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

Cell Reports (Nov 2024)

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

Journal volume & issue: Vol. 43, no. 11
p. 114914

Abstract

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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.

Published in Cell Reports

ISSN: 2211-1247 (Online)
Publisher: Elsevier
Country of publisher: Netherlands
LCC subjects: Science: Biology (General)
Website: http://www.cell.com/cell-reports/home

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