bioRxiv : the preprint server for biology最新文献

{"title":"NitrOFF: An engineered fluorescent biosensor to illuminate nitrate transport in living cells.","authors":"Mariah A Cook, Jonathan D Smailys, Ke Ji, Shelby M Phelps, Jasmine N Tutol, Wantae Kim, Whitney S Y Ong, Weicheng Peng, Caden Maydew, Y Jessie Zhang, Sheel C Dodani","doi":"10.1101/2025.03.22.644677","DOIUrl":"https://doi.org/10.1101/2025.03.22.644677","url":null,"abstract":"<p><p>The duality of nitrate is nowhere best exemplified than in human physiology - a detrimental pollutant but also a protective nutrient and signaling ion - particularly as connected to reactive nitrogen oxides. Aside from limited insights into nitrate uptake and storage, foundational nitrate biology has lagged. Genetically encoded fluorescent biosensors can address this gap with real-time imaging. However, imaging technologies for mammalian cell applications remain rare. Here, we set out to design and engineer a two-domain chimera fusing the split green fluorescent protein EGFP and the nitrate recognition domain NreA from <i>Staphylococcus carnosus</i> . Over 7 rounds of directed evolution, 15 mutations were accumulated resulting in the functional biosensor NitrOFF. NitrOFF has a high degree of allosteric communication between the domains reflected in a turn-off intensiometric response ( <i>K</i> _d ≈ 9 µM). This was further reinforced by X-ray crystal structures of apo and nitrate bound NitrOFF, which revealed that the two domains undergo a large-scale conformational rearrangement that changes the relative positioning of the EGFP and NreA domains by 68.4°. Such a dramatic difference was triggered by the formation of a long helix at the engineered linker connecting the two domains, peeling the β7 strand off the EGFP and thus extinguishing the fluorescence upon nitrate binding. Finally, as a proof-of-concept, we highlighted the utility of this first-generation biosensor to monitor exogenous nitrate uptake and modulation in a human embryonic kidney (HEK) 293 cell line.</p><p><strong>Abstract figure: </strong></p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11957115/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143757291","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"LetA defines a structurally distinct transporter family involved in lipid trafficking.","authors":"Cristina C Santarossa, Yupeng Li, Sara Yousef, Hale S Hasdemir, Carlos C Rodriguez, Max B Haase, Minkyung Baek, Nicolas Coudray, John G Pavek, Kimber N Focke, Annika L Silverberg, Carmelita Bautista, Johannes Yeh, Michael T Marty, David Baker, Emad Tajkhorshid, Damian C Ekiert, Gira Bhabha","doi":"10.1101/2025.03.21.644421","DOIUrl":"https://doi.org/10.1101/2025.03.21.644421","url":null,"abstract":"<p><p>Membrane transport proteins translocate diverse cargos, ranging from small sugars to entire proteins, across cellular membranes. A few structurally distinct protein families have been described that account for most of the known membrane transport processes. However, many membrane proteins with predicted transporter functions remain uncharacterized. We determined the structure of <i>E. coli</i> LetAB, a phospholipid transporter involved in outer membrane integrity, and found that LetA adopts a distinct architecture that is structurally and evolutionarily unrelated to known transporter families. LetA functions as a pump at one end of a ~225 Å long tunnel formed by its binding partner, MCE protein LetB, creating a pathway for lipid transport between the inner and outer membranes. Unexpectedly, the LetA transmembrane domains adopt a fold that is evolutionarily related to the eukaryotic tetraspanin family of membrane proteins, including TARPs and claudins. LetA has no detectable homology to known transport proteins, and defines a new class of membrane transporters. Through a combination of deep mutational scanning, molecular dynamics simulations, AlphaFold-predicted alternative states, and functional studies, we present a model for how the LetA-like family of membrane transporters may use energy from the proton-motive force to drive the transport of lipids across the bacterial cell envelope.