Internal Motility in Stiffening Actin-Myosin Networks

We present a study on filamentous actin solutions containing heavy meromyosin subfragments of myosin II motor molecules. We focus on the viscoelastic phase behavior and internal dynamics of such networks during ATP depletion. Upon simultaneously using micro-rheology and fluorescence microscopy as complementary experimental tools, we find a sol-gel transition accompanied by a sudden onset of directed filament motion. We interpret the sol-gel transition in terms of myosin II enzymology, and suggest a "zipping" mechanism to explain the filament motion in the vicinity of the sol-gel transition.Comment: 4 pages, 3 figure

Elasticity of Semiflexible Biopolymer Networks

We develop a model for gels and entangled solutions of semiflexible biopolymers such as F-actin. Such networks play a crucial structural role in the cytoskeleton of cells. We show that the rheologic properties of these networks can result from nonclassical rubber elasticity. This model can explain a number of elastic properties of such networks {\em in vitro}, including the concentration dependence of the storage modulus and yield strain.Comment: Uses RevTeX, full postscript with figures available at http://www.umich.edu/~fcm/preprints/agel/agel.htm

Hypertrophic cardiomyopathy mutations in the calponin-homology domain of ACTN2 affect actin binding and cardiomyocyte Z-disc incorporation

α-Actinin-2 (ACTN2) is the only muscle isoform of α-actinin expressed in cardiac muscle. Mutations in this protein have been implicated in mild to moderate forms of hypertrophic cardiomyopathy (HCM). We have investigated the effects of two mutations identified from HCM patients, A119T and G111V, on the secondary and tertiary structure of a purified actin binding domain (ABD) of ACTN2 by circular dichroism and X-ray crystallography, and show small but distinct changes for both mutations. We also find that both mutants have reduced F-actin binding affinity, although the differences are not significant. The full length mEos2 tagged protein expressed in adult cardiomyocytes shows that both mutations additionally affect Z-disc localization and dynamic behaviour. Overall, these two mutations have small effects on structure, function and behaviour, which may contribute to a mild phenotype for this disease

α-Actinin and Filamin Cooperatively Enhance the Stiffness of Actin Filament Networks

BACKGROUND: The close subcellular proximity of different actin filament crosslinking proteins suggests that these proteins may cooperate to organize F-actin structures to drive complex cellular functions during cell adhesion, motility and division. Here we hypothesize that alpha-actinin and filamin, two major F-actin crosslinking proteins that are both present in the lamella of adherent cells, display synergistic mechanical functions. METHODOLOGY/PRINCIPAL FINDINGS: Using quantitative rheology, we find that combining alpha-actinin and filamin is much more effective at producing elastic, solid-like actin filament networks than alpha-actinin and filamin separately. Moreover, F-actin networks assembled in the presence of alpha-actinin and filamin strain-harden more readily than networks in the presence of either alpha-actinin or filamin. SIGNIFICANCE: These results suggest that cells combine auxiliary proteins with similar ability to crosslink filaments to generate stiff cytoskeletal structures, which are required for the production of internal propulsive forces for cell migration, and that these proteins do not have redundant mechanical functions

Plastin increases cortical connectivity to facilitate robust polarization and timely cytokinesis.

The cell cortex is essential to maintain animal cell shape, and contractile forces generated within it by nonmuscle myosin II (NMY-2) drive cellular morphogenetic processes such as cytokinesis. The role of actin cross-linking proteins in cortical dynamics is still incompletely understood. Here, we show that the evolutionarily conserved actin bundling/cross-linking protein plastin is instrumental for the generation of potent cortical actomyosin contractility in the Caenorhabditis elegans zygote. PLST-1 was enriched in contractile structures and was required for effective coalescence of NMY-2 filaments into large contractile foci and for long-range coordinated contractility in the cortex. In the absence of PLST-1, polarization was compromised, cytokinesis was delayed or failed, and 50% of embryos died during development. Moreover, mathematical modeling showed that an optimal amount of bundling agents enhanced the ability of a network to contract. We propose that by increasing the connectivity of the F-actin meshwork, plastin enables the cortex to generate stronger and more coordinated forces to accomplish cellular morphogenesis

