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

Abstract

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 two-dimensional culture systems. Studying two absorptive lineage-determining TFs, we found 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 could indicate the progression of differentiation and modulate early versus late differentiation within a lineage.

Keywords: 2D enteroid monolayer culture; CP: Developmental biology; CP: Genomics; automated imaging; cellular feature extraction; cellular protein diffusion; cellular protein self-association; cluster analysis based on protein diffusion behavior; differentiation; intestinal absorptive lineage; live-cell single-molecule tracking; mouse small intestinal organoid; protein diffusion correlation; transcription factor.