GFAP filaments are core elements of the cytoskeleton in astrocytes, shaping cellular processes and influencing central nervous system function and pathology. How these filaments are built at the molecular level has remained unclear. Using cryo-electron microscopy and cryo-electron tomography, we show that GFAP filaments are helical polymers assembled from extended and twisted coiled-coil tetramers that interdigitate to form a complex filament tube. In the filament lumen, the low-complexity head domains aggregate to form a flexible fibre, while the tail domains extend from the surface and facilitate higher-order bundling of the filaments, indicating that both the low-complexity structural regions and the well-ordered coiled-coils of the filament tube contribute to the unique structure of GFAP filaments. Our findings provide molecular insights into the pathogenesis of Alexander Disease, localising multiple deleterious mutations to the critical interlock interaction between successive tetramers emergent in the final filament assembly.

Intermediate filaments (IFs) are central to the mechanical integrity of metazoan cells and play critical roles in various fundamental cellular and multicellular processes, including cell motility, signal transduction, and wound healing. To perform their functions, IF proteins self-assemble into nanoscale biopolymers, each exhibiting unique properties that are finely tuned to their specific roles across different tissue types. However, the 3D structure of IFs has remained largely unresolved due to their intrinsic flexibility and polymorphism. This chapter reviews recent advances in the structural analysis of IFs, with a focus on vimentin IFs (VIFs), which are featuring a helical tube with a central luminal fiber. We discuss how AlphaFold-based modeling, chemical cross-linking data, and cryo-electron microscopy (cryo-EM) reconstructions have been integrated to generate a detailed structural model of VIFs, highlighting key features such as the helical symmetry of the filaments and the nature of the luminal fiber. Additionally, we explore potential sources of IF polymorphism and their implications for the analysis of IF structures.

In the cell nucleus, chromatin is anchored to the nuclear lamina, a network of lamin filaments and binding proteins that underly the inner nuclear membrane. The nuclear lamina is involved in chromatin organization through the interaction of lamina-associated domains within the densely packed heterochromatin regions. Using cryo-focused ion beam milling in conjunction with cryo-electron tomography, we analyzed the distribution of nucleosomes at the lamin-chromatin interface at the nanometer scale. Depletion of lamins A and C reduced nucleosome concentration at the nuclear periphery, while B-type lamin depletion contributed to nucleosome density in proximity to the lamina but not further away. We then investigated whether specific lamins can mediate direct interactions with chromatin. Using cryo-electron microscopy, we identified a specific binding motif of the lamin A tail domain that interacts with nucleosomes, distinguishing it from the other lamin isoforms. Furthermore, we examined chromatin structure dynamics using a genome-wide analysis that revealed lamin-dependent macroscopic-scale alterations in gene expression and chromatin remodeling. Our findings provide detailed insights into the dynamic and structural interplay between lamin isoforms and chromatin, molecular interactions that shape chromatin architecture and epigenetic regulation.

Focal adhesions (FAs) are dynamic macromolecular assemblies that anchor the actin cytoskeleton to the extracellular matrix via integrin receptors, thereby regulating cell morphology and migration. Although FA maturation and organization have been extensively studied, it remains unclear how regulatory proteins influence the 3D architecture of FAs. Here, we show that loss of the vasodilator-stimulated phosphoprotein (VASP) impairs adhesion dynamics. We employed CRISPR/Cas9-mediated knockout of VASP and/or the mechanosensitive adaptor protein zyxin to investigate their respective roles in actin–adhesion coupling. Loss of VASP and zyxin correlates with altered FA morphology and impaired dynamics. Using cryo-electron tomography (cryo-ET), we resolved the polarity of individual actin filaments associated with FAs and identified a contractility-related actin layer enriched with tropomyosin. VASP and zyxin are required for the assembly of dense and aligned actin bundles with uniform polarity, oriented with their barbed ends towards the cell edge. In contrast, the tropomyosin-decorated dorsal actin layer remains unaffected by these perturbations. Our findings reveal distinct, layered architectures within FAs and underscore the cooperative role of VASP and zyxin in stabilizing the organization of actin filaments at functional adhesion sites. ### Competing Interest Statement The authors have declared no competing interest.

Intermediate filaments (IFs) are integral components of the cytoskeleton. They provide cells with tissue-specific mechanical properties and are involved in numerous cellular processes. Due to their intricate architecture, a 3D structure of IFs has remained elusive. Here we use cryo-focused ion-beam milling, cryo-electron microscopy and tomography to obtain a 3D structure of vimentin IFs (VIFs). VIFs assemble into a modular, intertwined and flexible helical structure of 40 α-helices in cross-section, organized into five protofibrils. Surprisingly, the intrinsically disordered head domains form a fiber in the lumen of VIFs, while the intrinsically disordered tails form lateral connections between the protofibrils. Our findings demonstrate how protein domains of low sequence complexity can complement well-folded protein domains to construct a biopolymer with striking mechanical strength and stretchability.

