Biology subjects

Anantakrishnan, S.

Publications and source records attributed to Anantakrishnan, S..

4 recordsLinked to original sources

bioRxiv2025

Distinct Sox9 single-molecule dynamics characterize adult differentiation and fetal-like reprogrammed states in intestinal organoids

Transcription factors (TFs) mediate gene expression changes during differentiation and development. However, how TF biophysical properties and abundance dynamically regulate specific cell state transitions remains poorly understood. Using automated live-cell single-molecule tracking (SMT) in intestinal organoid models, we revealed an expression level-independent decrease in the fraction of immobile Sox9 molecules during differentiation from [~]48% to [~]38%, largely dependent on DNA binding. Strikingly, long-term Sox9 overexpression caused organoids to transition from budding to spheroid morphology accompanied by increased proliferation and a loss in gene expression signatures for intestinal identity and function. In this fetal-like reprogrammed state, a larger fraction of partially self- interacting Sox9 molecules ([~]61%) binds to DNA. Our results suggest context-dependent Sox9 single-molecule dynamics during adult intestinal differentiation and fetal-like reversion in consequence to long-term Sox9 overexpression. Our work underpins the power of our automated live-cell SMT framework to generate testable hypotheses towards unraveling molecular mechanisms underlying tissue-level phenotypes. Highlights- Heterogenous diffusion behavior of Sox9 across intestinal differentiation states - Concentration-independent decrease in chromatin-bound Sox9 upon differentiation - Sox9 overexpression leads to fetal-like reversion and more immobile Sox9 molecules - Automated SMT in 2D monolayers reveals TF dynamics underlying organoid phenotypes

developmental biology↗

bioRxiv2024

Automated live-cell single-molecule tracking in enteroid monolayers reveals transcription factor dynamics probing lineage-determining function

Lineage transcription factors (TFs) provide one regulatory level of differentiation crucial for the generation and maintenance of healthy tissues. To probe TF function by measuring their dynamics during adult intestinal homeostasis, we established HILO-illumination-based live-cell single-molecule tracking (SMT) in mouse small intestinal enteroid monolayers recapitulating tissue differentiation hierarchies in vitro. To increase the throughput, capture cellular features, and correlate morphological characteristics with diffusion parameters, we developed an automated imaging and analysis pipeline, broadly applicable to 2D culture systems. Studying two absorptive lineage-determining TFs, we find an expression level-independent contrasting diffusive behavior: While Hes1, key determinant of absorptive lineage commitment, displays a large cell-to-cell variability and an average fraction of DNA-bound molecules of [~]32%, Hnf4g, conferring enterocyte identity, exhibits more uniform dynamics and a bound fraction of [~]56%. Our results suggest that TF diffusive behavior can indicate the progression of differentiation and modulate early versus late differentiation within a lineage. Highlights- Automated live-cell single-molecule tracking records hundreds of cells in enteroid monolayers - Cellular diffusion clustering and morphological feature correlation reveals subpopulations - Transcription factor dynamics regulate differentiation independent of expression level - Hes1 and Hnf4g display contrasting dynamics assisting early vs. late absorptive differentiation

developmental biology↗

bioRxiv2022

Thermodynamic Architecture and Conformational Plasticity of GPCRs

G-protein-coupled receptors (GPCRs) are ubiquitous integral membrane proteins involved in diverse cellular signaling processes and consequently serve as crucial drug targets. Here, we carry out the first large-scale ensemble thermodynamic study of 45 different ligand-free GPCRs employing a structure-based statistical mechanical framework and identify extensive conformational plasticity encompassing the seven transmembrane (TM) helices. Multiple partially structured states or intermediates co-exist in equilibrium in the native ensemble, with the TM helices 1, 6 and 7 displaying varied degrees of structure, and TM3 exhibiting the maximal stability. Active state GPCRs are characterized by reduced conformational heterogeneity with altered coupling-patterns distributed not just locally but throughout the structural scaffold. Strongly coupled residues are distributed across the structure in an anisotropic manner accounting for only 13% of the residues, highlighting that a large number of residues in GPCRs are inherently dynamic to enable structural motions critical for function. Our work thus uncovers the thermodynamic hallmarks of GPCR structure and activation, and how differences quantifiable only via higher-order coupling free energies provide insights into their exquisite structural specialization and the fluid nature of the intramolecular interaction network. The intricate landscapes and perturbation methodologies presented here lay the foundation for understanding allosteric mechanisms in GPCRs, location of structural-functional hot-spots, and effects of disease-causing mutations.

biophysics↗

bioRxiv2021

picCASO: A Cellular Automaton for Spatial Organisation

Microbial consortia exhibit spatial patterning across diverse environments. Since probing the self-organization of natural microbial communities is limited by their inherent complexity, synthetic models have emerged as attractive alternatives. In this study, we develop novel frameworks of bacterial communication and explore the emergent spatiotemporal organization of microbes. Specifically, we build quorum sensing-mediated models of microbial growth that are utilized to characterize the dynamics of communities from arbitrary initial configurations and establish the effectiveness of our communication strategies in coupling the growth rates of microbes. Our simulations indicate that the behavior of quorum sensing-coupled consortia can be most effectively modulated by the rates of secretion of AHLs. Such a mechanism of control enables the construction of desired relative populations of constituent species in spatially organized populations. Our models accurately recapitulate previous experiments that have investigated pattern formation in synthetic multi-cellular systems. Additionally, our software tool enables the easy implementation and analysis of our frameworks for a variety of initial configurations and simplifies the development of sophisticated gene circuits facilitating distributed computing. Overall, we demonstrate the potential of spatial organization as a tunable parameter in synthetic biology by introducing a communication paradigm based on the location and strength of coupling of microbial strains. Author SummaryInteracting microorganisms that coexist in a given environment tend to have well-defined spatial arrangements. While the emergence of such organization is seen across different microbiomes in nature, it is hitherto not well understood. Decoding the inherent spatial patterning of microbes is constrained by the complexity of their natural habitats. Here, we take advantage of synthetic models of microbial communities to study the dynamics of emergent spatial organization. Our framework of bacterial communication utilizes modular synthetic devices to couple growth rates. In addition to uncovering potential principles of spatial organization, this work enables the construction of complex genetic circuits distributed across communicating strains. Additionally, we have developed a software tool, picCASO, that streamlines the investigation of microbial systems communicating through such frameworks.

systems biology↗