A Conserved Mechanism for Bni1- and mDia1-induced Actin Assembly and Dual Regulation of Bni1 by Bud6 and Profilin (original) (raw)
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The Role of the FH1 Domain and Profilin in Formin-Mediated Actin-Filament Elongation and Nucleation
Current Biology, 2008
Background: Formin proteins nucleate actin filaments de novo and stay associated with the growing barbed end. Whereas the formin-homology (FH) 2 domains mediate processive association, the FH1 domains-in concert with the actin-monomer-binding protein profilin-increase the rate of barbed-end elongation. The mechanism by which this effect is achieved is not well understood. Results: We used total internal reflection fluorescence microscopy to measure the effect of profilin on the elongation of single actin filaments associated with FH1FH2 constructs (derived from the formin Bni1p from S. cerevisiae) with FH1 domains containing one to eight profilinbinding polyproline tracks. Over a large range of profilin concentrations (0.5-25 mM), the rate of barbed-end elongation increases with the number of polyproline tracks in the FH1 domain. The binding of profilin-actin to the FH1 domain is the rate-limiting step (up to rates of at least 88 s 21) in FH1-mediated transfer of actin subunits to the barbed end. Dissociation of formins from barbed ends growing in the presence of profilin is proportional to the elongation rate. Profilin profoundly inhibits nucleation by FH2 and FH1FH2 constructs, but profilin-actin bound to FH1 might contribute weakly to nucleation. Conclusions: To achieve fast elongation, formin FH1 domains bind profilin-actin complexes and deliver them rapidly to the barbed end associated with the FH2 domain. Because subunit addition promotes dissociation of FH2 domains from growing barbed ends, FH2 domains must pass through a state that is prone to dissociation during each cycle of actin subunit addition coupled to formin translocation.
A nucleator arms race: cellular control of actin assembly
Nature Reviews Molecular Cell Biology, 2010
For more than a decade the Arp2/3 complex, a handful of nucleation-promoting factors, and formins were the only molecules known to directly nucleate actin filament formation de novo. However, the past several years have brought a surge in the discovery of mammalian proteins with roles in actin nucleation and dynamics. Newly recognized nucleation-promoting factors, such as WASH, WHAMM, and JMY stimulate Arp2/3 complex activity at distinct cellular locations. Formin nucleators with additional biochemical and cellular activities have also been uncovered. Finally, the Spire, Cordon-bleu, and Leiomodin nucleators have revealed new ways of overcoming the kinetic barriers to actin polymerization. Actin is one of the most abundant and highly-conserved proteins in eukaryotic cells, and is structurally related to prokaryotic actin-like proteins, hinting at its ancient evolutionary origins (Box.1). A 42kDa monomeric ATP-binding protein, globular (G)-actin, can undergo cycles of self-assembly into filamentous (F)-actin, ATP hydrolysis, and depolymerization. Actin filaments contain dynamic barbed ends and less active pointed ends that are differentiated by their structural and biochemical characteristics. Filament turnover is controlled by many actinbinding proteins, including some that function in monomer sequestration or delivery, and others that promote filament nucleation, elongation, capping, severing, or depolymerization. The ability of this versatile cytoskeletal system to generate force, create structural scaffolds, and act as tracks for motor proteins makes it a critical participant in numerous cellular functions, including morphogenesis, migration, cytokinesis, and membrane transport (Fig.1). Box 1 The Actin Cytoskeleton: Ancient evolutionary origins and functions Several prokaryotic proteins have structural similarities to eukaryotic actin and assemble into filaments, suggesting that they represent an ancestral actin cytoskeleton. These bacterial factors include MreB (murein formation cluster E B) and ParM (partitioning M) (reviewed in Ref.159; see the figure). MreB is expressed primarily in rod-shaped bacteria, and is an ATPase, similar to eukaryotic actin. Also like actin, MreB subunits are preferentially incorporated into filaments in their ATP-bound form, and depolymerize mainly in the ADPbound form. However unlike F-actin, MreB subunits generally have a non-helical structure, and form short coils, spirals, and ribbons. These filaments help determine bacterial shape by forming a scaffold beneath the inner plasma membrane and positioning enzymes involved in cell wall biosynthesis. Another actin-like bacterial protein, ParM, is encoded on the E.coli R1 plasmid. ParM polymerizes in an ATP-dependent manner to form helical filaments like F-actin, but with the opposite handedness. Moreover, its assembly kinetics are drastically different from eukaryotic actin. ParM spontaneously nucleates more efficiently than actin and might not need nucleation factors. ParM filaments continuously cycle between phases of elongation and rapid catastrophic disassembly, a behavior that was first described for microtubules and is termed dynamic instability. Finally, ParM filaments
Developmental Cell, 2009
Normal cellular development and function require tight spatiotemporal control of actin assembly. Formins are potent actin assembly factors that protect the growing ends of actin filaments from capping proteins. However, it is unresolved how the duration of formin-mediated actin assembly events is controlled, whether formins are actively displaced from growing ends, and how filament length is regulated in vivo. Here, we identify Bud14 as a highaffinity inhibitor of the yeast formin Bnr1 that rapidly displaces the Bnr1 FH2 domain from growing barbed ends. Consistent with these activities, bud14D cells display fewer actin cables, which are aberrantly long, bent, and latrunculinA resistant, leading to defects in secretory vesicle movement. Moreover, bud14D suppressed mutations that cause abnormally numerous and shortened cables, restoring wild-type actin architecture. From these results, we propose that formin displacement factors regulate filament length and are required in vivo to maintain proper actin network architecture and function.
