Biology subjects

Gupta, S.

Publications and source records attributed to Gupta, S..

At least 217 records · Page 12Linked to original sources

bioRxiv2023

Identification of merozoite secreted repertoire and immuno-pharmacological inhibition of a novel host-parasite interaction to block malarial infection

BackgroundDuring the intra-erythrocytic proliferation of Plasmodium falciparum, the host erythrocyte invasion is regarded as a complex and tightly regulated process comprising multiple receptor-ligand interactions, and numerous secretory molecules. Proteins secreted sequentially from apical organelles of merozoites serve as adhesins that play a crucial role in RBC invasion and can serve as vaccine and therapeutic targets. MethodsPurified merozoites were triggered to discharge apical organelle contents by exposure to ionic conditions mimicking that of blood plasma. The secreted proteins were subjected to tandem mass spectrometry, and a well-characterized invasion ligand, RhopH3, was identified. A novel RhopH3 receptor, 14-3-3{square} was unearthed using a Bacterial two-hybrid approach. This interaction was confirmed using multiple biophysical and biochemical approaches. We were successful in disrupting this interaction using a de novo peptide binder of 14-3-3{square}, and we subsequently assessed its effect on merozoite invasion. ResultsA total of 66 proteins were identified in the secretory fraction with apical organellar or merozoite membrane localization. The well-known adhesin, RhopH3 was also identified and its interaction with the host phosphopeptide-binding protein, 14-3-3{square} was established. We also discovered a de novo peptide with the potency to disrupt this crucial interaction, thereby blocking merozoite invasion. ConclusionWe, for the first time, report the secretory repertoire of plasmodium merozoite. Our study shows the importance of the erythrocyte protein, 14-3-3{square} during the invasion process and paves the way for developing anti-malarial peptides or small molecules that inhibit the host-pathogen interaction, hence abrogating the invasion process.

microbiology↗

bioRxiv2023

Learning transcriptional and regulatory dynamics driving cancer cell plasticity using neural ODE-based optimal transport

While single-cell technologies provide snapshots of tumor states, building continuous trajectories and uncovering causative gene regulatory networks remains a significant challenge. We present Cflows, an AI framework that combines neural ODE networks with Granger causality to infer continuous cell state transitions and gene regulatory interactions from static scRNA-seq data. In a new 5-time point dataset capturing tumorsphere development over 30 days, Cflows reconstructs two types of trajectories leading to tumorsphere formation or apoptosis. Trajectory-based cell-of-origin analysis delineated a novel cancer stem cell profile characterized by CD44hiEPCAM+CAV1+, and uncovered a cell cycle-dependent enrichment of tumorsphere-initiating potential in G2/M or S-phase cells. Cflows uncovers ESRRA as a crucial causal driver of the tumor-forming gene regulatory network. Indeed, ESRRA inhibition significantly reduces tumor growth and metastasis in vivo. Cflows offers a powerful framework for uncovering cellular transitions and dynamic regulatory networks from static single-cell data.

cancer biology↗

bioRxiv2023

Representations of carbon dioxide in the mosquito antennal lobe

Carbon dioxide (CO2) is one of the prominent sensory cues used by mosquitoes to find hosts for blood-feeding. CO2 is detected on the maxillary palps by capitate peg sensory neurons, whose axons project to the antennal lobe in the brain. Behavioral studies have shown that mosquitoes prefer non-homogenous plumes of CO2 over homogenous plumes and CO2 greatly enhances the attractiveness of lactic acid, a skin volatile. However, the neural mechanisms underlying these behavioral preferences are not known. Using in vivo intracellular recordings from projection neurons and local neurons in the antennal lobe, along with single sensillum recordings from the maxillary palps, we checked the representations of CO2 in the first two layers of the Aedes aegypti olfactory system. We found that the preference to non-homogeneous plumes of CO2 and its synergistic attraction with lactic acid are encoded in the PN population responses. Our results provide a foundation for understanding CO2-mediated host-attraction in mosquitoes.

