regulatory proteins frequently manifests in changes of the morphology, dynamics, and ultrastructural architecture of actin filament-rich cell peripheral structures, such as lamellipodia or filopodia. However, the observed ultrastructural effects often remain subtle and require sufficiently large datasets for appropriate quantitative analysis. The acquisition of such large datasets has been enabled by recent advances in high-throughput cryo-electron tomography (cryo-ET) methods. This also necessitates the development of complementary approaches to maximize the extraction of relevant biological information. We have developed a computational toolbox for the semi-automatic quantification of segmented and vectorized fila- mentous networks from pre-processed cryo-electron tomograms, facilitating the analysis and cross-comparison of multiple experimental conditions. GUI-based components simplify the processing of data and allow users to obtain a large number of ultrastructural parameters describing filamentous assemblies. We demonstrate the feasibility of this workflow by analyzing cryo-ET data of untreated and chemically perturbed branched actin filament networks and that of parallel actin filament arrays. In principle, the computational toolbox presented here is applicable for data analysis comprising any type of filaments in regular (i.e. parallel) or random arrangement. We show that it can ease the identification of key differences between experimental groups and facilitate the in-depth analysis of ultrastructural data in a time-efficient manner. AU - Dimchev, Georgi A AU - Amiri, Behnam AU - Fäßler, Florian AU - Falcke, Martin AU - Schur, Florian KM ID - 14502 KW - cryo-electron tomography KW - actin cytoskeleton KW - toolbox TI - Computational toolbox for ultrastructural quantitative analysis of filament networks in cryo-ET data ER - TY - JOUR AB - Toscana virus is a major cause of arboviral disease in humans in the Mediterranean basin during summer. However, early virus-host cell interactions and entry mechanisms remain poorly characterized. Investigating iPSC-derived human neurons and cell lines, we found that virus binding to the cell surface was specific, and 50% of bound virions were endocytosed within 10 min. Virions entered Rab5a+ early endosomes and, subsequently, Rab7a+ and LAMP-1+ late endosomal compartments. Penetration required intact late endosomes and occurred within 30 min following internalization. Virus entry relied on vacuolar acidification, with an optimal pH for viral membrane fusion at pH 5.5. The pH threshold increased to 5.8 with longer pre-exposure of virions to the slightly acidic pH in early endosomes. Strikingly, the particles remained infectious after entering late endosomes with a pH below the fusion threshold. Overall, our study establishes Toscana virus as a late-penetrating virus and reveals an atypical use of vacuolar acidity by this virus to enter host cells. AU - Koch, Jana AU - Xin, Qilin AU - Obr, Martin AU - Schäfer, Alicia AU - Rolfs, Nina AU - Anagho, Holda A. AU - Kudulyte, Aiste AU - Woltereck, Lea AU - Kummer, Susann AU - Campos, Joaquin AU - Uckeley, Zina M. AU - Bell-Sakyi, Lesley AU - Kräusslich, Hans Georg AU - Schur, Florian Km AU - Acuna, Claudio AU - Lozach, Pierre Yves ID - 14255 IS - 8 JF - PLoS Pathogens SN - 1553-7366 TI - The phenuivirus Toscana virus makes an atypical use of vacuolar acidity to enter host cells VL - 19 ER - TY - JOUR AB - The next steps of deep space exploration are manned missions to Moon and Mars. For safe space missions for crew members, it is important to understand the impact of space flight on the immune system. We studied the effects of 21 days dry immersion (DI) exposure on the transcriptomes of T cells isolated from blood samples of eight healthy volunteers. Samples were collected 7 days before DI, at day 7, 14, and 21 during DI, and 7 days after DI. RNA sequencing of CD3+T cells revealed transcriptional alterations across all time points, with most changes occurring 14 days after DI exposure. At day 21, T cells showed evidence of adaptation with a transcriptional profile resembling that of 7 days before DI. At 7 days after DI, T cells again changed their transcriptional profile. These data suggest that T cells adapt by rewiring their transcriptomes in response to simulated weightlessness and that remodeling cues persist when reexposed to normal gravity. AU - Gallardo-Dodd, Carlos J. AU - Oertlin, Christian AU - Record, Julien AU - Galvani, Rômulo G. AU - Sommerauer, Christian AU - Kuznetsov, Nikolai V. AU - Doukoumopoulos, Evangelos AU - Ali, Liaqat AU - Oliveira, Mariana M. S. AU - Seitz, Christina AU - Percipalle, Mathias AU - Nikić, Tijana AU - Sadova, Anastasia A. AU - Shulgina, Sofia M. AU - Shmarov, Vjacheslav A. AU - Kutko, Olga V. AU - Vlasova, Daria D. AU - Orlova, Kseniya D. AU - Rykova, Marina P. AU - Andersson, John AU - Percipalle, Piergiorgio AU - Kutter, Claudia AU - Ponomarev, Sergey A. AU - Westerberg, Lisa S. ID - 14784 IS - 34 JF - Science Advances KW - Multidisciplinary SN - 2375-2548 TI - Exposure of volunteers to microgravity by dry immersion bed over 21 days results in gene expression changes and adaptation of T cells VL - 9 ER - TY - THES AB - The extracellular matrix (ECM) is a hydrated and complex three-dimensional network consisting of proteins, polysaccharides, and water. It provides structural scaffolding for the cells embedded within it and is essential in regulating numerous physiological processes, including cell migration and proliferation, wound healing, and stem cell fate. Despite extensive study, detailed structural knowledge of ECM components in physiologically relevant conditions is still rudimentary. This is due to methodological limitations in specimen preparation protocols which are incompatible with keeping large samples, such as the ECM, in their native state for subsequent imaging. Conventional electron microscopy (EM) techniques rely on fixation, dehydration, contrasting, and sectioning. This results in the alteration of a highly hydrated environment and the potential introduction of artifacts. Other structural biology techniques, such as nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography, allow high-resolution analysis of protein structures but only work on homogenous and purified samples, hence lacking contextual information. Currently, no approach exists for the ultrastructural and structural study of extracellular components under native conditions in a physiological, 3D environment. In this thesis, I have developed a workflow that allows for the ultrastructural analysis of the ECM in near-native conditions at molecular resolution. The developments I introduced include implementing a novel specimen preparation workflow for cell-derived matrices (CDMs) to render them compatible with ion-beam milling and subsequent high-resolution cryo-electron tomography (ET). To this end, I have established protocols to generate CDMs grown over several weeks on EM grids that are compatible with downstream cryo-EM sample preparation and imaging techniques. Characterization of these ECMs confirmed that they contain essential ECM components such as collagen I, collagen VI, and fibronectin I in high abundance and hence represent a bona fide biologically-relevant sample. I successfully optimized vitrification of these specimens by testing various vitrification techniques and cryoprotectants. In order to obtain high-resolution molecular insights into the ultrastructure and organization of CDMs, I established cryo-focused ion beam scanning electron microscopy (FIBSEM) on these challenging and complex specimens. I explored different approaches for the creation of thin cryo-lamellae by FIB milling and succeeded in optimizing the cryo-lift-out technique, resulting in high-quality lamellae of approximately 200 nm thickness. High-resolution Cryo-ET of these lamellae revealed for the first time the architecture of native CDM in the context of matrix-secreting cells. This allowed for the in situ visualization of fibrillar matrix proteins such as collagen, laying the foundation for future structural and ultrastructural characterization of these proteins in their near-native environment. In summary, in this thesis, I present a novel workflow that combines state-of-the-art cryo-EM specimen preparation and imaging technologies to permit characterization of the ECM, an important tissue component in higher organisms. This innovative and highly versatile workflow will enable addressing far-reaching questions on ECM architecture, composition, and reciprocal ECM-cell interactions. AU - Zens, Bettina ID - 12491 KW - cryo-EM KW - cryo-ET KW - FIB milling KW - method development KW - FIBSEM KW - extracellular matrix KW - ECM KW - cell-derived matrices KW - CDMs KW - cell culture KW - high pressure freezing KW - HPF KW - structural biology KW - tomography KW - collagen SN - 2663-337X TI - Ultrastructural characterization of natively preserved extracellular matrix by cryo-electron tomography ER - TY - JOUR AB - With more than 80 members worldwide, the Orthobunyavirus genus in the Peribunyaviridae family is a large genus of enveloped RNA viruses, many of which are emerging pathogens in humans and livestock. How orthobunyaviruses (OBVs) penetrate and infect mammalian host cells remains poorly characterized. Here, we