ABSTRACT Inositol hexakisphosphate kinases (IP6Ks) catalyse the synthesis of the inositol pyrophosphate 5-InsP 7 , and regulate diverse physiological processes. Mice lacking IP6K1 display reduced body weight despite normal food intake, a phenotype that is more apparent in juvenile mice during their rapid growth phase. Additionally, Ip6k1 -/- mice exhibit decreased serum albumin, elevated faecal protein, and reduced skeletal muscle mass compared to Ip6k1 +/+ mice, suggestive of a deficiency in protein digestion in the absence of IP6K1. We found that IP6K1 is expressed throughout the mouse gastrointestinal tract, and is especially enriched in the cytoplasm of chief cells in the stomach, which are responsible for the storage and secretion of digestive enzymes. Pepsinogen C (PGC) containing granules were sparse, and gastric lipase F (LIPF) granules were completely absent in the gastric glands of Ip6k1 -/- mice, despite normal expression levels of these enzymes, implicating IP6K1 in digestive enzyme granule biogenesis. Consequently, the level of the active protease pepsin C was decreased in the gastric lumen of Ip6k1 -/- mice compared with their wild type counterparts. CRISPR/Cas9-mediated deletion of IP6K1 in the gastric adenocarcinoma cell line AGS was able to recapitulate the phenotype of reduced PGC granule intensity seen in gastric chief cells of Ip6k1 -/- mice. PGC granule formation was restored in IP6K1 -/- AGS cells by the reintroduction of catalytically active or inactive IP6K1, indicating that IP6K1 supports the formation of secretory granules independent of its ability to synthesise 5-InsP 7 . The proteoglycan SDC4, identified as an interactor of IP6K1, was seen to co-localise and co-migrate with PGC granules in IP6K1 +/+ but not in IP6K1 -/- AGS cells. Our findings identify IP6K1 as a novel regulator of secretory granule biogenesis in gastric chief cells, to influence protein digestion in the mammalian stomach.

Personalized cancer therapies focus mostly on targeting driver alterations, such as oncogenic point mutations or oncogenic driver events within large somatic copy number alterations. However, these alterations are often not actionable or only present in a small subset of patients. We hypothesized that passenger events, specifically in amplified regions, could be therapeutically exploited by providing actionable molecules on the cell surface, serving as Trojan horses for specific therapy delivery. Applying a multiomics in silico approach, we identified MPZL1 ( Myelin protein zero-like 1 ), a glycosylated cell surface receptor located on chromosome 1q, as a promising candidate, which is amplified in up to 75% of cases in several solid cancers. Notably, immunohistochemistry of a wide range of human cancer tissues (n=2244 samples) as well as normal tissues revealed strong membranous MPZL1 expression in a majority of solid tumors (e.g. 48% of hepatocellular carcinomas or 89% of triple-negative breast cancers), whereas healthy tissues were mostly negative or just faintly positive for MPZL1. Next, we generated a highly specific monoclonal antibody directed to the extracellular domain of human MPZL1 protein and utilized this antibody to produce MPZL1 CAR-T cells. MPZL1 CAR-T cells showed high specificity as well as high sensitivity in targeting a multitude of human cancer cell lines (e.g. liver, breast, and lung cancer) with high MPZL1 expression in vitro. Finally, we demonstrate strong therapeutic efficiency of MPZL1 CAR-T cells not only in different human xenograft tumors in vivo but also in a unique autochthonous liver cancer mouse model. Our work provides a framework to target passenger events within large chromosomal amplifications, reveals MPZL1 as a new trojan horse entry point for therapies of 1q-amplified cancers, and as such opens a new avenue for innovative approaches in anti-cancer drug development.

Chickens ( Gallus gallus ) are uniquely suited for germline studies because their primordial germ cells (PGCs) can be propagated long-term in vitro and used for germline transmission. To develop a loss-of-function screening platform in chicken PGCs, we compared three perturbation methods: CRISPR/Cas9 knockout, CRISPR interference (CRISPRi), and shRNA-mediated knockdown. We found that CRISPR/Cas9 editing causes severe toxicity in PGCs, with DNA damage hypersensitivity over 20-fold greater than in somatic cells, and with distinct DNA damage checkpoint responses between male (ZZ) and female (ZW) lines. CRISPRi using dCas9-KRAB was ineffective in chicken—likely because of species-specific epigenetic constraints—whereas shRNA knockdown produced robust, nontoxic gene silencing. These results identify DNA damage hypersensitivity as a major barrier to nuclease-based editing in the germline and establish RNAi as a feasible platform for genome-wide functional screening in chicken PGCs.

