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This short review is a survey of the biochemical mechanisms of control of actin filament barbed end assembly in motile processes. Regulated filament treadmilling is at the origin of barbed end growth. Barbed end nucleating, signal-responsive machineries specify the sites of filament assembly at the membrane to elicit polarized migration and determine the number of force-producing filaments. The rate of barbed end growth is controlled both by barbed end-bound factors (leaky cappers, processive motors of actin assembly) and by proteins that associate with monomeric actin and modify the rate of actin association to barbed ends. The flux of assembly at barbed ends of the different complexes of monomeric actin itself is controlled by barbed end capping proteins and by proteins that affect the rate of pointed end depolymerization, which is rate-limiting in the treadmilling cycle. While many actin-binding proteins fulfill one defined regulatory function, some of them can combine two different functions, or switch from one function to the other in a regulated fashion. Understanding the full complexity of motile behavior of living cells requires the biochemical analysis of individual actin regulatory proteins and the development of biomimetic motile systems.

Actin depolymerizing factor (ADF) and cofilin are the founding members of a group of structurally and functionally related actin binding proteins now collectively known as the ADF/cofilin (AC) family. AC proteins are expressed in all eukaryotic organisms, and their unique ability to bind and dynamize filamentous actin renders them essential to all cellular processes dependent upon actin dynamics. Cell division, cell motility and neuronal pathfinding, membrane dynamics, and cell polarization could not proceed without the aid of these remarkable proteins.

This chapter reviews some aspects of the biochemistry and cellular function of profilin, focussing on its role as a control component of actin polymerization. Signalling-dependent changes in cell behaviour are direct consequences of a force-generating remodelling of the actin microfilament system at the inner surface of the plasma membrane. Characteristic for this sub-membraneous region is the enrichment of actin filaments in highly ordered bundles and sheets of filaments. These filaments, which have their fast polymerizing (+)-ends facing the lipid bilayer, are under constant turnover, with ATP-containing actin monomers being added at their (+)-ends, and ADP-actin monomers dissociating from the (−)-ends in a treadmilling process regulated by a number of actin-binding proteins. Here, profilin comes into play as one of the key regulators of actin filament formation. The protrusive surface activity, typically seen after receptor stimulation, is caused by local incorporation of actin from profilin-actin into the ends of growing filaments of filopodia and lamellipodia. Thus, the function of profilin is primarily integrated with the force-generating microfilament apparatus at the cell periphery. In addition to actin, profilin also binds polyphosphoinositides and proline-rich ligands.

Srv2/cyclase-associated protein (CAP) is a ubiquitously expressed actin monomer binding protein required for proper organization and rapid remodeling of cellular actin networks. CAP catalyzes the dissociation of cofilin-bound ADP-actin complexes, elevating cofilin levels available for filament disassembly. In addition, CAP and profilin promote exchange of nucleotide (ATP for ADP) on G-actin, then CAP releases profilin-bound ATP-G-actin to replenish the actin monomer pool. These functions are highly conserved, as expression of animal and plant CAPs complement cellular defects of yeast mutants. Unlike most actin monomer binding proteins, CAP oligomerizes, likely into hexamers. Within the high molecular weight complex formed, the C-terminal half of each CAP molecule binds one actin monomer. In addition, the N-terminus of CAP binds to cofilin-G-actin complexes and the middle region binds to Abpl and profilin. Abpl tethers CAP to filamentous actin networks. Cofilin and profilin function together with CAP to accelerate actin turnover, through a series of actin monomer handoffs guided by the changing nucleotide state of actin. Thus, the emerging view of CAP function is that it serves as a large molecular hub where multiple actin binding proteins interact to recycle actin monomers and cofilin. This macromolecular complex plays a key role in remodeling the actin cytoskeleton during events such as endocytosis, cell polarity, and cell motility.

Twinfilin family actin monomer-binding proteins are conserved in evolution from yeasts to mammals. They bind ADP-actin monomers with high affinity and prevent the assembly of actin monomers into filament ends. In addition to monomeric actin, twinfilins also bind and cap actin filament barbed ends, and interact direcdy with heterodimeric capping proteins. Interaction with capping protein is necessary for twinfilins localization to the cortical actin cytoskeleton at least in budding yeast. Genetic studies on yeast and demonstrate that twinfilin is intimately involved in the regulation of actin dynamics in cells, and that the lack of twinfilin results in uncontrolled actin filament assembly. Together, these data suggest that twinfilins play an important role in actin dynamics by preventing unwanted actin filament assembly in cells. However, the exact mechanism by which twinfilin regulates actin filament turnover and contributes to actin-dependent cellular processes remains to be elucidated.

