Biology subjects

Srinivasan, R.

Publications and source records attributed to Srinivasan, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Microbial diversity and insights into feeding behavior of the two-spot cotton leaf hopper (Amrasca biguttula)

The recent invasion of the two-spot cotton leaf hopper (TSCL) (Amrasca biguttula) into the U.S. now threatens row and vegetable crops as well as ornamentals. TSCL ecology and biology, including microbial diversity, are understudied. Insect-specific microbes could influence host biology and ecology including insect-plant interactions. Here, microbiota (bacteria and viruses) within TSCL along with its feeding behavior were characterized using high throughput sequencing, microscopy, and Electrical penetration graph (EPG). Results revealed low diversity of bacteria dominated by Wolbachia and confirm the absence of obligate (primary) symbionts. Two distinct and differentially abundant Wolbachia strains (B-supergroup) were found co-infecting midgut tissues of 60% of TSCL tested, and the remaining 40% were free of symbionts. Virome analysis uncovered a diverse complex of at least eight dsDNA genomes (12-17 kb) of Adintoviruses (Eupolintoviridae), but no RNA viruses commonly found in other leafhoppers were identified in TSCL. The mesophyll feeding nature of TSCL was identified by observing chlorophyll derived autofluorescence in midguts and higher abundance of chloroplast transcripts (photosystem I, II, Rubisco, cytochrome b6 and ATP synthase complex) in TSCL (whole insects) compared with thrips and aphids. EPG recordings show a cell-rupture style of feeding with differences between adults and nymphs. Lastly, consistent with its cell-rupturing feeding style, no evidence for the presence of plant-pathogenic bacteria or viruses that could induce hopperburn was found. These results overall provide insights into the microbial diversity and feeding pattern of TSCL and lay a foundation for future research. Key messageThis work characterizes the TSCL microbiome and its mesophyll feeding pattern. Symbionts within insects often complement the nutritional deficiencies of its host and can aid insect spread by boosting immunity. The findings of this study lay the foundation for future research on the biology, ecology, and management of this invasive pest.

microbiology↗

bioRxiv2026

A GATA2-PROX1-FOXC2 regulatory axis integrates shear stress signaling with lymphatic vascular development

The two-hit hypothesis provides a novel framework for incompletely penetrant vascular disorders. Emberger syndrome, caused by heterozygous loss-of-function mutations in mechanosensitive transcription factor (TF) GATA2, is associated with lymphedema in a subset of patients, suggesting that modifiers influence disease development. Heterozygous loss-of-function mutations in mechanosensitive TF FOXC2 also cause lymphedema. Complete deletion of either factor in lymphatic endothelial cells (LECs) causes severe, overlapping defects, including complete loss of lymphatic valves, whereas single heterozygous mutants exhibit milder phenotypes, suggesting genetic buffering. However, whether GATA2 and FOXC2 interact with each other and with shear stress-dependent transcriptional programs remains unclear. Here, we show that Gata2+/-;Foxc2+/- mice develop profound lymphatic vascular defects, including absent lymphatic valves, perinatal lethality, and valve dysfunction, revealing dosage-dependent cooperation between GATA2 and FOXC2. Using a Cre-dependent model of LEC-specific GATA2 overexpression, we found that increased GATA2 dosage downregulated PROX1 and disrupted lymphatic vascular development. ATAC-seq and bulk RNA-seq of shear-exposed human LECs showed that PROX1 governs shear-responsive genes, including KLF2 and KLF4, whereas FOXC2 modulates PROX1-dependent and independent gene subsets. Together, these findings identify PROX1 as a central integrator of shear stress-responsive transcriptional programs and reveal that balanced GATA2 and FOXC2 dosage preserves this network during lymphatic vascular development.

