How deep into the cell do mechanical signals reach? In this issue, Bhaskar Naidu et al. (https://doi.org/10.1083/jcb.202510026) show that the Golgi responds to cell-spreading force and tunes its secretory output in a loop that feeds back to support spreading.
Various pathogenic microorganisms produce toxins that create pores in cell membranes, causing cell damage and disrupting the host epithelial barrier. Recently, we reported that mice lacking the G protein-coupled receptor leukotriene B4 receptor 2 (BLT2), which is expressed in vascular endothelial and alveolar epithelial cells, are highly susceptible to pneumolysin (PLY), a pneumococci-generated toxin. Although we clarified the protective roles of BLT2 in vascular endothelial cells, those in alveolar epithelial cells have not been elucidated. Here, we report that lipid mediator 12-hydroxyheptadecatrienoic acid (12-HHT), which is produced by membrane-damaged epithelial cells, prevents cell death by promoting membrane repair through BLT2. BLT2 promoted the release of PLY-bound plasma membranes as extracellular vesicles in a sphingomyelinase-dependent manner. Additionally, BLT2 activated Rac1 and subsequent actin polymerization, leading to resistance to cell death. Furthermore, inhibition of 12-HHT production by aspirin and treatment with a BLT2 antagonist abolished the protective effect of BLT2. These findings provide a new therapeutic strategy for bacterial infection.
Non-vesicular lipid transfer at membrane contact sites (MCS) is proposed as a key principle in eukaryotic cells. While several lipid transfer proteins (LTPs) are localized at MCS, their physiological significance is poorly understood. Ceramide transfer protein (CERT) transfers ceramide between membranes in vitro. However, evidence for the in vivo significance of CERT function is limited. We find that loss of dcert, the only gene encoding CERT in Drosophila, results in elevated levels of short-chain ceramides along with reductions in the levels of its metabolite phosphoethanolamine ceramide in photoreceptors. Physiological analysis showed a reduced electrical response to light stimulation in dcert mutants. dcert mutants showed a reduction in the rate of phosphatidylinositol 4,5-bisphosphate (PIP2) resynthesis following light-induced phospholipase C (PLC) stimulation, associated with reduced ER-plasma membrane MCS density and function of the MCS-localized protein RDGB. Together, our findings suggest that CERT function regulates G-protein-coupled PLC signalling through modulation of MCS function in vivo.
How epithelial tissues remodel while preserving their barrier remains a fundamental question in epithelial biology. In this issue, Adhikary et al. (https://doi.org/10.1083/jcb.202510024) identify PAK4 as a dynamic regulator of vertex remodeling that fine-tunes actomyosin contractility to maintain tissue integrity.
In animal systems, the only case of third meiotic division without a prior round of DNA replication has been described in Drosophila males carrying mutations in roughex, a gene encoding an inhibitor of Cdk1 activity. Here, we describe another Drosophila gene, terno (teo), that regulates meiotic exit in males. In teo mutants, meiosis I and II are regular, but in many germline cysts, the 64 haploid spermatids undergo an extra division. Additionally, teo mutants are defective in sperm individualization. Teo interacts with cyclin A and B, Fizzy (Cdc20), and the anaphase-promoting complex/cyclosome components Cdc16 and Cdc27. In teo mutant testes, the cyclin A and B levels are higher than in wild type, and downregulation of either cyclin rescues the teo mutant phenotypes. These results suggest that Teo facilitates targeted degradation of the cyclins, so that in teo mutants, there is an increase in Cdk1-CycA and Cdk1-CycB activities, resulting in both the extra meiotic division and the sperm maturation defect.
Stressed cells can exchange mitochondria through intercellular tunneling nanotubes. In this issue of the JCB, Glover et al. (https://doi.org/10.1083/jcb.202511211) describe two functionally different tunnels: one for exporting dysfunctional mitochondria and another for retrieving respiration-active healthy mitochondria.
The potential for using therapeutic antisense oligonucleotides (ASOs) has been hampered by a lack of understanding of how they enter cells and subsequently access their targets. Endocytosis contributes to ASO uptake, but the machinery mediating subsequent ASO trafficking to permit suppression of their target mRNAs has not been described. Here, we show that direct ASO engagement with a scavenger receptor (CD44) activates the ERK-RSK axis to promote serine phosphorylation of a receptor tyrosine kinase (EPHA2). Serine phosphorylation of EPHA2 permits endocytosis, trafficking, and accumulation of ASOs in nuclear-captured endosomes. These endosomes are then subject to lipid peroxidation and become leaky, allowing ASOs to escape and effectively suppress target mRNA expression. Inhibition of stress granule-mediated repair of these leaky endosomes further enhances ASO effectiveness. These data identify an endocytic route to the nucleus which may be exploited to maximize the effectiveness of ASO-mediated therapies.
