Biology subjects

Kansal, K.

Publications and source records attributed to Kansal, K..

3 recordsLinked to original sources

bioRxiv2025

Prediction of Piconewton Receptor Tension Images using Deep Learning

Piconewton (pN) receptor forces govern many biological processes, but measuring these forces remains challenging. Molecular tension probes (MTPs) provide a sensitive means to measure pN cellular forces via fluorescence microscopy; however, MTPs are challenging to use and only forces transmitted through the probes are reported, complicating their use in heterogenous environments. Here, we present Tension Deep Learning (TensionDL), which leverages convolutional neural networks and image-to-image translation to predict pN receptor tension maps from images of cell morphology and the force-transducing protein vinculin. We validate the accuracy of TensionDL at the subcellular and cellular scales, demonstrate model accuracy across different substrate stiffnesses and cell types, and leverage TensionDL to make semi-quantitative predictions of cell mechanical output. Finally, TensionDL enables long-term mapping of pN receptor tension and infers tension distributions in heterogeneous environments in which some forces are not transduced through MTPs.

bioengineering↗

bioRxiv2025

Chimeric Antigen Receptors Transmit Co-stimulatory Domain Dependent Piconewton Forces to their Target

Chimeric antigen receptor (CAR) T cells promote tumor-specific cytotoxicity through engagement of a recombinant, synthetic receptor with target ligands expressed on cancer cells. Native T cells are mechanically active, both transmitting and sensing forces exceeding 19 piconewtons (pN) via transmembrane receptors, including the T cell receptor (TCR). Emerging evidence implicates mechanoactivity in CAR T cell biology, but CAR-transmitted T cell forces have not been directly measured. Here, we utilize DNA-based molecular tension probes (MTPs) conjugated to CAR target ligands, providing evidence of actin-polymerization dependent forces exceeding 4.7-19 pN borne by the CAR. We demonstrate force transmission by three clinically relevant CARs (CD123, CD33, and CD19), suggesting that these forces are generalizable across CAR targets and constructs. Additionally, we identify intracellular co-stimulatory domains as the main determinants of CAR-mediated forces, because first-generation CARs lacking co-stimulatory domains do not transmit measurable forces to their ligand. Finally, we demonstrate that CAR forces temporally precede Ca2+ signaling and are spatially correlated with phosphorylation of classical TCR-signaling machinery, indicating a link between CAR T cell forces and early biochemical signaling. Our study introduces CAR-mediated mechanobiology as a key correlate of early CAR T cell activation events. Significance StatementChimeric antigen receptor (CAR) T cell therapies have revolutionized treatment for several hematological malignancies. CARs are recombinant receptors containing domains derived from the T cell receptor complex machinery and other co-stimulatory proteins. Mechanical forces are believed to be important in T cell activation and antigen recognition. The role of mechanobiology in CAR T cell immunotherapy remains poorly understood. Here, using DNA-based molecular tension probes conjugated to CAR ligands, we provide direct evidence that CARs bear actin polymerization-dependent piconewton forces during antigen engagement that precede early signaling events. These forces depend on CAR co-stimulatory domains, and first-generation CARs lacking these domains fail to transmit detectable force. These findings suggest mechanobiology may be a key, tunable parameter for next-generation CAR T cells.

immunology↗

bioRxiv2024

Enteropathogenic E. coli effector Map regulates the depletion of the tight junction proteins occludin and claudin-4 via cathepsin B and Rab13-mediated mechanisms

Infections by Enteropathogenic E. coli (EPEC) cause acute diarrheal disease in infants accounting for severe morbidity and mortality. One of the underlying causes of the disease is the break-down of the intestinal barrier maintained by the tight junctions (TJs). EPEC uses a type 3 secretion system to translocate more than twenty effectors into infected cells which disrupt several functions of the host cells. The effectors EspF, Map, EspG1/G2 and NleA have been reported to disrupt the TJs and cause the leakage of charged ions and uncharged molecules through the barrier. We have reported earlier that EspF and Map cause the depletion of TJ proteins claudin-1, claudin-4 and occludin through both transcriptional and post-transcriptional mechanisms. Here, we show that the EPEC effector Map modulates the lysosomal protease, cathepsin B to deplete claudins and occludin. Expression of mutant Map proteins that lacked the mitochondrial targeting sequence (MTS) completely restored the total levels of occludin and its localization at the TJs and partially restored claudin-4 levels and its junctional localization. We also identified a novel interaction of Map with the GTPase Rab13. As Rab13 has been reported to mediate the recycling of occludin to the plasma membrane, its interaction with Map has important implications for the loss of TJ integrity in EPEC pathogenesis. Occludin regulates the passage of water and uncharged solutes through TJs and Map may block its recycling to compromise the TJs thus causing excessive leakage through the barrier.

cell biology↗