Julie Plastino

ActA and human zyxin harbour Arp2/3-independent actin-polymerization activity

The actin cytoskeleton is a dynamic network that is composed of a variety of F-actin structures. To understand how these structures are produced, we tested the capacity of proteins to direct actin polymerization in a bead assay in vitro and in a mitochondrial-targeting assay in cells. We found that human zyxin and the related protein ActA of Listeria monocytogenes can generate new actin structures in a vasodilator-stimulated phosphoprotein-dependent (VASP) manner, but independently of the Arp2/3 complex. These results are consistent with the concept that there are multiple actin-polymerization machines in cells. With these simple tests it is possible to probe the specific function of proteins or identify novel molecules that act upon cellular actin polymerization.

Stress release drives symmetry breaking for actin-based movement.

By using a simple assay composed of purified proteins, we studied the spontaneous polarization of actin networks polymerizing on spherical beads, which subsequently undergo movement. We show evidence that this symmetry breaking is based on the release of elastic energy, analogous to the fracture of polymer gels. The dynamics of this process and the thickness at which it occurs depend on the growth rate and mechanical properties of the actin gel. We explain our experimental results with a model based on elasticity theory and fracture mechanics.

Phosphorylation on Ser5 increases the F-actin-binding activity of L-plastin and promotes its targeting to sites of actin assembly in cells

L-plastin, a malignant transformation-associated protein, is a member of a large family of actin filament cross-linkers. Here, we analysed how phosphorylation of L-plastin on Ser5 of the headpiece domain regulates its intracellular distribution and its interaction with F-actin in transfected cells and in in vitro assays. Phosphorylated wild-type L-plastin localised to the actin cytoskeleton in transfected Vero cells. Ser5Ala substitution reduced the capacity of L-plastin to localise with peripheral actin-rich membrane protrusions. Conversely, a Ser5Glu variant mimicking a constitutively phosphorylated state, accumulated in actin-rich regions and promoted the formation of F-actin microspikes in two cell lines. Similar to phosphorylated wild-type L-plastin, this variant remained associated with cellular F-actin in detergent-treated cells, whereas the Ser5Ala variant was almost completely extracted. When compared with non-phosphorylated protein, phosphorylated L-plastin and the Ser5Glu variant bound F-actin more efficiently in an in vitro assay. Importantly, expression of L-plastin elicited collagen invasion in HEK293T cells, in a manner dependent on Ser5 phosphorylation. Based on our findings, we propose that conversely to other calponin homology (CH)-domain family members, phosphorylation of L-plastin switches the protein from a low-activity to a high-activity state. Phosphorylated L-plastin might act as an integrator of signals controlling the assembly of the actin cytoskeleton and cell motility in a 3D-space.

Actin-filament cross-linking protein T-plastin increases Arp2/3-mediated actin-based movement.

Increasing evidence suggests that actin cross-linking or bundling proteins might not only structure the cortical actin cytoskeleton but also control actin dynamics. Here, we analyse the effects of T-plastin/T-fimbrin, a representative member of an important actin-filament cross-linking protein by combining a quantitative biomimetic motility assay with biochemical and cell-based approaches. Beads coated with the VCA domain of the Wiskott/Aldrich-syndrome protein (WASP) recruit the actin-nucleating Arp2/3 complex, polymerize actin at their surface and undergo movement when placed in cell-free extracts. T-Plastin increased the velocity of VCA beads 1.5 times, stabilized actin comets and concomitantly displaced cofilin, an actin-depolymerizing protein. T-Plastin also decreased the F-actin disassembly rate and inhibited cofilin-mediated depolymerization of actin filaments in vitro. Importantly, a bundling-incompetent variant comprising the first actin-binding domain (ABD1) had similar effects. In cells, this domain induced the formation of long actin cables to which other actin-regulating proteins were recruited. Altogether, these results favor a mechanism in which binding of ABD1 controls actin turnover independently of cross-link formation. In vivo, this activity might contribute to the assembly and maintenance of the actin cytoskeleton of plasma-membrane protrusions.

