Abstract Extracellular vesicles (EVs) are evolutionarily conserved mediators of intercellular communication released by cells into biological fluids and the extracellular environment. Despite their growing relevance in biomedical and veterinary research, knowledge on EVs in marine bivalves remains limited. The aim of this study was to optimize tailored protocols for EV isolation from the hemolymph of the Manila clam ( Ruditapes philippinarum ) based on density gradient ultracentrifugation (dgUC) or size exclusion chromatography (SEC). EV-enriched fractions were identified through nanoparticle tracking analysis, protein quantification, transmission electron microscopy, and cryo-electron microscopy. Both methods successfully isolated small EVs (<200 nm). While dgUC yielded higher-purity preparations, SEC provided a higher recovery rate and compatibility with downstream metabolomic analyses. Metabolomics performed on SEC fractions and on hemolymph, revealed that EV-enriched fractions possessed a distinct metabolic signature including enrichment in metabolites associated with nucleotide metabolism, glycolysis, redox regulation, and energy metabolism. Furthermore, we performed a pilot investigation into the presence of EVs released into conditioned water by Manila clams. Using tangential flow filtration and ultrafiltration, EVs were successfully concentrated from water samples and characterized by nanoparticle tracking analysis, CONAN assay, atomic force microscopy, and electron microscopy. Our findings demonstrate the feasibility of isolating EVs both from Manila clam hemolymph and from conditioned water, providing the first evidence of water-derived EV recovery in aquatic animals. Although further methodological refinement is needed to improve the purity of EVs isolated from water, and additional characterization studies are required to better define the molecular composition of clam-derived EVs, these results establish a foundation for future investigations into the role of EVs in bivalve biology and their potential application as minimally invasive biomarkers for aquaculture, environmental monitoring, and ecosystem health assessment.

Spermatozoa strongly rely on their streamlined morphology to successfully ferti lize an oocyte. A striking example of a morphological defect resulting in infertility is acephalic spermatozoa syndrome, a rare but severe condition leading to detach ment of the sperm head and tail. Among the most common genetic causes for this syndrome are mutations in the linker of nucleo-and cytoskeleton (LINC) complex component SUN5. LINC complexes typically reside in the nuclear envelope, the double-membrane surrounding the nucleus, where they establish a physical bridge between nucleus and cytoplasm. This localization allows them to transduce mechan ical signals from the cytoplasm to the nucleus and to regulate nuclear morphology. In sperm, LINC complexes are essential for a multitude of morphological changes during sperm development, including the reshaping of the nucleus and establishing a stable head-tail junction. Here, using superresolution fluorescence microscopy, we find that sperm-specific SUN5 localizes to the base of the head in human, mouse, and boar sperm. By applying in situ cryoelectron tomography, we find an extensive hexagonal lattice in the nuclear envelope in this region. This lattice appears to main tain a consistent close apposition between the inner and outer nuclear membranes (ONM). Further structural analysis supports a model in which LINC complexes form this lattice by laterally interacting at the ONM. Overall, this study sheds light on nuclear envelope organization in the highly streamlined sperm cell, providing a structural basis for uniform nuclear envelope spacing maintained by a LINC lattice and rationalizing the disruptive effects of SUN5 mutations.

Structural biology is no longer confined to isolated macromolecules. Correlative light and electron microscopy workflows at cryogenic temperatures have been developed and optimised in cultured cell systems, opening the door for cellular structural biology. The next frontier is expanding these approaches to primary cells, tissues, biopsies, and patient material. The complexity of these samples means each presents unique challenges. Here, we review recent advances that enable the study of primary cells and complex samples. These examples demonstrate the diversity in available workflows and indicate that there is still no 'one solution for all'.

Abstract The manchette is a transient microtubule (MT)-based structure that is vital for the correct shaping of sperm during spermiogenesis. Throughout spermiogenesis, the manchette retains structural integrity for several days, raising the question of how its MTs are regulated. Here, using cryo-electron tomography of manchettes isolated from rat testes, we find that manchette MT ends are structurally diverse. We show that the MT-binding protein CLASP2 is present throughout the manchette and likely regulates both MT ends. Using cryo-electron microscopy single particle analysis and super-resolution microscopy, we reveal that SPACA9 and MNMIP1 (SH3D21) bind to the seam of manchette MTs from the luminal side. SPACA9 binds to both α- and β-tubulin of protofilament 1 but does not interact directly with protofilament 13, while MNMIP1 binds directly to protofilament 13. MNMIP1 further extends and threads through the MT lattice at the seam. Our study reveals a novel seam MT inner protein complex with a unique binding mode, providing a plausible explanation for MT regulation that maintains manchette integrity over an extended period.