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11957124/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143757435","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Single VLP lipid-mixing measurements confirm off-pathway state in dengue virus fusion mechanism.","authors":"Tasnim K Anika, Fiona Campbell, Bianca Linden, Connor J Criswell, Miranda Kimm, Priscilla Li-Ning Yang, Robert J Rawle","doi":"10.1101/2025.03.21.644571","DOIUrl":"https://doi.org/10.1101/2025.03.21.644571","url":null,"abstract":"<p><p>Dengue virus (DENV) is the causative agent of dengue fever and exerts a substantial healthcare burden worldwide. Like other flaviviruses, DENV must undergo membrane fusion with the host cell in order to initiate infection. This membrane fusion occurs following acidification during endocytosis and is pH dependent. Here, we interrogate whether the mechanism of DENV fusion contains an off-pathway state, such has been reported previously for two other flaviviruses - Zika virus and West Nile virus. To do this, we utilize single particle lipid mixing measurements of DENV virus-like particles (VLPs) to tethered liposomes, together with computational modeling inspired by chemical kinetics. By observing and then modeling the pH dependence of single VLP fusion kinetics, we provide evidence that the DENV fusion mechanism must contain an off-pathway state. Measuring the proportion of VLPs undergoing hemi-fusion over time, we also demonstrate that the off-pathway state appears to be slowly reversible over tens of minutes, at least for some virions. Additionally, we find that late endosomal anionic lipids do not appear to influence the off-pathway mechanism to any great extent. In conjunction with the prior reports on Zika virus and West Nile virus, this work indicates that an off-pathway fusion state may be a feature of flavivirus fusion more broadly. We also note that the platform and mechanistic model described in this study may be useful in elucidating the mechanism of action of small molecule inhibitors of flavivirus fusion developed by our group and others.</p><p><strong>Statement of significance: </strong>Dengue virus (DENV) causes dengue fever and infects an estimated hundreds of millions of people annually. To date, there are no specific antiviral drugs for DENV and limited vaccination options, highlighting the need to better understand this important pathogen. In this report, we investigate the mechanism of DENV membrane fusion, an early step in the viral infectious cycle, using a mix of experimental techniques and computer simulations. We find strong evidence that the DENV fusion mechanism contains an off-pathway state, in which it can get stalled prior to membrane fusion. Understanding this off-pathway state could be an avenue to develop antiviral strategies against DENV and other related viruses.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11957162/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143757589","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The <i>Legionella pneumophila</i> peptidoglycan recycling kinase, AmgK, is essential for survival and replication inside host alveolar macrophages.","authors":"Sushanta Ratna, Lina Pradhan, Marina P Vasconcelos, Aastha Acharya, Bella Carnahan, Alex Wang, Arit Ghosh, Abigail Bolt, Jacob Ellis, Stephen N Hyland, Ashlyn S Hillman, Joseph M Fox, April Kloxin, M Ramona Neunuebel, Catherine Leimkuhler Grimes","doi":"10.1101/2025.03.21.644609","DOIUrl":"https://doi.org/10.1101/2025.03.21.644609","url":null,"abstract":"&lt;p&gt;&lt;p&gt;Bacterial cells are surrounded by a dynamic cell wall which in part is made up of a mesh-like peptidoglycan (PG) layer that provides the cell with structural integrity and resilience. In Gram-positive bacteria, this layer is thick and robust, whereas in Gram-negative bacteria, it is thinner and flexible as the cell is supported by an additional outer membrane. PG undergoes continuous turnover, with degradation products being recycled to maintain cell wall homeostasis. Some Gram-negative species can bypass &lt;i&gt;de novo&lt;/i&gt; PG biosynthesis, relying instead on PG recycling to sustain growth and division. &lt;i&gt;Legionella pneumophila&lt;/i&gt; (hereafter &lt;i&gt;Legionella&lt;/i&gt; ), the causative agent of Legionnaires' disease, encodes such recycling machinery within its genome. This study investigates the biochemical, genetic, and pathogenic roles of PG recycling in &lt;i&gt;Legionella&lt;/i&gt; . Previously, we have shown that PG can be visualized in both model and native systems using a combination of &lt;i&gt;N&lt;/i&gt; -acetylmuramic acid (NAM) probes and PG recycling programs. Here, two PG recycling gene homologs in the &lt;i&gt;Legionella&lt;/i&gt; genome &lt;i&gt;lpg0296&lt;/i&gt; ( &lt;i&gt;amgK&lt;/i&gt; ) and &lt;i&gt;lpg0295&lt;/i&gt; ( &lt;i&gt;murU)&lt;/i&gt; were identified and characterized; chemical biology strategies were used to rigorously track the incorporation of \"click\"-PG-probes. Deletion of &lt;i&gt;amgK&lt;/i&gt; abolished PG labeling, while