SUMMARY Neurological diseases are frequently characterised by dysregulation of the microtubule cytoskeleton, which is critical for neuronal integrity and functioning. However, the precise mechanisms by which microtubule dynamics are regulated during the development of the nervous system remain poorly understood. Here, we use global phosphoproteomic screening to identify cytoskeletal substrates of Ser-Arg Protein Kinase (SRPK), which is implicated in several neurodevelopmental and neurodegenerative conditions. We show that SRPK directly phosphorylates Microtubule Associated Protein (MAP)1S at multiple sites in a C-terminal region involved in proteolytic maturation and microtubule binding. SRPK-dependent MAP1S phosphorylation modulates the affinity of the MAP1S microtubule binding domain for microtubules and MAP1S proteolytic processing by the Calpain (CAPN)10 protease. Finally, we show that MAP1S proteolytic processing occurs progressively during neurodevelopment via a specific CAPN10 expression switch, corresponding with MAP1S acquisition of microtubule binding activity. Our results demonstrate a role for SRPK in coordinating processing and functionalisation of a key microtubule regulatory protein during neurodevelopment and provide insight into mechanisms by which the microtubule cytoskeleton may be dysregulated in neurological diseases.

Human cytomegalovirus (HCMV) is a prevalent pathogen of the herpesvirus family, infecting most of the human population worldwide. Like all herpesviruses, HCMV can establish a latent infection that persists throughout the lifetime of the host. The HCMV immediate early (IE) proteins, IE1 and IE2, are viewed as master regulators of HCMV infection and are commonly assumed to play pivotal roles in regulating the balance between latent and lytic infection, as their repression is a hallmark of latency. However, it is still unclear whether their expression can indeed determine the establishment of productive infection and what functions, either related to viral gene expression or to cellular pathways, are involved in this activity. Using THP1 monocytes, ectopically expressing the HCMV receptor, PDGFRα to boost viral entry, we show that overexpression of either IE1 or IE2 significantly enhances productive infection, illustrating their critical role in determining infection outcome. Mechanistically, we show IE2 drives expression of the viral early genes at early stages of infection, whereas IE1 acts more broadly to enhance global viral gene expression. We further show that from the many functions of IE1, its ability to promote lytic infection is mainly linked to the disruption of PML nuclear bodies. Importantly, induction of either IE1 or IE2 expression in latently infected cells enhances viral reactivation, with IE1-mediated PML representing a central mechanism. Taken together, our findings elucidate the distinct and complementary roles of IE1 and IE2 in overcoming barriers to productive infection and reactivation.

ABSTRACT The human heart, originating from the splanchnic mesoderm, is the first functional organ to develop, co-evolving with the foregut endoderm through reciprocal signaling. Previously, cardioid models offered new insights on cardiovascular cell lineages and tissue morphogenesis during heart development, while mesoderm-endoderm crosstalk remain incompletely understood. Here, we integrated micropatterned cardioids, CRISPR-engineered reporter hiPSCs, deep-tissue imaging, and single-cell RNA sequencing (scRNA-seq) to explore synergistic mesoderm-endoderm co-development. scRNA-seq with PHATE trajectory mapping reconstructed lineage bifurcations of mesoderm-heart and endoderm-foregut lineages, identifying key cell types in cardiac and hepatic development. Ligand-receptor interaction analysis highlighted mesodermal cells enriched in non-canonical WNT, NRG, and TGF-β signaling, while endodermal cells exhibited VEGF and Hedgehog activity. We found that micropattern sizes influenced cellular composition, cardioid cavitation, contractile functions, and mesoderm-endoderm signaling crosstalk. The cardioids generated from 600 µm diameter circle patterns showed larger cavity formation resembling early heart chamber formation. Our findings establish micropatterned cardioids as a model for mesoderm-endoderm co-development, enhancing our understanding of heart-foregut synergy during early embryogenesis.

