An in vitro model system for cytoskeletal confinement

Abstract

The motility, shape, and functionality of the cell depend sensitively on cytoskeletal mechanics which in turn is governed by the properties of filamentous proteins - mainly actin, microtubules, and intermediate filaments. These biopolymers are confined in the dense cytoplasm and therefore experience strong geometric constraints on their equilibrium thermal fluctuations. To obtain a better understanding of the influence of confinement on cytoskeletal filaments we study the thermal fluctuations of individual actin filaments in a microfluidic in vitro system by fluorescence microscopy and determine the persistence length of the filaments by analyzing the radial distribution function. A unique feature of this method is that we obtain the persistence length without detailed knowledge of the complete contour of the filament which makes the technique applicable to a broad range of biological polymers, including those with a persistence length smaller than the optical resolution.

Hierarchical self-assembly of actin in micro-confinements using microfluidics

“…[9][10][11] Micropatterning of actin nucleation promoting factors has shown to strongly affect the self-organization of actin. 12 Also, self-assembly of actin bundles in confined geometries has been reported using narrow microfluidic channels.…”

Intermediate Filaments in Small Configuration Spaces

“…We are now in the position to test this scaling law experimentally by varying both free parameters d and L P . Since the latter is a material property, we include data of a second semiflexible polymer, filamentous actin, which has been studied extensively in confinement in the past [5,11,13,23]. Vimentin IFs and actin filaments are ideal candidates for such a comparison, since their persistence lengths differ by about 1 order of magnitude.…”

“…The confining channels are included in the experiment for three main reasons. First, biopolymers in the cell are typically strongly confined by other cell components [12,13]; thus, the channels mimic this crowded environment. Second, from a polymer physics point of view, predictions for the behavior of semiflexible polymers in confinement have existed for many years, like the scaling law for wormlike chains introduced some 30 years ago by Odijk [14].…”

Formation of Actin Networks in Microfluidic Concentration Gradients

“…The bending rigidity κ of a single actin filament can be calculated from its persistence length LP ≈ 13 μm (Köster and Pfohl, 2009) by κ = LPkBT ≈ 5.3 × 10 −26 Nm 2 with the Boltzmann's constant kB and the temperature T. To describe the bending of a bundle consisting of n filaments, two limiting types of F-actin bundle bending can be distinguished. In the decoupled case, bending shows no interfilament shearing as the interjacent crosslinks do not resist shear.…”