Actin, one of the main proteins of muscle and cytoskeleton, exists as a vaiety of highly conserved isoforms whose distribution in vertebrates is tissue-specific. Synthesis of specific actin isoforms is accompanied by their subcellular compartmentalization, with both processes being regulated by factors of cell proliferation and differentiation. Actin isoforms cannot substitute for each other, and the high-level synthesis of exogenous actins leads to alterations in cell organization and morphology. This indicates that the highly conserved actins are functionally specialized for the tissues in which they predominate. The first goal of this review is to analyze the data on the polymerizability of actin isoforms to show that cytoskeleton isoactins form less stable polymers than skeletal muscle actin. This difference correlates with the dynamics of actin microfilaments versus the stability of myofibrillar systems. The three-dimensional actin structure as well as progress in the analysis of conformational changes in both the actin monomer and the filament allows us to view the data on the structure and polymerization of isoactins in terms of structure function relationships within the actin molecule. Most of the amino acid substitutions that distinguish actin isoforms are located apart from actin contact sites in the polymer. We suggest that these substitutions can modulate the ability of actin monomers to form more or less stable polymers by long-range (allosteric) regulation of the contact sites.

Functional specificity of actin isoforms

Natural small-molecule inhibitors of actin cytoskeleton dynamics have long been recognized as valuable molecular probes for dissecting complex mechanisms of cellular function. More recently, their potential use as chemotherapeutic drugs has become a focus of scientific investigation. The primary focus of this review is the molecular mechanism by which different actin-targeting natural products function, with an emphasis on structural considerations of toxins for which high-resolution structural information of their interaction with actin is available. By comparing the molecular interactions made by different toxin families with actin, the structural themes of those that alter filament dynamics in similar ways can be understood. This provides a framework for novel synthetic-compound designs with tailored functional properties that could be applied in both research and clinical settings.

Actin-targeting natural products: structures, properties and mechanisms of action.

Actin has maintained an exquisite degree of sequence conservation over large evolutionary distances for reasons that are not understood. The desire to explain phenomena from muscle contraction to cytokinesis in mechanistic detail has driven the generation of an atomic model of the actin filament (F-actin). Here we use electron cryomicroscopy to show that frozen-hydrated actin filaments contain a multiplicity of different structural states. We show (at ∼10 A resolution) that subdomain 2 can be disordered and can make multiple contacts with the C terminus of a subunit above it. We link a number of disease-causing mutations in the human ACTA1 gene to the most structurally dynamic elements of actin. Because F-actin is structurally polymorphic, it cannot be described using only one atomic model and must be understood as an ensemble of different states.

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Structural Polymorphism in F-actin

To test the hypothesis that the myosin II motor domain (S1) preferentially binds to specific subsets of actin filaments in vivo, we expressed GFP-fused S1 with mutations that enhanced its affinity for actin in Dictyostelium cells. Consistent with the hypothesis, the GFP-S1 mutants were localized along specific portions of the cell cortex. Comparison with rhodamine-phalloidin staining in fixed cells demonstrated that the GFP-S1 probes preferentially bound to actin filaments in the rear cortex and cleavage furrows, where actin filaments are stretched by interaction with endogenous myosin II filaments. The GFP-S1 probes were similarly enriched in the cortex stretched passively by traction forces in the absence of myosin II or by external forces using a microcapillary. The preferential binding of GFP-S1 mutants to stretched actin filaments did not depend on cortexillin I or PTEN, two proteins previously implicated in the recruitment of myosin II filaments to stretched cortex. These results suggested that it is the stretching of the actin filaments itself that increases their affinity for the myosin II motor domain. In contrast, the GFP-fused myosin I motor domain did not localize to stretched actin filaments, which suggests different preferences of the motor domains for different structures of actin filaments play a role in distinct intracellular localizations of myosin I and II. We propose a scheme in which the stretching of actin filaments, the preferential binding of myosin II filaments to stretched actin filaments, and myosin II-dependent contraction form a positive feedback loop that contributes to the stabilization of cell polarity and to the responsiveness of the cells to external mechanical stimuli.

/pdf/stretching-actin-filaments-within-cells-enhances-their-52zpkdltcp.pdf

Stretching actin filaments within cells enhances their affinity for the myosin II motor domain.

High-speed atomic force microscopy was employed to observe structural changes in actin filaments induced by cofilin binding. Consistent with previous electron and fluorescence microscopic studies, cofilin formed clusters along actin filaments, where the filaments were 2-nm thicker and the helical pitch was ∼25% shorter, compared to control filaments. Interestingly, the shortened helical pitch was propagated to the neighboring bare zone on the pointed-end side of the cluster, while the pitch on the barbed-end side was similar to the control. Thus, cofilin clusters induce distinctively asymmetric conformational changes in filaments. Consistent with the idea that cofilin favors actin structures with a shorter helical pitch, cofilin clusters grew unidirectionally toward the pointed-end of the filament. Severing was often observed near the boundaries between bare zones and clusters, but not necessarily at the boundaries. DOI: 10.7554/eLife.04806.001

/pdf/cofilin-induced-unidirectional-cooperative-conformational-3gczv5v9ff.pdf

Cofilin-induced unidirectional cooperative conformational changes in actin filaments revealed by high-speed atomic force microscopy

The interaction between F-actin and soluble proteolytic fragments of myosin, heavy meromyosin and myosin subfragment 1 without ATP, has been studied by measuring the static anisotropy and the transient anisotropy decay of the fluorescent chromophore N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl) ethylenediamine bound to F-actin. In the presence of Ca2+ ions, the mobility of the chromophore was strongly decreased by adding heavy meromyosin or myosin subfragment 1, and this conformation change of F-actin showed a strong cooperativity; that is, a very small amount of myosin heads induced the maximum anisotropy change. On the other hand, in the presence of Mg2+ ions, the addition of a small amount of myosin subfragment 1 or of heavy meromyosin increased the mobility of labeled F-actin that reached a maximum at a molar ratio of about 1/25 or 1/50, respectively. With further addition of myosin heads, the mobility of the labeled actin decreased. From these studies, one concludes that F-actin undergoes a conformation change by interacting with myosin heads, which depends on the nature of the divalent cations present in the solution.

Jean Claude Auchet

Papers

Fluorescence anisotropy of labeled F-actin: influence of divalent cations on the interaction between F-actin and myosin heads.