Exploiting cell cycle-dependent dephosphorylation for mitosis-specific protein recruitment

Regulation of signalling cascades at the plasma membrane is often achieved through spatiotemporal control of key enzymes and their interactions with substrates, agonists or inhibitors. Inducible protein dimerization approaches enable the conditional recruitment of enzymes to the plasma membrane using chemogenetics or optogenetics, that is, small molecules or light. Although these tools are tremendously helpful for dissecting complex signalling networks and modifying the lipid composition of the plasma membrane, they rely on the use of exogenous triggers that can perturb normal cell physiology, which is particularly problematic when studying delicate processes such as mitosis.

We used MARS for mitosis-dependent plasma membrane recruitment of three functional enzymes, including class I phosphatidylinositol 3-OH kinase (PI3K), which produces phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P₃). MARS-directed localization of PI3K elevated PtdIns(3,4,5)P₃ levels at the plasma membrane specifically during mitosis, and this caused a selective delay of early mitosis progression up to the spindle assembly checkpoint but did not affect subsequent steps, revealing differential roles of PtdIns(3,4,5)P₃ at distinct stages of mitosis. Notably, these experiments were performed in an otherwise unperturbed bulk cell population, highlighting the application of MARS to edit the lipid content and modulate signalling events at the plasma membrane with precise spatiotemporal control and without external stimuli, including harsh synchronization reagents.