Actin filaments and bundles in flow

Actin, a protein and major component of the cytoskeleton, is organized in vivo into filaments, bundles, and networks which play an important role in mechanical stability and cellular motility. Aside from their biological relevance, actin filaments can be used as model systems for semiflexible polymers to answer fundamental physical questions in polymer science. In the presented thesis, results are discussed which consider the behavior of single semiflexible polymers in the field of microfluidics. Moreover, microfluidic tools are also used to study the bundling of actin filaments in vitro. In the first part of this thesis, the behavior of semiflexible actin filaments in flow inside microchannels is investigated. The filaments are analyzed at different channel positions along a cross-section for different flow velocities. To this end, fluorescently labeled actin filaments are observed by using fluorescence microscopy for which a laser illumination enables short exposure times. In order to gain results for which the semiflexible nature of actin filaments is significant, the channel dimensions (width and depth) are adapted to have approximately the same size as the characteristic lengths of the filaments (persistence length and contour length). The results indicate that the microflow causes either elongation or bending of filaments. Predominately, the filaments are elongated. The elongated filaments are nearly aligned in the flow direction. The elongation and alignment increase with larger flow velocities as it is seen in the end-to-end distance probability distributions and the angle probability distributions. The characteristic parameters of the filament orientation, the preferred angle and the width of the angle probability distribution, obey scaling laws which are known for strongly elongated or stiff polymers in simple shear flow. In addition to elongated filaments, tumbling of filaments is observed. The radii of curvature of bent filaments during tumbling decrease with increasing velocities. By balancing drag forces and bending forces, a scaling is derived with which the experimental values can be described. Additionally, bent filaments are also found at the channel center. In this region, the bent shapes are stable and can be quantified by parabolas. These bent conformations can be related to the non-monotonic parabolic velocity field, and the parabolic profiles can be reconstructed by modeling filament as elastic rods. Additionally to conformational and orientational studies of actin filaments in flow, an important point of interest is the channel positions along the cross-section at which filaments are most frequently found. The center-of-mass probability distributions show that the filaments are not equally distributed over the channel width. For large velocities, filaments are less frequently found near the channel center. Furthermore, depletion layers near walls are observed. Consequently, filaments migrate away from the channel center as well as away from the channel walls. The cross-streamline migration away from the channel center can be explained by a decrease of the filament diffusivity toward the walls due to an increase of the shear rate. Near walls, steric and hydrodynamic interactions with the walls lead to depletion layers. To quantify the spatially-varying diffusivity, the segment distributions of filaments at different channel positions as well as for different velocities are analyzed. Assuming proportionality between the diffusivity and the mean square deviation of segments from the center-ofmass streamline of filaments, the diffusivity at each channel position for a certain flow velocity can be determined. Using this diffusivity, a governing equation for the centerof-mass probability distribution is set up in which the spatially-varying diffusivity and hydrodynamic interactions with the walls can generate drift on the filaments. The calculated and measured distributions show the same essential characteristics like depletion layers at walls and the channel center. For large velocities, a nearly quantitative agreement is obtained. The second part of this thesis considers the actin bundling in the presence of linker molecules. Using microfluidic tools, a method is developed in order to observe the bundling of actin filaments in situ at a molecular level. The bundles are imaged by fluorescence microscopy and the intensity of the emitted light from a bundle is a measure for the number of filaments inside the bundle. Usage of a hydrodynamic focusing device enables a time-resolved visualization of the bundling from single filaments to thick bundles. As a result, it is shown that bundling is a diffusion-limited process. Furthermore, the analysis of thermal fluctuations of bundles characterizes their mechanical properties and a relation between the persistence length and the number of filaments is obtained. This relation suggests a weak coupling between filaments inside bundles, probably induced by the electrostatic nature of actin. The results presented in this thesis show that the combination of microfluidics and fluorescence microscopy is a powerful tool to investigate the kinetics of the actin bundling at a molecular scale. More generally, the time-resolved visualization of the step-by-step process has a large potential for studies of any bundling or network formation, and also for other time-dependent processes such as enzymatic reactions or polymerizations