The CRISPR-Cas system provides adaptive immunity in many bacteria and archaea by storing short fragments of viral DNA, known as spacers, in dedicated genomic arrays. A longstanding question in CRISPR-virus coevolution is the optimal number of spacers for each bacterium to maintain proper phage coverage. In this study, we investigate the optimal CRISPR memory size by combining steady-state immune models with dynamical antigenic traveling wave theory to obtain both analytic and numerical results of coevolutionary dynamics. We focus on two experimentally supported phenomena that shape immune dynamics: primed acquisition, where partial spacer-protospacer matches boost acquisition rates, and memory size fluctuations, where a short-term increase in memory size drives population dynamics. We find that under primed acquisition, longer optimal arrays benefit from maintaining multiple, partially matching spacers. In contrast, dynamic memory fluctuations favor shorter arrays by amplifying the fitness advantage of acquiring a few highly effective new spacers. Together, our results highlight that memory optimality is not fixed, but instead shaped by the interaction of acquisition dynamics and population-level immune pressures.

Natural killer (NK) cell-based immunotherapies represent a promising avenue for cancer treatment due to their ability to eliminate cancer cells independently of antigen presentation and potential for “off-the-shelf” use. However, the molecular determinants governing tumor cell susceptibility to NK cell-mediated cytotoxicity remain incompletely understood. Here we employed CRISPR activation (CRISPRa) screening to systematically identify cancer cell surface regulators of NK cell killing across multiple cancer types. Using a comprehensive surfaceome-focused library, we screened human and murine cancer cell lines co-cultured with NK cells, identifying both known and novel ligands that modulate NK cell cytotoxicity. Our screens revealed established factors including CD43 (encoded by SPN ), while uncovering previously uncharacterized regulators such as CD44, PDPN, and Siglec-1/CD169. Validation through complementary cDNA overexpression and genetic knockout approaches confirmed that disruption of CD43, CD44, PDPN, and Siglec-1 significantly altered cancer cell susceptibility to NK killing both in vitro and in humanized mouse models. Analysis of clinical datasets show that expression of identified factors correlates with patient survival outcomes in an NK-context dependent manner supporting their therapeutic relevance. Most notably, our mechanistic studies demonstrate that CD43-mediated NK cell resistance operates independently of its previously proposed interaction with Siglec-7 on NK cells. Furthermore, we find that targeting CD43 on either NK cells or engineered T cells substantially enhances their cytotoxic activity against leukemia cell lines. These results establish gain-of-function screening as a powerful approach for discovering immunoregulatory surface proteins and identify multiple promising targets for enhancing NK cell-based cancer immunotherapies.

Abstract Biologics produced in Escherichia coli BL21(DE3) require rigorous removal of lipopolysaccharide (LPS), also termed endotoxin, as its presence can elicit severe immune responses and life threatening complications in humans. Escherichia coli Nissle 1917 (EcN) is a clinically approved probiotic with unique biosafety characteristics due to its LPS-deficient phenotype, and has only 0.86% of the LPS activity observed in BL21(DE3), so its markedly lower immunostimulatory potential makes it an attractive chassis for low-LPS protein production. Using GFP as a model protein for comparison, its yield in EcN is only ~ 30% of that observed in BL21(DE3) owing to lower cell density (OD₆₀₀) and per-cell protein yield. Transcriptomic profiling revealed that heterologous protein induction in EcN suppresses key biosynthetic and energy-generating pathways—including ribosome biogenesis, translation, and the tricarboxylic acid (TCA) cycle—thereby constraining heterologous protein synthesis. To boost intracellular protein synthesis in EcN, we first inserted the T7 RNA polymerase gene into the chromosome, creating EcN::T7. We then used CRISPR/Cas9-mediated genome editing technology to delete ompT, iclR, and arcA, yielding the high-yield mutant EcN::T7Δ ompT Δ iclR Δ arcA . This engineered strain produced 3.2-fold more reporter protein than its parental strain EcN::T7, reaching ~ 70% of the yield observed in BL21(DE3). When applied to interferon α-2b (IFNα-2b) production, mutant EcN::T7Δ ompT Δ iclR Δ arcA achieved 89.3% of BL21(DE3)’s titer while exhibiting post-purification LPS levels comparable to BL21(DE3). Notably, the the IFNα-2b retained full biological activity. By eliminating the need of extensive LPS removal procedures, this strategy positions EcN as a cost-effective and clinically compliant platform for biomanufacturing.

