BEACH domain-containing proteins (BDCPs) represent a family of large membrane-associated transmembrane cargo adaptors. In the current study, we determined the cryo-EM structure of the full-length typical BDCP NBEAL2, revealing an N-terminal arch-like structure with C-terminal globular domains attached to its convex surface. Using structure-guided deletion mutants and protein chimeras as well as native alternatively spliced isoforms and disease-related point mutants, we show that the N-terminal α-solenoid/concanavalin A-like domain assembly of the typical BDCPs NBEAL1, NBEAL2, LYST, ALFY, LRBA, and NBEA functions as a modular membrane recruitment domain. We report that gray platelet syndrome-associated single aa mutations L388P or E643V within the membrane recruitment domain of NBEAL2 disrupt its membrane targeting in stably transfected cells, highlighting a potential structure-function mechanism by which failed membrane recruitment cause gray platelet syndrome or other BDCPs-related diseases.

The actin cytoskeleton plays a key integrative role in immunological synapse (IS) formation during T cell activation, but how these dynamics are altered in chimeric antigen receptor (CAR)-T cells remains unclear. Here, we used stimulated emission depletion (STED) microscopy to perform the first super-resolution analysis of actin remodeling at the IS in single- and dual (CD19/CD22) CAR-T cells, activated on supported lipid bilayers across different time points. Quantitative imaging reveals that CAR-T cells form structurally distinct synapses to untransduced cells, characterized by reduced actin-depleted regions, fewer actin foci, and persistent microvilli-like protrusions. These features indicate incomplete cytoskeletal contraction and impaired actin network reorganization, leading to partial synapse maturation. Our findings highlight fundamental differences in actin dynamics between CAR- and TCR-mediated signaling and suggest that defective actin remodeling may contribute to unstable synapse formation, altered signaling integration, and dysregulated responses or off-target effects. These insights could inform future CAR-T engineering strategies to enhance safety and efficacy.

Desmosomes are cell-cell adhesive junctions present in cardiac tissues and epithelial tissues such as the epidermis. These junctions anchor to the intermediate filament cytoskeleton, providing mechanical integrity. Our understanding of desmosome architecture has largely been influenced by observations of two-dimensional images obtained through conventional electron microscopy. Here, using focused ion beam scanning electron microscopy, we report the three-dimensional ultrastructure of desmosomes in A431 epidermoid carcinoma and S1 human mammary epithelial cells. We also reveal differences in desmosome ultrastructure at homotypic and heterotypic junctions of human airway epithelial cells. Quantitative analyses of these datasets reveal variations in desmosome size, shape, and organization. Importantly, we report the presence of discontinuities or "gaps" within the outer dense plaque, a novel feature observed in either one or both desmosome halves. This study provides the first comprehensive description of the epithelial desmosome as a three-dimensional structure and emphasizes the need to investigate the effects of dynamic morphogenetic processes and disease states on desmosome ultrastructure.

Cholesterol is essential for the organization of neurotransmitter release machinery, yet how it regulates the balance among different forms of synaptic exo- and endocytosis remains poorly understood. Moreover, which pre-synaptic processes rely on neuronal vs. astrocyte-derived cholesterol is unknown. Using nanoscale-precision imaging of single-vesicle release in hippocampal synapses, we demonstrate that astrocytic cholesterol is a critical determinant of both temporal and spatial aspects of presynaptic dynamics by differentially modulating the two main forms of synchronous release, univesicular (UVR) and multivesicular (MVR), effectively fine-tuning their balance. Moreover, astrocytic cholesterol determines the spatial distribution of vesicle release across the active zone and the balance of the two main forms of single-vesicle endocytosis, fast and ultrafast. Imaging of a cholesterol biosensor revealed that astrocyte-neuron cholesterol signaling is activity dependent. These findings suggest that astrocytic cholesterol release modulates synaptic strength in an activity-dependent manner to fine-tune the balance of different forms of synaptic vesicle exo- and endocytosis.

Malaria transmission relies on sporozoite formation in the mosquito midgut and subsequent salivary gland (SG) invasion. Despite their importance, the cell biology of these processes remains poorly understood. We apply mosquito tissue ultrastructure expansion microscopy (MoTissU-ExM), which physically expands infected mosquito tissues while preserving host and parasite ultrastructure. MoTissU-ExM reveals parasite structures and organelles, including features previously seen only by electron microscopy and novel structures not observed before. We use MoTissU-ExM to investigate sporozoite formation and SG invasion, focusing on rhoptries-secretory organelles critical for host cell invasion. We establish a timeline for rhoptry biogenesis, show that two rhoptries are consumed during SG invasion, and provide the first evidence that rhoptry pairs are specialized for different invasion events. We further characterize RON11 as the first protein involved in sporozoite rhoptry biogenesis; its disruption produces sporozoites that specifically fail to invade SG epithelial cells, blocking parasite transmission.

