Structure formation in active networks

Structure formation and constant reorganization of the actin cytoskeleton are key requirements for the function of living cells. Here we show that a minimal reconstituted system consisting of actin filaments, crosslinking molecules and molecular-motor filaments exhibits a generic mechanism of structure formation, characterized by a broad distribution of cluster sizes. We demonstrate that the growth of the structures depends on the intricate balance between crosslinker-induced stabilization and simultaneous destabilization by molecular motors, a mechanism analogous to nucleation and growth in passive systems. We also show that the intricate interplay between force generation, coarsening and connectivity is responsible for the highly dynamic process of structure formation in this heterogeneous active gel, and that these competing mechanisms result in anomalous transport, reminiscent of intracellular dynamics

Mitotic Spindle Orients Perpendicular to the Forces Imposed by Dynamic Shear

Orientation of the division axis can determine cell fate in the presence of morphogenetic gradients. Understanding how mitotic cells integrate directional cues is therefore an important question in embryogenesis. Here, we investigate the effect of dynamic shear forces on confined mitotic cells. We found that human epithelial cells (hTERT-RPE1) as well as MC3T3 osteoblasts align their mitotic spindle perpendicular to the external force. Spindle orientation appears to be a consequence of cell elongation along the zero-force direction in response to the dynamic shear. This process is a nonlinear response to the strain amplitude, requires actomyosin activity and correlates with redistribution of myosin II. Mechanosteered cells divide normally, suggesting that this mechanism is compatible with biological functions

Rheological Characterization of the Bundling Transition in F-Actin Solutions Induced by Methylcellulose

In many in vitro experiments Brownian motion hampers quantitative data analysis. Therefore, additives are widely used to increase the solvent viscosity. For this purpose, methylcellulose (MC) has been proven highly effective as already small concentrations can significantly slow down diffusive processes. Beside this advantage, it has already been reported that high MC concentrations can alter the microstructure of polymer solutions such as filamentous actin. However, it remains to be shown to what extent the mechanical properties of a composite actin/MC gel depend on the MC concentration. In particular, significant alterations might occur even if the microstructure seems unaffected. Indeed, we find that the viscoelastic response of entangled F-actin solutions depends sensitively on the amount of MC added. At concentrations higher than 0.2% (w/v) MC, actin filaments are reorganized into bundles which drastically changes the viscoelastic response. At small MC concentrations the impact of MC is more subtle: the two constituents, actin and MC, contribute in an additive way to the mechanical response of the composite material. As a consequence, the effect of methylcellulose on actin solutions has to be considered very carefully when MC is used in biochemical experiments

Analysis of the Localization of MEN Components by Live Cell Imaging Microscopy.

Mitotic exit is determined by multiple spatial and temporal cues from the spindle poles and the two compartments in a dividing yeast cell-the mother and the bud. These signals are ultimately integrated by the activation of the mitotic exit network (MEN) to promote persistent release of Cdc14 from the nucleolus. Live imaging analysis using fluorescent protein tags is invaluable to dissect this critical decision-making trigger. Here, we present protocols for routine yeast live cell microscopy applicable to this problem