Biology subjects

Thapa, A.

Publications and source records attributed to Thapa, A..

17 recordsLinked to original sources

bioRxiv2026

Trans-branching of polyubiquitin chains orchestrates the DNA replication stress response

Polyubiquitin chain geometry dictates functional consequences of ubiquitylation. Although branched polyubiquitin chains are abundant in cells, little is known about their functions. Here we show that branching on the DNA replication factor PCNA, mediated by the ubiquitin-conjugating enzyme UBE2K and involving lysines 63 and 48 of ubiquitin, orchestrates the sequence of events in response to replication stress. By inducing VCP-dependent extraction of PCNA from chromatin, branching promotes re-priming of stalled forks and necessitates a BRCA1-dependent pathway of daughter-strand gap repair. Our study identifies hyper-accumulation of daughter-strand gaps as the mechanistic basis underlying the toxicity of inhibitors of the PCNA-specific isopeptidase, USP1, in BRCA1-deficient cells. Moreover, an unexpected preference of UBE2K to operate in trans suggests a general timing mechanism to organize hierarchies amongst ubiquitin signals.

molecular biology↗

bioRxiv2026

Mucosally sourced complement factor B modulates the host response to colitis

Distinct host factors maintain intestinal homeostasis but are incompletely understood. The complement system is primarily liver-derived and serum-operative. However, there is growing recognition for complement-mediated host defense at mucosal surfaces. The alternative pathway, which is constitutively active at low levels and amplifies complement activation independent of antibodies, requires Complement Factor B (CFB). Despite its evolutionary conservation, the spatial, cellular, and functional roles of CFB in the intestine are poorly understood. Here, we show that CFB is produced in the human colon and is increased in patients with active inflammatory bowel disease. To isolate the role of local CFB in mucosal responses, we interrogated a mouse strain that has no circulating, liver-derived CFB but retains intact CFB expression in the gut. Global CFB-deficient mice succumb to colitis compared to these liver-specific knockout mice, suggesting that locally synthesized CFB mitigates colitis. Single-cell analyses identify enterocytes and fibroblasts as key CFB producers in the gut. Compartment-specific deletion of CFB from epithelial or stromal cells abrogates mucosal protection independent of circulating levels, which corroborates with pharmacological CFB inhibition. These findings redefine complement in the intestine as a locally regulated mucosal defense system and establish gut-derived CFB as a critical determinant of intestinal homeostasis. BRIEF SUMMARYThe role of local immune mediators in gut mucosal immune responses is still not entirely understood. In this study, we demonstrate a novel role for complement protein Factor B, a key component of the alternative pathway, which is locally sourced through epithelial and stromal cells. In vivo modeling of impaired local Factor B synthesis results in worse colitis, revealing a key role for mucosal sourced components of the alternative pathway.

immunology↗

bioRxiv2025

Trichoderma afroharzianum behaves differently with respect to the host transcriptome and microbial communities under varying iron availability in pea plants

AimsTrichoderma afroharzianum T22 is widely recognized for enhancing plant stress resilience, yet its effects in pea plants may vary depending on iron (Fe) availability. Methods and ResultsWe assessed the impact of T22 on pea grown under differential Fe status through integrated physiological and omics analyses. We found that the benefits of T22 are highly context dependent, providing significant improvements in photosynthesis and Fe/N accumulation under Fe deficiency but minimal effects under sufficiency. RNA-seq identified 262 DEGs under Fe deficiency and 555 DEGs under Fe sufficiency following T22 inoculation, with the latter primarily associated with basal metabolic functions, indicating potential colonization costs rather than adaptive responses. Particularly, T22 inoculation upregulated symbiosis-related genes (Nodule-specific GRPs, Major facilitator, sugar transporter-like), Fe transporters (NRAMPs, HMAs), and redox-associated genes (Glutathione S-transferase, Glutathione peroxidase) in the roots under Fe shortage, reflecting a coordinated response to enhance nutrient acquisition and stress tolerance. Microbiome profiling revealed that under Fe deficiency, T22 reshaped the root community by enriching several bacterial taxa such as Comamonadaceae, Pseudomonadaceae and Mitsuaria. These enriched bacterial taxa may act as potential helpers to T22 by providing complementary thereby amplifying its beneficial effects under Fe deficiency. In contrast, under Fe sufficiency, community restructuring was primarily limited to the enrichment of Rhizobiaceae, Pararhizobium. Fungal taxa showed minimal overall changes, with the exception of a significant enrichment of Paecilomyces in response to T22 under Fe-deficient soil conditions. ConclusionsThese findings indicate that T22 functions in a context-dependent manner, with bacterial enrichment varying with Fe availability, while fungal helper effects were not prominent following T22 inoculation in pea plants. Impact StatementBeneficial microbes do not function uniformly across environments. This study highlights how Fe availability determines whether Trichoderma acts as a strong mutualist or a neutral colonizer in pea plants. Our findings advance a precision bioinoculant framework for deploying microbial consortia specifically in Fe-deficient soils to enhance legume resilience and sustainable crop production.

