Leishmania profilin interacts with actin through an unusual structural mechanism to control cytoskeletal dynamics in parasites.

Diseases caused by Leishmania and Trypanosoma parasites are a major health problem in tropical countries. Because of their complex life cycle involving both vertebrate and insect hosts, and >1 billion years of evolutionarily distance, the cell biology of trypanosomatid parasites exhibits pronounced differences to animal cells. For example, the actin cytoskeleton of trypanosomatids is divergent when compared with other eukaryotes.

Reconstitution of actin-based cellular processes: Why encapsulation changes the rules.

While in vitro reconstitution of cellular processes is progressing rapidly, the encapsulation of biomimetic systems to reproduce the cellular environment is a major challenge. Here we review the difficulties, using reconstitution of processes dependent on actin polymerization as an example. Some of the problems are purely technical, due to the need for engineering strategies to encapsulate concentrated solutions in micrometer-sized compartments.

pH gradients guide ADF/cofilin isoforms in pollen tubes.

In a recent study, Wang et al. (https://doi.org/10.

Correction: Non-linear elastic properties of actin patches to partially rescue yeast endocytosis efficiency in the absence of the cross-linker Sac6.

Correction for 'Non-linear elastic properties of actin patches to partially rescue yeast endocytosis efficiency in the absence of the cross-linker Sac6' by Belbahri Reda , , 2022, , 1479-1488, https://doi.org/10.1039/D1SM01437D.

Specialization of actin isoforms derived from the loss of key interactions with regulatory factors.

A paradox of eukaryotic cells is that while some species assemble a complex actin cytoskeleton from a single ortholog, other species utilize a greater diversity of actin isoforms. The physiological consequences of using different actin isoforms, and the molecular mechanisms by which highly conserved actin isoforms are segregated into distinct networks, are poorly known. Here, we sought to understand how a simple biological system, composed of a unique actin and a limited set of actin-binding proteins, reacts to a switch to heterologous actin expression.

Non-linear elastic properties of actin patches to partially rescue yeast endocytosis efficiency in the absence of the cross-linker Sac6.

Clathrin mediated endocytosis is an essential and complex cellular process involving more than 60 proteins. In yeast, successful endocytosis requires counteracting a large turgor pressure. To this end, yeasts assemble actin patches, which accumulate elastic energy during their assembly.

Linking single-cell decisions to collective behaviours in social bacteria.

Social bacteria display complex behaviours whereby thousands of cells collectively and dramatically change their form and function in response to nutrient availability and changing environmental conditions. In this review, we focus on motility, which supports spectacular transitions based on prey availability across its life cycle. A large body of work suggests that these behaviours require sensory capacity implemented at the single-cell level.

A functional family of fluorescent nucleotide analogues to investigate actin dynamics and energetics.

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.

Amoeboid Swimming Is Propelled by Molecular Paddling in Lymphocytes.

Mammalian cells developed two main migration modes. The slow mesenchymatous mode, like crawling of fibroblasts, relies on maturation of adhesion complexes and actin fiber traction, whereas the fast amoeboid mode, observed exclusively for leukocytes and cancer cells, is characterized by weak adhesion, highly dynamic cell shapes, and ubiquitous motility on two-dimensional and in three-dimensional solid matrix. In both cases, interactions with the substrate by adhesion or friction are widely accepted as a prerequisite for mammalian cell motility, which precludes swimming.

Diversity from similarity: cellular strategies for assigning particular identities to actin filaments and networks.

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.

Force Production by a Bundle of Growing Actin Filaments Is Limited by Its Mechanical Properties.

Bundles of actin filaments are central to a large variety of cellular structures such as filopodia, stress fibers, cytokinetic rings, and focal adhesions. The mechanical properties of these bundles are critical for proper force transmission and force bearing. Previous mathematical modeling efforts have focused on bundles' rigidity and shape.

Mechanical stiffness of reconstituted actin patches correlates tightly with endocytosis efficiency.

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.

Sizes of actin networks sharing a common environment are determined by the relative rates of assembly.

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).

Tropomyosin Isoforms Specify Functionally Distinct Actin Filament Populations In Vitro.

Actin filaments assemble into a variety of networks to provide force for diverse cellular processes [1]. Tropomyosins are coiled-coil dimers that form head-to-tail polymers along actin filaments and regulate interactions of other proteins, including actin-depolymerizing factor (ADF)/cofilins and myosins, with actin [2-5]. In mammals, >40 tropomyosin isoforms can be generated through alternative splicing from four tropomyosin genes.

Architecture dependence of actin filament network disassembly.

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.

Cofilin-2 controls actin filament length in muscle sarcomeres.

ADF/cofilins drive cytoskeletal dynamics by promoting the disassembly of "aged" ADP-actin filaments. Mammals express several ADF/cofilin isoforms, but their specific biochemical activities and cellular functions have not been studied in detail. Here, we demonstrate that the muscle-specific isoform cofilin-2 promotes actin filament disassembly in sarcomeres to control the precise length of thin filaments in the contractile apparatus.

Dissecting principles governing actin assembly using yeast extracts.

In this chapter, we describe recent protocols that we have developed to trigger actin assembly and actin-based motility in yeast cell extracts. Our method allows for the fast preparation of yeast extracts that are competent in dynamic assembly of distinct actin filament structures of biologically appropriate protein composition. Compared to previous extract-based systems using other eukaryotic cell types, yeast provides a unique advantage for combining reconstituted assays with the preparation of extracts from genetically modified yeast strains.

Cell-cycle regulation of formin-mediated actin cable assembly.

Assembly of appropriately oriented actin cables nucleated by formin proteins is necessary for many biological processes in diverse eukaryotes. However, compared with knowledge of how nucleation of dendritic actin filament arrays by the actin-related protein-2/3 complex is regulated, the in vivo regulatory mechanisms for actin cable formation are less clear. To gain insights into mechanisms for regulating actin cable assembly, we reconstituted the assembly process in vitro by introducing microspheres functionalized with the C terminus of the budding yeast formin Bni1 into extracts prepared from yeast cells at different cell-cycle stages.

Membrane-sculpting BAR domains generate stable lipid microdomains.

Bin-Amphiphysin-Rvs (BAR) domain proteins are central regulators of many cellular processes involving membrane dynamics. BAR domains sculpt phosphoinositide-rich membranes to generate membrane protrusions or invaginations. Here, we report that, in addition to regulating membrane geometry, BAR domains can generate extremely stable lipid microdomains by "freezing" phosphoinositide dynamics.

Lsb1 is a negative regulator of las17 dependent actin polymerization involved in endocytosis.

The spatial and temporal regulation of actin polymerization is crucial for various cellular processes. Members of the Wiskott-Aldrich syndrome protein (WASP) family activate the Arp2/3-complex leading to actin polymerization. The yeast Saccharomyces cerevisiae contains only one WASP homolog, Las17, that requires additional factors for its regulation.

Actin cytoskeleton: a team effort during actin assembly.

Two recent studies highlight how tandems of previously described actin nucleators collaborate to produce new actin filaments. One key player in these collaborations is formin, which appears to function as a modulator of filament elongation.

Structure and activity of full-length formin mDia1.

Formins are a conserved family of actin assembly-promoting factors with essential and diverse biological roles. Most of our biochemical understanding of formin effects on actin dynamics is derived from studies using formin fragments. In addition, all structural information on formins has been limited to fragments.