Resolving cell cycle speed in one snapshot with a live-cell fluorescent reporter

Abstract

Cell proliferation changes concomitantly with fate transitions during reprogramming, differentiation, regeneration, and oncogenesis. Methods to resolve cell cycle length heterogeneity in real-time are currently lacking. Here, we describe a genetically encoded fluorescent reporter that captures live cell cycle speed using a single measurement. This reporter is based on the color-changing Fluorescent Timer (FT) protein, which emits blue fluorescence when newly synthesized before maturing into a red fluorescent protein. We generated a mouse strain expressing an H2B-FT fusion reporter from a universally active locus, and demonstrate that faster-cycling cells can be distinguished from slowercycling ones based on the intracellular fluorescence ratio between the FT's blue and red states. Using this reporter, we reveal the native cell cycle speed distributions of fresh hematopoietic cells, and demonstrate its utility in analyzing cell proliferation in solid tissues. This system is broadly applicable for dissecting functional heterogeneity associated with cell cycle dynamics in complex tissues.

The Erythropoietin Receptor Stimulates Rapid Cycling and Formation of Larger Red Cells During Mouse and Human Erythropoiesis

“…In the first 26 hours of culture, the difference in growth rate for the Bcl-xL-Epor -/and EpoR-Epor -/cultures indicated a substantial difference in doubling time, of 6.1 h for EpoR-Epor -/erythroblasts, compared with 8.6 h for Bcl-xL-Epor -/erythroblasts (95% confidence intervals: 5.6 to 7.1 h and 7.7 to 10.4 h, respectively ; Fig 2c, e). A cycle length of 6 hours for EpoR-Epor -/is in good agreement with our recent direct measurement of a 6 hour cell cycle duration in early erythroblasts in vivo 27 , and with an independent measurement using a fluorescent timer protein that found early erythroblasts to have the shortest cell cycle amongst bone marrow hematopoietic progenitors 28 .…”

“…First, erythroblasts underwent slower and fewer cell cycles, generating far fewer red cells, showing that EpoR is essential for the rapid cycling of early erythroblasts. We confirmed this using a mouse transgenic for a fluorescent reporter of cell cycle length, finding that Epo stimulation shortened cell cycle length in vivo in early erythroblasts, cells that are already amongst the fastest cycling cells in the bone marrow 27,28 . Second, we found that, unexpectedly, despite its stimulation of rapid erythroblast cycling, EpoR signaling increases cell size in both erythroblasts and red cells.…”

“…To test whether EpoR stimulation altered cell cycle length in vivo, we used a recently -engineered mouse expressing a live-cell fluorescent reporter of cell cycle speed 28 . Specifically, the mouse expresses a transgene encoding histone H2B fused to a fluorescent timer protein (H2B-FT, Fig 2f).…”

“…In contrast, the red-fluorescent H2B-FT accumulates to higher levels in cells of long cycles due to its extensive stability. The ratio of blue to red fluorescence was found to be a sensitive indicator of cell cycle length in vivo in diverse cell types including bone marrow progenitors 28 .…”

Cell cycle dynamics in the reprogramming of cellular identity

“…Analysis of 100 proteins in human cancer cells revealed that the abundance of proteins with a long half‐life depends on dilution through cell division . Indeed, the concept that stable proteins are preferentially retained in slow‐dividing or nondividing cells could be recapitulated by transgenically expressed reporter proteins . These data support the hypothesis that cell cycle length can be opted to control and alter the concentration of molecular species dependent on their inherent half‐lives.…”