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Abstract

The cytoskeleton consists of a network of three protein filaments: actin filaments, microtubules and intermediate filaments. In coordination they regulate a multitude of essential cell functions including cell locomotion, cell shape stabilization, mitosis and cargo transport. The cytoskeleton is physically linked to the extracellular environment by integrins, a protein family of cell surface receptors. To this day, the re-organization and interplay of the cytoskeleton in migrating cells is often studied by manipulation of regulatory signalling pathways far upstream from the cytoskeleton proteins. Direct cytoskeletal reorganization through crosslinking of the different filaments has not been achieved and the study of crosslinker properties is limited by those of naturally occurring binders. Hence, immediate binders for actin, tubulin and integrin, could be engineered as crosslinkers of the cytoskeleton to regulate cell migration. In particular, synthetic cytoskeleton binders, would provide an orthogonal toolbox for mechanical perturbations of the cytoskeleton, that are decoupled from biochemical effectors. In my thesis, I investigated designed ankyrin repeat proteins (DARPins) as potential binders of cytoskeletal elements and integrins with the long-term goal of fabricating modular tools for cytoskeleton manipulation. DARPins can be selected for binding different targets by ribosome display from a large protein library. I tested DARPins selected for binding actin, tubulin or the cytoplasmic tail of β3 integrins for their target-binding ability in living cells. While the integrin- and tubulin-binding DARPins did not efficiently label their target proteins, I identified five DARPins that labeled actin structures in motile cells, albeit with distinct patterns. I focused my studies on them, analyzed their actin-binding dynamics and found that the difference in their localization was affected by inherent dynamics of the actin cytoskeleton. Furthermore, I identified one DARPin that showed superior actin-labelling efficiency of filopodia compared to the widely used actin-label LifeAct. Next, the actin-binding DARPins were explored as building blocks for actin cross-linkers and engineered to dimerize upon illumination with blue light. For this, I demonstrated actin bundling in the presence of a covalent, homo-bifunctional DAPRin. Then, DARPins were fused to the iLid-nano system for optogenetic control of their dimerization. Association dynamics of the iLid-DARPins were quantified and dimerized actin-binding DARPins accumulated in the targeted subcellular region. An actin crosslinker based on a DARPin with slow actin-binding dynamics caused re-organization of F-actin in living cells. Finally, DARPins without optogenetic control were applied in giant unilamellar vesciles to bundle or link actin to the membrane. I conclude that DARPins are promising new labels of the actin-cytoskeleton in living cells and provide potential, orthogonal strategies for mechanical actin perturbations. For example, they can be used to study the influence of actin crosslinker properties such as binding-dynamics independently from size and structure, due to their origin from a common protein scaffold. I anticipate that DARPins will be recognized as a versatile, modular tool for interdisciplinary research in a wide scope of applications.