investigated the entry mechanisms of the OBV Germiston (GERV). Viral particles were visualized by cryo-electron microscopy and appeared roughly spherical with an average diameter of 98 nm. Labeling of the virus with fluorescent dyes did not adversely affect its infectivity and allowed the monitoring of single particles in fixed and live cells. Using this approach, we found that endocytic internalization of bound viruses was asynchronous and occurred within 30-40 min. The virus entered Rab5a+ early endosomes and, subsequently, late endosomal vacuoles containing Rab7a but not LAMP-1. Infectious entry did not require proteolytic cleavage, and endosomal acidification was sufficient and necessary for viral fusion. Acid-activated penetration began 15-25 min after initiation of virus internalization and relied on maturation of early endosomes to late endosomes. The optimal pH for viral membrane fusion was slightly below 6.0, and penetration was hampered when the potassium influx was abolished. Overall, our study provides real-time visualization of GERV entry into host cells and demonstrates the importance of late endosomal maturation in facilitating OBV penetration. AU - Windhaber, Stefan AU - Xin, Qilin AU - Uckeley, Zina M. AU - Koch, Jana AU - Obr, Martin AU - Garnier, Céline AU - Luengo-Guyonnot, Catherine AU - Duboeuf, Maëva AU - Schur, Florian KM AU - Lozach, Pierre-Yves ID - 10639 IS - 5 JF - Journal of Virology KW - virology KW - insect science KW - immunology KW - microbiology SN - 0022-538X TI - The Orthobunyavirus Germiston enters host cells from late endosomes VL - 96 ER - TY - JOUR AB - The potential of energy filtering and direct electron detection for cryo-electron microscopy (cryo-EM) has been well documented. Here, we assess the performance of recently introduced hardware for cryo-electron tomography (cryo-ET) and subtomogram averaging (STA), an increasingly popular structural determination method for complex 3D specimens. We acquired cryo-ET datasets of EIAV virus-like particles (VLPs) on two contemporary cryo-EM systems equipped with different energy filters and direct electron detectors (DED), specifically a Krios G4, equipped with a cold field emission gun (CFEG), Thermo Fisher Scientific Selectris X energy filter, and a Falcon 4 DED; and a Krios G3i, with a Schottky field emission gun (XFEG), a Gatan Bioquantum energy filter, and a K3 DED. We performed constrained cross-correlation-based STA on equally sized datasets acquired on the respective systems. The resulting EIAV CA hexamer reconstructions show that both systems perform comparably in the 4–6 Å resolution range based on Fourier-Shell correlation (FSC). In addition, by employing a recently introduced multiparticle refinement approach, we obtained a reconstruction of the EIAV CA hexamer at 2.9 Å. Our results demonstrate the potential of the new generation of energy filters and DEDs for STA, and the effects of using different processing pipelines on their STA outcomes. AU - Obr, Martin AU - Hagen, Wim J.H. AU - Dick, Robert A. AU - Yu, Lingbo AU - Kotecha, Abhay AU - Schur, Florian KM ID - 11155 IS - 2 JF - Journal of Structural Biology KW - Structural Biology SN - 1047-8477 TI - Exploring high-resolution cryo-ET and subtomogram averaging capabilities of contemporary DEDs VL - 214 ER - TY - JOUR AB - One hallmark of plant cells is their cell wall. They protect cells against the environment and high turgor and mediate morphogenesis through the dynamics of their mechanical and chemical properties. The walls are a complex polysaccharidic structure. Although their biochemical composition is well known, how the different components organize in the volume of the cell wall and interact with each other is not well understood and yet is key to the wall’s mechanical properties. To investigate the ultrastructure of the plant cell wall, we imaged the walls of onion (Allium cepa) bulbs in a near-native state via cryo-focused ion beam milling (cryo-FIB milling) and cryo-electron tomography (cryo-ET). This allowed the high-resolution visualization of cellulose fibers in situ. We reveal the coexistence of dense fiber fields bathed in a reticulated matrix we termed “meshing,” which is more abundant at the inner surface of the cell wall. The fibers adopted a regular bimodal angular distribution at all depths in the cell wall and bundled according to their orientation, creating layers within the cell wall. Concomitantly, employing homogalacturonan (HG)-specific enzymatic digestion, we observed changes in the meshing, suggesting that it is—at least in part—composed of HG pectins. We propose the following model for the construction of the abaxial epidermal primary