plant biology↗

bioRxiv2025

Bacillus subtilis reprograms host transcriptome and rhizosphere microbiome via systemic signaling to confer alkaline stress tolerance in garden pea

Soil alkalinity severely limits legume growth, but the role of Bacillus subtilis in alkaline stress tolerance remains unclear in garden pea. We found that multiple garden pea genotypes inoculated with B. subtilis under alkaline stress showed host-specific improvements in growth parameters. Mechanistic analysis conducted on Sugar Snap showed improved nodulation, mineral status, and photosystem efficiency, while split-root assays showed responses consistent with systemic effects of B. subtilis in alkaline tolerance. Further, FeEDDHA partially reduced alkaline stress symptoms but did not fully restore nodulation. In contrast, B. subtilis increased rhizosphere Fe-chelating activity and improved nodulation, leading to stronger symbiotic recovery than inorganic Fe alone. This suggests that factors associated with B. subtilis inoculation, beyond Fe availability alone, may contribute to the observed recovery of nodulation. This is further supported by in vitro co-culture experiments showing enhanced growth of R. leguminosarum in the presence of B. subtilis under alkaline conditions, indicating potential microbial compatibility for coping with stress. RNA-seq analysis identified 958 upregulated and 1,134 downregulated genes in roots inoculated with B. subtilis under alkaline conditions. The upregulated genes were mostly involved in the sugar-mediated symbiotic association (SWEET and GLUT), pH homeostasis (cation/H+ exchanger and ATPase), and nutrient assimilation (Ammonium transporter and Zn/Fe permease). Microbial community analysis revealed that B. subtilis significantly altered bacterial alpha diversity under alkaline stress, whereas fungal alpha diversity remained unaffected. Further, B. subtilis reshaped the rhizosphere microbial community and enriched taxa such as Pseudomonas, Pseudorhizobium, and Chaetomium, which were potentially associated with responses to alkaline stress. Taken together, microbial interventions such as B. subtilis offer an effective strategy to boost legume tolerance to alkaline soils.

plant biology↗

bioRxiv2025

SHLD2 loss is a synthetic vulnerability to Polθ inhibition combined with radiotherapy

DNA polymerase theta (Pol{theta}) plays a crucial role in the repair of DNA double-strand breaks (DSBs) by microhomology-mediated end joining (MMEJ). We previously demonstrated that Pol{theta} inhibition (Pol{theta}i) is an effective and well-tolerated approach to sensitise tumours to radiotherapy (RT). Here, we profiled 54 cancer cell lines and found that Pol{theta}i induces significant radiosensitisation in most models, though with marked variability not explained by indicators of Pol{theta} activity. To pinpoint molecular determinants of radiosensitisation by Pol{theta}i, we performed a CRISPR knockout screen which revealed loss of the TP53BP1/Shieldin pathway component SHLD2 (FAM35A) as a vulnerability to Pol{theta}i combined with RT. We demonstrated that SHLD2 loss not only increases sensitivity to RT alone, but also enhances the radiosensitising effect of Pol{theta}i, both in vitro and in vivo. Importantly, we found that SHLD2 is deleted in a subset of human prostate cancers, often co-occurring with PTEN loss, an adverse prognostic factor. Furthermore, we show that SHLD2-deficient cancer cells are more reliant on Pol{theta} to prevent DSB accumulation and chromosomal instability. In summary, we discovered SHLD2 loss as a novel collateral vulnerability that can be exploited through combined treatment with Pol{theta}i and RT.

