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

Abstract

SummaryLineage 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 hierarchiesin 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 earlyversuslate 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 earlyvs.late absorptive differentiation

Single-molecule live imaging of subunit interactions and exchange within cellular regulatory complexes

“…We engineered an automated system to perform fSMT measurements with higher throughput (see also refs. 53,54 ). Using code written in the Nikon Elements macro language and Python (https://github.com/tgwgraham/autosmt_v4tg2), we programmed a Nikon Ti-E microscope to raster-scan a rectangular grid of different fields of view in a sample, which were spaced far enough apart to avoid photobleaching of adjacent fields of view.…”

“…In PAPA, excitation of a "sender" fluorophore reactivates a nearby "receiver" fluorophore from a photochemically-induced dark state (Figure 1 and Movie S1). [52][53][54] This proximity-dependent reactivation can be used to detect interaction between a pair of proteins labeled with sender and receiver fluorophores. Unlike previous approaches, such as single-molecule FRET and two-color colocalization, PAPA has a flexible distancedependence, is not impeded by spectral crosstalk, and can efficiently detect intracellular protein interactions at physiological concentrations in fast single-molecule tracking (fSMT), making it possible to localize and measure the mobility of complexes containing a specific pair of subunits in living cells.…”