Developmental Biology↗

bioRxiv2026

Wnt stimulation and inhibition in the development and phenotype of patient-derived gallbladder organoids

Gallbladder cancer (GBC) is a highly lethal malignancy with limited experimental models to study disease biology or evaluate therapeutic responses. Although canonical Wnt activation is commonly used for patient-derived organoid (PDO) development and expansion, gallbladder PDOs has also been generated under Wnt-inhibitory conditions. No comparative assessment has determined how Wnt pathway modulation influences gallbladder PDO development, phenotype or drug response. This study systematically compared the impact of canonical Wnt activation (WNTAct medium containing CHIR99021) versus inhibition (WNTInh medium containing DKK1) on the establishment, propagation, molecular features and therapeutic responses of PDOs generated from malignant or non-malignant gallbladder tissues derived from the same patient. Both media supported successful PDO generation with comparable efficiency, preserving biliary epithelial functions and marker expression. Transcriptomic profiling confirmed selective enrichment of canonical Wnt target genes in PDOs generated in WNTAct cultures. WNTAct conditions enabled markedly superior long-term propagation, whereas WNTInh cultures more consistently retained the dysplastic features in malignant samples. Gemcitabine response assays demonstrated significantly greater drug sensitivity in PDOs grown in WNTAct medium, a phenotype reversible upon media switching but requiring extended adaptation, indicating a dynamic and context-dependent influence of Wnt signaling on chemotherapeutic vulnerability. Collectively, the findings reveal a trade-off between long-term propagation and histological fidelity in gallbladder PDOs and show that Wnt signaling modulates gemcitabine sensitivity in a reversible manner. This comparative framework provides practical guidance for selecting culture conditions for gallbladder PDO based disease modelling and precision oncology applications.

cell biology↗

bioRxiv2025

Comparative genomics and interactions of vector thysanopterans and transmitted viruses

We analyzed the genomes of nine thysanopteran (thrips) species, with one newly generated, and examined their relationships with 23 representative orthotospoviruses from the family Tospoviridae (order Bunyavirales). Thrips can be agricultural pests, contributing to measurable yield reductions in economically valuable crops and ornamentals. Some thrips species are confirmed orthotospovirus vectors, while most of the [~]7,000 identified species are unknown in their vector status. We conducted in silico protein-protein interaction predictions for several thrips proteins, including an endocuticle protein previously reported to bind to orthotospovirus glycoproteins. In most ecologically observed vector-virus pairs, the predicted protein-protein interactions were confirmed, and additional plausible vector-virus transmission interactions emerged from our analyses. These results expand our understanding of vector-virus co-evolution and highlight candidate molecular interfaces that could be targeted to disrupt virus transmission in agricultural systems.

bioinformatics↗

bioRxiv2025

Distinct, asynchronous processes underlie cultural and genetic variation in a bird species with a complex song repertoire

Dialects in birdsongs represent population clusters and cultural diversity. Cultural and genetic variation in animals, however, do not always evolve along the same trajectory. Few studies have tested this in a wild songbird with a continuous learning strategy. Songbirds (oscine Passeriformes) with a continual learning window exhibit great song diversity and may show cultural differentiation that outpaces genetic divergence. We investigate cultural and genetic variation in a tropical endemic songbird in a topographically and climatically complex sky-island landscape of the Western Ghats of India. We combine fine-scale acoustic, behavioural, and genetic data: from 90 individuals (10,816 songs), analysed using frequency-temporal parameters and a linguistic method for syntax; a playback experiment with 136 trials to compare behavioural responses to local and non-local songs; and blood samples from 242 individuals (10,638 SNPs) to infer genetic population structure. We find no difference in songs across locations and individuals in the time-frequency domain, but only in syntax. One-third of notes, the smallest vocal units, are widely shared across all individuals in their distribution. Longer note combinations result in greater individual differences within populations, but only form small differences between populations. Geographic distance strongly predicts genetic but not song syntactic differences, and playback experiments show equal responses to local and non-local songs. Thus, strong individual signatures but weak local-level dialects are generated by a shared note repertoire and associated combinatorial syntax. We highlight that cultural transmission may operate at fine social scales, decoupled from genetic structure, which is strongly driven by isolation-by-distance. Significance statementWhether learned behaviours exhibit patterns similar to those of inherited genes remains a long-standing question in evolutionary biology. Birdsong has a strong cultural component paralleling human language, yet most knowledge comes from birds with a narrow learning window. Open-ended learners, who acquire new songs throughout life like humans, are poorly documented in the wild. We studied such a songbird restricted to the tropical cloud montane forests of southern India. Songs appear acoustically similar across populations but differ in how basic units are ordered (syntax), while individuals are more distinct from each other in longer note combinations than populations are. Genetic similarity decreased with geographic distance. Culturally learned traits can thus evolve independently of genetic structure, showing how learning decouples behaviour from geography-driven genetic ancestry.