Centrioles are essential for centrosome and cilium formation. In fly spermatocytes, they undergo dramatic elongation to form giant centrioles that support sperm development. The conserved centriole protein Ana1/Cep295 is known to promote this elongation, yet the underlying mechanism remains poorly understood. Here, we show that Ana1 regulates centriole length through two distinct mechanisms. It directly interacts with Cep135 to promote centriole elongation and to recruit Cep135 into the proximal centriole-like structure. Ana1 also binds and bundles microtubules in vitro, and this activity is required for centriole elongation in vivo. Importantly, these two interactions are not mutually exclusive and both contribute to Ana1-induced centriole overelongation. Together, our findings reveal that Ana1 is involved in centriole elongation through interactions with distinct centriolar components during fly spermatogenesis.

Embryonic, adult, and cancer cells can exchange cytoplasmic proteins and even organelles using long, slender nanotubular interconnections. These cytoplasmic extensions, termed cytonemes, tunneling nanotubes, or tumor microtubes, span a spectrum of sizes. They have a structural core of F-actin filaments and can transport proteins and organelles ranging in size from small vesicles to mitochondria. These nanotubular interconnections can support embryonic development and tissue-tissue interactions as well as cancer cell defense against hypoxia, metabolic stressors, and chemotherapy. For example, tunneling nanotubes can transfer mitochondria to immune cells from non-malignant or tumor cells to promote or disable tumor cell killing. Numerous molecules initiate and maintain these nanotubular structures, suggesting cell type specificity. Many unanswered conceptual and mechanistic questions remain concerning the molecular and regulatory mechanisms driving their generation, structural and cell type-specific versus universal properties, and potential for novel therapeutic interventions-all providing exciting new opportunities in this rapidly expanding field of cell biology.

Cells respond to various stressors by inhibiting global translation and forming stress granules (SGs), cytoplasmic organelles enriched in certain RNA-binding proteins, and RNA. Genotoxic stress also induces SG assembly, but it is unclear how nuclear stress signals are transmitted to trigger cytoplasmic responses. We show that DNA-damaging agents that activate a nuclear poly(ADP-ribose) polymerase (PARP), PARP1, stall translation and induce SGs. We find that PARP1 activation depletes NAD+, which depletes cellular ATP, activating ATP-sensor AMPK and inhibiting mTORC1 via Raptor phosphorylation. Subsequent hypophosphorylation of 4EBP1 inhibits translation. These effects are suppressed by PAR-metabolism regulators, XRCC1, PARG, and Nudix5, and reversed by NAD+ precursor supplementation. Cells lacking SG scaffolds, G3BP1 and G3BP2, show reduced viability after genotoxic stress, which is rescued by G3BP1 overexpression. These findings link PARP1 activity to translational control and SG formation, which may protect against cell death following DNA damage. These mechanisms provide insight into PARP1- and stress granule-associated diseases, including cancer and neurodegeneration.

The Golgi complex serves as a critical hub for cellular homeostasis, yet its communication with the nucleus remains largely unexplored. By analyzing and siRNA-validating localization data from the Human Protein Atlas, we uncovered substantial proteome interconnectivity between the Golgi and nucleus, including an unexpected enrichment for DNA repair factors. We identify a cluster of DNA damage response (DDR) proteins occupying distinct sub-Golgi compartments that redistribute dynamically between the Golgi and nucleus in response to genotoxic stress, with the type of DNA lesion shaping the direction of redistribution. Focusing on the homologous recombination (HR) regulator RAD51C, we show that DNA damage triggers ataxia telangiectasia mutated (ATM)-dependent release of a giantin-tethered Golgi RAD51C pool, with subsequent importin-β-dependent nuclear import, where repair-associated foci form. Giantin depletion prematurely releases RAD51C, producing aberrant nuclear foci lacking key DDR markers, reducing ATM activation and HR efficiency, elevating genome instability, and accelerating proliferation. The Golgi thus acts as a spatiotemporal coordination node for DDR factors safeguarding genomic stability.

