Cell cycle proliferation decisions: the impact of single cell analyses

Abstract

Cell proliferation is a fundamental requirement for organismal development and homeostasis. The mammalian cell division cycle is tightly controlled to ensure complete and precise genome duplication and segregation of replicated chromosomes to daughter cells. The onset of DNA replication marks an irreversible commitment to cell division, and the accumulated efforts of many decades of molecular and cellular studies have probed this cellular decision, commonly called the restriction point. Despite a long-standing conceptual framework of the restriction point for progression through G1 phase into S phase or exit from G1 phase to quiescence (G0), recent technical advances in quantitative single cell analysis of mammalian cells have provided new insights. Significant intercellular heterogeneity revealed by single cell studies and the discovery of discrete subpopulations in proliferating cultures suggests the need for an even more nuanced understanding of cell proliferation decisions. In this review, we describe some of the recent developments in the cell cycle field made possible by quantitative single cell experimental approaches.

Keywords: G0; G1; G2; biosensor; cell cycle; cell division; quiescence; restriction point; review; single cell.

Figure 1. Advantages of single cell analysis

(A) Hypothetical molecular signal in individual cells of an artificially-synchronized population. Black dots represent cell divisions. Cells are synchronized in the first cell cycle, but within two to three cell cycles the population is completely asynchronous. (B, Left) Single cell analysis identifies and tracks a representative molecular signal in coexisting cell populations where different subpopulations adopt different cell cycle fates. (B, Right) Ensemble analysis of the same coexisting populations reports only the average signal that may not represent either subpopulation’s cell cycle fate.

Figure 2. The restriction point during re-entry to G1 from quiescence

(A) The prevailing paradigm of the restriction point. Mitogens activate Ras signaling, downstream MAP Kinases, and ultimately Cyclin D transcription. Cyclin D complexes with CDK4 and CDK6 to partially phosphorylate Rb, and this hypophosphorylation causes release from E2F and partially activates Cyclin E expression. Cyclin E complexes with CDK2 to hyperphosphorylate Rb, creating a positive feedback loop where progressively increasing Cyclin E levels increase Rb phosphorylation leading to increased Cyclin E transcription and S phase commitment. (B) An alternate model for the restriction point. Cyclin D-CDK complexes only monophosphorylate Rb on 14 unique sites. The monophosphorylated Rb remains E2F-bound, but at least some aspects of Rb-mediated gene repression are relieved moving cells from G0 to G1. Multiple sustained mitogen-dependent inputs (p27 degradation, origin licensing, etc.) activate Cyclin E-CDK2 complexes which rapidly hyperphosphorylate Rb in a switch-like fashion to drive commitment to S phase.

Figure 3. Multiple proliferation decisions in actively dividing cells

Actively dividing cells have two distinct proliferation decision points. The first is a window during G2, in which cells with CDK activity above a critical threshold pre-commit to completing the next cell cycle with a short G1. A subpopulation of G2 cells with reduced mitogen signaling and/or low Cyclin-CDK activity do not commit to the next cell cycle. These cells have a long, variable length subsequent G1/G0-like phase and are presented with a second proliferation decision at the G1 restriction point. Sustained mitogen signaling promotes increased CDK activity above the commitment threshold which is self-sustaining through multiple feedback and feedforward relationships. In the absence of sustained signaling and increasing CDK activity, cells exit to G0. The probability of cells committing to the cell cycle or exiting to G0 is the cumulative probability of multiple molecular events.