evolutionary biology↗

bioRxiv2025

Mitochondrial DNA damage in substantia nigra parscompacta astrocytes exacerbates dopaminergic neuron lossin a 6-hydroxydopamine mouse model of parkinsonism

Parkinsons disease (PD) is the fastest growing neurological disorder with no known cure. Our ability to develop disease-modifying treatments that slow down the loss of substantia nigra pars compacta (SNc) dopaminergic (DA) neurons is hindered by a dearth of knowledge on roles for non-neuronal elements such as astrocytes during PD pathogenesis. More specifically, the extent to which mitochondrial DNA (mtDNA) damage in SNc astrocytes contributes to SNc DA neuron loss during PD remains unknown. To address this knowledge gap, we utilized an adeno-associated virus (AAV) called Mito-PstI that expresses the restriction enzyme PstI as an approach to damage mtDNA in SNc astrocytes and assess the effect of astrocytic mtDNA damage on SNc DA neuron function and viability in mice. Mito-PstI-induced mtDNA damage in SNc astrocytes disrupted mitochondrial morphology, caused increased wrapping of SNc astrocytic processes around SNc DA neurons, and abnormally increased dopamine release by SNc DA neuron axonal terminals within the dorsolateral striatum (DLS). In addition, mice injected with Mito-PstI in the SNc showed increased spontaneous and apomorphine-induced rotations contralateral to the side with SNc Mito-PstI injections. In further experiments, we used a parkinsonian mouse model with low dose 6-hydroxydopamine (6-OHDA) injection into the DLS to show that Mito-PstI expression in SNc astrocytes caused a worsening of 6-OHDA-induced spontaneous contralateral rotational behavior, and exacerbated SNc DA neuron loss. These results suggest that mitochondria in SNc astrocytes are not only critical for the function of SNc DA neurons, but are also a new target for developing disease-modifying strategies against PD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=157 SRC="FIGDIR/small/673287v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@b4a98aorg.highwire.dtl.DTLVardef@e6e2fcorg.highwire.dtl.DTLVardef@402b93org.highwire.dtl.DTLVardef@ecdfd2_HPS_FORMAT_FIGEXP M_FIG C_FIG Main PointsO_LIMito-PstI expression in mouse SNc astrocytes increases dopamine release C_LIO_LIMito-PstI expression in SNc astrocytes worsens contralateral rotational behavior in mice C_LIO_LIMito-PstI expression in SNc astrocytes increases DA neuron loss in 6-OHDA mice C_LI

neuroscience↗

bioRxiv2025

An improved deep learning model for immunogenic B epitope prediction

The recognition of B epitopes by B cells of the immune system initiates an immune response that leads to the production of antibodies to combat bacterial and viral infections. Computational methods for predicting the epitopes on antigens have shown promising results in the development of subunit vaccines and therapeutics. Recently, the use of protein language models (pLMs) for epitope prediction has led to a substantial increase in prediction accuracy. However, further improvements in precision are necessary for practical applications. Here, we develop and evaluate a series of models using different combinations of features and feature fusion techniques on a curated independent test set. Our results show that the models that use protein embeddings along with structural features are better at predicting both linear and conformational B epitopes when compared to a baseline model that uses only protein embeddings as features. Additionally, we show that the embeddings of ESM-2, an evolutionary scale model, likely capture T-B reciprocity.