Intermediate filaments (IFs) are integral to the cell cytoskeleton, supporting cellular mechanical stability. Unlike other cytoskeletal components, the detailed structure of assembled IFs has yet to be resolved. This review highlights new insights, linking the complex IF hierarchical assembly to their mechanical properties and impact on cellular functions. While we focus on vimentin IFs, we draw comparisons to keratins, showcasing the distinctive structural and mechanical features that underlie their unique mechanical responses.

Physical interactions of cells with the underlying extracellular matrix (ECM) play key roles in multiple cellular processes. The actin cytoskeleton is a central driver and regulator of cellular dynamics, that produces membrane-protrusions such as lamellipodia and filopodia. Here, we examined actin organization in expanding lamellipodia during early stages of cell spreading. To gain insight into the 3D actin organization, we plated fibroblasts on galectin-8 coated EM grids, an ECM protein presents in disease states. We then combined cryo-electron tomography with advanced image processing tools for reconstructing the structure of F-actin in the lamellipodia. This approach enabled us to resolve the polarity and orientation of filaments, and the structure of the Arp2/3 complexes associated with F-actin branches. We show that F-actin in lamellipodial protrusions forms a dense network with three distinct sub-domains. One consists primarily of radial filaments, with their barbed ends pointing towards the membrane, the other is enriched with parallel filaments that run between the radial fibers, in addition to an intermediate sub-domain. Surprisingly, a minor, yet significant (~10%) population of actin filaments, are oriented with their barbed-ends towards the cell center. Our results provide structural insights into F-actin assembly and dynamic reorganization in the leading edge of spreading cells.

ABSTRACT Lamins are the major constituent of the nuclear lamina, a protein meshwork underlying the inner nuclear membrane. Nuclear lamins are type V intermediate filaments that assemble into ~3.5 nm thick filaments. To date, only the conditions for the in vitro assembly of Caenorhabditis elegans lamin (Ce-lamin) are known. Here, we investigated the assembly of Ce-lamin filaments by cryo-electron microscopy and tomography. We show that Ce-lamin is composed of ~3.5 nm protofilaments that further interact in vitro and are often seen as 6–8 nm thick filaments. We show that the assembly of lamin filaments is undisturbed by the removal of flexible domains, that is, the intrinsically unstructured head and tail domains. In contrast, much of the coiled-coil domains are scaffold elements that are essential for filament assembly. Moreover, our results suggest that Ce-lamin helix 1A has a minor scaffolding role but is important to the lateral assembly regulation of lamin protofilaments.

During activation the platelet cytoskeleton is reorganized, inducing adhesion to the extracellular matrix and cell spreading. These processes are critical for wound healing and clot formation. Initially, this task relies on the formation of strong cellular-extracellular matrix interactions, exposed in subendothelial lesions. Despite the medical relevance of these processes, there is a lack of high-resolution structural information on the platelet cytoskeleton controlling cell spreading and adhesion. Here, we present in situ structural analysis of membrane receptors and the underlying cytoskeleton in platelet protrusions by applying cryoelectron tomography to intact platelets. We utilized three-dimensional averaging procedures to study receptors at the plasma membrane. Analysis of substrate interaction-free receptors yielded one main structural class resolved to 26 Å, resembling the αIIbβ3 integrin folded conformation. Furthermore, structural analysis of the actin network in pseudopodia indicates a nonuniform polarity of filaments. This organization would allow generation of the contractile forces required for integrin-mediated cell adhesion.

The LMNA gene encodes the A-type lamins, which polymerize into ∼3.5-nm-thick filaments and, together with B-type lamins and associated proteins, form the nuclear lamina. Mutations in LMNA cause a wide variety of pathologies. In this study, we analyzed the nuclear lamina of embryonic fibroblasts from LmnaH222P/H222P mice, which develop cardiomyopathy and muscular dystrophy. Although the organization of the lamina appeared unaltered, there were changes in chromatin and B-type lamin expression. An increase in nuclear size and consequently a relative reduction in heterochromatin near the lamina allowed for a higher resolution structural analysis of lamin filaments using cryo-electron tomography. This was most apparent when visualizing lamin filaments in situ and using a nuclear extraction protocol. Averaging of individual segments of filaments in LmnaH222P/H222P mouse fibroblasts resolved two polymers that constitute the mature filaments. Our findings provide better views of the organization of lamin filaments and the effect of a striated muscle disease-causing mutation on nuclear structure.

