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.

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.

The actin cytoskeleton has the particularity of being assembled into many functionally distinct filamentous networks from a common reservoir of monomeric actin. Each of these networks has its own geometrical, dynamical and mechanical properties, because they are capable of recruiting specific families of actin-binding proteins (ABPs), while excluding the others. This review discusses our current understanding of the underlying molecular mechanisms that cells have developed over the course of evolution to segregate ABPs to appropriate actin networks. Segregation of ABPs requires the ability to distinguish actin networks as different substrates for ABPs, which is regulated in three different ways: (1) by the geometrical organization of actin filaments within networks, which promotes or inhibits the accumulation of ABPs; (2) by the identity of the networks' filaments, which results from the decoration of actin filaments with additional proteins such as tropomyosin, from the use of different actin isoforms or from covalent modifications of actin; (3) by the existence of collaborative or competitive binding to actin filaments between two or multiple ABPs. This review highlights that all these effects need to be taken into account to understand the proper localization of ABPs in cells, and discusses what remains to be understood in this field of research.

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