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Division and cell proliferation is an essence of life. A human cell has in its core a verysimple yet a very complex machinery to coordinate cell cycle activities and events. Asomatic human cell has as a base four different cell cycle stages: a preparatory cellgrowth stage (G1) a synthetic stage where genomic material is replicated (S) a secondgrowth and preparation stage (G2) and a mitotic stage, where genetic material issegregated in two new cells (M). The main driver of these phase shifts is the oscillatorybehavior of cell cycle proteins called Cyclins, which are being produced and degraded ina periodic manner. Cyclins steer kinase activity, and function together with other cellcyclekinases as Polo-like kinase 1 (Plk1). On top of the cell cycle regulation a cell hasimportant mechanisms to sense and repair DNA damage, a so-called DNA damageresponse. DNA damage occurs regularly because of intrinsic factors related to cellularactivities e.g. genome replication, metabolism or exogenous factors like solar radiation.Therefore, the response to DNA damage is an inherent part of the cell cycle and its mainaction is to halt cell cycle progression and establish a checkpoint. A cell has severalcheckpoints throughout the cell cycle: a G1/S checkpoint, an intra-S checkpoint, a G2/Mcheckpoint and a M-checkpoint. At these positions a cell can stop or slow down in caseof unfavorable conditions, stress or DNA damage. To take care of DNA damage, repairmechanisms are put in place and if possible, a cell eventually continues proliferation(checkpoint recovery) or exits the cell cycle.In this thesis I focused on the regulation that precedes mitotic entry – the regulation ofG2 phase during both normal mitotic entry and after checkpoint activation. I furtherfocused on the activity of the protein Plk1 that is important but not essential to entermitosis in a normal cell cycle, but becomes indispensable for mitotic entry after DNAdamage.For this study I employed a biosensor for Plk1 activity, Plk1-FRET, and developed asetup that allows to follow single cells expressing the sensor over several cell cycles andlater quantify the signals. To study protein behaviors we further developed a techniquethat allows to elucidate dynamics of the cell cycle proteins from fixed cells growing onmicropatterns. Using this approach combined with endogenously tagged Cyclin A andCyclin B cell lines and a Plk1-FRET biosensor, we find that activities that precede mitoticentry are in place several hours before mitosis, at the completion of S phase, contrary tothe previous belief that the mitotic entry network is activated less than an hour beforemitosis. We further employed two different model systems and find that Cdk1 andPlk1/Plx1 coordinate degradation of Bora, a protein important for Plk1 activation. Wefind that in human cells Plk1-induced Bora degradation starts about two hours beforemitosis, at the time when Plk1 activity reaches the cytoplasm. Moreover, a small pool ofBora is not degraded and is stabilized in mitosis, providing the possibility to keep Plk1active in mitosis.Lastly, using a micropatterning approach and Plk1-FRET biosensor in combination witha probe for APC/C activation I show that upon checkpoint activation in G2 there is adecision point marked by a threshold of Plk1 activity. Activity above this thresholdcorrelates with progression to mitosis, whereas activity below it correlates with cellcycle exit. Furthermore, cells damaged in S phase can exit the cell cycle in two positionsin G2, with and without upregulating Plk1 activity, indicating that Plk1 activity is notrequired for cell cycle exit. Likewise, G1 cells that crossed the G1/S border afterreceiving DNA damage can exit the cell cycle in G2 phase, in a similar manner as cellsreceiving DNA damage in S-phase.Taken together our results shed light on the activities underlying the G2/M transitionboth in an unperturbed cell cycle and after DNA damage

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET