When cells respond to light All you need is LOV

This thesis is part of a project with the goal of establishing a simple artificial cell containing a minimum of required components to control microtubule growth and adjust the morphology of the encapsulating membrane in vitro. First, we focused on creating a working system in vivo. Regulation of protein activity in the system was achieved through the use of optogenetics. All signaling is subjected to mechanisms such as diffusion, even the optogenetic systems, which is why we altered the diffusion rate of components in chapter 2. We developed a strategy with different types of membrane anchors which decrease lateral diffusion and were able to demonstrate their effect through light-induced recruitment of signaling proteins to the plasma membrane. Chapter 3 addresses the problem of combining FRET sensors with blue light sensitive optogenetic domains through redshifted fluorescent proteins. With this method we explored the effect of photoregulatable Rac1 on stathmin and microtubules. In chapter 4 we created a photoregulatable kinase that directly phosphorylates stathmin, in order to reduce the amount of intermediates between stimuli and microtubule response. Finally, in chapter 5 we offer a way to directly regulate the stathmin molecule itself via blue light. This lead to the first light-induced, reversible mechanism to capture and release endogenous tubulin at will. These tools are a first step towards the creation of a minimalistic membrane regulating in vitro system.