cancer biology↗

bioRxiv2025

Iron retention coupled with trade-offs in localized symbiotic effects confers tolerance to combined iron deficiency and drought in soybean

Iron (Fe) and water availability are closely interlinked, with deficiencies in both adversely affecting soybean growth. However, the strategies employed by soybean to tolerate such conditions remain poorly understood. This study elucidates the interactions of host factors, and microbial associations using multi-omics approaches in Clark (tolerant) and Arisoy (sensitive) genotypes exposed to Fe deficiency and drought. Clark exhibited resilience to stress through sustained osmotic regulation, nutrient uptake, and photosynthetic activity, in contrast to Arisoy. Particularly, Fe retention in Clark, accompanied by the upregulation of ferritin-like proteins, may mitigate oxidative stress by reducing Fenton reactions. Furthermore, higher jasmonic and salicylic acid levels in Clark may contribute to its enhanced stress adaptation compared to Arisoy. RNA-seq analysis revealed 818 and 500 upregulated, along with 931 and 361 downregulated genes, in the roots of Clark and Arisoy, respectively, under stress. We observed the upregulation of symbiotic genes, such as Chalcone-flavonone isomerase 1 and SWEET10, accompanied by increased rhizosphere siderophore and root flavonoid in Clark. This indicates a significant role of microbes in mediating differential stress tolerance in soybean. Particularly, the combined stress led to distinct root and nodule microbiome dynamics, with Clark recruiting beneficial microbes such as Variovorax and Paecilomyces, whereas Arisoy exhibited the opposite pattern. In addition, Clark maintained nodule Bradyrhizobium and tissue nitrogen status, supported by ammonium retention and induction of Ammonium transporter 1 in the roots. Furthermore, in vitro compatibility between V. paradoxus and P. lilacinus suggests a synergistic interaction, with their localized signals benefiting Clark. Remarkably, enriched microbiomes significantly improved growth parameters, accompanied by elevated rhizosphere siderophore in sensitive genotypes under stress. This study is the first to uncover mechanisms of dual stress tolerance in soybean that may offer promising targets for breeding programs and microbiome-based biofertilizer strategies to improve combined stress tolerance in soybean and other legumes. HighlightIron retention coupled with symbiotic associations driven by the enrichment of Variovorax and Paecilomyces in the roots confers tolerance to combined iron deficiency and drought in soybean.

plant biology↗

bioRxiv2024

Vocal species are more central in Eastern Himalayan Mixed-Species bird flocks

Mixed-species flocks (MSF) represent an important form of social organisation in bird communities worldwide. Despite its likely importance in flock formation and cohesion, the role of vocal communication in the formation and maintenance of MSF in birds is hitherto understudied. In this study, we examine if a species centrality within a mixed-species flock is influenced by its vocal behaviour during the dawn chorus, i.e., the time of MSF formation. Using acoustic sampling and field observations, we studied the bird species found in MSF in the Eastern Himalayas. Our results show differential vocal activity patterns among MSF-forming bird species and suggest a positive correlation between calling rates and closeness centrality (species importance in a flock) in understory MSFs. We also found a more synchronised vocalisation pattern in the understory MSFs, with a consistent peak in vocal activity in the early morning hours, whereas no consistent vocal pattern was found for canopy flocks. Overall, our results suggest a potential mechanism that drives MSF formation wherein the vocal activity of central species precedes and likely attracts participation from other attendant species.

ecology↗

bioRxiv2024

Transcriptional Responses and Microbiome Shifts in Pea Roots Under High pH Stress