neuroscience↗

bioRxiv2023

HLA3DB: comprehensive annotation of peptide/HLA complexes enables blind structure prediction of T cell epitopes

The class I proteins of the major histocompatibility complex (MHC-I) display epitopic peptides derived from endogenous proteins on the cell surface for immune surveillance. Accurate modeling of peptide/HLA (pHLA, the human MHC) structures has been mired by conformational diversity of the central peptide residues, which are critical for recognition by T cell receptors. Here, analysis of X-ray crystal structures within a curated database (HLA3DB) shows that pHLA complexes encompassing multiple HLA allotypes present a discrete set of peptide backbone conformations. Leveraging these representative backbones, we employ a regression model trained on terms of a physically relevant energy function to develop a comparative modeling approach for nonamer peptide/HLA structures named RepPred. Our method outperforms the top pHLA modeling approach by up to 19% in terms of structural accuracy, and consistently predicts blind targets not included in our training set. Insights from our work provide a framework for linking conformational diversity with antigen immunogenicity and receptor cross-reactivity.

bioinformatics↗

bioRxiv2023

Deletion of smooth muscle O-GlcNAc transferase prevents development of atherosclerosis in western diet-fed hyperglycemic ApoE-/- mice in vivo

Accumulating evidence highlights protein O-GlcNAcylation as a putative pathogenic contributor of diabetic vascular complications. We previously reported that elevated protein O-GlcNAcylation correlates with increased atherosclerotic lesion formation and VSMC proliferation in response to hyperglycemia. However, the role of O-GlcNAc transferase (OGT), regulator of O-GlcNAc signaling, in evolution of diabetic atherosclerosis remains elusive. The goal of this study was to determine whether smooth muscle OGT (smOGT) plays a direct role in hyperglycemia-induced atherosclerotic lesion formation and SMC de-differentiation. Using tamoxifen-inducible Myh11-CreERT2 and OGTfl/fl mice, we generated smOGTWT and smOGTKO mice, with and without ApoE-null backgrounds. Following STZ-induced hyperglycemia, smOGTWT and smOGTKO mice were kept on standard laboratory diet for study duration. In a parallel study, smOGTWTApoE-/- and smOGTKOApoE-/- were kept on Western diet beginning 8-wks-age. Animals harvested at 14-16-wks-age were used for plasma and tissue collection. Loss of smOGT augmented SM contractile marker expression in aortic vessels of STZ-induced hyperglycemic smOGTKO mice. Consistently, smOGT deletion attenuated atherosclerotic lesion lipid burden (Oil red O), plaque area (H&E), leukocyte (CD45) and smooth muscle cell (ACTA2) abundance in Western diet-induced hyperglycemic smOGTKOApoE-/- mice. This was accompanied with increased SM contractile markers, and reduced inflammatory and proliferative marker expression. Further, smOGT deletion attenuated YY1 and SRF expression (transcriptional regulators of SM contractile genes) in hyperglycemic smOGTKOApoE-/- and smOGTKO mice. These data uncover an atheroprotective outcome of smOGT loss-of-function and suggest a direct regulatory role of OGT-mediated O-GlcNAcylation in VSMC de-differentiation in hyperglycemia.

pathology↗

bioRxiv2023

Growth-inhibiting effects of the unconventional plant APYRASE 7 of Arabidopsis thaliana influences the LRX1/FER/RALF growth regulatory module

Plant cell growth involves coordination of numerous processes and signaling cascades among the different cellular compartments to concomitantly enlarge the protoplast and the surrounding cell wall. In Arabidopsis thaliana, the cell wall integrity-sensing process involves the extracellular LRX (LRR-Extensin) proteins that bind RALF (Rapid ALkalinization Factor) peptide hormones and, in vegetative tissues, interact with the transmembrane receptor kinase FERONIA (FER). This LRX/RALF/FER signaling module influences cell wall composition and regulates cell growth. The numerous proteins involved in or influenced by this module are beginning to be characterized. In a genetic screen, mutations in Apyrase 7 (APY7) were identified to suppress growth defects observed in lrx1 and fer mutants. APY7 encodes a Golgi-localized NTP-diphosphohydrolase, but opposed to other apyrases of Arabidopsis, APY7 revealed to be a negative regulator of cell growth. APY7 modulates the growth-inhibiting effect of RALF1, influences the cell wall architecture, and alters the pH of the extracellular matrix, all of which affect cell growth. Together, this study reveals a function of APY7 in cell wall formation and cell growth that is connected to growth processes influenced by the LRX/FER/RALF signaling module.

