Cell cycle regulation and DNA damage response : a record of polo-like kinase 1 activity

Abstract

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.