STAT5A is unique in its diverse expression in different cells. Here, we show that STAT5A deficiency results in reduced expression of the actin-bundling protein α-actinin-1, which alters cytoskeletal reorganization, including loss of actin bundles, reduction of cellular motility, and clustering of mitochondria and endoplasmic reticulum in the perinuclear region. These changes in cellular architecture led to production of ROS by the mitochondria, which may be explained by reduced peroxisome abundance. This, in turn, results in dsDNA breaks and formation of cytoplasmic micronuclei, and activating the cGAS-STING pathway, and mediating type I IFN production and the expression of IFN-stimulated genes. The ectopic expression of α-actinin-1 or STAT5A in STAT5A knockout cells is sufficient to restore actin bundle formation and nullifies all downstream effects. Conversely, inhibiting downstream steps suppresses only subsequent events in the pathway. STAT5A knockout results in a similar phenotype as seen with cytochalasin B, an inhibitor of actin polymerization. Overall, we show that STAT5A-α-actinin-1 links cytoskeleton integrity to mitochondrial immune regulation.
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.
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.
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.
Centriolar satellites (CS) are dynamic and heterogeneous granular assemblies that concentrate around centrosomes and contribute to ciliogenesis. In this issue, Begar et al. (https://doi.org/10.1083/jcb.202509238) examine the CS scaffold protein PCM1 to dissect CS assembly and structure during the cell cycle and ciliogenesis.
Nucleotide excision repair is essential for genome stability; however, enzymatic scissors that remove damage must be tightly regulated to prevent erroneous DNA cleavage. In this issue, Muniesa-Vargas et al. (https://doi.org/10.1083/jcb.202602121) used live-cell imaging to uncover how cutting DNA by XPG endonuclease is controlled during repair.
Unorthodox AQP12-type channels are poorly understood intracellular aquaporins localized in the endoplasmic reticulum and zymogen granules (ZGs) of pancreatic acinar cells. Despite connections to major diseases, their biophysical properties and intracellular trafficking regulation remain largely unknown. Here, we show that heterologously expressed plant and metazoan AQP12-related channels specifically localize to the intracellular yolk platelet (YP) membrane of frog oocytes, a feature that is recapitulated in vivo for invertebrate and vertebrate orthologs. Using native YP membranes, we show that the vertebrate channels are mercury-sensitive polytransporters with intracellular trafficking regulated by Ca2+ and cAMP signaling pathways. We identify a novel pan-vertebrate C-terminal YP-targeting domain (YPD) in AQP12, which also drives orthodox aquaporin chimeras and truncated channels to YPs and ZGs. In cultured pancreatic cells, the YPD and Ca2+-induced AQP12 N-terminal phosphorylation coregulate secretagogue-triggered channel transport to the ZGs for enzyme secretion. These findings uncover conserved signaling mechanisms for AQP12 trafficking to intracellular protein storage vesicles, and open unexpected avenues for targeted delivery systems.
Actin’s transition from monomers (G-actin) to polymers (F-actin) and then into bundled and branched networks underlies many cellular and system functions. Yet, how these networks are dynamically assembled and disassembled is incompletely understood—including why F-actin is often simultaneously and redundantly bundled by different proteins. Here, we focus on fascin and espin, two bundlers that often coexist and robustly bundle F-actin. We find that they synergistically bundle F-actin compared to equal amounts of each one alone. However, we show that bundles containing these two proteins are robustly destabilized by a synergism between Mical and cofilin, indicating mechanisms of how complex bundles are disassembled and remodeled. Yet, our results also reveal that together fascin and espin protect F-actin from this disassembly more effectively than each one alone—including to regulate F-actin disassembly and cellular remodeling in vivo. These findings reveal mechanisms for assembling and disassembling complex networks of bundled F-actin, including a synergism between different bundlers and disassemblers in these processes.
The centromere is the chromosomal site of kinetochore assembly, defined by the histone H3 variant CENP-A. In each cell cycle, the assembly and maintenance of CENP-A is functionally critical for chromosome segregation. In Drosophila male meiosis, CID (fly CENP-A) is assembled in two phases: prophase of meiosis I and postmeiosis II. Here, we investigate the dynamics of the assembly components CAL1 and CENP-C in prophase I and determine the requirements for the second assembly phase. In early prophase I, CENP-C functions with CAL1 to maintain the centromere. In late prophase I, CAL1 is undetectable at centromeres and CENP-C is not critical for centromere maintenance. Instead, CENP-C is crucial for meiotic kinetochore recruitment and function. This CENP-C pool also functions in CID assembly postmeiosis II, which is independent of CAL1. In addition to different functional pools of CENP-C, distinct pools of the CID protein persist in the male germline, and the synthesis of each pool is uncoupled from its cell cycle deposition timing.