bioinformatics↗

bioRxiv2025

Dual Inhibition of CDK4/6 and mTORC1 Establishes a Preclinical Strategy for Translocation Renal Cell Carcinoma

PurposeTranslocation renal cell carcinoma (tRCC) is a rare and aggressive subtype of kidney cancer driven by an oncogenic fusion involving a transcription factor in the MiT/TFE gene family, most commonly TFE3. Treatment of tRCC currently lacks a clear standard of care, underscoring the pressing need to nominate new therapeutic targets with mechanistic rationale in this cancer. Experimental DesignIn this study, we applied integrative genomic approaches to identify activation of the cyclin-dependent kinase 4/6 (CDK4/6) and mammalian target of rapamycin complex 1 (mTORC1) pathways in tRCC. We tested the activity of CDK4/6 inhibitors (CDK4/6i), alone or in combination with mTORC1-selective inhibition, using in vitro and in vivo models of tRCC. ResultstRCC tumors displayed multiple genomic and transcriptional features associated with activation of the CDK4/6 and mTORC1 signaling pathways. Genetic or pharmacologic inhibition of CDK4/6 suppressed tRCC cell growth and induced cell cycle arrest in vitro but was not cytotoxic, with rapid cell regrowth observed after drug withdrawal. The mTORC1-selective inhibitor, RMC-5552, potently reduced translation of Cyclin D1, which complexes with CDK4/6 proteins to regulate G1-S cell cycle progression. Combined treatment with the CDK4/6 inhibitor, palbociclib, and RMC-5552 resulted in synergistic suppression of tRCC cell viability and increased markers of apoptosis in vitro. The combination of palbociclib and RMC-5552 in a tRCC xenograft model showed greater eOicacy than either single agent while also being well-tolerated. ConclusionsOur study indicates the therapeutic potential of combined CDK4/6 and mTORC1 inhibition in tRCC, providing the rationale for further clinical evaluation of this strategy. Translational RelevanceTranslocation renal cell carcinoma (tRCC) is a rare and aggressive form of kidney cancer that accounts for 2-5% of all RCCs in adults and around 50% of RCCs in children. tRCC lacks effective therapies and represents a significant unmet clinical need. Therapies approved for clear cell RCC (ccRCC), including VEGF/multikinase inhibitors and immune checkpoint blockade, typically show decreased efficacy in tRCC. Via an integrative genomic analysis, we nominate combined inhibition of the cyclin-dependent kinase 4/6 (CDK4/6) and mammalian target of rapamycin complex 1 (mTORC1) pathways in tRCC. We demonstrate that combining the CDK4/6 inhibitor palbociclib with the mTORC1-selective inhibitor RMC-5552 synergistically inhibits the growth of tRCC cells in vitro and of tRCC xenografts in vivo, where the combination is well-tolerated. This proof-of-concept study provides preclinical evidence supporting CDK4/6 inhibitor-based combination regimens tailored to tRCC biology, offering a foundation for future clinical studies in tRCC, which currently has no clear standard of care.

cancer biology↗

bioRxiv2025

Loss of Nucleotide Sugar Transporter (AtNST) gene function in the Golgi membranes impairs pollen development and embryo sac progression in Arabidopsis thaliana