Linker of nucleo- and cytoskeleton (LINC) complexes reside in the nuclear envelope, the double-membrane surrounding the nucleus, where they establish a physical bridge between nucleus and cytoplasm. LINC complexes are conserved throughout the tree of life and are present in most nucleated cell types in the human body. They play a major role in signal transduction across the nuclear envelope and in regulating nuclear morphology. One of the most drastic nuclear remodeling events occurs during sperm maturation. Multiple sperm-specific LINC complexes are essential for the sperm cell to adapt its highly streamlined nuclear shape and to secure a stable connection between sperm head and tail. Importantly, mutations in the LINC protein SUN5 result in head-tail detachment, also referred to as acephalic spermatozoa syndrome, rendering affected individuals infertile. Here, using super-resolution fluorescence microscopy we find that sperm-specific SUN5 localizes to the base of the human sperm head and by applying in situ cryo-electron tomography we find an extensive two-dimensional lattice in the nuclear envelope at this region. Moreover, this lattice appears to maintain a consistent close apposition between the inner and outer nuclear membranes. Further structural analysis supports a model in which SUN5 forms trimers that laterally interact at the outer nuclear membrane. Overall, this study sheds light on nuclear envelope organization in the highly streamlined sperm cell, providing mechanistic insights into uniform nuclear envelope spacing maintained by a LINC lattice and rationalizing disruptive effects of SUN5 mutations on the sperm head-tail junction. ### Competing Interest Statement The authors have declared no competing interest. European Research Council, https://ror.org/0472cxd90, 101088673

Reproduction, development and homeostasis depend on motile cilia, whose rhythmic beating is powered by a microtubule-based molecular machine called the axoneme. Although an atomic model of the axoneme is available for the alga Chlamydomonas reinhardtii1, structures of mammalian axonemes are incomplete1-5. Furthermore, we do not fully understand how molecular structures of axonemes vary across motile-ciliated cell types in the body. Here we use cryoelectron microscopy, cryoelectron tomography and proteomics to resolve the 96-nm modular repeat of axonemal doublet microtubules (DMTs) from both sperm flagella and epithelial cilia of the oviduct, brain ventricles and respiratory tract. We find that sperm DMTs are the most specialized, with epithelial cilia having only minor differences across tissues. We build a model of the mammalian sperm DMT, defining the positions and interactions of 181 proteins including 34 newly identified proteins. We elucidate the composition of radial spoke 3 and uncover binding sites of kinases associated with regeneration of ATP and regulation of ciliary motility. We discover a sperm-specific, axoneme-tethered T-complex protein ring complex (TRiC) chaperone that may contribute to construction or maintenance of the long flagella of mammalian sperm. We resolve axonemal dyneins in their prestroke states, illuminating conformational changes that occur during ciliary movement. Our results illustrate how elements of chemical and mechanical regulation are embedded within the axoneme, providing valuable resources for understanding the aetiology of ciliopathy and infertility, and exemplifying the discovery power of modern structural biology.

The TRiC chaperonin is responsible for folding ~5%-10% of the proteome in eukaryotic cells. Our recent cryo-electron microscopy studies of axonemes from diverse mammalian cell types led to the surprising discovery that a fully assembled TRiC chaperonin is a structural component of mammalian sperm flagella, where it is tethered to the radial spokes of doublet microtubules. In contrast, axoneme-tethered TRiC is not observed in mammalian epithelial cilia, nor in any of the non-mammalian sperm flagella studied to date. In this Perspective, we explore several hypotheses for the potential functions of axoneme-tethered TRiC in mature sperm.

The development of correctly shaped sperm cells is crucial for male reproductive health and fertility. The manchette is a transient microtubule-based structure that assembles during spermiogenesis and contributes to sperm head shaping. Defects in the manchette can cause sperm deformations and subsequent infertility. Previous studies have suggested that the manchette acts as a cellular transport platform, distributing proteins and vesicles during spermiogenesis in a process known as intra-manchette transport. The manchette and intra-manchette transport are still poorly understood, as high-resolution imaging is missing. Here, we used cryo-electron tomography and proteomics to visualize the manchette and identify some of its transport components. We characterize the overall architecture of the manchette and show that its perinuclear ring thickens as the structure constricts. We observed for the first time dynein directly interacting with the manchette. We further find F-actin as single filaments and filament clusters intercalating with the manchette microtubules. Our results provide new insights into the manchette's architecture and potential role as a transport scaffold, highlighting its significance for the shaping of sperm cells during spermiogenesis.

