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.