Abstract
During late mitosis and the early G1 phase, the origins of replication are licensed by binding to double hexamers of MCM2-7. In this study, we investigated how licensing and proliferative commitment are coupled in the epithelium of the small intestine. We developed a method for identifying cells in intact tissue containing DNA-bound MCM2-7. Interphase cells above the transit-amplifying compartment had no DNA-bound MCM2-7, but still expressed the MCM2-7 protein, suggesting that licensing is inhibited immediately upon differentiation. Strikingly, we found most proliferative Lgr5+ stem cells are in an unlicensed state. This suggests that the elongated cell-cycle of intestinal stem cells is caused by an increased G1 length, characterized by dormant periods with unlicensed origins. Significantly, the unlicensed state is lost in Apc-mutant epithelium, which lacks a functional restriction point, causing licensing immediately upon G1 entry. We propose that the unlicensed G1 phase of intestinal stem cells creates a temporal window when proliferative fate decisions can be made.
Highlights
Cell division is necessary for homeostasis of adult tissue
To establish the overall MCM2–7 protein abundance along intestinal crypts, we first examined the expression of MCM2–7 proteins in the epithelium of the small intestines of adult murine by high-resolution immunofluorescence microscopy
We focused on Mcm2 as a surrogate for all the members of the MCM2–7 complex, based on their similar function and localization
Summary
Cell division is necessary for homeostasis of adult tissue It allows for the replacement of aged or damaged cells and provides specialized cells critical for tissue function. The rapidly renewing intestinal epithelium replenishes its cellular content every 4–5 d This high turnover rate is maintained primarily by Lgr5+ intestinal stem cells in the crypt base, which are thought to be continually proliferative (Basak et al, 2014) as confirmed by proteomic and transcriptomic analysis (Muñoz et al, 2012). There is a quiescent stem cell population that can reengage with the cell cycle to repopulate the Lgr5+ cell population if it becomes depleted. Lgr5+ stem cells can divide to form transit-amplifying (TA) cells, which undergo several rounds of cell division before differentiating and losing proliferative competency (Potten and Loeffler, 1990)
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