plant biology↗

bioRxiv2023

Modeling transcriptional regulation of the cell cycle using a novel cybernetic-inspired approach

Quantitative understanding of cellular processes, such as cell cycle and differentiation, is impeded by various forms of complexity ranging from myriad molecular players and their multilevel regulatory interactions, cellular evolution with multiple intermediate stages, lack of elucidation of cause-effect relationships among the many system players, and the computational complexity associated with the profusion of variables and parameters. In this paper, we present an elegant modeling framework based on the cybernetic concept that biological regulation is inspired by objectives embedding entirely novel strategies for dimension reduction, process stage specification through the system dynamics, and innovative causal association of regulatory events with the ability to predict the evolution of the dynamical system. The elementary step of the modeling strategy involves stage-specific objective functions that are computationally-determined from experiments, augmented with dynamical network computations involving end point objective functions, mutual information, change point detection, and maximal clique centrality. We demonstrate the power of the method through application to the mammalian cell cycle, which involves thousands of biomolecules engaged in signaling, transcription, and regulation. Starting with a fine-grained transcriptional description obtained from RNA sequencing measurements, we develop an initial model, which is then dynamically modeled using the cybernetic-inspired method (CIM), utilizing the strategies described above. The CIM is able to distill the most significant interactions from a multitude of possibilities. In addition to capturing the complexity of regulatory processes in a mechanistically causal and stage-specific manner, we identify the functional network modules, including novel cell cycle stages. Our model is able to predict future cell cycles consistent with experimental measurements. We posit that this state-of-the-art framework has the promise to extend to the dynamics of other biological processes, with a potential to provide novel mechanistic insights. STATEMENT OF SIGNIFICANCECellular processes like cell cycle are overly complex, involving multiple players interacting at multiple levels, and explicit modeling of such systems is challenging. The availability of longitudinal RNA measurements provides an opportunity to "reverse-engineer" for novel regulatory models. We develop a novel framework, inspired using goal-oriented cybernetic model, to implicitly model transcriptional regulation by constraining the system using inferred temporal goals. A preliminary causal network based on information-theory is used as a starting point, and our framework is used to distill the network to temporally-based networks containing essential molecular players. The strength of this approach is its ability to dynamically model the RNA temporal measurements. The approach developed paves the way for inferring regulatory processes in many complex cellular processes.

systems biology↗

bioRxiv2023

Universal open MHC-I molecules for rapid peptide loading and enhanced complex stability across HLA allotypes