The β-thymosins are a family of highly conserved polar peptides consisting of 40 to 44 amino acid residues. All β-thymosins bind monomeric G-actin in a 1:1 complex. The dissociation constant of the complex is in the micromolar range and allows for fast binding and release of G-actin. Because of the high intracellular concentration of β-thymosins (up to 500 µM) in most vertebrate cells, β-thymosins are considered the main intracellular G-actin sequestering peptides. Thymosin β binds to G-actin in an extended conformation, and folds into a stable conformation upon binding. The N- and C-termini of thymosin β contact the barbed and pointed ends of the monomeric actin. Thymosin β is present in the nucleus as well as the cytoplasm and might be responsible for sequestering nuclear actin. Even minor cell damage might be responsible for the release of β-thymosins detectable in the extracellular fluids. Extracellular β-thymosins affect matrix metallo-proteinases, chemotaxis, angiogenesis and wound healing. However, only very little is known about the molecular mechanisms mediating the effects attributed to extracellular β-thymosins.

Multirepeat β-thymosins contain multiple copies of the β-thymosin actin binding module. This family is mainly distributed within lower metazoan species and, with one exception, absent in mammals in which the classical single repeat β-thymosins appear dominant. The repeated nature in combination with sequence variation in the consecutive modules renders these proteins different actin modulating capacities as compared to the classical β-thymosins. These properties are discussed in function of recent structural models indicating how these proteins contact actin. The importance of the multirepeat β-thymosins is underscored by their crucial role in neuronal development and reproduction.

The dynamic turnover of actin filaments generates the forces driving cellular motile processes. A key factor of actin polymerization is the de novo nucleation and elongation of actin filaments, which can be catalysed by a limited number of proteins or protein complexes, the best studied of which is the Arp2/3-complex. The activity of the Arp2/3 complex is tightly regulated and controlled through signal-dependent association with nucleation promotion factors, like the WASP and WAVE family of proteins. An emerging common theme for these factors is that they act as coincident detectors of a variety of signaling pathways through the formation of large multi-molecular complexes. These complexes impose a strict spatial and temporal control on the activities of WASP and WAVE family proteins within the cells. They further contribute to fine tune Arp2/3-mediated branched actin filament elongation so as to adapt its biochemical activity to a vast array of diverse cellular functions. In this chapter we will provide an overview of the most recent finding defining the composition and mode of regulation of the WAVE-, WASP- and N/WASP-based complexes in mediating distinct actin dynamics-based cellular processes.

Verprolin is an actin-binding protein first identified in budding yeast . The yeast verprolin is needed for actin polymerisation during polarised growth and during endocytosis. In vertebrate cells, three genes encoding orthologues have been identified: and . The mammalian verprolins have been implicated in the regulation of actin dynamics either by binding directly to actin, by binding the WASP family of proteins or by binding to other actin regulating proteins. This review article will bring up to discussion the current understanding of the mechanisms underlying verprolin-dependent mobilisation of the actin filament system.

The dynamic remodeling of the actin cytoskeleton plays an essential role in many cellular processes, including cell motility, cytokinesis, and intracellular transport. A large number of actin-binding proteins (ABPs) participate in this process, regulating the assembly of actin filaments into functional networks. ABPs are extremely diverse, both structurally and functionally, but they most seem to share a common binding area on the actin surface, consistent of the cleft between actin subdomains 1 and 3. Actin itself is thought to interact in this cleft in the filament. As a result, part of the cleft becomes buried in F-actin by inter-subunit contacts, whereas another part remains exposed and mediates the interactions of various filamentous actin-binding proteins. The convergence of actin-binding proteins into a common binding area imposes enormous constraints on their interactions and could serve a regulatory function. Because the cleft falls near the hinge for domain motions in actin, binding in this area is an effective way for ABPs to “sense” the conformation of actin, in particular conformational changes resulting from ATP hydrolysis by actin or from the G- to F-actin transition.