cell wall: the cell deposits successive layers of cellulose fibers at −45° and +45° relative to the cell’s long axis and secretes the surrounding HG-rich meshing proximal to the plasma membrane, which then migrates to more distal regions of the cell wall. AU - Nicolas, William J. AU - Fäßler, Florian AU - Dutka, Przemysław AU - Schur, Florian KM AU - Jensen, Grant AU - Meyerowitz, Elliot ID - 11351 IS - 11 JF - Current Biology KW - General Agricultural and Biological Sciences KW - General Biochemistry KW - Genetics and Molecular Biology SN - 0960-9822 TI - Cryo-electron tomography of the onion cell wall shows bimodally oriented cellulose fibers and reticulated homogalacturonan networks VL - 32 ER - TY - JOUR AB - Inositol hexakisphosphate (IP6) is an assembly cofactor for HIV-1. We report here that IP6 is also used for assembly of Rous sarcoma virus (RSV), a retrovirus from a different genus. IP6 is ~100-fold more potent at promoting RSV mature capsid protein (CA) assembly than observed for HIV-1 and removal of IP6 in cells reduces infectivity by 100-fold. Here, visualized by cryo-electron tomography and subtomogram averaging, mature capsid-like particles show an IP6-like density in the CA hexamer, coordinated by rings of six lysines and six arginines. Phosphate and IP6 have opposing effects on CA in vitro assembly, inducing formation of T = 1 icosahedrons and tubes, respectively, implying that phosphate promotes pentamer and IP6 hexamer formation. Subtomogram averaging and classification optimized for analysis of pleomorphic retrovirus particles reveal that the heterogeneity of mature RSV CA polyhedrons results from an unexpected, intrinsic CA hexamer flexibility. In contrast, the CA pentamer forms rigid units organizing the local architecture. These different features of hexamers and pentamers determine the structural mechanism to form CA polyhedrons of variable shape in mature RSV particles. AU - Obr, Martin AU - Ricana, Clifton L. AU - Nikulin, Nadia AU - Feathers, Jon-Philip R. AU - Klanschnig, Marco AU - Thader, Andreas AU - Johnson, Marc C. AU - Vogt, Volker M. AU - Schur, Florian KM AU - Dick, Robert A. ID - 9431 IS - 1 JF - Nature Communications KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Physics and Astronomy KW - General Chemistry TI - Structure of the mature Rous sarcoma virus lattice reveals a role for IP6 in the formation of the capsid hexamer VL - 12 ER - TY - JOUR AB - The small cellular molecule inositol hexakisphosphate (IP6) has been known for ~20 years to promote the in vitro assembly of HIV-1 into immature virus-like particles. However, the molecular details underlying this effect have been determined only recently, with the identification of the IP6 binding site in the immature Gag lattice. IP6 also promotes formation of the mature capsid protein (CA) lattice via a second IP6 binding site, and enhances core stability, creating a favorable environment for reverse transcription. IP6 also enhances assembly of other retroviruses, from both the Lentivirus and the Alpharetrovirus genera. These findings suggest that IP6 may have a conserved function throughout the family Retroviridae. Here, we discuss the different steps in the viral life cycle that are influenced by IP6, and describe in detail how IP6 interacts with the immature and mature lattices of different retroviruses. AU - Obr, Martin AU - Schur, Florian KM AU - Dick, Robert A. ID - 10103 IS - 9 JF - Viruses KW - virology KW - infectious diseases SN - 1999-4915 TI - A structural perspective of the role of IP6 in immature and mature retroviral assembly VL - 13 ER - TY - JOUR AB - A precise quantitative description of the ultrastructural characteristics underlying biological mechanisms is often key to their understanding. This is particularly true for dynamic extra- and intracellular filamentous assemblies, playing a role in cell motility, cell integrity, cytokinesis, tissue formation and maintenance. For example, genetic manipulation or modulation of actin regulatory proteins frequently manifests in changes of the morphology, dynamics, and ultrastructural architecture of actin filament-rich cell peripheral structures, such as lamellipodia or filopodia. However, the observed ultrastructural effects often remain subtle and require sufficiently large datasets for appropriate quantitative analysis. The acquisition of such large datasets has been enabled by recent advances in high-throughput cryo-electron tomography (cryo-ET) methods. This also necessitates the development of complementary approaches to maximize the extraction of relevant biological information. We have developed a computational toolbox for the semi-automatic