genetic complementation restored labeling. Additionally, copper-free click chemistry with ultra-fast tetrazine-NAM probes enabled live-cell PG labeling. The data suggest that &lt;i&gt;amgK&lt;/i&gt; contributes to the pathogenicity of the organism, as &lt;i&gt;amgK&lt;/i&gt; deletion increased &lt;i&gt;Legionella&lt;/i&gt; 's susceptibility to antibiotics and significantly reduced &lt;i&gt;Legionella'&lt;/i&gt; s ability to replicate in host alveolar macrophages. An intracellular replication assay demonstrated that while PG recycling is not essential for internalization, successful replication of &lt;i&gt;Legionella&lt;/i&gt; within MH-S murine alveolar macrophages requires functional &lt;i&gt;amgK&lt;/i&gt; . These findings underscore the essential role of AmgK in &lt;i&gt;Legionella&lt;/i&gt; 's intracellular survival, emphasizing the importance of PG recycling in pathogenicity, and establish a foundation for developing novel &lt;i&gt;Legionella&lt;/i&gt; -specific antibiotic strategies.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Author summary: &lt;/strong&gt;In this work, we evaluated the functionality of the peptidoglycan kinase, AmgK, from &lt;i&gt;Legionella&lt;/i&gt; . AmgK homologs from other organisms have been shown to play a role in peptidoglycan (PG) recycling. Here we performed a series of chemical biology, genetic and biochemical experiments to show that &lt;i&gt;Legionella&lt;/i&gt; carries a functional AmgK. The data show that without AmgK &lt;i&gt;Legionella&lt;/i&gt; becomes hypersensitive to Fosfomycin, an antibiotic that targets bacterial cell wall biosynthesis. Moreover, PG recycling is required for &lt;i&gt;Legionella&lt;/i&gt; to successfully replicate within a host macrophage. Collectively, the data point to a role of PG recycling in pathogenic","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11957156/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143757473","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Dependence of cell fate potential and cadherin switching on primitive streak coordinate during differentiation of human pluripotent stem cells.","authors":"Ye Zhu, Aryeh Warmflash","doi":"10.1101/2025.01.31.635963","DOIUrl":"10.1101/2025.01.31.635963","url":null,"abstract":"<p><p>During gastrulation, the primitive streak (PS) forms and begins to differentiate into mesendodermal subtypes. This process involves an epithelial-mesenchymal transition (EMT), which is marked by cadherin switching, where E-Cadherin is downregulated, and N-Cadherin is upregulated. To understand the relationships between differentiation, EMT, and cadherin switching, we made measurements of these processes during differentiation of human pluripotent stem cells (hPSCs) to PS and subsequently to mesendoderm subtypes using established protocols, as well as variants in which signaling through key pathways including Activin, BMP, and Wnt were modulated. We found that perturbing signaling so that cells acquired identities ranging from anterior to posterior PS had little impact on the subsequent differentiation potential of cells but strongly impacted the degree of cadherin switching. The degree of E-Cadherin downregulation and N-Cadherin upregulation were uncorrelated and had different dependence on signaling. The exception to the broad potential of cells throughout the PS was the loss of definitive endoderm potential in cells with mid to posterior PS identities. Thus, cells induced to different PS coordinates had similar potential within the mesoderm but differed in cadherin switching. Consistently, E-Cadherin knockout did not alter cell fates outcomes during differentiation. Overall, cadherin switching and EMT are modulated independently of cell fate commitment in mesendodermal differentiation.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11838492/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143461710","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bacterial ubiquitin ligase engineered for small molecule and protein target identification.","authors":"James S Ye, Abir Majumdar, Brenden C Park, Miles H Black, Ting-Sung Hsieh, Adam Osinski, Kelly A Servage, Kartik Kulkarni, Jacinth Naidoo, Neal M Alto, Margaret M Stratton, Dominique Alfandari, Joseph M Ready, Krzysztof Pawłowski, Diana R Tomchick, Vincent S Tagliabracci","doi":"10.1101/2025.03.20.644192","DOIUrl":"https://doi.org/10.1101/2025.03.20.644192","url":null,"abstract":"<p><p>The <i>Legionella</i> SidE effectors ubiquitinate host proteins independently of the canonical E1-E2 cascade. Here we engineer the SidE ligases to develop a modular proximity