O_LINucleotide Sugar Transporters (NSTs) are transmembrane proteins which are localized in the Golgi membranes and transport nucleotide sugars from cytosol to Golgi lumen. Transported nucleotide-sugars serve as donors in post-translational modification of proteins/ lipids, and donate sugar moiety to growing carbohydrate chain on nascent protein/lipid molecule, a reaction catalyzed by the enzyme glycosyltransferase. C_LIO_LIHere we reported that a mutation in the Arabidopsis thaliana gene At3g11320 coding for NST protein causes defects in male gametophyte development including collapsed, nonviable pollen. This mutation also caused impairment in the female gametophyte progression by arresting it at the functional megaspore (FM) stage. C_LIO_LIFurther, the mutant phenotype including silique size and seed set was reverted when the cDNA of AtNST gene was over-expressed in the mutant back ground. No abortive ovules were found in the siliques from the complemented plants. C_LIO_LIThe results suggest that AtNST (At3g11320) gene might be responsible for maintaining and regulating transport of nucleotide-sugars from cytoplasm to the Golgi lumen for the glycosylation of essential proteins/carbohydrates/lipids etc., which are necessary for both male and female gametophyte development stages. This study shed further light on the role of such nucleotide sugar transporters in plant reproductive development processes, at least in Arabidopsis. C_LI

plant biology↗

bioRxiv2025

Gcn4 mediates SAM allocations and methylation balance between phospholipids and histones

Methionine availability alters the methylation status of various cellular molecules, and thereby controls metabolic state, via its metabolite S-adenosyl methionine (SAM). In yeast cells, supplementing excess methionine drives cell growth and proliferation, as well as phospholipid biogenesis. High methionine stabilizes the transcription factor Gcn4 (Atf4), which drives an anabolic transcription program to sustain high growth. Here, we investigated whether Gcn4 regulates the allocation of SAM towards distinct methylation outputs, during this methionine-dependent anabolic program. We find that methionine induced Gcn4 increases the expression of phospholipid methyltransferases during methionine supplementation. In{Delta} gcn4 cells, the expression of these methyltransferases significantly decreases, and results in reduced SAM allocations towards phospholipid methylation, alongside increased intracellular SAM, disrupting the balance in the SAM/SAH ratio in these cells. Excess SAM pools are re-allocated to histones, resulting in their hyper-methylation. Collectively, we uncover that Gcn4 modulates SAM allocations towards phospholipid methyltransferases, and the overall methyl allocation between phospholipids and histones. Our study reveals a novel role for Gcn4 in SAM dependent phospholipid biosynthesis in cells growing in methionine sufficiency.

cell biology↗

bioRxiv2025

Developing in vitro patient derived CERvical Cancer OrganoidS (CERCOS) as a potential preclinical tool in cervical cancer research

Advanced cervical cancer remains a major cause of mortality in women worldwide as it has limited treatment options and recurrence is very common. This highlights the necessity to develop patient derived organoids (PDOs) as preclinical models, that can recapitulate the clinical heterogeneity of the cancer in terms of molecular features, and genetic background. The PDOs have potential for guiding personalized treatment in clinical practice. In this study, we have established patient-derived cervical cancer organoids or CERvical Cancer OrganoidS (CERCOS) from biopsy samples of five patients having two different subtypes (squamous cell carcinoma and adenocarcinoma) using a modified protocol. The CERCOS were characterized to assess their genetic and phenotypic similarity with the parent tissue. The organoids developed in vitro showed preservation of the histopathological and somatic mutational profile of the parent tissue. Expression of cervical cancer-related genes, including PIK3CA, MET, and LRP1B, was found to be comparable between the organoids and the parent tissue. Moreover, characterization of the PDOs post cryopreservation showed the maintenance of the morphological and histopathological features of the parent tissue. This study demonstrates that the CERvical Cancer OrganoidS (CERCOS), established using the current protocol, preserves genetic and phenotypic similarity and can serve as a platform for precision medicine and biobanking purposes.

cancer biology↗

bioRxiv2025

Genetically encoded nAChR upregulation is neuroprotective in female parkinsonian mice