quantification of segmented and vectorized filamentous networks from pre-processed cryo-electron tomograms, facilitating the analysis and cross-comparison of multiple experimental conditions. GUI-based components simplify the processing of data and allow users to obtain a large number of ultrastructural parameters describing filamentous assemblies. We demonstrate the feasibility of this workflow by analyzing cryo-ET data of untreated and chemically perturbed branched actin filament networks and that of parallel actin filament arrays. In principle, the computational toolbox presented here is applicable for data analysis comprising any type of filaments in regular (i.e. parallel) or random arrangement. We show that it can ease the identification of key differences between experimental groups and facilitate the in-depth analysis of ultrastructural data in a time-efficient manner. AU - Dimchev, Georgi A AU - Amiri, Behnam AU - Fäßler, Florian AU - Falcke, Martin AU - Schur, Florian KM ID - 10290 IS - 4 JF - Journal of Structural Biology KW - Structural Biology SN - 1047-8477 TI - Computational toolbox for ultrastructural quantitative analysis of filament networks in cryo-ET data VL - 213 ER - TY - JOUR AB - De novo loss of function mutations in the ubiquitin ligase-encoding gene Cullin3 lead to autism spectrum disorder (ASD). In mouse, constitutive haploinsufficiency leads to motor coordination deficits as well as ASD-relevant social and cognitive impairments. However, induction of Cul3 haploinsufficiency later in life does not lead to ASD-relevant behaviors, pointing to an important role of Cul3 during a critical developmental window. Here we show that Cul3 is essential to regulate neuronal migration and, therefore, constitutive Cul3 heterozygous mutant mice display cortical lamination abnormalities. At the molecular level, we found that Cul3 controls neuronal migration by tightly regulating the amount of Plastin3 (Pls3), a previously unrecognized player of neural migration. Furthermore, we found that Pls3 cell-autonomously regulates cell migration by regulating actin cytoskeleton organization, and its levels are inversely proportional to neural migration speed. Finally, we provide evidence that cellular phenotypes associated with autism-linked gene haploinsufficiency can be rescued by transcriptional activation of the intact allele in vitro, offering a proof of concept for a potential therapeutic approach for ASDs. AU - Morandell, Jasmin AU - Schwarz, Lena A AU - Basilico, Bernadette AU - Tasciyan, Saren AU - Dimchev, Georgi A AU - Nicolas, Armel AU - Sommer, Christoph M AU - Kreuzinger, Caroline AU - Dotter, Christoph AU - Knaus, Lisa AU - Dobler, Zoe AU - Cacci, Emanuele AU - Schur, Florian KM AU - Danzl, Johann G AU - Novarino, Gaia ID - 9429 IS - 1 JF - Nature Communications KW - General Biochemistry KW - Genetics and Molecular Biology TI - Cul3 regulates cytoskeleton protein homeostasis and cell migration during a critical window of brain development VL - 12 ER - TY - JOUR AB - Cryo electron tomography with subsequent subtomogram averaging is a powerful technique to structurally analyze macromolecular complexes in their native context. Although close to atomic resolution in principle can be obtained, it is not clear how individual experimental parameters contribute to the attainable resolution. Here, we have used immature HIV-1 lattice as a benchmarking sample to optimize the attainable resolution for subtomogram averaging. We systematically tested various experimental parameters such as the order of projections, different angular increments and the use of the Volta phase plate. We find that although any of the prominently used acquisition schemes is sufficient to obtain subnanometer resolution, dose-symmetric acquisition provides considerably better outcome. We discuss our findings in order to provide guidance for data acquisition. Our data is publicly available and might be used to further develop processing routines. AU - Turoňová, Beata AU - Hagen, Wim J.H. AU - Obr, Martin AU - Mosalaganti, Shyamal AU - Beugelink, J. Wouter AU - Zimmerli, Christian E. AU - Kräusslich, Hans Georg AU - Beck, Martin ID - 7511 JF - Nature Communications TI - Benchmarking tomographic acquisition schemes for high-resolution structural biology VL - 11 ER - TY - JOUR AB - The actin-related protein (Arp)2/3 complex nucleates branched actin filament networks pivotal for cell migration, endocytosis and pathogen infection. Its activation is tightly regulated and involves complex structural rearrangements and actin filament binding, which are yet to be understood. Here, we report a 9.0 Å resolution structure of the actin filament Arp2/3 