Derivation of a Novel G2 Reporter System

Abstract Progression through G2 phase of the cell cycle is a technically difficult area of cell biology to study due to the lack of physical markers specific to this phase. The FUCCI system uses the biology of the cell cycle to drive fluorescence in select phases of the cell cycle. Similarly, a commercially available system has used a fluorescent analog of the Cyclin B1 protein to visualize cells from late S phase to the metaphase– anaphase transition. We have modified these systems to use the promoter and destruction box elements of Cyclin B1 to drive a cyan fluorescent protein. We demonstrate here that this is a useful tool for measuring the length of G2 phase without perturbing any aspect of cell cycle progression

The Tuberin and Cyclin B1 complex functions as a novel G2/M sensor of serum conditions and Akt signaling.

A great deal of ground breaking work has determined that the Tuberin and Hamartin Complex function as a negative regulator of protein synthesis and cell cycle progression through G1/S. This is largely attributed to the GTPase activity of Tuberin that indirectly inhibits the mammalian target of rapamycin (mTOR). During times of ample nutrition Tuberin is inhibited by growth factor signaling, including direct phosphorylation by Akt/PKB, allowing for activation of mTOR and subsequent protein synthesis. It is well rationalized that maintaining homeostasis requires communication between cell growth (mTOR signaling) and cell division (cell cycle regulation), however how this occurs mechanistically has not been resolved. This work demonstrates that in the presence of high serum, and/or Akt signaling, direct binding between Tuberin and the G2/M cyclin, Cyclin B1, is stabilized and the rate of mitotic entry is decreased. Importantly, we show that this results in an increase in cell size. We propose that this represents a novel cell cycle checkpoint linking mitotic onset with the nutritional status of the cell to control cell growth