Mechanism and Function of Formins in the Control of Actin Assembly
Annual Review of Biochemistry, 2007
Formins are a widely expressed family of proteins that govern cell shape, adhesion, cytokinesis, and morphogenesis by remodeling the actin and microtubule cytoskeletons. These large multidomain proteins associate with a variety of other cellular factors and directly nucleate actin polymerization through a novel mechanism. The signature formin homology 2 (FH2) domain initiates filament assembly and remains persistently associated with the fast-growing barbed end, enabling rapid insertion of actin subunits while protecting the end from capping proteins. On the basis of structural and mechanistic work, an integrated model is presented for FH2 processive motion. The adjacent FH1 domain recruits profilin-actin complexes and accelerates filament elongation. The most predominantly expressed formins in animals and fungi are autoinhibited through intramolecular interactions and appear to be activated by Rho GTPases and additional factors. Other classes of formins lack the autoinhibitory and/or Rho-binding domains and thus are likely to be controlled by alternative mechanisms.
Bni1p, a yeast formin linking cdc42p and the actin cytoskeleton during polarized morphogenesis
Science, 1997
The Saccharomyces cerevisiae BNI1 gene product (Bni1p) is a member of the formin family of proteins, which participate in cell polarization, cytokinesis, and vertebrate limb formation. During mating pheromone response, bni1 mutants showed defects both in polarized morphogenesis and in reorganization of the underlying actin cytoskeleton. In two-hybrid experiments, Bni1p formed complexes with the activated form of the Rho-related guanosine triphosphatase Cdc42p, with actin, and with two actin-associated proteins, profilin and Bud6p (Aip3p). Both Bni1p and Bud6p (like Cdc42p and actin) localized to the tips of mating projections. Bni1p may function as a Cdc42p target that links the pheromone response pathway to the actin cytoskeleton.
Mechanism and cellular function of Bud6 as an actin nucleation-promoting factor
Molecular Biology of the Cell, 2011
Formins are a conserved family of actin assembly–promoting factors with diverse biological roles, but how their activities are regulated in vivo is not well understood. In Saccharomyces cerevisiae, the formins Bni1 and Bnr1 are required for the assembly of actin cables and polarized cell growth. Proper cable assembly further requires Bud6. Previously it was shown that Bud6 enhances Bni1-mediated actin assembly in vitro, but the biochemical mechanism and in vivo role of this activity were left unclear. Here we demonstrate that Bud6 specifically stimulates the nucleation rather than the elongation phase of Bni1-mediated actin assembly, defining Bud6 as a nucleation-promoting factor (NPF) and distinguishing its effects from those of profilin. We generated alleles of Bud6 that uncouple its interactions with Bni1 and G-actin and found that both interactions are critical for NPF activity. Our data indicate that Bud6 promotes filament nucleation by recruiting actin monomers to Bni1. Geneti...
Journal of Integrative Plant Biology, 2012
Formin is a major protein responsible for regulating the nucleation of actin filaments, and as such, it permits the cell to control where and when to assemble actin arrays. It is encoded by a multigene family comprising 21 members in Arabidopsis thaliana. The Arabidopsis formins can be separated into two phylogenetically-distinct classes: there are 11 class I formins and 10 class II formins. Significant questions remain unanswered regarding the molecular mechanism of actin nucleation and elongation stimulated by each formin isovariant, and how the different isovariants coordinate to regulate actin dynamics in cells. Here, we characterize a class II formin, AtFH19, biochemically. We found that AtFH19 retains all general properties of the formin family, including nucleation and barbed end capping activity. It can also generate actin filaments from a pool of actin monomers bound to profilin. However, both the nucleation and barbed end capping activities of AtFH19 are less efficient compared to those of another well-characterized formin, AtFH1. Interestingly, AtFH19 FH1FH2 competes with AtFH1 FH1FH2 in binding actin filament barbed ends, and inhibits the effect of AtFH1 FH1FH2 on actin. We thus propose a mechanism in which two quantitatively different formins coordinate to regulate actin dynamics by competing for actin filament barbed ends.
Specification of Architecture and Function of Actin Structures by Actin Nucleation Factors
Annual Review of Biophysics, 2015
The actin cytoskeleton is essential for diverse processes in mammalian cells; these processes range from establishing cell polarity to powering cell migration to driving cytokinesis to positioning intracellular organelles. How these many functions are carried out in a spatiotemporally regulated manner in a single cytoplasm has been the subject of much study in the cytoskeleton field. Recent work has identified a host of actin nucleation factors that can build architecturally diverse actin structures. The biochemical properties of these factors, coupled with their cellular location, likely define the functional properties of actin structures. In this article, we describe how recent advances in cell biology and biochemistry have begun to elucidate the role of individual actin nucleation factors in generating distinct cellular structures. We also consider how the localization and orientation of actin nucleation factors, in addition to their kinetic properties, are critical to their abi...