The cell plasma membrane is more than just a rigid barrier that protects the interior of the cell. It is involved in numerous important cell functions such as cell motility, cell adhesion, transduction, signaling, etc. To further investigate these membranes, fluorescent membrane probes have emerged as a powerful tool to analyze the different functions of the plasma membrane, as they are non-invasive, relatively easy to use and allow for live cell imaging. Our group has introduced so called flippers, that are fluorescent probes to image physical forces in model and cellular membranes. The flippers operate by twisting around a two dithienothiophene, whereas in liquid disordered membranes the flipper is more twisted than in ordered or lipid rafts. This results in a red shift of excitation maximum and an increase in fluorescence lifetime.
Although the Flipper-TR can distinguish between the different domains in membranes by fluorescence lifetime, it lacks a selective partitioning into a phase of a lipid bilayer. The main topic of this thesis resolved around the selective partitioning of flipper probes by introducing hydrophobic interfacers of different lengths, unsaturation, bulkiness and cholesterol The flippers had improved hydrophobic matching with the surrounding lipid environment, in which the bulkier flippers clearly preferred the disordered phase and the saturated long acyl chain flippers selectively stained the ordered phase. The original Flipper-TR, short acyl chain flippers and cholesteryl flipper did not have a preference over the phases. Although these flippers had a selective partitioning into phases of giant unilamellar vesicles (GUVs), in cellular environments they would either precipitate or internalize into the cell and stain organelles inside.
To overcome the internalization of the flippers into the cells, the polar headgroup was changed from one negative charge to 4 formal negative charges, by a dendritic-like expansion. The flipper was equipped with a long acyl chain (C16), and the headgroup was expanded to four glutamic acids compared to one. The probe had similar characteristics in model membranes, clearly preferring the liquid ordered phase, but the delivery to the membrane through micelle formation resulted in an increase in fluorescence intensity as well as fluorescence lifetime. Although the dendritic flipper had notable internalization, the fluorescence from internal organelles was decreased significantly.