High soil pH severely impacts plant growth and productivity, yet the transcriptomic changes and microbial dynamics underlying stress adaptation in garden pea (Pisum sativum ssp. hortense) remain unclear. This study demonstrates that high soil pH leads to stunted growth, reduced biomass, impaired photosynthesis, and nutrient status in garden pea. Further, disruption in key nitrogen-fixing bacteria (Rhizobium indicum, R. leguminosarum, and R. redzepovicii), along with the downregulation of NifA and NifD genes and upregulation of NifH in nodules highlight the critical role of micronutrient balance in legume-microbe symbiosis and a compensatory response to maintain nitrogen status. RNA-seq analysis revealed extensive transcriptional reprogramming in roots, characterized by the upregulation of oxidative stress response genes (e.g., oxidoreductase and glutathione transferase activities, metal ion transporters) and the downregulation of genes related to ammonia-lyase activity and ion binding, reflecting broader disruptions in nutrient homeostasis. KEGG pathway analysis identified enrichment of MAPK signaling pathway, likely interacting with other pathways associated with stress tolerance, metabolic adjustment, and structural reorganization as part of adaptive responses to high pH. Root microbiome analysis showed significant enrichment of Variovorax, Shinella, and Chaetomium, suggesting host-driven recruitment under high pH stress. Stable genera such as Pseudomonas, Novosphingobium, Mycobacterium, Herbaspirillum, and Paecilomyces displayed resilience to stress conditions, potentially forming core microbiome components for adaptation to high pH. In a targeted study, inoculation of plants with an enriched microbiome, particularly C. globosum, under high pH conditions improved growth parameters and increased the abundance of Stenotrophomonas and Pseudomonas in the roots. It suggests that these bacterial genera may act as helper microbes to C. globosum, collectively promoting stress resilience in pea suffering from high pH. These findings provide a foundation for microbiome-aided breeding programs and the development of microbial consortia to enhance the adaptation of pea plants to high pH conditions.

plant biology↗

bioRxiv2024

Fe deficiency causes transcriptional shift in roots leading to disruption of drought tolerance in soybean

Soil alkalinity and drought collectively cause severe growth retardation in crops. However, the adaptive responses and transcriptional changes under such conditions remain unclear in soybean. In this growth incubator study, soil alkalinity and drought stress led to significant reductions in plant biomass, chlorophyll, and nutrient uptake in soybean. However, the photochemical efficiency of photosystem II remained stable, suggesting the activation of protective mechanisms to maintain photosynthetic functions. RNA-seq analysis demonstrated 357 upregulated and 799 downregulated genes in roots due to combined soil alkalinity and drought. Analysis revealed a complex response, with upregulation of genes predominantly involved in mineral homeostasis (Iron dehydrogenase, Sulfurylase), reactive oxygen species scavenging (Glutamate synthase, L-Ascorbate peroxidase) and hormonal signaling. Particularly, several ethylene-responsive genes, including the Transcription factors TF5 and TF018, were upregulated, indicating the activation of stress-related signaling pathways. In a targeted study, plants supplemented with an ethylene precursor showed significant improvements in morpho-physiological traits and Fe status under combined stress. However, ethylene precursor applied in non-stressed plants led to reduced growth, and Fe levels, suggesting an involvement of the context-dependent role of ethylene in promoting stress tolerance. Furthermore, ethylene precursors caused an increase in root flavonoid and rhizosphere siderophore while restoring bacterial and fungal microbial cells in roots under combined stress in soil. However, in healthy plants, flavonoid and siderophore levels decreased, accompanied by a reduction in microbial cells to control levels. This suggests that elevated ethylene may regulate root exudates to recruit microbes dominated by host response, aiding soybean plants cope with combined stresses, although this effect may not occur in non-stressed plants. This is the first report on the transcriptional response and physiological adjustments in soybean exposed to alkalinity and drought, potentially advancing knowledge for genetic and agronomic interventions to improve stress resilience in legume crops.

plant biology↗

bioRxiv2024

Trichoderma afroharzianum induces rhizobia and flavonoid through systemic signaling to combat iron deficiency in garden pea