Parkinsons disease is projected to rise to pandemic proportions by 2050, which has resulted in an urgent need for disease-modifying treatments. In this regard, we previously showed that in a mouse model of parkinsonism with unilateral 6-hydroxydopamine (6-OHDA) injection into the dorsolateral striatum (DLS), low doses of the neuronal nicotinic acetylcholine receptor (nAChR) partial agonist and smoking cessation drug, cytisine exerts sex-specific neuroprotection in substantia nigra pars compacta (SNc) dopaminergic (DA) neurons of only female mice by reducing apoptotic endoplasmic reticulum (ER) stress. Although these data suggest that neuroprotection might occur via cytisine-mediated upregulation of {beta}2 subunit-containing ({beta}2*) nAChRs in SNc DA neurons, there is no direct evidence to support this idea. Therefore, this study asks the critical question of whether upregulation of {beta}2* nAChRs in SNc DA neurons alone is sufficient to reduce apoptotic ER stress and exert neuroprotection in a preclinical unilateral DLS mouse model of 6-OHDA-induced parkinsonism. To address this question, we generate and characterize a novel {beta}2-upregulated transgenic mouse line. These transgenic mice possess mutations in the M3-M4 intracytoplasmic loop of {beta}2 subunits that cause constitutive upregulation of {beta}2* nAChRs without nicotinic ligands. Surprisingly, when compared to wild-type littermates, only female {beta}2-upregulated transgenic mice demonstrate upregulation of {beta}2* nAChRs in SNc DA neurons as assessed by significant increases in Sec24D-containing ER exit sites (Sec24D-ERES). Using the optogenetic calcium and dopamine sensors, GCaMP6f and GRABDA respectively, we found significant increases in dihydro-beta-erythroidine (Dh{beta}E)-sensitive {beta}2* nAChR-mediated calcium influx in SNc DA neuron dendrites and Dh{beta}E-sensitive acetylcholine (ACh)-evoked dopamine release at SNc DA neuron terminals of the DLS in female transgenic mice. We then used four independent readouts to assess neuroprotection of SNc DA neurons following unilateral 6-OHDA injection into the DLS, viz., contralateral apomorphine-induced rotations, preservation of SNc DA neurons, inhibition of a major proapoptotic ER stress protein, C/EBP homologous protein (CHOP) and glial fibrillary acid protein (GFAP) expression in SNc astrocytes. In all four readouts, female {beta}2-upregulated transgenic mice showed significant neuroprotection. From a clinical perspective, this study shows that upregulation without nicotinic ligand-mediated activation of {beta}2* nAChRs in SNc DA neurons can be a translationally viable disease-modifying strategy for Parkinsons disease. In addition, we envision that the novel transgenic {beta}2-upregulated mice created in this study will provide a valuable tool for understanding the role of nAChR upregulation in major neurological disorders such as addiction, anxiety, depression and dementia.

neuroscience↗

bioRxiv2025

SKiD: A Structure-Oriented Kinetics Database of Enzyme-Substrate Interactions

Enzymes are essential biological catalysts that drive nearly all biochemical reactions. Understanding their efficiency and specificity involves studying enzyme kinetics, particularly the parameters kcat and Km. However, there is limited data linking these kinetic parameters with the three-dimensional (3D) structures of enzyme-substrate complexes. Since enzyme function is determined by its structure, such mapping enhances insight into structural basis of enzymatic function and supports applications in enzyme design, synthetic biology and metabolic engineering. To address this critical gap, this work presents SKiD (Structure-oriented Kinetics Database), a comprehensive, structured database integrating kcat and Km values with the corresponding 3D structural data. This is accomplished by integrating data from existing bioinformatics resources using automated programs to process the data and enhancing it with computational predictions. The erroneous data encountered during data integration is manually resolved. Metadata such as literature and assay conditions (e.g., pH and temperature) are preserved. The 3D coordinates of the modelled enzyme-substrate complexes are provided along with their UniProtKB identifier. The database is freely accessible from: https://doi.org/10.5281/zenodo.15355031.

bioinformatics↗

bioRxiv2025

Insights into gallbladder cancer pathogenesis from a living organoid gallbladder cholangiocyte biorepository

