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Actin and serum response factor transduce physical cues from the microenvironment to regulate epidermal stem cell fate decisions

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Abstract

Epidermal homeostasis depends on a balance between stem cell renewal and differentiation and is regulated by extrinsic signals from the extracellular matrix (ECM)1,2. A powerful approach to analysing the pathways involved is to engineer single-cell microenvironments in which individual variables are precisely and quantitatively controlled3,4,5. Here, we employ micropatterned surfaces to identify the signalling pathways by which restricted ECM contact triggers human epidermal stem cells to initiate terminal differentiation. On small (20 μm diameter) circular islands, keratinocytes remained rounded, and differentiated at higher frequency than cells that could spread on large (50 μm diameter) islands. Differentiation did not depend on ECM composition or density. Rather, the actin cytoskeleton mediated shape-induced differentiation by regulating serum response factor (SRF) transcriptional activity. Knockdown of SRF or its co-factor MAL inhibited differentiation, whereas overexpression of MAL stimulated SRF activity and involucrin expression. SRF target genes FOS and JUNB were also required for differentiation: c-Fos mediated serum responsiveness, whereas JunB was regulated by actin and MAL. Our findings demonstrate how biophysical cues are transduced into transcriptional responses that determine epidermal cell fate.

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Figure 1: Regulation of keratinocyte shape and differentiation on micropatterned substrates.
Figure 2: Limited adhesion promotes loss of stem cell markers.
Figure 3: The actin cytoskeleton mediates shape-induced terminal differentiation.
Figure 4: Influence of MAL/SRF signalling on shape-induced differentiation.
Figure 5: Regulation of AP-1 signalling and role in differentiation.

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Acknowledgements

This work was funded by the Wellcome Trust, the Medical Research Council (MRC), Cancer research UK (CRUK) and the TUMIC European Union FP7 network. J.T.C. is the recipient of an NIH (National Institutes of Health) postdoctoral fellowship. We thank R. Treisman and G. Posern for providing reagents and the core facilities of the Wellcome Trust Centre for Stem Cell Research for technical assistance.

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J.T.C. designed and conducted experiments, developed the substrates and wrote the manuscript. J.E.G. and B.T. developed substrates and carried out experiments. D.W.T performed single-cell PCR. G.D. performed chromatin immunoprecipitation experiments. W.T.S.H. and F.M.W. consulted on experimental design. F.M.W. co-wrote the manuscript.

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Correspondence to Fiona M. Watt.

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Connelly, J., Gautrot, J., Trappmann, B. et al. Actin and serum response factor transduce physical cues from the microenvironment to regulate epidermal stem cell fate decisions. Nat Cell Biol 12, 711–718 (2010). https://doi.org/10.1038/ncb2074

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