Iron (Fe) deficiency is a limiting factor for legumes, yet the role of beneficial fungi in mitigating Fe deficiency in garden pea is understudied. In this study, Trichoderma afroharzianum T22 colonized the roots of garden pea cultivars exposed to alkaline-exposed Fe-deficiency in a host-specific manner. In Fe-deficient Sugar Snap, T22 improved growth parameters, consistent with increased tissue mineral content, particularly Fe and Mn, as well as enhanced rhizosphere siderophore levels. The split-root assay demonstrated that the beneficial effects of T22 on Fe deficiency mitigation are the result of a whole-plant association rather than localized root-specific effects. RNA-seq analysis showed 575 and 818 differentially expressed genes upregulated and downregulated in the roots inoculated with T22 under Fe deficiency. The upregulated genes were mostly involved in the flavonoid biosynthetic pathway (monooxygenase activity, ammonia-lyase activity, 4-coumarate-CoA ligase), along with genes related to mineral transport and redox homeostasis. Further, a flavonoid precursor restored plant health even in the absence of T22, confirming the role of microbial symbiosis in mitigating Fe deficiency. Interestingly, T22 restored the abundance of rhizobia, particularly Rhizobium leguminosarum and Rhizobium indicum, along with the induction of NifA, NifD, and NifH in nodules, suggesting a connection between T22 and rhizobia under Fe-starvation. Further, the elevated rhizosphere siderophore, root flavonoid, expression of PsCoA (4-coumarate-CoA ligase) as well as the relative abundance of TaAOX1 and R. leguminosarum diminished when T22 was substituted with exogenous Fe. This suggests that exogenous Fe eliminates the need for microbiome-driven Fe mobilization, while T22-mediated Fe mitigation depends on flavonoid-driven symbiosis and R. leguminosarum abundance. It was further supported by the positive interaction of T22 on R. leguminosarum growth in Fe-deficient media. Thus, the beneficial effect of T22 on rhizobia likely stems from their interactions, not just improved Fe status in plants. This study provides the first mechanistic insights into T22 interactions with host and rhizobia, advancing microbiome strategies to alleviate Fe deficiency in peas and other legumes.

plant biology↗

bioRxiv2023

Understanding inter-individual social networks in mixed-species bird flocks

Mixed-species flocks (MSFs) are an important form of social organisation in forest bird communities worldwide. MSFs provide participants with the benefits of reduced predation risk and/or enhanced foraging efficiency. Recent work has shown that participation in MSFs confers long-term survival benefits in the face of anthropogenic change. However, our understanding of MSFs mainly comes from studies that examine species-level networks, where each node is a unique species and the edges or connections between nodes are associations/interactions between species. While valuable, such approaches might not allow us to understand and investigate the mechanisms that drive MSF formation and structure because social interactions and their effects occur at the individual-level. Empirical studies on multi-species, individual-level MSF social networks have seldom been undertaken due to the various complexities and logistical challenges involved. In this study, we use mist-netting and colour-ringing followed by a standardised observation protocol to construct individual-level social networks in MSFs at 2000m ASL in Eaglenest Wildlife Sanctuary, Arunachal Pradesh, Eastern Himalaya, India. First, we found two separate flocktypes at our study site, comprising two distinct sets of understorey species. The mechanisms contributing to individual-level co-occurrences are likely to differ between these flocktypes, and with MSFs in the Neotropics. The Rusty-fronted Barwing (the nuclear species of one flocktype) shows spatially disjunct territories for each flock while the Yellow-throated Fulvetta (nuclear species of the other flocktype) shows large spatial overlap in its MSF networks, which is likely driven by non-individual-specific benefits such as predation risk dilution. Further, the addition of associating individuals to the social networks has opposite impacts on the two networks. The addition of Coral-billed Scimitar Babblers to the barwing networks greatly reduces network modularity because the associating individuals bridge two modules of barwings (both spatially and in the social network). On the contrary, territorial Rufous-capped Babblers and Grey-cheeked Warblers increase the modularity of the spatially overlapping fulvetta network. Our study provides novel insights into flock formation mechanisms in the Eastern Himalaya, likely applicable to other multi-species flock systems in the Old World.

ecology↗

bioRxiv2023

Constitutively synergistic multiagent drug formulations targeting MERTK, FLT3, and BCL-2 for treatment of AML