Gallbladder cancer (GBC) while rare worldwide has a high prevalence in India. Pathogenesis is unclear and outcomes poor. Gallbladder cholangiocyte organoids (GCOs) or gallbladder carcinoma organoids (GBCOs) were developed and serially propagated from surgically resected gallbladder tissues with benign or malignant diseases, respectively. Patient derived organoids (PDOs) were derived from 15 normal; 58 inflamed; 12 xanthogranulomatous cholecystitis (XGC); 5 pre-invasive neoplasm and 13 invasive malignant gallbladder pathologies. Protocol optimisation achieved 58% (69/119) success in organoid generation and expansion. Organoids maintained tight junction integrity; P-gp pump and enzymatic activity; preserved tissue-specific gene and protein marker expression; histological features and genetic variations. Cryopreserved organoids from 62 patients with primary tissue and high-quality DNA, RNA and protein derivatives have been banked. In gene expression analyses of tissue, XGC samples clustered with malignant subtypes, separate from benign pathologies. Derived XGC organoids showed a similar clustering. Enriched hallmark pathways in XGC support neoplastic change through chronic inflammation. PDOs generated from different gallbladder pathologies are a promising model to investigate the pathogenesis of GBC. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/651639v1_ufig1.gif" ALT="Figure 1"> View larger version (69K): org.highwire.dtl.DTLVardef@465f3dorg.highwire.dtl.DTLVardef@16c5f0org.highwire.dtl.DTLVardef@11d4807org.highwire.dtl.DTLVardef@163d072_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Distinct filament morphology and membrane tethering features of the dual FtsZs in Odinarchaeota

The deep-branching Asgard archaea have emerged as a model to study Eukaryogenesis as they harbor homologs of many of the major eukaryotic protein families. In this study, we use Candidatus Odinarchaeota FtsZs as representatives to understand the probable origin, evolution, and assembly of the FtsZ-Tubulin protein superfamily in Asgard archaea. We have performed a comparative analysis of the biochemical properties and cytoskeletal assembly of FtsZ1 and FtsZ2, the two FtsZ isoforms in the Odinarchaeota metagenome. Our electron microscopy analysis reveals that OdinFtsZ1 assembles into curved single protofilaments, while OdinFtsZ2 forms stacked spiral ring-like structures. Upon sequence analysis, we identified an N-terminal amphipathic helix in OdinFtsZ1, which mediates direct membrane tethering. In contrast, OdinFtsZ2 is recruited to the membrane by the membrane-anchor OdinSepF via OdinFtsZ2s C-terminal tail. Overall, we report the presence of two distant evolutionary paralogs of FtsZ in Odinarchaeota, with distinct filament assemblies and differing modes of membrane targeting mechanisms, namely direct vs SepF-mediated membrane binding. Our findings highlight the diversity of FtsZ proteins in the transitional archaeal phylum Odinarchaeota, providing valuable insights into the evolution and differentiation of tubulin family proteins.

biochemistry↗

bioRxiv2024

Chemically-inducible CRISPR/Cas9 circuits for ultra-high dynamic range gene perturbation

CRISPR/Cas9 technologies provide unique capabilities for modeling disease and understanding gene-to-phenotype connections. In cultured cells, chemical-mediated control of Cas9 activity can limit off-target effects and enable mechanistic study of essential genes. However, widely-used Tet-On systems often show "leaky" Cas9 expression, leading to unintended edits, as well as weak activity upon induction. Leakiness can be distinctly problematic in the context of Cas9 nuclease activity, which may result in cumulative DNA damage and degradation of the target cell genome over time. To overcome these deficiencies, we established transgenic platforms that minimize Cas9 functionality in the off-state along with maximized and uncompromised on-state gene editing efficiency. By combining conditional destabilization and inhibition of Cas9, we developed an all-in-one (one or multiple guide RNAs and Cas9) ultra-tight, Tet-inducible system with exceptional dynamic range (on vs. off-state) across various cell lines and targets. As an alternative to Tet-mediated induction, we created a branaplam-regulated splice switch module for low-baseline and robust Cas9 activity control. Lastly, for circumstances where DNA damage needs to be avoided, we constructed a dual-control, Tet-inducible CRISPRi module for tight and potent transcriptional silencing. This upgraded suite of inducible CRISPR systems has broad applications for numerous cell types and experimental conditions.

