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

Molecular Rotors Provide Insights into Microscopic Structural Changes During Protein Aggregation

Changes in microscopic viscosity represent an important characteristic of structural transitions in soft matter systems. Here we demonstrate the use of molecular rotors to explore the changes in microrheology accompanying the transition of proteins from their soluble states into a gel phase composed of amyloid fibrils. The formation of beta-sheet rich protein aggregates, including amyloid fibrils, is a hallmark of a number of neurodegenerative disorders, and as such, the mechanistic details of this process are actively sought after. In our experiments, molecular rotors report an increase in rigidity of approximately three orders of magnitude during the aggregation reaction. Moreover, phasor analysis of the fluorescence decay signal from the molecular rotors suggests the presence of multiple distinct mechanistic stages during the aggregation process. Our results show that molecular rotors can reveal key microrheological features of protein systems not observable through classical fluorescent probes operating in light switch mode

Dimeric Metal-Salphen Complexes Which Target Multimeric G‑Quadruplex DNA

G-Quadruplex DNA structures have attracted increasing attention due to their biological roles and potential as targets for the development of new drugs. While most guanine-rich sequences in the genome have the potential to form monomeric G-quadruplexes, certain sequences have enough guanine-tracks to give rise to multimeric quadruplexes. One of these sequences is the human telomere where tandem repeats of TTAGGG can lead to the formation of two or more adjacent G-quadruplexes. Herein we report on the modular synthesis via click chemistry of dimeric metal-salphen complexes (with NiII and PtII) bridged by either polyether or peptide linkers. We show by circular dichroism (CD) spectroscopy that they generally have higher selectivity for dimeric vs monomeric G-quadruplexes. The emissive properties of the PtII-salphen dimeric complexes have been used to study their interactions with monomeric and dimeric G-quadruplexes in vitro as well as to study their cellular uptake and localization