Abstract
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.