The actin cytoskeleton plays a fundamental role in numerous cellular processes, such as cell motility, cytokinesis, and adhesion to the extracellular matrix. Revealing the polarity of individual actin filaments in intact cells would foster an unprecedented understanding of cytoskeletal processes and their associated mechanical forces. Cryo-electron tomography provides the means for high-resolution structural imaging of cells. However, the low signal-to-noise ratio of cryo-tomograms obscures the high frequencies, and therefore the polarity of actin filaments cannot be directly measured. Here, we developed a method that enables us to determine the polarity of actin filaments in cellular cryo-tomograms. We applied it to reveal the actin polarity distribution in focal adhesions, and show a linear relation between actin polarity and distance from the apical boundary of the adhesion site.

ABSTRACTIntermediate filaments are integral components of the cytoskeleton in metazoan cells. Due to their specific viscoelastic properties they are principal contributors to flexibility and tear strength of cells and tissues. Vimentin, an intermediate filament protein expressed in fibroblasts and endothelial cells, assembles into ~11 nm thick biopolymers, that are involved in a wide variety of cellular functions in health and disease. Here, we reveal the structure of in-situ polymerized vimentin filaments to a subnanometer resolution by applying cryo-electron tomography to mouse embryonic fibroblasts grown on electron microscopy grids. We show that vimentin filaments are tube-like assemblies with a well-defined helical symmetry. Their structure is comprised of five octameric, spring-like protofibrils harboring 40 vimentin polypeptide chains in cross-section. The protofibrils are connected by the intrinsically disordered head and helix 1A domains of vimentin. Individual filaments display two polymerization states characterized by either the presence or absence of a luminal density along the helical axis. The structure of vimentin filaments unveils the generic building plan of the intermediate filament superfamily in molecular details.

Keratin intermediate filaments are an essential and major component of the cytoskeleton in epithelial cells. They form a stable yet dynamic filamentous network extending from the nucleus to the cell periphery, which provides resistance to mechanical stresses. Mutations in keratin genes are related to a variety of epithelial tissue diseases. Despite their importance, the molecular structure of keratin filaments remains largely unknown. In this study, we analyzed the structure of keratin 5/keratin 14 filaments within ghost mouse keratinocytes by cryo-electron microscopy and cryo-electron tomography. By averaging a large number of keratin segments, we have gained insights into the helical architecture of the filaments. Two-dimensional classification revealed profound variations in the diameter of keratin filaments and their subunit organization. Computational reconstitution of filaments of substantial length uncovered a high degree of internal heterogeneity along single filaments, which can contain regions of helical symmetry, regions with less symmetry and regions with significant diameter fluctuations. Cross-section views of filaments revealed that keratins form hollow cylinders consisting of multiple protofilaments, with an electron dense core located in the center of the filament. These findings shed light on the complex and remarkable heterogenic architecture of keratin filaments, suggesting that they are highly flexible, dynamic cytoskeletal structures.

Vinculin plays a fundamental role in integrin-mediated cell adhesion. Activated by talin, it interacts with diverse adhesome components, enabling mechanical coupling between the actin cytoskeleton and the extracellular matrix. Here we studied the interactions of activated full-length vinculin with actin and the way it regulates the organization and dynamics of the Arp2/3 complex-mediated branched actin network. Through a combination of surface patterning and light microscopy experiments we show that vinculin can bundle dendritic actin networks through rapid binding and filament crosslinking. We show that vinculin promotes stable but flexible actin bundles having a mixed-polarity organization, as confirmed by cryo-electron tomography. Adhesion-like synthetic design of vinculin activation by surface-bound talin revealed that clustered vinculin can initiate and immobilize bundles from mobile Arp2/3-branched networks. Our results provide a molecular basis for coordinate actin bundle formation at nascent adhesions.

Most systemic viral gene therapies have been limited by sequestration and degradation of virions, innate and adaptive immunity, and silencing of therapeutic genes within the target cells. Here we engineer a high-affinity protein coat, shielding the most commonly used vector in clinical gene therapy, human adenovirus type 5. Using electron microscopy and crystallography we demonstrate a massive coverage of the virion surface through the hexon-shielding scFv fragment, trimerized to exploit the hexon symmetry and gain avidity. The shield reduces virion clearance in the liver. When the shielded particles are equipped with adaptor proteins, the virions deliver their payload genes into human cancer cells expressing HER2 or EGFR. The combination of shield and adapter also increases viral gene delivery to xenografted tumors in vivo, reduces liver off-targeting and immune neutralization. Our study highlights the power of protein engineering for viral vectors overcoming the challenges of local and systemic viral gene therapies.