The explosion of cryo-electron microscopy (cryo-EM) over the last decade has brought with it a range of new approaches for gaining high-resolution structural information on previously inaccessible biological systems. Cryo-EM single-particle analysis (SPA) approaches typically entail overexpression and purification of the target protein. Larger and more complex molecular assemblies often require extensive optimization of the expression, purification and reconstitution procedures. Additionally, prior knowledge of the composition of the structure of interest is required. Approaches employing cryo-focused ion beam (FIB) milling and cryo-electron tomography (cryo-ET) have proven incredibly useful for exploring protein structures within cells while maintaining near-native conditions. Such strategies avoid purification of the target protein or protein complex, yet are often still limited in throughput and achievable resolution. Here, we highlight recent studies demonstrating that the range of samples suitable for SPA is expanding towards increasingly more native samples. We specifically focus on studies investigating complex macromolecular assemblies where tailored sample-preparation strategies made them amenable for SPA, while still keeping them in close-to-native conditions. These examples show that SPA has become a discovery tool for de novo protein identification and complex stoichiometry in more complex and thicker samples.

Abstract Study question How can we make the human sperm connecting piece accessible for structural analysis by cryo-electron tomography (cryo-ET)? Summary answer Preparation of sperm by chemical thinning or production of lamellae via cryo-focused ion beam (cryo-FIB) milling allows direct imaging of the human sperm connecting piece. What is known already Previous studies from our group and others have demonstrated the versatility of cryo-ET to advance our understanding of sperm biology. Structures of macromolecular complexes have been resolved in vitro and in situ, enabling a functional assessment at molecular or even atomic level. Resolving structures at high resolution further allowed identification of proteins previously not known to have a role in mediating male fertility. Such insights are instrumental to further improve diagnosis and treatment of male infertility. Study design, size, duration In our research study we use sperm samples from patients undergoing IVF treatment or donor material, which we receive through an ongoing collaboration with the Division of Woman and Baby, University Medical Centre Utrecht (UMCU). These samples are processed in our own laboratory and subsequently imaged by cryo-ET at the Electron Microscopy Centre of Utrecht University. Participants/materials, setting, methods Due to its thickness, the connecting piece cannot be directly analyzed by cryo-ET. Thus, we designed two strategies to achieve thinning of this area and allow collection of tomograms for structural analysis. While the first approach involves incubation of sperm with a detergent, a reducing agent and heparin, the second approach utilizes cryo-FIB milling to produce thin lamellae of the connecting piece. Main results and the role of chance Both strategies developed to access the connecting piece with cryo-ET are effective and allowed us a glimpse of the molecular landscape of the link between sperm head and tail. Chemical thinning of the connecting piece enabled imaging of the proximal and distal centriole while still embedded in the segmented columns or outer dense fibers, respectively. Furthermore, electron density within the microtubules of both centrioles indicates that they are decorated by microtubule inner proteins. Data obtained from lamellae produced by cryo-FIB milling complements these findings, displaying regular densities along the microtubules extending from the proximal centriole. Analyzing tomograms collected from cryo-FIB lamellae we further found a regular protein density that appears to be connecting the nuclear envelope to the base plate. Moreover, cryo-ET of lamellae allowed a detailed view of nuclear pore complexes and the postacrosomal sheath in their native environment. Limitations, reasons for caution Altering the native state of the sperm cell by chemical preparation is a limitation of this approach. Cryo-FIB milling retains the native state of the cell but is limited in throughput. To improve our cryo-FIB milling approach, we will implement automated milling procedures. Wider implications of the findings Chemical preparation and cryo-FIB milling of sperm enable the in-depth analysis of the connecting piece by cryo-ET. These strategies can now be applied to provide structural details at the molecular level and thus advance our understanding of male infertility related to defects of the head-tail junction. Trial registration number not applicable

