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Actin polymerization provides force for vital processes of the eukaryotic cell, but our understanding of actin dynamics and energetics remains limited due to the lack of high-quality probes. Most current probes affect dynamics of actin or its interactions with actin-binding proteins (ABPs), and cannot track the bound nucleotide. Here, we identify a family of highly sensitive fluorescent nucleotide analogues structurally compatible with actin. We demonstrate that these fluorescent nucleotides bind to actin, maintain functional interactions with a number of essential ABPs, are hydrolyzed within actin filaments, and provide energy to power actin-based processes. These probes also enable monitoring actin assembly and nucleotide exchange with single-molecule microscopy and fluorescence anisotropy kinetics, therefore providing robust and highly versatile tools to study actin dynamics and functions of ABPs., Actin polymerization provides force for vital processes of the eukaryotic cell, but our understanding of actin dynamics and energetics remains limited due to the lack of high-quality probes. Here authors identify a family of highly sensitive fluorescent nucleotide analogues which bind to actin and provide energy to power actin-based processes.

International audience; Turnover of actin networks in cells requires the fast disassembly of aging actin structures. While ADF/cofilin and Aip1 have been identified as central players, how their activities are modulated by the architecture of the networks remains unknown. Using our ability to reconstitute a diverse array of cellular actin organizations, we found that ADF/cofilin binding and ADF/cofilin-mediated disassembly both depend on actin geometrical organization. ADF/cofilin decorates strongly and stabilizes actin cables, whereas its weaker interaction to Arp2/3 complex networks is correlated with their dismantling and their reorganization into stable architectures. Cooperation of ADF/cofilin with Aip1 is necessary to trigger the full disassembly of all actin filament networks. Additional experiments performed at the single-molecule level indicate that this cooperation is optimal above a threshold of 23 molecules of ADF/cofilin bound as clusters along an actin filament. Our results indicate that although ADF/cofilin is able to dismantle selectively branched networks through severing and debranching, stochastic disassembly of actin filaments by ADF/cofilin and Aip1 represents an efficient alternative pathway for the full disassembly of all actin networks. Our data support a model in which the binding of ADF/cofilin is required to trigger a structural change of the actin filaments, as a prerequisite for their disassembly by Aip1.

The actin cytoskeleton is an assembly of organized polymer structures. In cells, actin contributes to their internal organization, their rigidity, and their ability to exert forces. The properties of the actin networks are regulated by multiple families of actin binding proteins (ABPs). In this work, we focus on Arp2/3-branched networks, which are implicated in a variety of cellular functions such as motility or endocytosis. We propose to examine the relationship between the mechanical properties of actin networks and the biochemical composition of these gels.While atomic force microscopy and micropipette techniques have been successfully used to probe the mechanics of actin gels in vitro, their main drawback for our purpose is the limited amount of measurements per experiment. To overcome this limitation, we use instead a quantitative high-throughput system of magnetic colloids (Pujol et al., 2012). Actin networks are assembled around the colloidal particles from sets of purified proteins (bottom-up approach) or from yeast protein extracts. The advantage of these extracts is that proteins can be genetically removed one-by-one, in order to test for their functions in a near-physiological environment (top-down approach) (Michelot and Drubin, 2014).In this first study, we focus on the impact of crosslinkers, which create attachment points between neighboring filaments. The absence of two crosslinkers Sac6 (fimbrin) and Scp1 (calponin) softens and modifies the long-term evolution of actin gels assembled from extracts. Indeed, networks’ structural integrity is not recovered after load. In agreement with previous data, the addition of purified fimbrin in the bottom-up approach increases the elastic modulus of actin gels in a sigmoidal dose-dependent manner. Moreover, the system seems to evolve from little plasticity and non-linear behavior under load to a more plastic and less non-linear response.

Within the cytoplasm of a single cell, several actin networks can coexist with distinct sizes, geometries, and protein compositions. These actin networks assemble in competition for a limited pool of proteins present in a common cellular environment. To predict how two distinct networks of actin filaments control this balance, the simultaneous assembly of actin-related protein 2/3 (Arp2/3)-branched networks and formin-linear networks of actin filaments around polystyrene microbeads was investigated with a range of actin accessory proteins (profilin, capping protein, actin-depolymerizing factor [ADF]/cofilin, and tropomyosin). Accessory proteins generally affected actin assembly rates for the distinct networks differently. These effects at the scale of individual actin networks were surprisingly not always correlated with corresponding loss-of-function phenotypes in cells. However, our observations agreed with a global interpretation, which compared relative actin assembly rates of individual actin networks. This work supports a general model in which the size of distinct actin networks is determined by their relative capacity to assemble in a common and competing environment., A biomimetic assay using polystyrene beads compares the rates of actin assembly on linear and branched networks, revealing how the size of rival actin networks in cells is regulated by their relative capacity to assemble in a common environment.

Clathrin-mediated endocytosis involves the sequential assembly of more than 60 proteins at the plasma membrane. An important fraction of these proteins regulates the assembly of an actin-related protein 2/3 (Arp2/3)-branched actin network, which is essential to generate the force during membrane invagination. We performed, on wild-type (WT) yeast and mutant strains lacking putative actin crosslinkers, a side-by-side comparison of in vivo endocytic phenotypes and in vitro rigidity measurements of reconstituted actin patches. We found a clear correlation between softer actin networks and a decreased efficiency of endocytosis. Our observations support a chain-of-consequences model in which loss of actin crosslinking softens Arp2/3-branched actin networks, directly limiting the transmission of the force. Additionally, the lifetime of failed endocytic patches increases, leading to a larger number of patches and a reduced pool of polymerizable actin, which slows down actin assembly and further impairs endocytosis., This study uses in vitro reconstitution of endocytic actin patches and mechanical measurements with chains of superparamagnetic microbeads to reveal a tight correlation between the stiffness of actin networks and the efficiency of endocytosis in yeast.