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Abstract: A major structural component of the cell is the actin cytoskeleton, in which actin subunits are polymerised into actin filaments. These networks can be cross-linked by various types of ABPs (Actin Binding Proteins), such as Filamin A. In this paper, the passive response of cross-linked actin filament networks is evaluated, by use of a numerical and continuum network model. For the numerical model, the influence of filament length, statistical dispersion, cross-link compliance (including that representative of Filamin A) and boundary conditions on the mechanical response is evaluated and compared to experimental results. It is found that the introduction of statistical dispersion of filament lengths has a significant influence on the computed results, reducing the network stiffness by several orders of magnitude. Actin networks have previously been shown to have a characteristic transition from an initial bending-dominated to a stretching-dominated regime at larger strains, and the cross-link compliance is shown to shift this transition. The continuum network model, a modified eight-chain polymer model, is evaluated and shown to predict experimental results reasonably well, although a single set of parameters cannot be found to predict the characteristic dependence of filament length for different types of cross-links. Given the vast diversity of cross-linking proteins, the dependence of mechanical response on cross-link compliance signifies the importance of incorporating it properly in models to understand the roles of different types of actin networks and their respective tasks in the cell. [Copyright &y& Elsevier]

Abstract: Tropomyosin is a two-chained α-helical coiled coil that binds along the length of the actin filament and regulates its function. The paper addresses the question of how a “simple” coiled-coil sequence encodes the information for binding and regulating the actin filament, its universal target. Determination of the tropomyosin sequence confirmed Crick’s predicted heptapeptide repeat of hydrophobic interface residues and revealed additional features that have been shown to be important for its function: a 7-fold periodicity predicted to correspond to actin binding sites and interruptions of the canonical interface with destabilizing residues, such as Ala. Evidence from published work is summarized, leading to the proposal of a paradigm that binding of tropomyosin to the actin filament requires local instability as well as regions of flexibility. The flexibility derives from bends and local unfolding at regions with a destabilized coiled-coil interface, as well as from the dynamic end-to-end complex. The features are required for tropomyosin to assume the form of the helical actin filament, and to bind to actin monomers along its length. The requirement of instability/flexibility for binding may be generalized to the binding of other coiled coils to their targets. [Copyright &y& Elsevier]

Abstract: Pleckstrin-2 (PLEK2) has been implicated to be regulated by phosphatidylinositol (PI) 3-kinase, while pleckstrin1 (PLEK1) has been suggested to be a major PKC substrate in platelets. In this paper, we confirmed that PLEK2 specifically bound to the PI 3-kinase products in vitro and explored its behavior. PLEK2 was found to be expressed in various adherent cell lines, while PLEK1 expression was restricted to non-adherent cells in the protein level. Expression of PLEK2 in COS1 cells induced formation of protrusive F-actin structure and enhanced the actin rearrangements induced on collagen- or fibronectin-coated plates. A PLEK2 mutant incapable of binding to the PI 3-kinase products did not show any effect on actin rearrangement. Knockdown of PLEK2 by shRNA inhibited spreading of HCC2998 adenocarcinoma cells. PLEK2 colocalized with Rac and was suggested to be oligomerized. These results suggest that PLEK2 is involved in actin rearrangement in a PI 3-kinase dependent manner. [Copyright &y& Elsevier]