The polymorphic nature and intrinsic instability of class I major histocompatibility complex (MHC-I) and MHC-like molecules loaded with suboptimal peptides, metabolites, or glycolipids presents a fundamental challenge for identifying disease-relevant antigens and antigen-specific T cell receptors (TCRs), hindering the development of autologous therapeutics. Here, we leverage the positive allosteric coupling between the peptide and light chain ({beta}2 microglobulin, {beta}2m) subunits for binding to the MHC-I heavy chain (HC) through an engineered disulfide bond bridging conserved epitopes across the HC/{beta}2m interface, to generate conformationally stable, open MHC-I molecules. Biophysical characterization shows that open MHC-I molecules are properly folded protein complexes of enhanced thermal stability compared to the wild type, when loaded with low- to intermediate-affinity peptides. Using solution NMR, we characterize the effects of the disulfide bond on the conformation and dynamics of the MHC-I structure, ranging from local changes in {beta}2m interacting sites of the peptide binding groove to long-range effects on the 2-1 helix and 3 domain. The interchain disulfide bond stabilizes empty MHC-I molecules in a peptide-receptive, open conformation to promote peptide exchange across multiple human leucocyte antigen (HLA) allotypes, covering representatives from five HLA-A, six HLA-B supertypes, and oligomorphic HLA-Ib molecules. Our structural design, combined with conditional {beta}-peptide ligands, provides a universal platform for generating ready-to-load MHC-I systems of enhanced stability, enabling a range of approaches to screen antigenic epitope libraries and probe polyclonal TCR repertoires in the context of highly polymorphic HLA-I allotypes, as well as oligomorphic nonclassical molecules. Significance StatementWe outline a structure-guided approach for generating conformationally stable, open MHC-I molecules with enhanced ligand exchange kinetics spanning five HLA-A, all HLA-B supertypes, and oligomorphic HLA-Ib allotypes. We present direct evidence of positive allosteric cooperativity between peptide binding and {beta}2m association with the heavy chain by solution NMR and HDX-MS spectroscopy. We demonstrate that covalently linked {beta}2m serves as a conformational chaperone to stabilize empty MHC-I molecules in a peptide-receptive state, by inducing an open conformation and preventing intrinsically unstable heterodimers from irreversible aggregation. Our study provides structural and biophysical insights into the conformational properties of MHC-I ternary complexes, which can be further applied to improve the design of ultra-stable, universal ligand exchange systems in a pan-HLA allelic setting.

biochemistry↗

bioRxiv2023

PERK-mediated induction of miR-5p and miR-3p arms of miR-616 regulates cell growth by targeting c-MYC

C/EBP homologous protein (CHOP), also known as DNA damage-inducible transcript 3 (DDIT3), is a member of CCAAT/enhancer-binding protein (C/EBP) family. The expression of CHOP is upregulated during unfolded protein response (UPR), and sustained CHOP activity plays an important role in UPR-induced apoptosis. MicroRNA-616 is localized in an intron of the CHOP gene. However, regulation of miR-616 expression during UPR and its function in breast cancer is not clearly understood. We show that miR-5p/-3p arms of miR-616 are expressed with levels of 5p arm higher than 3p arm. During conditions of UPR, the expression of miR-5p and miR-3p arms of miR-616 and its host gene (CHOP) was concordantly increased primarily in a PERK-dependent manner. We show that ectopic expression of miR-616 significantly suppressed cell growth and colony formation, whereas knockout of miR-616 increased it. We identified that MYC proto-oncogene (c-MYC) gene is repressed during the UPR and targeted by miR-616. Further, we show that expression of miR-616 and CHOP is downregulated in human breast cancer, where expression of miR-616 was associated with poor overall survival (OS) in luminal A subtype and better OS HER2 subtype of breast cancer. In summary, our results suggest a dual function for the DDIT3 locus, where CHOP protein and miR-616 can co-operate to regulate cancer progression.

cancer biology↗

bioRxiv2023

Characterization of the pearl millet cultivation environments in India: status and perspectives enabled by expanded data analytics and digital tools

The cultivation of pearl millet in India is experiencing important transformations due to changes in weather, socio-economic trends, and technological progress. In this scope, we propose a new characterization of the pearl millet production environment in India using the latest available data and methodology. For that, we constructed a database incorporating data on various aspects of pearl millet cultivation at the district level from 1998 to 2017. We complemented this analysis using extensive pearl millet agri-system simulations to evaluate crop models abilities to reconstruct and analyse the system at an unprecedented scale. We also proposed a new method to infer system parameters from crop model data. Our results show important differences compared to the characterization currently used. The East part of the pearl millet tract (East Rajasthan, Haryana, Uttar Pradesh, and Madhya Pradesh) emerges as the only region where pearl millet cultivation has grown with potential surplus that is likely exported. Important reductions of pearl millet cultivated area in Gujarat, Maharashtra and Karnataka are potentially due to economy-driven transition to other more pro table crops like cotton, maize, or castor bean. The data used also point toward a constant increase of the rain during the growing season which could have major consequences on the future of this crop, with potential positive effects like extra yield but also negative like extra pressure due to more intense and erratic rainfall or transition to more pro table crops requiring more water. Despite difficulties to predict pearl millet yield in rapidly changing environments, the tested crop models reflected reasonably well the pearl millet production system, thus, setting the base for effective system design in future climatic scenarios. Our data and results have been gathered in an open-source interactive online application.