A bstract Whole genome doubling (WGD) is a frequent event in tumourigenesis that promotes chromosomal instability and tumour evolution. WGD is sensed indirectly due to the presence of extra centrosomes, which activate the PIDDosome to induce a p53-dependent G1-arrest. Here we uncouple WGD from centrosome amplification and show that p53 still arrests tetraploid cells, but via the mitotic stopwatch; a p53/53BP1/USP28-dependent pathway that causes G1-arrest following an extended mitotic delay. Mitotic timing is unaffected by WGD, but the threshold mitotic delay needed to invoke a G1-arrest is reduced. This sensitivity to the stopwatch mechanism is not associated with altered levels of mitotic stopwatch components, but instead, is associated with enhanced p21 concentrations prior to mitosis. Similar effects are observed in diploid cells treated with CDK4/6 inhibitors to double their size, implicating G1 delays and cell size as key determinants of stopwatch sensitivity. This ability of the mitotic stopwatch to arrest proliferation after increases to genome or cell size has important implications for the progression and treatment of cancer. It also demonstrates that the stopwatch pathway can sense more than just mitotic delays.

Neuropilin-1 (NRP-1) is a versatile transmembrane protein expressed in numerous cell types and tissues, both in health and in disease. In particular, it is expressed by activated T cells, where it has been shown to play an inhibitory role, dampening their response to tumor cells. Chimeric antigen receptor (CAR) T cells have had considerable success in treating cancers such as B cell leukemias, but face several obstacles in the treatment of solid tumors. We hypothesized that NRP-1 may contribute to dampening CAR T cell responses to cancer. While NRP-1 blockade either through neutralizing antibodies or through CRISPR/Cas9-mediated deletion did not improve CAR T cell cytotoxic activity in an in vitro solid tumor model, NRP-1 was found to have a protective effect against CAR T cells when expressed by the tumor cells, in line with previous literature implicating NRP-1 as a pro-tumor factor involved in their growth and invasiveness. We found that NRP-1 was transferred from target cells to CAR T cells via trogocytosis, i.e. the “nibbling” and subsequent display of membrane proteins from one cell by another. CD19, the target antigen in this model, was also transferred to CAR T cells, raising interesting questions about trogocytosis as a marker for effective tumor cell killing through direct interaction. On the contrary, trogocytosis may impede effective CAR T function by promoting fratricide due to their display of target antigen. While no conclusive mechanisms for NRP-1 activity were found, several avenues of research have been opened up to further our understanding of the multifaceted roles of NRP-1 in CAR T cell activity and interactions with their targets.

Genetic variation within species shapes phenotypes, but identifying the specific genes and variants that cause phenotypic differences is costly and challenging. Here, we introduce CRI-SPA-Map, a genetic mapping strategy combining CRISPR-Cas9 genome engineering, selective ploidy ablation (SPA), and high-throughput phenotyping for precise genetic mapping with or without genotyping in the yeast Saccharomyces cerevisiae . In CRI-SPA-Map, a donor strain carrying SPA machinery is mated to a genetically different recipient strain harboring a genome-integrated selectable cassette. In the resulting diploid, CRISPR-Cas9 cuts the cassette for replacement with DNA from the homologous donor chromosome. Donor chromosomes are then removed using SPA to yield haploid recombinant strains. To establish CRI-SPA-Map, we mated a W303 SPA strain to 92 strains from the BY4742 yeast knockout collection that carry gene deletion cassettes on the left arm of chromosome XIV and created 1,451 recombinant isolates. Whole-genome sequencing verified that deletion cassette replacement introduced short donor DNA tracts of variable length, resulting in a finely recombined mapping population. Using only the known location of the gene deletions, which marks where donor DNA is introduced, we identified a 6.5 kb-region shaping yeast growth. Further dissection of this region pinpointed two causal variants in two genes, MKT1 and SAL1 . Engineering these variants alone and in combination revealed gene-by-environment interactions at both genes, as well as epistatic interactions between them that were in turn dependent on the environment. CRI-SPA-Map is a cost-effective strategy for creating high-resolution recombinant panels of yeast strains for identifying the genetic basis of phenotypic variation.