Although high-dose, multi-agent chemotherapy has improved leukemia survival rates in recent years, treatment outcomes remain poor in high-risk subsets, including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) in infants. Development of new, more effective therapies for these patients is therefore an urgent, unmet clinical need. To address this challenge, we developed a nanoscale combination drug formulation that exploits ectopic expression of MERTK tyrosine kinase and dependency on BCL-2 family proteins for leukemia cell survival in pediatric AML and MLL-rearranged precursor B-cell ALL (infant ALL). In a novel, high-throughput combination drug screen, the MERTK/FLT3 inhibitor MRX-2843 synergized with venetoclax and other BCL-2 family protein inhibitors to reduce AML cell density in vitro. Neural network models based on drug exposure and target gene expression were used to identify a classifier predictive of drug synergy in AML. To maximize the therapeutic potential of these findings, we developed a combination monovalent liposomal drug formulation that maintains ratiometric drug synergy in cell-free assays and following intracellular delivery. The translational potential of these nanoscale drug formulations was confirmed in a genotypically diverse set of primary AML patient samples and both the magnitude and frequency of synergistic responses were not only maintained but were improved following drug formulation. Together, these findings demonstrate a systematic, generalizable approach to combination drug screening, formulation, and development that maximizes therapeutic potential, was effectively applied to develop a novel nanoscale combination therapy for treatment of AML, and could be extended to other drug combinations or diseases in the future.

bioengineering↗

bioRxiv2023

Microbial stimulation of oxytocin release from the intestinal epithelium via secretin signaling

Intestinal microbes impact the health of the intestine and organs distal to the gut. Limosilactobacillus reuteri is a human intestinal microbe that promotes normal gut transit1, the anti-inflammatory immune system2-4, wound healing5-7, normal social behavior in mice8-10, and prevents bone reabsorption11-17. Each of these functions is impacted by oxytocin18-22, and oxytocin signaling is required for L. reuteri-mediated wound healing5 and social behavior9; however, the initiating events in the gut that lead to oxytocin stimulation and related beneficial functions remain unknown. Here we found evolutionarily conserved oxytocin production in the intestinal epithelium through analysis of single-cell RNA-Seq datasets and imaging of human and mouse intestinal tissues. Moreover, human intestinal organoids produce oxytocin, demonstrating that the intestinal epithelium is sufficient to produce oxytocin. We subsequently found that L. reuteri facilitates oxytocin secretion directly from human intestinal tissue and human intestinal organoids. Finally, we demonstrate that stimulation of oxytocin secretion by L. reuteri is dependent on the gut hormone secretin, which is produced in enteroendocrine cells23, while oxytocin itself is produced in enterocytes. Altogether, this work demonstrates that oxytocin is produced and secreted from enterocytes in the intestinal epithelium in response to secretin stimulated by L. reuteri. This work thereby identifies oxytocin as an intestinal hormone and provides mechanistic insight into avenues by which gut microbes promote host health.

microbiology↗

bioRxiv2020

TET2 drives 5hmc marking of GATA6 and epigenetically defines pancreatic ductal adenocarcinoma transcriptional subtypes

Background and AimsPancreatic ductal adenocarcinoma (PDAC) is characterised by advanced disease stage at presentation, aggressive disease biology and resistance to therapy resulting in extremely poor five-year survival <10%. PDAC is classified into transcriptional subtypes with distinct survival characteristics, although how these arise is not known. Epigenetic deregulation, rather than genetics, has been proposed to underpin progression but exactly why is unclear and hindered by analysis of clinical samples. MethodsGenome-wide epigenetic mapping of DNA modifications 5-hydroxymethylcytosine (5mc) and 5-hydroxymethylcytosine (5hmc) using oxidative bisulphite sequencing (oxBS). Bioinformatics using iCluster and mutational profiling to identify overlap with transcriptional signatures in FFPE from resected patients and confirmation in vivo. ResultsWe find that more aggressive squamous-like PDAC subtypes result from epigenetic inactivation of loci including GATA6 that promote differentiated classical-pancreatic subtypes. We show that squamous-like PDAC transcriptional subtypes are associated with greater loss of 5hmc due to reduced expression of the 5mc-hydroxylase TET2. Furthermore, we find that SMAD4 directly supports TET2 levels in the pancreas and classical-pancreatic tumors and loss of SMAD4 expression is associated reduced 5hmc, GATA6 and squamous-like tumors. Importantly, enhancing TET2 stability using Metformin and VitaminC/ascorbic acid (AA) restores 5hmc and GATA6 levels, reverting squamous-like tumor phenotypes and WNT-dependence in vitro and in vivo. ConclusionsWe identify epigenetic deregulation of pancreatic differentiation as an underpinning event behind the emergence of transcriptomic subtypes in PDAC. Our data shows that restoring epigenetic control increases biomarkers of classical-pancreatic tumors and raises the possibility that combination of Vitamin C and Metformin may prolong survival in patients with squamous-like pancreatic cancer.