Current Influenza virus vaccines primarily induce antibody responses against variable epitopes in hemagglutinin (HA), necessitating frequent updates. However, antibodies against neuraminidase (NA) can also confer protection against influenza, making NA an attractive target for the development of novel vaccines. In this study, we aimed to enhance the immunogenicity of recombinant NA antigens by presenting them multivalently on a nanoparticle carrier. Soluble tetrameric NA antigens of the N1 and N2 subtypes, confirmed to be correctly folded by cryo-electron microscopy structural analysis, were conjugated to Mi3 self-assembling protein nanoparticles using the SpyTag-SpyCatcher system. Immunization of mice with NA-Mi3 nanoparticles induced higher titers of NA-binding and -inhibiting antibodies and improved protection against a lethal challenge compared to unconjugated NA. Additionally, we explored the co-presentation of N1 and N2 antigens on the same Mi3 particles to create a mosaic vaccine candidate. These mosaic nanoparticles elicited antibody titers that were similar or superior to the homotypic nanoparticles and effectively protected against H1N1 and H3N2 challenge viruses. The NA-Mi3 nanoparticles represent a promising vaccine candidate that could complement HA-directed approaches for enhanced potency and broadened protection against influenza A virus.

The manchette is a transient microtubule based structure that plays a vital role in nuclear shaping during spermiogenesis. It comprises thousands of microtubules (MTs) that build a scaffold around the distal half of the nucleus. The manchette distributes proteins and vesicles during spermiogenesis in a process called intra-manchette transport (IMT). The current hypothesis is that IMT shares many similarities with intra-flagellar transport (IFT) and utilizes both MTs and filamentous actin (F-actin). However, IMT is still poorly understood as direct visualization of IMT complexes is missing, and the presence of F-actin has not been experimentally shown. Here, we use proteomics and cryogenic-electron tomography (cryo-ET) to identify and visualize IMT components. We find that F-actin is an integral part of the manchette with two different spatial organizations, namely bundles and single filaments, providing tracks for transport as well as having structural and mechanical roles. We further uncover that IMT on MTs is mediated by two distinct transport machineries: dynein mediated transport of soluble cargo and dynein independent transport for vesicles. Our results provide new insights into the manchette's function as a transport scaffold, highlighting its significance for the polarization of spermatids during spermiogenesis. ### Competing Interest Statement The authors have declared no competing interest.

Sperm cells are terminally differentiated cells that are essential for reproduction in sexually reproducing species. Consistent with their highly specialized function, sperm cells harbor a unique proteome containing many proteins not expressed in somatic cells. In contrast, the post-translational landscape of the sperm proteome remains largely unexplored, limiting our understanding of how modifications such as glycosylation impact sperm function and sperm-egg interactions. Here, we used glycopeptide-centric glycoproteomics to comprehensively characterize protein N-glycosylation in sperm from three mammalian species, revealing clear conservation of glycosylation profiles. We find that glycosylation patterns in sperm proteins are distinct from those in plasma, with as clear distinctive features less sialyation and more paucimannosylation in sperm. Moreover, based on their subcellular location, sperm protein glycosylation varies, with paucimannose species enriched in the acrosomal vesicle, oligomannose species in the sperm head membrane, and complex glycan species in the acrosomal membrane.

Vesicle-encapsulated nonenveloped viruses are a recently recognized alternate form of nonenveloped viruses that can avoid immune detection and potentially increase systemic transmission. Avian orthoreoviruses (ARVs) are the leading cause of various disease conditions among birds and poultry. However, whether ARVs use cellular vesicle trafficking routes for egress and cell-to-cell transmission is still poorly understood. We demonstrated that fusogenic ARV-infected quail cells generated small (~100 nm diameter) extracellular vesicles (EVs) that contained electron-dense material when observed by transmission electron microscope. Cryo-EM tomography indicated that these vesicles did not contain ARV virions or core particles, but the EV fractions of OptiPrep gradients did contain a small percent of the ARV virions released from cells. Western blotting of detergent-treated EVs revealed that soluble virus proteins and the fusogenic p10 FAST protein were contained within the EVs. Notably, virus particles mixed with the EVs were up to 50 times more infectious than virions alone. These results suggest that EVs and perhaps fusogenic FAST-EVs could contribute to ARV virulence.