ligation approach for the identification of targets of small molecules and proteins, which we call SidBait. We validate the method with known small molecule-protein interactions and use it to identify CaMKII as an off-target interactor of the breast cancer drug ribociclib. Structural analysis and activity assays confirm that ribociclib binds the CaMKII active site and inhibits its activity. We further customize SidBait to identify protein-protein interactions, including substrates for enzymes, and discover the F-actin capping protein (CapZ) as a target of the <i>Legionella</i> effector RavB during infection. Structural and biochemical studies indicate that RavB allosterically binds CapZ and decaps actin, thus functionally mimicking eukaryotic CapZ interacting proteins. Collectively, our results establish SidBait as a reliable tool for identifying targets of small molecules and proteins.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11957136/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143757197","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Heterozygosity at a conserved candidate sex determination locus is associated with female development in the clonal raider ant (<i>Ooceraea biroi</i>).","authors":"Kip D Lacy, Jina Lee, Kathryn Rozen-Gagnon, Wei Wang, Thomas S Carroll, Daniel J C Kronauer","doi":"10.1101/2025.01.24.634795","DOIUrl":"10.1101/2025.01.24.634795","url":null,"abstract":"<p><p>Sex determination is a developmental switch that triggers sex-specific developmental programs. This switch is \"flipped\" by the expression of genes that promote male- or female-specific development. Many lineages have evolved sex chromosomes that act as primary signals for sex determination. However, haplodiploidy (males are haploid and females are diploid), which occurs in ca. 12% of animal species, is incompatible with sex chromosomes. Haplodiploid taxa must, therefore, rely on other strategies for sex determination. One mechanism, \"complementary sex determination\" (CSD), uses heterozygosity as a proxy for diploidy. In CSD, heterozygosity at a sex determination locus triggers female development, while hemizygosity or homozygosity permits male development. CSD loci have been mapped in honeybees and two ant species, but we know little about their evolutionary history. Here, we investigate sex determination in the clonal raider ant, <i>Ooceraea biroi</i>. We identified a 46kb candidate CSD locus at which all females are heterozygous, but most diploid males are homozygous for either allele. As expected for CSD loci, the candidate locus has more alleles than most other loci, resulting in a peak of nucleotide diversity. This peak negligibly affects the amino acid sequences of protein-coding genes, suggesting that heterozygosity of a non-coding genomic sequence triggers female development. This locus is distinct from the CSD locus in honeybees but homologous to a CSD locus mapped in two distantly related ant species, implying that this molecular mechanism has been conserved since a common ancestor that lived approximately 112 million years ago.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11838215/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143461754","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"AnnSQL: A Python SQL-based package for fast large-scale single-cell genomics analysis using minimal computational resources.","authors":"Kenny Pavan, Arpiar Saunders","doi":"10.1101/2024.11.02.621676","DOIUrl":"10.1101/2024.11.02.621676","url":null,"abstract":"<p><p>As single-cell genomics technologies continue to accelerate biological discovery, software tools that use elegant syntax and minimal computational resources to analyze atlas-scale datasets are increasingly needed. Here we introduce AnnSQL, a Python package that constructs an AnnData-inspired database using the in-process DuckDb engine, enabling orders-of-magnitude performance enhancements for parsing single-cell genomics datasets with the ease of SQL. We highlight AnnSQL functionality and demonstrate transformative runtime improvements by comparing AnnData or AnnSQL operations on a 4.4 million cell single-nucleus RNA-seq dataset: AnnSQL-based operations were executed in minutes on a laptop for which equivalent AnnData operations largely failed (or were ∼700x slower) on a high-performance computing cluster. AnnSQL lowers computational barriers for large-scale single-cell/nucleus RNA-seq analysis on a personal computer, while demonstrating a promising computational infrastructure extendable for complete single-cell workflows across various genome-wide measurements.