SummaryCell cycle kinetics are crucial to cell fate decisions. Although live imaging has provided extensive insights into this relationship at the single-cell level, the limited number of fluorescent markers that can be used in a single experiment has hindered efforts to link the dynamics of individual proteins responsible for decision making directly to cell cycle progression. Here, we present fluorescently tagged endogenous proliferating cell nuclear antigen (PCNA) as an all-in-one cell cycle reporter that allows simultaneous analysis of cell cycle progression, including the transition into quiescence, and the dynamics of individual fate determinants. We also provide an image analysis pipeline for automated segmentation, tracking, and classification of all cell cycle phases. Combining the all-in-one reporter with labeled endogenous cyclin D1 and p21 as prime examples of cell-cycle-regulated fate determinants, we show how cell cycle and quantitative protein dynamics can be simultaneously extracted to gain insights into G1 phase regulation and responses to perturbations.

Summary Cellular redox states regulate the balance between stem cell maintenance and activation. Increased levels of intracellular reactive oxygen species (ROS) are linked to proliferation and lineage specification. In contrast to this general principle, we here show that in the hippocampus of adult mice, quiescent neural precursor cells (NPCs) maintain the highest ROS levels (hiROS). Classifying NPCs on the basis of cellular ROS content identified distinct functional states. Shifts in ROS content primed cells for a subsequent state transition, with lower ROS content marking proliferative activity and differentiation. Physical activity, a physiological activator of adult hippocampal neurogenesis, recruited hiROS NPCs into proliferation via a transient Nox2-dependent ROS surge. In the absence of Nox2, baseline neurogenesis was unaffected, but the activity-induced increase in proliferation disappeared. These results provide a metabolic classification of NPC functional states and describe a mechanism linking the modulation of cellular ROS by behavioral cues to the activation of adult NPCs.

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The pancreatic beta-cells control glucose homeostasis by secreting insulin in response to nutrient intake. The number of beta-cells is under tight metabolic control, as this number increases with higher nutrient intake. However, the signaling pathways matching nutrition with beta-cell mass plasticity remain poorly defined. By applying pharmacological and genetic manipulations, we show that reactive oxygen species (ROS) regulate dose-dependently beta-cell proliferation in vivo and in vitro. In particular, reducing ROS levels in beta-cells blocks their proliferation in response to nutrients. Using a non-invasive genetic sensor of intracellular hydrogen peroxide (H2O2), we reveal that glucose can directly increase the levels of H2O2. Furthermore, a moderate increase in H2O2 levels can stimulate beta-cell proliferation. Interestingly, while high H2O2 levels are inhibitory to beta-cell proliferation, they expand beta-cell mass in vivo by inducing rapid beta-cell neogenesis. Our study thus reveals a ROS-level-dependent mechanism linking nutrients with beta-cell mass plasticity. Hence, given the requirement of ROS for beta-cell mass expansion, antioxidant therapies should be applied with caution in diabetes.

Intracellular redox states regulate the balance between stem cell maintenance and activation. Increased levels of reactive oxygen species (ROS) are linked to proliferation and lineage specification. In contrast to this general principle, we show that in the hippocampus of adult mice it is the quiescent neural stem cells (NSCs) that maintain the highest ROS levels (hiROS). Classifying NSCs based on intracellular ROS content identified subpopulations with distinct molecular profiles, corresponding to functional states. Shifts in ROS content primed cells for a subsequent transition of cellular state, with lower cellular ROS content marking activity and differentiation. Physical activity, a known physiological activator of adult hippocampal neurogenesis, recruited the quiescent hiROS NSCs into proliferation via a transient Nox2-dependent ROS surge. In the absence of Nox2, baseline neurogenesis was unaffected, but the activity-induced increase in proliferation disappeared. These results describe a novel mechanism linking the modulation of cellular ROS by behavioral cues to the maintenance and activation of adult NSCs.