Visualizing Actin Packing and the Effects of Actin Attachment on Lipid Membrane Viscosity Using Molecular Rotors

The actin cytoskeleton and its elaborate interplay with the plasma membrane participate in and control numerous biological processes in eukaryotic cells. Malfunction of actin networks and changes in their dynamics are related to various diseases, from actin myopathies to uncontrolled cell growth and tumorigenesis. Importantly, the biophysical and mechanical properties of actin and its assemblies are deeply intertwined with the biological functions of the cytoskeleton. Novel tools to study actin and its associated biophysical features are, therefore, of prime importance. Here we develop a new approach which exploits fluorescence lifetime imaging microscopy (FLIM) and environmentally sensitive fluorophores termed molecular rotors, acting as quantitative microviscosity sensors, to monitor dynamic viscoelastic properties of both actin structures and lipid membranes. In order to reproduce a minimal actin cortex in synthetic cell models, we encapsulated and attached actin networks to the lipid bilayer of giant unilamellar vesicles (GUVs). Using a cyanine-based molecular rotor, DiSC₂(3), we show that different types of actin bundles are characterized by distinct packing, which can be spatially resolved using FLIM. Similarly, we show that a lipid bilayer-localized molecular rotor can monitor the effects of attaching cross-linked actin networks to the lipid membrane, revealing an increase in membrane viscosity upon actin attachment. Our approach bypasses constraints associated with existing methods for actin imaging, many of which lack the capability for direct visualization of biophysical properties. It can therefore contribute to a deeper understanding of the role that actin plays in fundamental biological processes and help elucidate the underlying biophysics of actin-related diseases.