molecular biology↗

bioRxiv2024

Optogenetic inhibition of light-captured alcohol-taking striatal engrams facilitates extinction and suppresses reinstatement

BackgroundAlcohol use disorder (AUD) is a complex condition, and it remains unclear which specific neuronal substrates mediate alcohol-seeking and -taking behaviors. Engram cells and their related ensembles, which encode learning and memory, may play a role in this process. We aimed to assess the precise neural substrates underlying alcohol-seeking and -taking behaviors and determine how they may affect one another. MethodsUsing FLiCRE (Fast Light and Calcium-Regulated Expression; a newly developed technique which permits the trapping of acutely activated neuronal ensembles) and operant-self administration (OSA), we tagged striatal neurons activated during alcohol-taking behaviors. We used FLiCRE to express an inhibitory halorhodopsin in alcohol-taking neurons, permitting loss-of-function manipulations. ResultsWe found that the inhibition of OSA-tagged alcohol-taking neurons decreased both alcohol-seeking and -taking behaviors in future OSA trials. In addition, optogenetic inhibition of these OSA-tagged alcohol-taking neurons during extinction training facilitated the extinction of alcohol-seeking behaviors. Furthermore, inhibition of these OSA-tagged alcohol-taking neurons suppressed the reinstatement of alcohol-seeking behaviors, but, interestingly, it did not significantly suppress alcohol-taking behaviors during reinstatement. ConclusionsOur findings suggest that alcohol-taking neurons are crucial for future alcohol-seeking behaviors during extinction and reinstatement. These results may help in the development of new therapeutic approaches to enhance extinction and suppress relapse in individuals with AUD.

neuroscience↗

bioRxiv2024

Heterogeneous brain region-specific responses to astrocytic mitochondrial DNA damage in mice

Astrocytes use Ca2+ signals to regulate multiple aspects of normal and pathological brain function. Astrocytes display context-specific diversity in their functions, and in their response to noxious stimuli between brain regions. Indeed, astrocytic mitochondria have emerged as key players in governing astrocytic functional heterogeneity, given their ability to dynamically adapt their morphology to regional demands on their ATP generation and Ca2+ buffering functions. Although there is reciprocal regulation between mitochondrial dynamics and mitochondrial Ca2+ signaling in astrocytes, the extent of this regulation into the rich diversity of astrocytes in different brain regions remains largely unexplored. Brain-wide, experimentally induced mitochondrial DNA (mtDNA) loss in astrocytes showed that mtDNA integrity is critical for proper astrocyte function, however, few insights into possible diverse responses to this noxious stimulus from astrocytes in different brain areas were reported in these experiments. To selectively damage mtDNA in astrocytes in a brain-region-specific manner, we developed a novel adeno-associated virus (AAV)-based tool, Mito-PstI, which expresses the restriction enzyme PstI, specifically in astrocytic mitochondria. Here, we applied Mito-PstI to two distinct brain regions, the dorsolateral striatum, and the hippocampal dentate gyrus, and we show that Mito-PstI can induce astrocytic mtDNA loss in vivo, but with remarkable brain-region-dependent differences on mitochondrial dynamics, spontaneous Ca2+ fluxes and astrocytic as well as microglial reactivity. Thus, AAV-Mito-PstI is a novel tool to explore the relationship between astrocytic mitochondrial network dynamics and astrocytic mitochondrial Ca2+ signaling in a brain-region-selective manner.

neuroscience↗