plant biology↗

bioRxiv2023

Sex differences in patch-leaving foraging decisions in rats

The ubiquity, importance, and sophistication of foraging behavior makes it an ideal platform for studying naturalistic decision making in animals. We developed a spatial patch-foraging task for rats, in which subjects chose how long to remain in one foraging patch as the rate of food earnings steadily decreased. The cost of seeking out a new location was varied across sessions. The behavioral task was designed to mimic the structure of natural foraging problems, where distinct spatial locations are associated with different reward statistics, and decisions require navigation and movement through space. Male and female Long-Evans rats generally followed the predictions of theoretical models of foraging, albeit with a consistent tendency to persist with patches for too long compared to behavioral strategies that maximize food intake rate. The tendency to choose overly-long patch residence times was stronger in male rats. We also observed sex differences in locomotion as rats performed the task, but these differences in movement only partially accounted for the differences in patch residence durations observed between male and female rats. Together, these results suggest a nuanced relationship between movement, sex, and foraging decisions. Significance statementForaging behavior offers a naturalistic way of studying temporal investment amongst different choice options, a translationally-important form of decision making. Previous laboratory investigations have relied on foraging tasks that require little movement from subjects, which could affect the strategies that animals use and the neural mechanisms that support them. We developed a spatial foraging task for rats. Behavior generally matched the predictions of theoretical models, although rats remained in patches for longer than prescribed. Male rats exhibited a stronger tendency to overharvest patches than female rats. Sex differences in movement did not account for sex differences in foraging. These data highlight the interplay between movement and decision making, and demonstrate the utility of spatial tasks for studies of foraging.

animal behavior and cognition↗

bioRxiv2023

Foreign RNA spike-ins enable accurate allele-specific expression analysis at scale

MotivationAnalysis of allele-specific expression is strongly affected by the technical noise present in RNA-seq experiments. Previously, we showed that technical replicates can be used for precise estimates of this noise, and we provided a tool for correction of technical noise in allele-specific expression analysis. This approach is very accurate but costly due to the need for two or more replicates of each library. Here, we develop a spike-in approach that is highly accurate at only a small fraction of the cost. ResultsWe show that a distinct RNA added as a spike-in before library preparation reflects technical noise of the whole library and can be used in large batches of samples. We experimentally demonstrate the effectiveness of this approach using combinations of RNA from species distinguishable by alignment, namely, mouse, human, and C.elegans. Our new approach, controlFreq, enables highly accurate and computationally efficient analysis of allele-specific expression in (and between) arbitrarily large studies at an overall cost increase of ~ 5%. AvailabilityAnalysis pipeline for this approach is available at GitHub as R package controlFreq (github.com/gimelbrantlab/controlFreq). Contactagimelbrant@altius.org

bioinformatics↗

bioRxiv2023

How cells wrap around virus-like particles using extracellular filamentous protein structures

Nanoparticles, such as viruses, can enter cells via endocytosis. During endocytosis, the cell surface wraps around the nanoparticle to effectively eat it. Prior focus has been on how nanoparticle size and shape impacts endocytosis. However, inspired by the noted presence of extracellular vimentin affecting viral and bacteria uptake, as well as the structure of coronaviruses, we construct a computational model in which both the cell-like construct and the virus-like construct contain filamentous protein structures protruding from their surfaces. We then study the impact of these additional degrees of freedom on viral wrapping. We find that cells with an optimal density of filamentous extracellular components (ECCs) are more likely to be infected as they uptake the virus faster and use relatively less cell surface area per individual virus. At the optimal density, the cell surface folds around the virus, and folds are faster and more efficient at wrapping the virus than crumple-like wrapping. We also find that cell surface bending rigidity helps generate folds, as bending rigidity enhances force transmission across the surface. However, changing other mechanical parameters, such as the stretching stiffness of filamentous ECCs or virus spikes, can drive crumple-like formation of the cell surface. We conclude with the implications of our study on the evolutionary pressures of virus-like particles, with a particular focus on the cellular microenvironment that may include filamentous ECCs.

