Proteasomes are fundamental for protein homeostasis and genome integrity and essential in spermatogenesis and fertilization. However, their presence, composition and role within the sperm nucleus are a subject of debate. Here we use in situ cryo-electron tomography in human sperm cells to elucidate the molecular architecture of nuclear proteasomes, which cluster in DNA-free, nuclear cavities within the sperm nucleus. We show that the main population of proteasomes consists of 20S core particles, with a smaller fraction of 20S capped by PA200 activator. Using single-particle cryo-electron microscopy of purified native human sperm proteasomes, we elucidate the features of the essential testis-specific subunit α4s, reporting the presence of a unique splice variant. We resolve a native peptide in the catalytic β2 subunit, providing insight into the proteolysis mechanism and PA200-mediated enhancement of trypsin activity. We show nuclear enrichment of proteasomes during sperm-cell differentiation in human testis tissue, with 20S and PA200 clustering following meiosis, at the spermatid stage. Our findings shed light on the organization and compositional diversity of nuclear proteasomes in human sperm cells, as well as their catalytic function.

Structurally diverse tau filaments form proteinaceous aggregates in a heterogeneous group of neurodegenerative diseases called tauopathies. The factors extrinsic to the highly ordered core structure that influence tau filament stability are not well understood. Here, we found that polyubiquitinated tau filaments from Alzheimer disease and vacuolar tauopathy human brain tissue exhibit distinct seeding patterns in mice, in association with differences in tau filament ultrastructure determined by cryo-electron microscopy. Chemical modulation of the polarity of polyubiquitin adjacent to the tau core with the small molecule ubistatin B resulted in the repositioning of poorly structured densities toward positively charged residues on the highly structured core filament, leading to shifting of the protofilament-protofilament interface of certain vacuolar tauopathy tau filaments. These results suggest that the structure of tau filaments that are associated with different seeding activities in vivo can be influenced by post-translational modifications.

Mitochondrial proteostasis depends on precise N-terminal processing of imported precursor proteins. Defects in this maturation step are implicated in disease, yet the functional impact in humans remains unclear. Here we show that the intermediate cleaving peptidase ICP55, which removes a single amino acid, acts as a key stabilizer of multimeric mitochondrial protein complexes. Using proteomics and complexome profiling, we identify over 100 human ICP55 substrates and demonstrate that loss of ICP55 triggers widespread destabilization of protein assemblies, with a global shift toward smaller subcomplexes. Thus, we uncover a conserved, post-translational mechanism that safeguards mitochondrial proteostasis by regulating complex integrity through a single amino-acid cleavage, and we reveal N-terminal proteoform control as an unexpected layer of organellar homeostasis.

G protein-coupled receptors (GPCRs) regulate human physiology and are major drug targets. Although cryo-electron microscopy has accelerated GPCR structural biology, inactive-state structures remain difficult because current fusion-based strategies often require extensive experimental screening to identify rigid constructs suitable for high-resolution reconstruction. Here we introduce a universal pipeline that integrates an in silico fusion construct screening program, NOAH (nonexperimental, artificial-intelligence-assisted, high-throughput construct screening for structural analysis), with a de novo designed fusion protein, ARK1 (artificially designed fiducial marker). NOAH enabled structure determination of vasopressin V2 receptor bound to the antagonist tolvaptan or partial agonist OPC51803 and bradykinin B2 receptor bound to the antagonist icatibant, revealing receptor activation and inhibition mechanisms. Coupling NOAH to ARK1 improved the V2 receptor-tolvaptan map and enabled high-resolution structures of lysophosphatidic acid receptor 2 bound to Ki16425 and free fatty acid receptor 2 bound to GLPG0974. NOAH-ARK1 minimizes trial-and-error construct optimization and provides a broadly applicable route for GPCR structural analysis and drug discovery.

N-methyl-D-aspartate receptors (NMDARs) mediate excitatory signaling essential for synaptic plasticity and memory. Unlike GluN2-containing NMDARs, GluN3-containing receptors are activated solely by glycine and exhibit profound desensitization and paradoxical potentiation by GluN1-selective antagonists, including CGP-78608 (CGP). Although GluN3A-containing NMDARs regulate synapse pruning and excitotoxicity, and are associated with schizophrenia, autism and stroke, their native stoichiometry and gating mechanism are poorly defined. Here, using single-molecule pull-down analysis, we show that native GluN3A-containing receptors are diheteromeric assemblies. Cryogenic-electron microscopy analysis of GluN1-GluN3A receptors in antagonist-bound, preactive, active and desensitized states, augmented by electrophysiology and pharmacology experiments, show how glycine activates the receptor solely via GluN3A-dependent conformational changes, opening the gate with two-fold symmetry, and induces a roughly four-fold symmetric desensitized state. CGP-bound GluN1 restricts GluN3A rotation, promoting glycine-induced activation by blocking desensitization. These findings illuminate how CGP potentiates GluN1-GluN3A receptor activity, place the receptor gating mechanism on a structural foundation and define the molecular basis for pharmacological modulation.