cancer biology↗

bioRxiv2020

Conserved Arginine Residues in Synaptotagmin 1 Regulate Fusion Pore Expansion Through Membrane Contact

Synaptotagmin 1 is a vesicle-anchored membrane protein that functions as the Ca2+ sensor for synchronous neurotransmitter release. In this work, an arginine containing region in the second C2 domain of synaptotagmin 1 (C2B) is shown to control the expansion of the fusion pore and thereby the concentration of neurotransmitter released. This arginine apex, which is opposite the Ca2+ binding sites, interacts with membranes or membrane reconstituted SNAREs; however, only the membrane interactions occur under the conditions in which fusion takes place. Other regions of C2B influence the probably and kinetics of fusion but do not control the expansion of the fusion pore. These data indicate that the C2B domain has at least two distinct molecular roles in the fusion event, and the data are consistent with a novel model where the arginine apex of C2B positions the domain at the curved membrane surface of the expanding fusion pore.

neuroscience↗

bioRxiv2019

Phosphatidylinositol 4,5 bisphosphate controls the cis and trans interactions of synaptotagmin 1

Synaptotagmin 1 acts as the Ca2+-sensor for synchronous neurotransmitter release; however, the mechanism by which it functions is not understood and is presently a topic of considerable interest. Here we describe measurements on full-length membrane reconstituted synaptotagmin 1 using site-directed spin labeling where we characterize the linker region as well as the cis (vesicle membrane) and trans (cytoplasmic membrane) binding of its two C2 domains. In the full-length protein, the C2A domain does not undergo membrane insertion in the absence of Ca2+; however, the C2B domain will bind to and penetrate in trans to a membrane containing phosphatidylinositol 4,5 bisphosphate (PIP2), even if phosphatidylserine (PS) is present in the cis membrane. In the presence of Ca2+, the Ca2+-binding loops of C2A and C2B both insert into the membrane interface; moreover, C2A preferentially inserts into PS containing bilayers and will bind in a cis configuration to membranes containing PS even if a PIP2 membrane is presented in trans. The data are consistent with a bridging activity for Syt1 where the two domains bind to opposing vesicle and plasma membranes. The failure of C2A to bind membranes in the absence of Ca2+ and the long unstructured segment linking C2A to the vesicle membrane indicates that synaptotagmin 1 could act to significantly shorten the vesicle-plasma membrane distance with increasing levels of Ca2+.

neuroscience↗

bioRxiv2018

Brief Freezing Steps Lead to Robust Immunofluorescence in Drosophila Larval Brains

Drosophila melanogaster possess complex neuronal networks regulating sophisticated behavioral outputs that aid in studying the molecular mechanisms of neuronal function and neurodegenerative disease. Immunofluorescence (IF) techniques provide a way to visualize the spatiotemporal organization of these networks, permitting observation of their development, functional location, remodeling, and eventually - degradation. However, general immunostaining techniques do not always result in sufficient antibody penetration through the brain, and techniques used to enhance permeability can compromise structural integrity. We have found that freezing larval brains facilitates permeability with no apparent loss of antibody specificity or structural integrity. To demonstrate the advantage of this freezing technique, we compared results to two commonly used permeation methods: Detergent alone (Basic) and proteolytic degradation (Collagenase) techniques.\n\nSummaryHere we compare four different immunofluorescence techniques demonstrating that freezing Drosophila brains results in robust staining of small neurons in the larval brain without compromising structural integrity.

cell biology↗