Cryo-electron tomography reveals a detailed view of the structural organization of the lamin meshwork within the lamina of the mammalian cell nucleus. In the cell nucleus, the lamina is a mesh of intermediate filament proteins called lamins that connects the nuclear envelope to chromatin. It provides structural stability to the nucleus and has a role in chromatin organization, gene transcription and DNA replication. Ohad Medalia and colleagues use cryo-electron tomography to investigate the structural organization of the lamina in the mammalian nucleus. Their analysis of individual lamin filaments provides information on the appearance and macromolecular assembly of these filaments, and finds some notable structural differences from other elements of the cytoskeleton. The nuclear lamina is a fundamental constituent of metazoan nuclei. It is composed mainly of lamins, which are intermediate filament proteins that assemble into a filamentous meshwork, bridging the nuclear envelope and chromatin1,2,3,4. Besides providing structural stability to the nucleus5,6, the lamina is involved in many nuclear activities, including chromatin organization, transcription and replication7,8,9,10. However, the structural organization of the nuclear lamina is poorly understood. Here we use cryo-electron tomography to obtain a detailed view of the organization of the lamin meshwork within the lamina. Data analysis of individual lamin filaments resolves a globular-decorated fibre appearance and shows that A- and B-type lamins assemble into tetrameric filaments of 3.5 nm thickness. Thus, lamins exhibit a structure that is remarkably different from the other canonical cytoskeletal elements. Our findings define the architecture of the nuclear lamin meshworks at molecular resolution, providing insights into their role in scaffolding the nuclear lamina.

Platelets are essential for hemostasis and wound healing. They are involved in fundamental processes of vascular biology such as angiogenesis, tissue regeneration, and tumor metastasis. Upon activation, platelets shed small plasma membrane vesicles termed platelet-derived microparticles (PMPs). PMPs include functional cell adhesion machinery that comprises transmembrane receptors (most abundant are the αIIbβ3 integrins), cytoskeletal systems and a large variety of adapter and signaling molecules. Glanzmann thrombasthenia (GT) is a condition characterized by platelets that are deficient of the integrin αIIbβ3 heterodimer. Here, we use cryo-electron tomography (cryo-ET) to study the structural organization of PMPs (in both healthy and GT patients), especially the cytoskeleton organization and receptor architecture. PMPs purified from GT patients show a significantly altered cytoskeletal organization, characterized by a reduced number of filaments present, compared to the healthy control. Furthermore, our results show that incubating healthy PMPs with manganese ions (Mn(2+)), in the presence of fibrinogen, induces a major conformational change of integrin receptors, whereas thrombin activation yields a moderate response. These results provide the first insights into the native molecular organization of PMPs.

Nuclear pore complexes (NPCs) perforate the nuclear envelope and allow the exchange of macromolecules between the nucleus and the cytoplasm. To acquire a deeper understanding of this transport mechanism, we analyse the structure of the NPC scaffold and permeability barrier, by reconstructing the Xenopus laevis oocyte NPC from native nuclear envelopes up to 20 Å resolution by cryo-electron tomography in conjunction with subtomogram averaging. In addition to resolving individual protein domains of the NPC constituents, we propose a model for the architecture of the molecular gate at its central channel. Furthermore, we compare and contrast this native NPC structure to one that exhibits reduced transport activity and unveil the spatial properties of the NPC gate.

Structural analysis of macromolecular assemblies in their physiological environment is a challenging task that is instrumental in answering fundamental questions in cellular and molecular structural biology. The continuous development of computational and analytical tools for cryo-electron tomography (cryo-ET) enables the study of these assemblies at a resolution of a few nanometers. Through the implementation of thinning procedures, cryo-ET can now be applied to the reconstruction of macromolecular structures located inside thick regions of vitrified cells and tissues, thus becoming a central tool for structural determinations in various biological disciplines. Here, we focus on the successful in situ applications of cryo-ET to reveal structures of macromolecular complexes within eukaryotic cells.

In eukaryotic cells, cotranslational protein translocation across the endoplasmic reticulum (ER) membrane requires an elaborate macromolecular machinery. While structural details of ribosomes bound to purified and solubilized constituents of the translocon have been elucidated in recent years, little structural knowledge of ribosomes bound to the complete ER protein translocation machinery in a native membrane environment exists. Here, we used cryoelectron tomography to provide a three-dimensional reconstruction of 80S ribosomes attached to functional canine pancreatic ER microsomes in situ. In the resulting subtomogram average at 31 Å resolution, we observe direct contact of ribosomal expansion segment ES27L and the membrane and distinguish several membrane-embedded and lumenal complexes, including Sec61, the TRAP complex and another large complex protruding 90 Å into the lumen. Membrane-associated ribosomes adopt a preferred three-dimensional arrangement that is likely specific for ER-associated polyribosomes and may explain the high translation efficiency of ER-associated ribosomes compared to their cytosolic counterparts.