Organization of the Cytoskeleton: Studies in Microfluidic Drops

The cytoskeleton is a cellular protein system, which acts as a scaffold for cells. The major fiber-forming constituents of the cytoskeleton are microtubules, actin filaments and intermediate filaments, which build networks and bundles. One fundamental property of these proteins is their charge which gives rise to the electrostatic interaction with oppositely charged counterions. Here, the behavior of vimentin intermediate filaments in the presence of small counterions is investigated in vitro. This interaction process is studied from the experimental and theoretical point of view. For the experimental part, vimentin filaments are brought into contact with counterions in microfluidic drops. To study the interaction of fluorescently tagged vimentin and magnesium ions, a microfluidic device is developed to image drops containing fluorescently tagged vimentin filaments and counterions at tunable concentrations in an equilibrium state. The drops are produced, stored over time and the content of the drops is imaged using fluorescence microscopy. This way, the bulk threshold concentration of the magnesium ions is determined, upon which the counterions mediate the aggregation of vimentin filaments. In addition to this device, another microfluidic device is presented which allows for temporal resolution of the aggregation process, starting rapidly after the filaments have contact to the multivalent counterions for the first time. Using this technique, a typical time scale of ten minutes for the aggregation process from unconnected filaments to networks is identified. During the experiments, the multivalent counterions compete with monovalent potassium ions for binding to the vimentin filaments. Considering the interaction process from the theoretical point of view, a model for the competitive binding of the counterions to the vimentin filaments is applied. The counterion composition on the surfaces is determined at the onset of aggregation mediated by di- and trivalent counterions. When the vimentin filaments aggregate in the experiments, about half of the counterions on the surface are multivalent. Therefore, we hypothesize that these multivalent counterions correlate with each other, leading to a filament – filament attraction on the basis of non-specific electrostatic interaction. This principle might also be transferable to other intermediate filaments, as they share the same structural tripartite buildup