Collective endothelial migration during vascular development relies on dynamic cell-cell contacts, yet how the junctions between leader and follower cells are organized remains poorly understood. Using Cryo-SIM followed by FIB-SEM, we investigated the nanoscale 3D ultrastructure of asymmetric adherens junctions (AAJs), revealing a wide spectrum of membrane folds at the contact interface between migrating endothelial cells. By performing a junction localization screen, we identified a variety of membrane curvature-sensing BAR domain proteins selectively enriched at AAJs. Among these, BIN1 and SNX9 emerged as novel regulators of front-to-rear polarity in follower cells during endothelial collective migration. The spatiotemporal recruitment of these individual BAR proteins associated with distinct phases of AAJ remodeling. Furthermore, depletion of SNX9 disturbed VE-cadherin dynamics, endothelial cell directionality, and sheet migration in the common cardinal vein of zebrafish. These findings highlight junctional nanoscale membrane curvatures as hubs for the spatiotemporal recruitment of a repertoire of BAR proteins to remodel AAJs and guide collective endothelial migration during vascular development.

Cells face diverse mechanical stimuli that vary with cell type, state, and pathological conditions. Mechanobiology investigates how cells sense and respond to these forces. While most work has focused on the cell surface and nucleus as primary mechanosensors, how intracellular organelles adapt to extracellular mechanical forces remains largely unknown. Here, we show that extracellular mechanical signals influence the secretory function of the Golgi apparatus. By subjecting adherent cells to mechanical challenges-cell spreading on different ligands, altered substrate stiffness, or equibiaxial strain-we reveal that extracellular forces modulate Golgi-to-cell surface carrier biogenesis, thereby regulating exocytosis. Together with changes in Golgi membrane tension, we identify molecular determinants of the mechanotransduction pathway, including microtubule acetylation, diacylglycerol production, and protein kinase D activity. In turn, inhibition of Golgi export suppresses this mechanoresponse and causes impaired cell spreading. These findings uncover a bidirectional mechanotransduction axis in which extracellular mechanics tune Golgi secretory output, providing a framework for investigating organelle-based mechanoadaptation in physiology and disease.

Retinal photoreceptors transmit light signals to their postsynaptic neurons with high precision, speed and without fatigue. This high-throughput neurotransmission relies on a sophisticated molecular machinery centered on a presynaptic organelle, the synaptic ribbon (SR). A hallmark of SRs is the recruitment of synaptic vesicles (SVs) from the cytoplasmic SV pool via "tethering". However, the identity of the tether and the mechanism underlying SV tethering are unknown. Here, we show that cell-specific deletion of the SR-associated protein Piccolino from rod photoreceptors disrupts SR morphology and ablates SV tethering. Nanoscale epitope mapping suggests that Piccolino acts as an SV tether by extending its N terminus away from the SR into the SV-filled terminal cytoplasm. With in silico modeling and protein lipid-binding assays, we demonstrate that an amphipathic liquid packing sensor motif (ALPS) at the N terminus of Piccolino binds SV-like liposomes, implicating this interaction as the mechanism underlying SV tethering. Together, our findings identified Piccolino as the molecular link between the SR and SVs.

Efficient neurotransmission relies on the precise integration of synaptic vesicle (SV) recycling at the presynaptic terminal. While SV recycling is essential for neurotransmission, the molecular mechanisms coordinating vesicle dynamics at presynaptic terminals remain poorly understood. Here we report that the active zone proteins, CAST and ELKS, play a crucial role in maintaining functional SV pool size through direct interaction with endophilin-A family proteins. In cultured hippocampal neurons, disruption of CAST binding to endophilin-A resulted in a reduced number of SVs available for release, mislocalization of endophilin-A, and altered presynaptic localization of clathrin light chain. Furthermore, reduction of endophilin-A affected expression levels of active zone proteins. Collectively, these findings redefine the role of CAST/ELKS beyond active zone structural organization, demonstrating that their interaction with endophilin-A contributes to maintaining the SV pool required for sustained synaptic transmission at the presynaptic active zone.

Primordial germ cells (PGCs) are the first cells specified in the Drosophila embryo and are precursors to the germline. Their formation requires suppression of somatic fates, achieved by degrading the receptor tyrosine kinase Torso at the posterior pole through the ubiquitin ligase adaptor germ cell-less (GCL). Although Torso is known to antagonize PGC formation, the underlying mechanisms remained unclear. Here, we combine optogenetic Ras activation and Ras effector loop mutants to show that Ras suppresses PGC formation independently of the canonical Raf/MEK/ERK pathway. We identify an unexpected early role for Torso in activating phosphoinositide 3-kinase (PI3K), generating membrane domains enriched in phosphatidylinositol (3,4,5)-trisphosphate (PIP3). Elevated PI3K activity disrupts PGC formation, while reduced PI3K activity creates ectopic PGCs. We demonstrate that GCL remodels the posterior pole membrane by suppressing Torso-dependent PI3K activation. Clearing PIP3 enables myosin II enrichment, allowing for PGC formation. Together, our findings reveal how antagonistic Torso and GCL activities establish the soma-germline boundary by organizing cortical lipids.