Abstract
Cultured human epidermal keratinocyte stem cells (holoclones) are crucial for regenerative medicine for burns and genetic disorders. In serial culture, holoclones progressively lose their proliferative capacity to become transient amplifying cells with limited growth (paraclones), a phenomenon termed clonal conversion. Although it negatively impacts the culture lifespan and the success of cell transplantation, little is known on the molecular mechanism underlying clonal conversion. Here, we show that holoclones and paraclones differ in their actin filament organization, with actin bundles distributed radially in holoclones and circumferentially in paraclones. Moreover, actin organization sets the stage for a differing response to epidermal growth factor (EGF), since EGF signalling induces a rapid expansion of colony size in holoclones and a significant reduction in paraclones. Furthermore, inhibition of PI3K or Rac1 in holoclones results in the reorganization of actin filaments in a pattern that is similar to that of paraclones. Importantly, continuous Rac1 inhibition in holoclones results in clonal conversion and reduction of growth potential. Together, our data connect loss of stem cells to EGF-induced colony dynamics governed by Rac1.
Highlights
Actin filament organization and actomyosin contractility are critical for cell shape changes and movements during many developmental processes including gastrulation, tissue morphogenesis and remodelling (Gorfinkiel & Blanchard, 2011; Mason & Martin, 2011)
Because epidermal growth factor receptor (EGFR) signalling is important for the successful expansion of diploid human keratinocytes in culture (Barrandon & Green, 1987b), we have further explored how keratinocyte colonies respond to epidermal growth factor (EGF)
Time-lapse imaging showed that the expansion of a growing colony in response to EGF was caused by two mechanisms: a centrifugal migration of the peripheral cells with maintenance of cell–cell contact, and a flattening of the cells located at the center of the colony (Supporting Information Movie S1)
Summary
Actin filament organization and actomyosin contractility are critical for cell shape changes and movements during many developmental processes including gastrulation, tissue morphogenesis and remodelling (Gorfinkiel & Blanchard, 2011; Mason & Martin, 2011). The epidermis is a superb model system to study the role of actin filament dynamics in tissue homeostasis because it constantly renews thanks to keratinocyte stem/progenitor cells located in the epithelial basal layer, and in epidermal appendages. Actin filaments are reorganized during terminal differentiation of epidermal keratinocytes (Connelly et al, 2010; Lewis et al, 1987; Vaezi et al, 2002), through a molecular mechanism mediated by RhoA and Rac (Benitah et al, 2005; Vaezi et al, 2002), the small Rho GTPases that function downstream of epidermal growth factor receptor (EGFR) signalling, and other tyrosine kinase receptor pathways (Raftopoulou & Hall, 2004). The impact of actin filament reorganization in epidermal keratinocyte stem cells remains unknown
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