To grow and divide cells must tightly coordinate anabolic programs with the availability of nutrients and growth factors. This balance is especially critical during postnatal development, when biosynthetic and energetic demands are high, and nutrient supply and neonates have to adapt to periods of fasting. These conditions place acute stress on the proteostasis network, making autophagy essential for nutrient recycling. We found that the chaperone aryl hydrocarbon receptor-interacting protein (AIP) supports both arms of this metabolic balance: promoting anabolic PI3K-AKT signaling for mTORC1 activation and enabling catabolic processes such as proteasomal degradation and autophagy. Loss of AIP causes a severe neonatal metabolic disorder, where affected infants fail to thrive postnatally. Our findings establish AIP as a central regulator of neonatal metabolic adaptation and cellular homeostasis. One Sentence Summary AIP integrates nutrient sensing and protein recycling to sustain neonatal survival.

Abstract Salmonella is one of the most prevalent and highly transmissible food-borne pathogens, making rapid and accurate screening essential for safeguarding human health and ensuring food safety. This study introduces a one-tube nested PCR mediated CRISPR-Cas12a for ultrasensitive visual screening of Salmonella spp. using fluorescent lateral flow strip. By leveraging the simultaneous dual-segment amplification capability of the designed one-tube nested PCR and the collateral activated trans -cleavage activity of CRISPR-Cas12a, the method achieves a detection limit of ‌10 1 CFU/mL‌, with no cross-reactivity against other common food-borne pathogens. This approach employs the fluorophore-labeled DNA reporters that are cleaved by activated Cas12a, allowing for rapid and on-site visualization of detection results. Validation in different food matrices yields satisfactory results, demonstrating robustness against matrix interference. Comparative analysis revealed a ‌10-fold sensitivity improvement‌ over traditional single-primer PCR protocols, attributed to the dual amplification efficiency of designed one-tube nested PCR and the collateral activated cleavage specificity of CRISPR-Cas12a. The portability, rapid visual readout, and ultrasensitive performance of the method enable real-time, on-site screening of Salmonella in diverse food supply chains, even in resource-limited settings. Its high specificity, robustness against matrix effects, and minimal equipment requirements make it a transformative, user-friendly tool for enhancing global food safety surveillance and preventing outbreaks.

Phytophthora nicotianae is an oomycete pathogen that severely threatens tobacco production worldwide. Though several dominant resistance (R) genes against P. nicotianae are used in tobacco breeding, they often fail due to rapid emergence of new virulent strains. Instead, targeting plant susceptibility (S) genes offers a promising approach for durable and broad resistance. Evidence from various plant species demonstrates that loss of the S gene DMR6 enhances disease resistance without compromising yield, emphasizing its value for resistance breeding. In this study, we identified and functionally characterized two DMR6 orthologs in tobacco (Nicotianan tobaccum), NtDMR6T and NtDMR6S, which were both induced upon P. nicotianae infection. Targeted mutagenesis of NtDMR6T and NtDMR6S using CRISPR/Cas9 demonstrated that single-gene knockouts conferred enhanced resistance to P. nicotianae, while double mutants exhibited an additive resistance effect. Notably, all mutant lines showed no obvious growth or developmental defects under greenhouse or field conditions. RT-qPCR analysis indicated that NtDMR6s negatively regulate tobacco resistance by modulating multiple defense pathways, including the MAPK signaling cascade. Collectively, our findings demonstrate that NtDMR6T and NtDMR6S act as negative regulators of resistance in allotetraploid tobacco and represent promising S gene targets for the development of P. nicotianae resistant cultivars, thereby providing a new strategy for tobacco disease resistance breeding.

The precise insertion of DNA sequences into plant genomes is a fundamental goal of modern biotechnology. This capability has the potential to accelerate crop improvement and expand the possibilities in synthetic biology. Recent advancements, driven by CRISPR-Cas systems, have initiated an era of programmable DNA integration. This progress has resulted in a diverse set of tools, including enhanced gene targeting (GT), prime editing (PE), and innovative platforms using transposases and recombinases. These tools are no longer just theoretical; they enable a wide range of applications, such as in-locus protein tagging, engineering of cis-regulatory elements, and the targeted integration of multi-gene cassettes for stacking complex traits. However, significant challenges remain. The most notable issues include low efficiency in inserting large DNA fragments, ongoing delivery challenges to elite cultivars, and evolving regulatory frameworks. In this review, we critically synthesize the development of these technologies, starting from early random integration methods to the latest CRISPR-based programmable systems. We evaluate their underlying mechanisms and identify the key technical barriers that currently hinder their routine application. Finally, we discuss emerging solutions, including next-generation editors, tissue-culture-free delivery systems, and strategies for regulatory harmonization, which are paving the way for efficient and predictable DNA insertion necessary for next-generation plant breeding and biotechnology.