Sperm motility is crucial to reproductive success in sexually reproducing organisms. Impaired sperm movement causes male infertility, which is increasing globally. Sperm are powered by a microtubule-based molecular machine—the axoneme—but it is unclear how axonemal microtubules are ornamented to support motility in diverse fertilization environments. Here, we present high-resolution structures of native axonemal doublet microtubules (DMTs) from sea urchin and bovine sperm, representing external and internal fertilizers. We identify >60 proteins decorating sperm DMTs; at least 15 are sperm associated and 16 are linked to infertility. By comparing DMTs across species and cell types, we define core microtubule inner proteins (MIPs) and analyze evolution of the tektin bundle. We identify conserved axonemal microtubule-associated proteins (MAPs) with unique tubulin-binding modes. Additionally, we identify a testis-specific serine/threonine kinase that links DMTs to outer dense fibers in mammalian sperm. Our study provides structural foundations for understanding sperm evolution, motility, and dysfunction at a molecular level.

Electron tomography at cryogenic temperatures is becoming a prominent tool in cell biology. It provides three-dimensional detailed information on cellular organization. Advanced image processing of tomogram sub-volumes allows near-atomic in-cell macromolecules structure determination. Here we describe how cells and tissues are prepared for imaging and how data is collected and processed. Selected examples are given to demonstrate the breadth of samples that can be processed thus demonstrating the contribution of cryogenic electron tomography to cell biology; prospects for future developments are discussed.

Enteroviruses are globally prevalent human pathogens responsible for many diseases. The nonstructural protein 2C is a AAA+ helicase and plays a key role in enterovirus replication. Drug repurposing screens identified 2C-targeting compounds such as fluoxetine and dibucaine, but how they inhibit 2C is unknown. Here, we present a crystal structure of the soluble and monomeric fragment of coxsackievirus B3 2C protein in complex with (S)-fluoxetine (SFX), revealing an allosteric binding site. To study the functional consequences of SFX binding, we engineered an adenosine triphosphatase (ATPase)–competent, hexameric 2C protein. Using this system, we show that SFX, dibucaine, HBB [2-(α-hydroxybenzyl)-benzimidazole], and guanidine hydrochloride inhibit 2C ATPase activity. Moreover, cryo–electron microscopy analysis demonstrated that SFX and dibucaine lock 2C in a defined hexameric state, rationalizing their mode of inhibition. Collectively, these results provide important insights into 2C inhibition and a robust engineering strategy for structural, functional, and drug-screening analysis of 2C proteins.

To find and fuse with the egg, mammalian sperm must complete an arduous voyage through the female reproductive tract. The sperm cell’s remarkable odyssey is powered by its flagellum, a microtubule-based molecular machine ornamented with accessory structures that stabilize the sperm tail in viscous media. Recently, cryo-electron tomography (cryo-ET) revealed that mammalian sperm flagella are further reinforced at the molecular scale with sperm-specific microtubule inner proteins (sperm-MIPs), but the identities of these sperm-MIPs are unknown. Here, we use cryo-electron microscopy to resolve structures of native bovine sperm doublet microtubules, thus identifying most sperm-MIPs. In the A-tubule, several copies of testis-specific Tektin-5 contribute to an extended protein network spanning nearly the entire microtubule lumen. Different copies of Tektin-5 adopt a range of conformations and organizations based on their local interactions with other MIPs. The B-tubule is in turn stabilized by sperm-MIPs that bind longitudinally along and laterally across protofilaments. We further resolve structures of endpiece singlet microtubules, revealing MIPs shared between singlets and doublets. Our structures shed light on the molecular diversity of cilia across different cell types of the vertebrate body and provide a structural framework for understanding the molecular underpinnings of male infertility.

Mitochondria-cytoskeleton interactions modulate cellular physiology by regulating mitochondrial transport, positioning, and immobilisation. However, there is very little structural information defining mitochondria-cytoskeleton interfaces in any cell type. Here, we use cryo-focused ion beam milling-enabled cryo-electron tomography to image mammalian sperm, where mitochondria wrap around the ciliary cytoskeleton. We find that mitochondria are tethered to their neighbours through inter-mitochondrial linkers and are anchored to the cytoskeleton through ordered arrays on the outer mitochondrial membrane.

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) exist in a variety of oligomeric forms, each with defined cellular and subcellular distributions. Although crystal structures of AChE and BChE have been available for many years, structures of the physiologically relevant ChE tetramer were only recently solved by cryo-electron microscopy (cryo-EM) single-particle analysis. Here, we briefly review how these structures contribute to our understanding of cholinesterase oligomerization, highlighting the advantages of using cryo-EM to resolve structures of protein assemblies that cannot be expressed recombinantly. We argue that the next frontier in cholinesterase structural biology is to image membrane-anchored ChE oligomers directly in their native environment-the cell.