Active multistage coarsening of actin networks driven by myosin motors.

In cells, many vital processes involve myosin-driven motility that actively remodels the actin cytoskeleton and changes cell shape. Here we study how the collective action of myosin motors organizes actin filaments into contractile structures in a simplified model system devoid of biochemical regulation. We show that this self-organization occurs through an active multistage coarsening process. First, motors form dense foci by moving along the actin network structure followed by coalescence. Then the foci accumulate actin filaments in a shell around them. These actomyosin condensates eventually cluster due to motor-driven coalescence. We propose that the physical origin of this multistage aggregation is the highly asymmetric load response of actin filaments: they can support large tensions but buckle easily under piconewton compressive loads. Because the motor-generated forces well exceed this threshold, buckling is induced on the connected actin network that resists motor-driven filament sliding. We show how this buckling can give rise to the accumulation of actin shells around myosin foci and subsequent coalescence of foci into superaggregates. This new physical mechanism provides an explanation for the formation and contractile dynamics of disordered condensed actomyosin states observed in vivo.

Encapsulation of active cytoskeletal protein networks in cell-sized liposomes.

We demonstrate that cytoskeletal actin-myosin networks can be encapsulated with high efficiency in giant liposomes by hydration of lipids in an agarose hydrogel. The liposomes have cell-sized diameters of 10-20 µm and a uniform actin content. We show by measurements of membrane fluorescence intensity and bending rigidity that the majority of liposomes are unilamellar. We further demonstrate that the actin network can be specifically anchored to the membrane by biotin-streptavidin linkages. These protein-filled liposomes are useful model systems for quantitative studies of the physical mechanisms by which the cytoskeleton actively controls cell shape and mechanics. In a broader context, this new preparation method should be widely applicable to encapsulation of proteins and polymers, for instance, to create polymer-reinforced liposomes for drug delivery.

Growing actin networks form lamellipodium and lamellum by self-assembly.

Many different cell types are able to migrate by formation of a thin actin-based cytoskeletal extension. Recently, it became evident that this extension consists of two distinct substructures, designated lamellipodium and lamellum, which differ significantly in their kinetic and kinematic properties as well as their biochemical composition. We developed a stochastic two-dimensional computer simulation that includes chemical reaction kinetics, G-actin diffusion, and filament transport to investigate the formation of growing actin networks in migrating cells. Model parameters were chosen based on experimental data or theoretical considerations. In this work, we demonstrate the system's ability to form two distinct networks by self-organization. We found a characteristic transition in mean filament length as well as a distinct maximum in depolymerization flux, both within the first 1-2 microm. The separation into two distinct substructures was found to be extremely robust with respect to initial conditions and variation of model parameters. We quantitatively investigated the complex interplay between ADF/cofilin and tropomyosin and propose a plausible mechanism that leads to spatial separation of, respectively, ADF/cofilin- or tropomyosin-dominated compartments. Tropomyosin was found to play an important role in stabilizing the lamellar actin network. Furthermore, the influence of filament severing and annealing on the network properties is explored, and simulation data are compared to existing experimental data.

Nonequilibrium fluctuations of a remodeling in vitro cytoskeleton.

Motor proteins actively contract the actin cytoskeleton of cells and thereby give rise to nonequilibrium fluctuations as well as changes in the architecture of the cytoskeleton. Here, we show, by video microrheology of a reconstituted cytoskeleton, that motors generate time-dependent nonequilibrium fluctuations, which evolve as the network is remodeled. At earlier times, the fluctuation spectrum is dominated by strong non-Gaussian fluctuations, which arise from large displacements. At later times, directed displacements are infrequent and finally disappear. We show that these effects are due to contractile coarsening of the network into large actin-myosin foci.

Robust organizational principles of protrusive biopolymer networks in migrating living cells.

Cell migration is associated with the dynamic protrusion of a thin actin-based cytoskeletal extension at the cell front, which has been shown to consist of two different substructures, the leading lamellipodium and the subsequent lamellum. While the formation of the lamellipodium is increasingly well understood, organizational principles underlying the emergence of the lamellum are just beginning to be unraveled. We report here on a 1D mathematical model which describes the reaction-diffusion processes of a polarized actin network in steady state, and reproduces essential characteristics of the lamellipodium-lamellum system. We observe a steep gradient in filament lengths at the protruding edge, a local depolymerization maximum a few microns behind the edge, as well as a differential dominance of the network destabilizer ADF/cofilin and the stabilizer tropomyosin. We identify simple and robust organizational principles giving rise to the derived network characteristics, uncoupled from the specifics of any molecular implementation, and thus plausibly valid across cell types. An analysis of network length dependence on physico-chemical system parameters implies that to limit array treadmilling to cellular dimensions, network growth has to be truncated by mechanisms other than aging-induced depolymerization, e.g., by myosin-associated network dissociation at the transition to the cell body. Our work contributes to the analytical understanding of the cytoskeletal extension's bisection into lamellipodium and lamellum and sheds light on how cells organize their molecular machinery to achieve motility.

Optical neuronal guidance.

We present a novel technique to noninvasively control the growth and turning behavior of an extending neurite. A highly focused infrared laser, positioned at the leading edge of a neurite, has been found to induce extension/turning toward the beam's center. This technique has been used successfully to guide NG108-15 and PC12 cell lines [Ehrlicher, A., Betz, T., Stuhrmann, B., Koch, D. Milner, V. Raizen, M. G., and Kas, J. (2002). Guiding neuronal growth with light. Proc. Natl. Acad. Sci. USA 99, 16024-16028], as well as primary rat and mouse cortical neurons [Stuhrmann, B., Goegler, M., Betz, T., Ehrlicher, A., Koch, D., and Kas, J. (2005). Automated tracking and laser micromanipulation of cells. Rev. Sci. Instr. 76, 035105]. Optical guidance may eventually be used alone or with other methods for controlling neurite extension in both research and clinical applications.