ABSTRACT Phosphoinositides (PIs) are a family of seven low abundance membrane lipids, each with distinct signaling functions. The phosphoinositide kinase PIKfyve generates phosphoinositide-3,5-bisphosphate (PI(3,5)P₂) and PI5P. Emerging evidence implicates PIKfyve in key cellular processes, including autophagy, phagocytosis, endosomal trafficking, lysosomal maintenance, and melanosome formation. Complete loss of PIKfyve function is embryonic lethal in model organisms. In humans, heterozygous mutations in PIKFYVE are associated with Fleck corneal dystrophy and congenital cataracts. In this study, we investigate the role of PIKfyve in photoreceptors and the adjacent retinal pigment epithelium (RPE), host to dynamic endolysosomal pathways required for enduring the high oxidative stress environment, transporting metabolites and phototransduction components, and the breakdown of outer segment discs. To assess PIKfyve function in the retina and RPE in our zebrafish model, we employed CRISPR/Cas9-mediated gene editing and pharmacological inhibition using the specific PIKfyve inhibitor apilimod. Loss of PIKfyve activity leads to RPE expansion characterized by the accumulation of LC3- and LAMP1-positive vacuoles, along with defects in phagosome degradation and minor changes to melanosome biogenesis. Photoreceptors deprived of PIKfyve function develop a single large vacuole in the inner segment, while the OS remains largely intact over the timespan analyzed. Electroretinography (ERG) recordings revealed complete visual impairment in pikfyve crispant larvae, and significantly reduced visual function in larvae treated with apilimod post embryogenesis. These findings highlight the critical role of PIKfyve in the development and homeostasis of the RPE and retina.

Glycolipids constitute an important component of the plasma membrane based on both abundance as well as function. Gangliosides, being a class of structurally diverse and functionally varied glycolipids, can act both as a receptor as well as a ligand and therefore is established as a crucial player in several normal cellular processes. In certain diseases, and in particular cancer, select gangliosides are overexpressed often leading to disease manifestation. GM2-synthase, the enzyme responsible for the formation of a pro-tumorigenic ganglioside, GM2 is well reported to be over-expressed across various cancer tissues and cell lines. This over-expression of GM2-synthase has been linked with increased migration, invasion and epithelial to mesenchymal transition (EMT) as well as induction of a local and systemic host immune suppression in cancer. Despite only a handful of studies demonstrating an epigenetic regulation underlying the transcriptional regulation of GM2-synthase (B4GalNT1) gene, the detailed mechanism still remains unclear. Here we identified the total proteome associated with the GM2-synthase promoter through a modified CRISPR-dCas9 based proteome profiling approach by categorizing all the identified proteins leading to a detailed elucidation of the molecular drivers behind GM2-synthase transcription. While the previous study identified an acetylation-dependent de-repression of the transcription factor SP1 causing GM2-synthase activation, the underlying molecular mechanism driving its activation wasn’t clear. This study demonstrated that the histone acetyl transferase (1), p300 acts as a pivotal factor which on one hand cause acetylation-mediated degradation of SP1, and on the other hand activates SMAD2/4 to have a direct positive impact on GM2 synthase gene transcription. We identified p300 to have an activator role in GM2 synthase gene transcription through knock out, knock down and over-expression experiments. Furthermore, SP1 degradation, SMAD activation and their DNA binding patterns show the reciprocal role of p300 on SP1 and SMAD complexes. Altogether we have identified SMAD 2/4 as an activator complex, p300 as a positive regulator and uncovered a critical p300-SMAD-SP1 regulatory axis in GM2-synthase transcriptional regulation.