Actin polymerization or myosin contraction: two ways to build up cortical tension for symmetry breaking

Cells use complex biochemical pathways to drive shape changes for polarization and movement. One of these pathways is the self-assembly of actin filaments and myosin motors that together produce the forces and tensions that drive cell shape changes. Whereas the role of actin and myosin motors in cell polarization is clear, the exact mechanism of how the cortex, a thin shell of actin that is underneath the plasma membrane, can drive cell shape changes is still an open question. Here, we address this issue using biomimetic systems: the actin cortex is reconstituted on liposome membranes, in an ‘outside geometry’. The actin shell is either grown from an activator of actin polymerization immobilized at the membrane by a biotin–streptavidin link, or built by simple adsorption of biotinylated actin filaments to the membrane, in the presence or absence of myosin motors. We show that tension in the actin network can be induced either by active actin polymerization on the membrane via the Arp2/3 complex or by myosin II filament pulling activity. Symmetry breaking and spontaneous polarization occur above a critical tension that opens up a crack in the actin shell. We show that this critical tension is reached by growing branched networks, nucleated by the Arp2/3 complex, in a concentration window of capping protein that limits actin filament growth and by a sufficient number of motors that pull on actin filaments. Our study provides the groundwork to understanding the physical mechanisms at work during polarization prior to cell shape modifications.

Cell biology: Actin filaments up against a wall

The front of motile cells is thought to be pushed out by branched filaments of actin protein abutting the cell membrane. New work challenges this textbook view, showing that actin branches grow away from, or obliquely to, a surface.

WAVE binds Ena/VASP for enhanced Arp2/3 complex-based actin assembly.

A dual in vitro/in vivo approach is used to show that WAVE directly binds Ena/VASP, coordinating its activity with that of the Arp2/3 complex for enhanced actin assembly.

Reconstituting the actin cytoskeleton at or near surfaces in vitro.

Actin filament dynamics have been studied for decades in pure protein solutions or in cell extracts, but a breakthrough in the field occurred at the turn of the century when it became possible to reconstitute networks of actin filaments, growing in a controlled but physiological manner on surfaces, mimicking the actin assembly that occurs at the plasma membrane during cell protrusion and cell shape changes. The story begins with the bacteria Listeria monocytogenes, the study of which led to the reconstitution of cellular actin polymerization on a variety of supports including plastic beads. These studies made possible the development of liposome-type substrates for filament assembly and micropatterning of actin polymerization nucleation. Based on the accumulated expertise of the last 15 years, many exciting approaches are being developed, including the addition of myosin to biomimetic actin networks to study the interplay between actin structure and contractility. The field is now poised to make artificial cells with a physiological and dynamic actin cytoskeleton, and subsequently to put these cells together to make in vitro tissues. This article is part of a Special Issue entitled: Mechanobiology.

Invading, Leading and Navigating Cells in Caenorhabditis elegans: Insights into Cell Movement in Vivo

Highly regulated cell migration events are crucial during animal tissue formation and the trafficking of cells to sites of infection and injury. Misregulation of cell movement underlies numerous human diseases, including cancer. Although originally studied primarily in two-dimensional in vitro assays, most cell migrations in vivo occur in complex three-dimensional tissue environments that are difficult to recapitulate in cell culture or ex vivo. Further, it is now known that cells can mobilize a diverse repertoire of migration modes and subcellular structures to move through and around tissues. This review provides an overview of three distinct cellular movement events in Caenorhabditis elegans—cell invasion through basement membrane, leader cell migration during organ formation, and individual cell migration around tissues—which together illustrate powerful experimental models of diverse modes of movement in vivo. We discuss new insights into migration that are emerging from these in vivo studies and important future directions toward understanding the remarkable and assorted ways that cells move in animals.

Actin takes its hat off to dynamin

A major player in membrane traffic, the large GTPase dynamin, is known in the actin dynamics field for its indirect association with the actin cytoskeleton via actin‐binding proteins (ABPs) like cortactin. In this issue of The EMBO Journal , Gu et al extend this picture by revealing a direct interaction between dynamin and F‐actin. They further show that oligomerized dynamin kicks off the gelsolin cap at the barbed ends of filaments. This study not only provides an interesting link between actin remodelling and membrane dynamics via dynamin, but also sheds light on the long‐standing mystery of how barbed ends are liberated from the high‐affinity capping protein gelsolin.