Automated live-cell single-molecule tracking in enteroid monolayers reveals transcription factor dynamics probing lineage-determining function
Nike Walther,
Sathvik Anantakrishnan,
Thomas G.W. Graham,
Gina M. Dailey,
Robert Tjian,
Xavier Darzacq
Affiliations
Nike Walther
Department of Molecular and Cell Biology, Li Ka Shing Center for Biomedical and Health Sciences, California Institute for Regenerative Medicine (CIRM) Center of Excellence, University of California, Berkeley, Berkeley, CA 94720, USA; Corresponding author
Sathvik Anantakrishnan
Department of Molecular and Cell Biology, Li Ka Shing Center for Biomedical and Health Sciences, California Institute for Regenerative Medicine (CIRM) Center of Excellence, University of California, Berkeley, Berkeley, CA 94720, USA; Biophysics Graduate Group, University of California, Berkeley, Berkeley, CA 94720, USA
Thomas G.W. Graham
Department of Molecular and Cell Biology, Li Ka Shing Center for Biomedical and Health Sciences, California Institute for Regenerative Medicine (CIRM) Center of Excellence, University of California, Berkeley, Berkeley, CA 94720, USA
Gina M. Dailey
Department of Molecular and Cell Biology, Li Ka Shing Center for Biomedical and Health Sciences, California Institute for Regenerative Medicine (CIRM) Center of Excellence, University of California, Berkeley, Berkeley, CA 94720, USA
Robert Tjian
Department of Molecular and Cell Biology, Li Ka Shing Center for Biomedical and Health Sciences, California Institute for Regenerative Medicine (CIRM) Center of Excellence, University of California, Berkeley, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, Berkeley, CA 94720, USA; Corresponding author
Xavier Darzacq
Department of Molecular and Cell Biology, Li Ka Shing Center for Biomedical and Health Sciences, California Institute for Regenerative Medicine (CIRM) Center of Excellence, University of California, Berkeley, Berkeley, CA 94720, USA; Corresponding author
Summary: 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.
