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

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 controlled(3-5). 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 mu m diameter) circular islands, keratinocytes remained rounded, and differentiated at higher frequency than cells that could spread on large (50 mu m 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.