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

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

Functional characterization of the pleckstrin homology domain of a cellulose synthase from the oomycete Saprolegnia monoica

Abstract not availableJohanna Fugelstad, Christian Brown, Elvira Hukasova, Gustav Sundqvist, Arne Lindqvist, Vincent Bulon

Assessing kinetics from fixed cells reveals activation of the mitotic entry network at the S/G2 transition

The ultimate aim of the cell cycle is to create an identical daughter cell. Therefore, correct progression through the different phases of the cell cycle is crucial to ensure faithful cell division. Successful execution of the different processes in the cell cycle is achieved by the coordinated action of a complex network of protein kinases and phosphatases at the centre of which stand Cyclin-Cdk complexes. Human cells possess a variety of cyclins and Cdks, which form complexes that regulate cell cycle transitions. In an unperturbed cell cycle, preparing a cell for mitosis requires faithful DNA replication and reorganisation of the cell’s structures and organelles. In this scenario, cells initiate successive waves of Cdk activity that orchestrate the timely and spatially controlled phosphorylation of a multitude of targets. In contrast, upon DNA damage cells must halt cell cycle progression in order to prevent mitotic entry of damaged cells and subsequently avoid potential propagation of mutations. Strict control of Cyclin-Cdk complexes is, therefore, essential both for correct cell division and to maintain genome integrity. However, the exact mechanisms underlying the activation of Cyclin- Cdk complexes in these different scenarios remain largely unknown. In this thesis, I have investigated several aspects of the regulation of Cdk activity both in the unperturbed cell cycle and during a DNA damage response. To address Cdk activity in the unperturbed cell cycle we established a novel quantitative immunofluorescence method and assessed the dynamics of cyclin accumulation and Cdk target phosphorylation in the unperturbed cell cycle. We found that the mitotic entry network first becomes activated at the S/G2 transition. This finding shifts the classical view of an abrupt Cdk activation at mitotic entry to an earlier and more gradual activation. Furthermore, it provides a potential link between S phase and mitosis, suggesting the existence of a mechanism that maintains pro-mitotic activities under a certain threshold until DNA replication is completed (Paper I). Interestingly, in parallel to an increase of pro-mitotic activities at the S/G2 transition, we observed a change in the localisation of Cyclin A2. Using genome-edited cell lines that express endogenous Cyclin A2-eYFP we were able to determine the cell cycle-dependent localisation of Cyclin A2 to the cytoplasm. Interestingly, despite coinciding with an increase of Cdk activity in the cell cycle we found that the cytoplasmic accumulation Cyclin A2 is modulated by p21 and the presence rather than activity of Cdk1. These findings suggest that complex formation and interaction with Cdk inhibitor proteins (CKI) might regulate Cyclin A2 localisation throughout the cell cycle (Paper IV). Despite not having uncovered a role for cytoplasmic Cyclin A2, we hypothesise that the cell cycle-dependent localisation of cyclins may be an important step to regulate Cdk activity. In order to understand how cells modulate Cdk activity upon DNA damage we made use of endogenously tagged cell lines expressing Cyclin B1-eYFP. We found that upon DNA damage cells continue to accumulate Cyclin B1 until reaching levels that are normally present in G2 phase. At this point, cells translocate Cyclin B1 to the nucleus in a p21 and p53- dependent manner where it is degraded by APC/CCdh1. We identified nuclear translocation and degradation of Cyclin B1 as a restriction point in the cell cycle when cells irreversibly exit the cell cycle and become senescent (Paper II). Senescence is regarded as an early barrier for tumorigenesis as it prevents the propagation of cells with damaged DNA. Our findings in Paper II suggested a link between mitotic inducers and the induction of senescence; therefore we decided to investigate the role of Cdk activity in terminal cell cycle exit. We found that upon DNA damage cells preserve low levels of Cdk activity to ensure that damaged cells continue to progress through the cell cycle until they reach a point where they can be forced into senescence. In this context, we found that Cdk activity induces p21 expression in a p53-independent manner to promote nuclear translocation and degradation of Cyclin B1 and other mitotic inducers (Paper III). Altogether, the data presented in this thesis points towards the existence of a link between the mitotic entry network and the DNA damage response to modulate the activity of Cyclin-Cdk complexes in time and space to trigger ensure correct progression to mitosis or, when needed, to trigger senescence

Centmitor-1, a Novel Acridinyl-Acetohydrazide, Possesses Similar Molecular Interaction Field and Antimitotic Cellular Phenotype as Rigosertib, ON 01910.Na

Mitosis is an attractive target for the development of new anticancer drugs. In a search for novel mitotic inhibitors, we virtually screened for low molecular weight compounds that would possess similar steric and electrostatic features, but different chemical structure than rigosertib (ON 01910.Na), a putative inhibitor of phosphoinositide 3-kinase (PI3K) and polo-like kinase 1 (Plk1) pathways. Highest scoring hit compounds were tested in cell-based assays for their ability to induce mitotic arrest. We identified a novel acridinyl-acetohydrazide, here named as Centmitor-1 (Cent-1), that possesses highly similar molecular interaction field as rigosertib. In cells, Cent-1 phenocopied the cellular effects of rigosertib and caused mitotic arrest characterized by chromosome alignment defects, multipolar spindles, centrosome fragmentation, and activated spindle assembly checkpoint. We compared the effects of Cent-1 and rigosertib on microtubules and found that both compoundsmodulated microtubule plus-ends and reduced microtubule dynamics. Also, mitotic spindle forces were affected by the compounds as tension across sister kinetochores was reduced in mitotic cells. Our results showed that both Cent-1 and rigosertib target processes that occur during mitosis as they had immediate antimitotic effectswhen added to cells during mitosis. Analysis of Plk1 activity in cells using a Forster resonance energy transfer (FRET)-based assay indicated that neither compound affected the activity of the kinase. Taken together, these findings suggest that Cent-1 and rigosertib elicit their antimitotic effects by targeting mitotic processes without impairment of Plk1 kinase activity