Summary Neural circuits are composed of different neuron types that exhibit distinctly different computational properties resulting from the sets of ion channels expressed. Profound insight exists into how neural computations arise from the precise regulation of ion channels (Armstrong et al., 1998; Lai, Jan, 2006; Nusser et al., 2012), how degenerate channel properties support similar computations (Marder, Prinz, 2002; Marder, Goaillard, 2006), and how channelopathies affect brain function (Kullmann, 2010). However, it remains elusive why neurons express many more channels, and isoforms thereof, than required to tune their specific excitabilities. Here, we employ an experiment-theory approach pairing electrophysiology with Drosophila genetics, and mathematical modelling to show that the variance in membrane properties that results from ion channel diversity promotes the robustness of neuron-type specific functions. Specifically, we show that the robustness of flight motoneuron coding properties to internal and external perturbations is significantly enhanced by the diversity of calcium channel splice isoforms expressed. Importantly, increased excitability robustness to perturbations of outward currents or temperature does not require adjustments in calcium channel mean properties. Instead, increases of the variance of calcium channel gating properties that result from channel isoform diversity broaden the dynamic input range the neuron can compute without reaching depolarization block. This broadens our concept of the functional consequences of the tremendous variety and diversity of ion channels expressed in brains. One Sentence Summary The variance of calcium channel gating properties is increased by channel isoform diversity and aids neuronal coding and excitability robustness.

ABSTRACT Neuron-specific expression of particular gap junction channel components defines the configuration and functional properties of electrical synapses. However, how a neuron utilises multiple, simultaneously expressed channel proteins - connexins or innexins -to make meaningful connections with distinct synaptic partners remains largely unknown. Using the posterior mechanosensory circuit in C. elegans, we discovered that individual electrical synapses can be formed by clustering together molecularly distinct gap junction channel-types made of three different innexin proteins, INX-1, UNC-7, and UNC-9. In this previously unknown configuration, which we term as heterochannel synapses, molecularly distinct gap junction channel types functionally collaborate to regulate posterior touch sensory behaviour, enhancing functional robustness. We show that the synaptic trafficking of the molecularly different channel types within a heterochannel synapse is independently regulated by discrete and conserved kinesin motor proteins, while distinct molecular pathways involving channel-specific retrograde kinesins regulate their turnover. These independent, channel-specific regulations also make individual synapse-level alterations in the composition of heterochannel synapses possible under altered environmental conditions, providing a novel mechanism for electrical synapse plasticity. Finally, we present evidence of heterochannel electrical synapses in C. elegans locomotory circuits and in the cerebellar Purkinje neurons of zebrafish larvae. Altogether, we demonstrate a novel heterochannel organization of electrical synapses, their regulation, and functional importance, which may be a conserved feature of metazoan nervous system.

A major challenge in human genetics is to identify all distal regulatory elements and determine their effects on target gene expression in a given cell type. To this end, large-scale CRISPR screens have been conducted to perturb thousands of candidate enhancers. Using these data, predictive models have been developed that aim to generalize such findings to predict which enhancers regulate which genes across the genome. However, existing CRISPR methods and large-scale datasets have limitations in power, scale, or selection bias, with the potential to skew our understanding of the properties of distal regulatory elements and confound our ability to evaluate predictive models. Here, we develop a new framework for highly powered, unbiased CRISPR screens, including an optimized experimental method (Direct-Capture Targeted Perturb-seq (DC-TAP-seq)), a random design strategy, and a comprehensive analytical pipeline that accounts for statistical power. We applied this framework to survey 1,425 randomly selected candidate regulatory elements across two human cell lines. Our results reveal fundamental properties of distal regulatory elements in the human genome. Most element-gene regulatory interactions are estimated to have small effect sizes (<10%), which previous experiments were not powered to detect. Most cis -regulatory interactions occur over short genomic distances (<100 kb). A large fraction of the discovered regulatory elements bind CTCF but do not show chromatin marks typical of classical enhancers. Housekeeping genes have similar frequencies of distal regulatory elements compared to other genes, but with 2-fold weaker effect sizes. Comparisons to the predictions of the ENCODE-rE2G model suggest that, while performance is similar across two cell types, new models will be needed to detect elements with weaker effect sizes, regulatory effects of CTCF sites, and enhancers for housekeeping genes. Overall, this study describes the first unbiased, perturbation-based survey of thousands of distal regulatory element-gene connections, and provides a framework for expanding such efforts to build more complete maps of distal regulation in the human genome.