Cytoplasmic dynein drives minus-end-directed motility along microtubules by converting ATP hydrolysis into coordinated structural changes but how this process produces directional stepping remains unresolved. The dynamics of dynein stepping have previously been characterized by tracking flexible regions of the motor with limited resolution. Here we site-specifically labeled the microtubule-binding domain of yeast dynein using a cysteine-light mutant and tracked stepping with submillisecond, nanometer precision at physiological ATP using minimal fluorescence photon flux (MINFLUX). We show that dynein hydrolyzes one ATP per step and moves in multiples of 8 nm. Steps are preceded by a transient plus-end-directed displacement ('dip'), corresponding to microtubule release upon ATP binding and diffusion of the stepping head around its partner. A slow ATP-hydrolyzing mutant shows more frequent dips, supporting a model in which ATP hydrolysis produces net forward movement. These results clarify the sequence of mechanochemical events underlying productive dynein stepping.

The balance between membrane fusion and fission dictates the size and shape of intracellular organelles: excess fusion causes their enlargement, and predominant fission leads to fragmentation and resorption. These processes are differentially affected by membrane tension. Fusion is favored by increased tension, and the opposite is true for fission. Variations in membrane tension therefore underlie organellar remodeling. In this Review, we analyze the central role of hydrostatic pressure generated by osmotic imbalance in dictating membrane traffic. Using the endocytic pathway as a framework, we describe the predominant role of ions and their transport pathways in establishing intraorganellar osmolarity. We illustrate the role of ion transport regulation in organellar volume homeostasis by analyzing the mechanism underlying the massive vacuolation that results from inhibition of phosphatidylinositol 3,5-bisphosphate synthesis by PIKfyve.

Nuclear envelope (NE) reformation after mitosis is essential for daughter cell viability and requires tightly coordinated nuclear pore complex (NPC) assembly and nuclear membrane reformation. Here we combined acute molecular perturbations in live cells with correlative three-dimensional electron tomography or MINFLUX super-resolution microscopy to show that degrading Nup62 during mitosis arrests NPC assembly at an intermediate step with smaller membrane pores and removes the whole central transport channel. Molecular dynamics simulations predicted that 32 copies of the central channel subcomplex, recruited into the previously unoccupied pore center, can self-associate through hydrophobic interactions to occupy and expand pore volume, exerting an outward-pushing force; indeed, disrupting these interactions during NPC assembly blocked pore dilation. Later in mitotic exit, perturbed cells exhibited impaired nuclear import, smaller nuclei and looser NE spacing. Acute inhibition of nuclear import recapitulated these NE defects without affecting NPC assembly. Together, our findings reveal a two-step molecular mechanism linking NPC assembly and NE reformation. First, hydrophobic FG-nucleoporins dilate the assembling nuclear pore by forming the central transport channel, which then allows nuclear-import-driven nuclear expansion, leading to tight, regular NE membrane spacing.

Eukaryotic sequence-specific transcription factors (TFs) must find their cognate DNA targets hidden in genomic chromatin amid an excess of nonspecific sequences and degenerate motifs. Although static TF interactions with nucleosomal targets have been elucidated, how TFs efficiently search for cognate sites within native gene-sized chromatin domains has been unclear. Here we used purified Saccharomyces cerevisiae HIS3 minichromosomes and single-molecule imaging to compare association and dissociation kinetics of transcription activator GCN4 on chromatin and naked genomic DNA. GCN4 displays widespread and stable off-target binding on bare DNA because of entrapment by degenerate sites and interactions with nonspecific DNA of increasing length, indicative of one-dimensional (1D) diffusion. Nucleosome organization on the minichromosome reduces promiscuous GCN4 residence times by obstructing TF association and restricting 1D target search within nucleosome-free regions. Furthermore, the intrinsically disordered GCN4 activation domain independently enhances targeting efficiency and specificity by accelerating association-dissociation kinetics in vitro and in living cells. Altogether, both nucleosome organization and activation domains independently suppress promiscuous GCN4 binding, which, if unchecked, may cause aberrant cryptic transcription known to occur upon chromatin disruptions.

Damage-associated molecular patterns (DAMPs) are endogenous danger signals. They can be preformed molecules released upon membrane rupture and stress-induced or newly generated factors arising during cell death. These signals link cellular demise to diverse host responses. Rather than passive by-products, DAMPs are actively mobilized through membrane-remodeling proteins, vesicular trafficking and metabolic regulation. Conformational changes, oligomerization and post-translational modifications shape their release and immunogenicity, as illustrated by redox-dependent DAMP states, pore-forming gasdermins and MLKL, and NINJ1-mediated membrane rupture. At the sensing interface, receptors such as TLR4, P2X7 and AGER, together with cytosolic STING1 pathways, translate DAMP recognition into downstream signaling through assembly-driven mechanisms. Cross-talk with metabolic pathways and membrane repair systems, including ESCRT-III and autophagy, further refines DAMP signaling dynamics. Here, we survey and contextualize recent literature to provide a structural and molecular framework for understanding how DAMPs encode immune outcomes and highlight opportunities for targeted therapeutic intervention.