Adhesions Assemble!—Autoinhibition as a Major Regulatory Mechanism of Integrin-Mediated Adhesion

The advent of cell-cell and cell-extracellular adhesion enabled cells to interact in a coherent manner, forming larger structures and giving rise to the development of tissues, organs and complex multicellular life forms. The development of such organisms required tight regulation of dynamic adhesive structures by signaling pathways that coordinate cell attachment. Integrin-mediated adhesion to the extracellular matrix provides cells with support, survival signals and context-dependent cues that enable cells to run different cellular programs. One mysterious aspect of the process is how hundreds of proteins assemble seemingly spontaneously onto the activated integrin. An emerging concept is that adhesion assembly is regulated by autoinhibition of key proteins, a highly dynamic event that is modulated by a variety of signaling events. By enabling precise control of the activation state of proteins, autoinhibition enables localization of inactive proteins and the formation of pre-complexes. In response to the correct signals, these proteins become active and interact with other proteins, ultimately leading to development of cell-matrix junctions. Autoinhibition of key components of such adhesion complexes—including core components integrin, talin, vinculin, and FAK and important peripheral regulators such as RIAM, Src, and DLC1—leads to a view that the majority of proteins involved in complex assembly might be regulated by intramolecular interactions. Autoinhibition is relieved via multiple different signals including post-translation modification and proteolysis. More recently, mechanical forces have been shown to stabilize and increase the lifetimes of active conformations, identifying autoinhibition as a means of encoding mechanosensitivity. The complexity and scope for nuanced adhesion dynamics facilitated via autoinhibition provides numerous points of regulation. In this review, we discuss what is known about this mode of regulation and how it leads to rapid and tightly controlled assembly and disassembly of cell-matrix adhesion

Actin binding domains direct actin-binding proteins to different cytoskeletal locations

<p>Abstract</p> <p>Background</p> <p>Filamin (FLN) and non-muscle α-actinin are members of a family of F-actin cross-linking proteins that utilize Calponin Homology domains (CH-domain) for actin binding. Although these two proteins have been extensively characterized, little is known about what regulates their binding to F-actin filaments in the cell.</p> <p>Results</p> <p>We have constructed fusion proteins consisting of green fluorescent protein (GFP) with either the entire cross-linking protein or its actin-binding domain (ABD) and examined the localization of these fluorescent proteins in living cells under a variety of conditions. The full-length fusion proteins, but not the ABD's complemented the defects of cells lacking both endogenous proteins indicating that they are functional. The localization patterns of filamin (GFP-FLN) and α-actinin (GFP-αA) were overlapping but distinct. GFP-FLN localized to the peripheral cell cortex as well as to new pseudopods of unpolarized cells, but was observed to localize to the rear of polarized cells during cAMP and folate chemotaxis. GFP-αA was enriched in new pseudopods and at the front of polarized cells, but in all cases was absent from the peripheral cortex. Although both proteins appear to be involved in macropinocytosis, the association time of the GFP-probes with the internalized macropinosome differed. Surprisingly, the localization of the GFP-actin-binding domain fusion proteins precisely reflected that of their respective full length constructs, indicating that the localization of the protein was determined by the actin-binding domain alone. When expressed in a cell line lacking both filamin and α-actinin, the probes maintain their distinct localization patterns suggesting that they are not functionally redundant.</p> <p>Conclusion</p> <p>These observations strongly suggest that the regulation of the binding of these proteins to actin filaments is built into the actin-binding domains. We suggest that different actin binding domains have different affinities for F-actin filaments in functionally distinct regions of the cytoskeleton.</p