biophysics↗

bioRxiv2023

Regulation of replication origin licensing by ORC phosphorylation reveals a two-step mechanism for Mcm2-7 ring closing

Eukaryotic DNA replication must occur exactly once per cell cycle to maintain cell ploidy. This outcome is ensured by temporally separating replicative helicase loading (G1 phase) and activation (S phase). In budding yeast, helicase loading is prevented outside of G1 by cyclin-dependent kinase (CDK) phosphorylation of three helicase-loading proteins: Cdc6, the Mcm2-7 helicase, and the origin recognition complex (ORC). CDK inhibition of Cdc6 and Mcm2-7 are well understood. Here we use single-molecule assays for multiple events during origin licensing to determine how CDK phosphorylation of ORC suppresses helicase loading. We find that phosphorylated ORC recruits a first Mcm2-7 to origins but prevents second Mcm2-7 recruitment. Phosphorylation of the Orc6, but not of the Orc2 subunit, increases the fraction of first Mcm2-7 recruitment events that are unsuccessful due to the rapid and simultaneous release of the helicase and its associated Cdt1 helicase-loading protein. Real-time monitoring of first Mcm2-7 ring closing reveals that either Orc2 or Orc6 phosphorylation prevents Mcm2-7 from stably encircling origin DNA. Consequently, we assessed formation of the MO complex, an intermediate that requires the closed-ring form of Mcm2-7. We found that ORC phosphorylation fully inhibits MO-complex formation and provide evidence that this event is required for stable closing of the first Mcm2-7. Our studies show that multiple steps of helicase loading are impacted by ORC phosphorylation and reveal that closing of the first Mcm2-7 ring is a two-step process started by Cdt1 release and completed by MO-complex formation. Significance StatementEach time a eukaryotic cell divides (by mitosis) it must duplicate its chromosomal DNA exactly once to ensure that one full copy is passed to each resulting cell. Both under-replication or over-replication result in genome instability and disease or cell death. A key mechanism to prevent over-replication is the temporal separation of loading of the replicative DNA helicase at origins of replication and activation of these same helicases during the cell division cycle. Here we define the mechanism by which phosphorylation of the primary DNA binding protein involved in these events inhibits helicase loading. Our studies identify multiple steps of inhibition and provide new insights into the mechanism of helicase loading in the uninhibited condition.

molecular biology↗

bioRxiv2022

STOmics-GenX: CRISPR based approach to improve cell identity specific gene detection from spatially resolved transcriptomics

The spatial organisation of cells defines the biological functions of tissue ecosystems from development to disease. Recently, an array of technologies have been developed to query gene expression in a spatial context. These include techniques such as employing barcoded oligonucleotides, single-molecule fluorescence in situ hybridization (smFISH), and DNA nanoball (DNB)-patterned arrays. However, resolution and efficiency vary across platforms and technologies. To obtain spatially relevant biological information from spatially resolved transcriptomics, we combined the Stereo-seq workflow with CRISPRclean technology to develop the STOmics-GenX pipeline. STOmics-GenX not only allowed us to reduce genomic, mitochondrial, and ribosomal reads, but also lead to a [~]2.1-fold increase in the number of detected genes when compared to conventional Stereo-seq (STOmics). Additionally, the STOmics-GenX pipeline resulted in an improved detection of cell type specific genes, thereby improving cellular annotations. Most importantly, STOmics-GenX allowed for enhanced detection of clinically relevant biomarkers such as Alpha-fetoprotein (AFP), enabling the identification of two spatially distinct subsets of hepatocytes in hepatocellular carcinoma tissue. Thereby, combining CRISPRclean technology with STOmics not only allowed improved gene detection but also paved the way for spatial precision oncology by improved detection of clinically relevant biomarkers.

