Optical Control of the Actin Cytoskeleton

• Applicant: Dr. Christoph Etling
• Subject Area: Organic Molecular Chemistry - Synthesis and Characterisation ; Biological and Biomimetic Chemistry
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 545910666

The actin cytoskeleton consists of actin polymers (comprised of G-actin monomers) and associated actin-binding proteins. These include the Arp2/3 complex, which initiates the formation of branched actin networks, and myosin II, a motor protein that moves along actin polymers. The dynamic interplay between these protein assemblies is crucial for essential cellular processes, including cellular locomotion, cell division and organelle dynamics. Understanding the molecular bases of these processes is fundamental to progress studies in cell biology, and to develop strategies for the diagnosis and treatment of diseases associated with their dysfunction. The study of the actin cytoskeleton relies on tools that allow scientists to control actin network construction and actin-dependent processes. Actin-associated processes are highly dynamic and ubiquitous within cells. Thus, equally dynamic molecular tools that enable the precise temporal and spatial control of these processes would be invaluable to study their roles in specific cellular functions throughout the cell cycle and within distinct cellular locations. Inhibitors whose activity can be controlled through a defined external stimulus could provide such dynamic and precise control. Due to its non-invasive nature, its high precision and its straightforward applicability, light is an ideal stimulus for this purpose. The objective of this project is to develop actin cytoskeleton inhibitors, whose activity can be precisely and reversibly controlled using light. To achieve this, I will synthesize variants of the G-actin-binding latrunculins, the Arp2/3 inhibitor CK-666, and the myosin II inhibitor blebbistatin that feature azobenzene photoswitches inside their structures. The photoswitches have two photoisomers that differ in molecular geometry and can be converted into one another by light irradiation. These will be incorporated into the structures of the inhibitors such that one isomeric form is biologically active, while the other one is inactive. This will allow applying scientists to control the compounds’ activity ("switch" it "on" or "off") in a spatiotemporally defined manner with light (optical control). To enable a rational design of potent and selective photoswitchable actin cytoskeleton inhibitors, this project will combine the power of chemical synthesis and structural biology. Libraries of photoswitchable actin cytoskeleton inhibitors will be synthesized and tested for light-dependent bioactivity. The binding of successful candidates to their target proteins will be investigated by cryo-electron microscopy and protein crystallography. The so gained structural information will enable the rational design of new and improved photoswitchable inhibitors as the project progresses. This project will thus contribute to the goal of providing a comprehensive "toolbox" of photoswitchable inhibitors that will enable investigations of the actin cytoskeleton with unprecedented precision.

• DFG Programme: WBP Fellowship
• International Connection: USA
• Host: Professor Dr. Dirk Trauner