</p><p><strong>Availability and implementation: </strong>AnnSQL is a pip installable package that can be found at https://github.com/ArpiarSaundersLab/annsql along with documentation at https://docs.annsql.com .</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11661128/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142879199","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Trehalose dimycolate inhibits phagosome maturation and promotes intracellular <i>M. tuberculosis</i> growth via noncanonical SNARE interaction.","authors":"Carolina Santamaria, Kyle J Biegas, Pamelia N Lim, Jessica Cabral, Christi Y Kim, James R Lee, Ishani V Gaidhane, Casey Papson, Kyla Gomard-Henshaw, Alissa C Rothchild, Benjamin M Swarts, M Sloan Siegrist","doi":"10.1101/2024.12.16.627577","DOIUrl":"https://doi.org/10.1101/2024.12.16.627577","url":null,"abstract":"<p><p>Mycobacterial cell envelopes are rich in unusual lipids and glycans that play key roles during infection and vaccination. The most abundant envelope glycolipid is trehalose dimycolate (TDM). TDM compromises the host response to mycobacterial species via multiple mechanisms, including inhibition of phagosome maturation. The molecular mechanism by which TDM inhibits phagosome maturation has been elusive. We find that a clickable, photoaffinity TDM probe recapitulates key phenotypes of native TDM in macrophage host cells and binds several host SNARE proteins, including VTI1B, STX8, and VAMP2. VTI1B and STX8 normally promote endosome fusion by forming a complex with VAMP8. However, in the presence of <i>Mycobacterium tuberculosis</i> , VTI1B and STX8 complex with VAMP2, which in turn decreases VAMP8 binding. VAMP2 acts together with mycolate structure to inhibit phagosome maturation and promotes intracellular <i>M. tuberculosis</i> replication. Thus one mechanism by which TDM constrains the innate immune response to <i>M. tuberculosis</i> is via non-canonical SNARE complexation.</p><p><strong>Significance statement: </strong>Glycolipids from the <i>Mycobacterium tuberculosis</i> cell envelope, particularly trehalose dimycolate (TDM), play major roles in subverting the immune response to this intracellular pathogen. How subversion occurs is often unclear because glycans and lipids are technically challenging to study in cells. We discovered that a TDM-mimicking chemical probe interacts with three host SNARE proteins, including two that regulate endosome fusion and one that does not. The presence of TDM or <i>M. tuberculosis</i> triggers abnormal binding of these SNAREs, which in turn inhibits the fusion of <i>M. tuberculosis</i> -containing phagosomes with lysosomes and promotes <i>M. tuberculosis</i> replication. Our work provides an unusual example of a bacterial pathogen restricting the immune response via glycolipid-SNARE interactions.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11702582/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143757608","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Three-photon population imaging of subcortical brain regions.","authors":"Hadas Frostig, Amy Monasterio, Hongjie Xia, Urvi Mishra, Baldwin Britton, John T Giblin, Jerome Mertz, Benjamin B Scott","doi":"10.1101/2025.03.21.644611","DOIUrl":"https://doi.org/10.1101/2025.03.21.644611","url":null,"abstract":"<p><p>Recording activity from large cell populations in deep neural circuits is essential for understanding brain function. Three-photon (3P) imaging is an emerging technology that allows for imaging of structure and function in subcortical brain structures. However, increased tissue heating, as well as the low repetition rate sources inherent to 3P imaging, have limited the fields of view (FOV) to areas of ≤0.3 mm ² . Here we present a Large Imaging Field of view Three-photon (LIFT) microscope with a FOV of [gt]3 mm ² . LIFT combines high numerical aperture (NA) optimized sampling, using a custom scanning module, with deep learning-based denoising, to enable population imaging in deep brain regions. We demonstrate non-invasive calcium imaging in the mouse brain from >1500 cells across CA1, the surrounding white matter, and adjacent deep layers of the cortex, and show population imaging with high signal-to-noise in the rat cortex at a depth of 1.2 mm. The LIFT microscope was built with all off-the-shelf components and allows for a flexible choice of imaging scale and rate.</p>","PeriodicalId":519960,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11957121/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143757625","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}