complex branch junction in cells using cryo-electron tomography and subtomogram averaging. This allows us to generate an accurate model of the active Arp2/3 complex in the branch junction and its interaction with actin filaments. Notably, our model reveals a previously undescribed set of interactions of the Arp2/3 complex with the mother filament, significantly different to the previous branch junction model. Our structure also indicates a central role for the ArpC3 subunit in stabilizing the active conformation. AU - Fäßler, Florian AU - Dimchev, Georgi A AU - Hodirnau, Victor-Valentin AU - Wan, William AU - Schur, Florian KM ID - 8971 JF - Nature Communications KW - General Biochemistry KW - Genetics and Molecular Biology KW - General Physics and Astronomy KW - General Chemistry SN - 2041-1723 TI - Cryo-electron tomography structure of Arp2/3 complex in cells reveals new insights into the branch junction VL - 11 ER - TY - JOUR AB - Retrovirus assembly is driven by the multidomain structural protein Gag. Interactions between the capsid domains (CA) of Gag result in Gag multimerization, leading to an immature virus particle that is formed by a protein lattice based on dimeric, trimeric, and hexameric protein contacts. Among retroviruses the inter- and intra-hexamer contacts differ, especially in the N-terminal sub-domain of CA (CANTD). For HIV-1 the cellular molecule inositol hexakisphosphate (IP6) interacts with and stabilizes the immature hexamer, and is required for production of infectious virus particles. We have used in vitro assembly, cryo-electron tomography and subtomogram averaging, atomistic molecular dynamics simulations and mutational analyses to study the HIV-related lentivirus equine infectious anemia virus (EIAV). In particular, we sought to understand the structural conservation of the immature lentivirus lattice and the role of IP6 in EIAV assembly. Similar to HIV-1, IP6 strongly promoted in vitro assembly of EIAV Gag proteins into virus-like particles (VLPs), which took three morphologically highly distinct forms: narrow tubes, wide tubes, and spheres. Structural characterization of these VLPs to sub-4Å resolution unexpectedly showed that all three morphologies are based on an immature lattice with preserved key structural components, highlighting the structural versatility of CA to form immature assemblies. A direct comparison between EIAV and HIV revealed that both lentiviruses maintain similar immature interfaces, which are established by both conserved and non-conserved residues. In both EIAV and HIV-1, IP6 regulates immature assembly via conserved lysine residues within the CACTD and SP. Lastly, we demonstrate that IP6 stimulates in vitro assembly of immature particles of several other retroviruses in the lentivirus genus, suggesting a conserved role for IP6 in lentiviral assembly. AU - Dick, Robert A. AU - Xu, Chaoyi AU - Morado, Dustin R. AU - Kravchuk, Vladyslav AU - Ricana, Clifton L. AU - Lyddon, Terri D. AU - Broad, Arianna M. AU - Feathers, J. Ryan AU - Johnson, Marc C. AU - Vogt, Volker M. AU - Perilla, Juan R. AU - Briggs, John A. G. AU - Schur, Florian KM ID - 7464 IS - 1 JF - PLOS Pathogens SN - 1553-7374 TI - Structures of immature EIAV Gag lattices reveal a conserved role for IP6 in lentivirus assembly VL - 16 ER - TY - DATA AB - Cryo-electron microscopy (cryo-EM) of cellular specimens provides insights into biological processes and structures within a native context. However, a major challenge still lies in the efficient and reproducible preparation of adherent cells for subsequent cryo-EM analysis. This is due to the sensitivity of many cellular specimens to the varying seeding and culturing conditions required for EM experiments, the often limited amount of cellular material and also the fragility of EM grids and their substrate. Here, we present low-cost and reusable 3D printed grid holders, designed to improve specimen preparation when culturing challenging cellular samples directly on grids. The described grid holders increase cell culture reproducibility and throughput, and reduce the resources required for cell culturing. We show that grid holders can be integrated into various cryo-EM workflows, including micro-patterning approaches to control cell seeding on grids, and for generating samples for cryo-focused ion beam milling and cryo-electron tomography experiments. Their adaptable design allows for the generation of specialized grid holders customized to a large variety of applications. AU - Schur, Florian KM ID - 14592 TI - STL-files for 3D-printed grid holders described in Fäßler F, Zens B, et al.; 3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy ER -