SPAtial Cell Exploration ( SPACE ) is a novel subcellular imaging-based method that couples large-scale perturbations with whole transcriptome single-cell multi-omics readout, while preserving spatial context, providing unprecedented insights into tissue organization and microenvironmental interactions at very low cost. As a demonstration, we performed SPACE in hundreds of spheroids and discovered novel biology related to CAF-tumor interactions using unbiased approaches. This scalable, cost-effective technology has broad applications in translational research and drug discovery, offering a transformative approach to high-throughput spatial perturbation studies.

BACKGROUND Reliable detection of huntingtin (HTT) is essential for understanding Huntington’s disease (HD) biology and evaluating therapeutic strategies. However, high-quality monoclonal antibodies (mAbs) against the HTT C-terminal domain remain limited. OBJECTIVE We sought to generate and validate novel monoclonal antibodies targeting the HTT C-terminal HEAT-containing domain to better detect HTT independently of potential effects of polyglutamine length that can impact some N-terminally targeted antibodies. METHODS We immunized mice with a highly purified, well-characterized recombinant protein corresponding to the HTT C-terminal domain. We generated monoclonal antibody-producing hybridoma cell lines and characterized the antibodies using parental and HTT-knockout cell lines in common immuno-applications. RESULTS Three novel, independent hybridoma lines producing anti-HTT monoclonal antibodies were derived. Using CRISPR-edited HTT knockout cell lines we identified one clone, anti-HTT [2F8], that was specific and effective across Western blot, immunofluorescence, and ELISA assays. All antibodies bound full-length HTT irrespective of HAP40 interaction or polyQ length and showed no cross-reactivity to the N-terminal HEAT domain. CONCLUSIONS These C-terminal HTT mAbs are thus valuable additional tools for studying endogenous HTT function in both normal and disease contexts.

Proteasomes reversibly form foci bodies in a liquid-liquid phase separation (LLPS)-dependent manner upon stress. We previously reported that internalized protein aggregates were targeted by proteasome-dense foci 1 , and proposed that such transient aggregate-associated droplets (TAADs) may facilitate aggregate removal 2 . Here we use quantitative imaging to show that TAADs represent a novel type of gel-like proteasome condensate. TAADs are irregular in shape and slow to disperse, sequestering proteasomes in agreement with our observation of confined diffusion 3 . We demonstrate that TAADs co-localize with cytosolic alpha-synuclein aggregates to facilitate their clearance. Inhibition of proteasome- or ubiquitination activity abolishes this aggregate clearance. We identify RAD23B necessary for TAAD formation, amid other co-localizing chaperones and (co-)proteins of the ubiquitin-proteasomes system. TAAD formation is associated with higher proteasomal substrate turnover whilst retaining overall catalytic efficiency, suggestive of altered degradation mechanisms upon aggregate engagement. Proteomics analysis reveals impact on key mitochondrial-associated processes even after TAAD-aggregate disengagement. Similar TAAD-aggregate co-localizations are found in iPSC-differentiated neurons and in disease-relevant samples, with no detection of compromised proteasome activity. Together, our results indicate a model where TAADs concentrate local proteasome activity, which facilitates aggregate clearance in healthy ageing cells. Potentially, should pathological aggregates persist, TAADs may remain engaged and conceivably sequester proteasomes from physiological activities, thus contributing to neurodegenerative disorders.

Abstract Protein acetylation regulates essential processes across eukaryotes. In trypanosomatids, stage-specific acetylation suggests roles in parasite differentiation. Here, we functionally characterized zinc-dependent lysine deacetylases (DAC1, DAC3, DAC4, and DAC5) in Leishmania mexicana . CRISPR-Cas9-mediated disruption revealed that DAC1 and DAC3 are essential for procyclics, while DAC4 and DAC5 are dispensable. DAC1 and DAC5 are localized in the cytoplasm, and DAC3 and DAC4 in the nucleus. Functional analysis implicates DAC1, DAC3, and DAC5 in procyclic proliferation, whereas DAC1 and DAC5 drive promastigote-to-metacyclic differentiation. DAC5 was required for metacyclogenesis in the sand flies, the promastigote–amastigote transition, and amastigote intracellular replication. Notably, DAC5-null parasites failed to induce lesions in mice, displaying an attenuated phenotype. Proteomic profiling uncovered altered acetylation patterns in DAC mutants, linking DAC5 to cytoskeleton regulation and cell cycle control. These findings identify acetylation as a central regulator of Leishmania stage differentiation and highlight DAC5 as a key factor in parasite virulence.