molecular biology↗

bioRxiv2022

Transcription regulates bleb formation and stability independent of nuclear rigidity

Chromatin is an essential component of nuclear mechanical response and shape that maintains nuclear compartmentalization and function. The biophysical properties of chromatin alter nuclear shape and stability, but little is known about whether or how major genomic functions can impact the integrity of the nucleus. We hypothesized that transcription might affect cell nuclear shape and rupture through its effects on chromatin structure and dynamics. To test this idea, we inhibited transcription with the RNA polymerase II inhibitor alpha-amanitin in wild type cells and perturbed cells that present increased nuclear blebbing. Transcription inhibition suppresses nuclear blebbing for several cell types, nuclear perturbations, and transcription inhibitors. Furthermore, transcription is necessary for robust nuclear bleb formation, bleb stabilization, and bleb-based nuclear ruptures. These morphological effects appear to occur through a novel biophysical pathway, since transcription does not alter either chromatin histone modification state or nuclear rigidity, which typically control nuclear blebbing. We find that active/phosphorylated RNA pol II Ser5, marking transcription initiation, is enriched in nuclear blebs relative to DNA. Thus, transcription initiation is a hallmark of nuclear blebs. Polymer simulations suggest that motor activity within chromatin, such as that of RNA pol II, can generate active forces that deform the nuclear periphery, and that nuclear deformations depend on motor dynamics. Our data provide evidence that the genomic function of transcription impacts nuclear shape stability, and suggests a novel mechanism, separate and distinct from chromatin rigidity, for regulating large-scale nuclear shape and function.

cell biology↗

bioRxiv2022

Precise temporal control of neuroblast migration through combined regulation and feedback of a Wnt receptor

Many developmental processes depend on precise temporal control of gene expression. We have previously established a theoretical framework for regulatory strategies that can govern such high temporal precision, but experimental validation of these predictions was still lacking. Here, we use the time-dependent expression of a Wnt receptor that controls neuroblast migration in C. elegans as a tractable system to study a robust, cell-intrinsic timing mechanism in vivo. Single molecule mRNA quantification showed that the expression of the receptor increases non-linearly, a dynamic that is predicted to enhance timing precision over an unregulated, linear increase in timekeeper abundance. We show that this upregulation depends on transcriptional activation, providing in vivo evidence for a model in which the timing of receptor expression is regulated through an accumulating activator that triggers expression when a specific threshold is reached. This timing mechanism acts across a cell division that occurs in the neuroblast lineage, and is influenced by the asymmetry of the division. Finally, we show that positive feedback of receptor expression through the canonical Wnt pathway enhances temporal precision. We conclude that robust cell-intrinsic timing can be achieved by combining regulation and feedback of the timekeeper gene.

developmental biology↗

bioRxiv2022

GESIAP: A Versatile Genetically Encoded Sensor-based Image Analysis Program

Intercellular communication mediated by a large number of neuromodulators diversifies physiological actions, yet neuromodulation remains poorly understood despite the recent upsurge of genetically encoded transmitter sensors. Here, we report the development of a versatile genetically encoded sensor-based image analysis program (GESIAP) that utilizes MATLAB-based algorithms to achieve high-throughput, high-resolution processing of sensor-based functional imaging data. GESIAP enables delineation of fundamental properties (e.g., transmitter spatial diffusion extent, quantal size, quantal content, release probability, pool size, and refilling rate at single release sites) of transmission mediated by various transmitters (i.e., monoamines, acetylcholine, neuropeptides, and glutamate) at various cell types (i.e., neurons, astrocytes, and other non-neuronal cells) of various animal species (i.e., mouse, rat, and human). Our analysis appraises a dozen of newly developed transmitter sensors, validates a conserved model of restricted non-volume neuromodulatory synaptic transmission, and accentuates a broad spectrum of presynaptic release properties that variegate neuromodulation.

neuroscience↗