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Doctoral thesis
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Spatial and Temporal Control of Plk1 inhibition: Regulating Mitosis with Light

Imprimatur date2024
Defense date2024-03-15
Abstract

Cell division is a fundamental process that is highly regulated by different proteins. One of the key regulators is the conserved mitotic kinase Polo-like kinase 1 (Plk1). Plk1 is necessary for several processes during cell division, such as mitotic entry, bipolar spindle assembly and cytokinesis. The kinase’s activity is regulated during mitosis and localizes to different mitotic structures. During development, it plays a crucial role in asymmetric division of C. elegans embryos.

The aim of this work was to develop tool compounds that enable precise spatial and temporal control of Plk1. This was done to enable studying the functions of this mitotic kinase both on subcellular and on organismal levels during development.

To achieve temporal control over Plk1, we photocaged the small molecule Plk1 inhibitor BI2536 at a position crucial for interaction with Plk1. The p-ethoxystyryl derivatized 7-diethylaminocoumarin was chosen as a photocage for its favorable photochemical properties, which enable activation of the inhibitor with 488 nm light. The caged Plk1 inhibitor cPlk1i does not inhibit Plk1 and it can be rapidly uncaged and activated with 488 nm light. Once uncaged, the inhibitor efficiently interferes with Plk1 activity.

To improve spatial control of the inhibitor, we employed two strategies: one to enhance spatial control on a cellular level and another to gain better control on a subcellular level. For subcellular targeting, we explored a chemical-genetics method to target the caged Plk1 inhibitor to the HaloTag and luciferase-containing protein chimera HLuc at various subcellular locations. We showed that we can uncage the Plk1 inhibitor bound to HLuc through BRET-induced bioluminolysis. However, Plk1 was not inhibited when the inhibitor was bound to HLuc expressed in RPE cells. The second aim was to improve spatial control over Plk1 inhibition on a cellular level, to enable spatially defined control of Plk1 inhibition in a multicellular setting. To prevent diffusion of the inhibitor out of cells, we introduced esters on the Plk1 inhibitor. After hydrolysis, the resulting carboxylate prevents cell-to-cell diffusion. We could show that introducing a photocaged carboxyl ester on the Plk1 inhibitor enables precise spatial and temporal control of the hydrolysis reaction. This allows for spatially defined inhibition in a specific region of a 250-micron large spheroid.

In the future, this tool will enable the dissection of Plk1 roles at different locations and in individual cells and regions of developing organisms and organoids. This novel Plk1 inhibitor has potential applications in biological research, particularly in the study of spatially defined processes in development.

eng
Keywords
  • Cell division
  • Plk1
  • Chemical Biology
  • Photocage
  • Coumarin
Citation (ISO format)
VON GLASENAPP, Victoria Franziska. Spatial and Temporal Control of Plk1 inhibition: Regulating Mitosis with Light. 2024. doi: 10.13097/archive-ouverte/unige:177432
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Creation05/29/2024 3:02:43 PM
First validation05/30/2024 12:17:14 PM
Update time05/30/2024 12:17:14 PM
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