Formin proteins nucleate actin filaments, remaining processively associated with the fast-growing barbed ends. Although formins possess common features, the diversity of functions and biochemical activities raised the possibility that formins differ in fundamental ways. Further, a recent study suggested that profilin and ATP hydrolysis are both required for processive elongation mediated by the formin mDia1. We used total internal reflection fluorescence microscopy to observe directly individual actin filament polymerization in the presence of two mammalian formins (mDia1 and mDia2) and two yeast formins (Bni1p and Cdc12p). We show that these diverse formins have the same basic properties: movement is processive in the absence or presence of profilin; profilin accelerates elongation; and actin ATP hydrolysis is not required for processivity. These results suggest that diverse formins are mechanistically similar, but the rates of particular assembly steps vary.
Formins are a conserved class of proteins expressed in all eukaryotes, with known roles in generating cellular actin-based structures. The mammalian formin, FRL␣, is enriched in hematopoietic cells and tissues, but its biochemical properties have not been characterized. We show that a construct composed of the C-terminal half of FRL␣ (FRL␣-C) is a dimer and has multiple effects on muscle actin, including tight binding to actin filament sides, partial inhibition of barbed end elongation, inhibition of barbed end binding by capping protein, acceleration of polymerization from monomers, and actin filament severing. These multiple activities can be explained by a model in which FRL␣-C binds filament sides but prefers the topology of sides at the barbed end (end-sides) to those within the filament. This preference allows FRL␣-C to nucleate new filaments by side stabilization of dimers, processively advance with the elongating barbed end, block interaction between C-terminal tentacles of capping protein and filament end-sides, and sever filaments by preventing subunit re-association as filaments bend. Another formin, mDia1, does not reduce the barbed end elongation rate but does block capping protein, further supporting an end-side binding model for formins. Profilin partially relieves barbed end elongation inhibition by FRL␣-C. When non-muscle actin is used, FRL␣-C's effects are largely similar. FRL␣-C's ability to sever filaments is the first such activity reported for any formin. Because we find that mDia1-C does not sever efficiently, severing may not be a property of all formins.Non-muscle cells contain a variety of actin filament-based structures, including lamellipodia, ruffles, filopodia, microvilli, and sarcomeric contractile structures (including cytokinetic actin rings and stress fibers). Assembly mechanisms for these structures are being vigorously investigated. Spontaneous nucleation of actin monomers occurs very slowly (1), and specific actin-associated proteins that promote rapid actin assembly are required for creating each actin-based structure. Arp2/3 1 complex is a well characterized nucleation factor, forming networks of branched actin filaments that are present in lamellipodia and ruffles (2). In contrast, the proteins controlling assembly of many other actin-based structures have not been identified.Formins are a conserved class of actin-associated proteins that have been found in all eukaryotes examined and accelerate filament assembly independently of Arp2/3 complex (3). Two unifying structural features of formins are the Formin Homology 1 and 2 (FH1, FH2) domains, generally found in the C-terminal half of the protein. The FH1 domain contains proline-rich sequences capable of binding profilin and SH3 (Src homology 3) domain-containing proteins. The FH2 domain forms a multimeric structure (4, 5).Budding yeast formins Bni1p and Bnr1p are required for the assembly of actin cables and cytokinetic actin rings in vivo (6, 7). Bni1p has barbed end nucleation activity in vitro, for which the FH2 domain...
SummaryEndothelial cells form cell-cell adhesive structures, called adherens and tight junctions, which maintain tissue integrity, but must be dynamic for leukocyte transmigration during the inflammatory response and cellular remodeling during angiogenesis. This review will focus on Vascular Endothelial (VE)-cadherin, an endothelial-specific cell-cell adhesion protein of the adherens junction complex. VE-cadherin plays a key role in endothelial barrier function and angiogenesis, and consequently VE-cadherin availability and function are tightly regulated. VE-cadherin also participates directly and indirectly in intracellular signaling pathways that control cell dynamics and cell cycle progression. Here we highlight recent work that has advanced our understanding of multiple regulatory and signaling mechanisms that converge on VE-cadherin and have consequences for endothelial barrier function and angiogenic remodeling.
Formin proteins are regulators of actin dynamics, mediating assembly of unbranched actin filaments. These multidomain proteins are defined by the presence of a Formin Homology 2 (FH2) domain. Previous work has shown that FH2 domains bind to filament barbed ends and move processively at the barbed end as the filament elongates. Here we report that two FH2 domains, from mammalian FRL1 and mDia2, also bundle filaments, whereas the FH2 domain from mDia1 cannot under similar conditions. The FH2 domain alone is sufficient for bundling. Bundled filaments made by either FRL1 or mDia2 are in both parallel and anti-parallel orientations. A novel property that might contribute to bundling is the ability of the dimeric FH2 domains from both FRL1 and mDia2 to dissociate and recombine. This property is not observed for mDia1. A difference between FRL1 and mDia2 is that FRL1-mediated bundling is competitive with barbed end binding, whereas mDia2-mediated bundling is not. Mutation of a highly conserved isoleucine residue in the FH2 domain does not inhibit bundling by either FRL1 or mDia2, but inhibits barbed end activities. However, the severity of this mutation varies between formins. For mDia1 and mDia2, the mutation strongly inhibits all effects of barbed end binding, but affects FRL1 much less strongly. Furthermore, our results suggest that the Ile mutation affects processivity. Taken together, our data suggest that the bundling activities of FRL1 and mDia2, while producing phenotypically similar bundles, differ in mechanistic detail.Formin proteins have emerged as important actin filament assembly factors. Mammals have 15 formin isoforms, which segregate into seven distinct groups based on Formin Homology 2 (FH2) 3 domain phylogeny (1). The FH2 domain is the defining structural feature of this family, and is responsible for most formin effects on actin. A key functional feature is that the FH2 domain is dimeric (2-5). Studies on the Bni1p FH2 domain from budding yeast show that this dimer has no measurable ability to dissociate (5).For all formins studied to date, the FH2 domain binds tightly to the actin filament barbed end, and moves processively as the barbed end elongates. This ability appears to mediate three effects: 1) polymerization acceleration, 2) alteration of elongation/depolymerization rates, and 3) protection of barbed ends from capping protein (reviewed in Ref. 6). In addition to barbed end binding, some formins can also bind actin filament sides (3) and bundle filaments (7,8).Recent structural studies have greatly advanced our understanding of how formins associate with filament barbed ends. The crystal structure of the Bni1p FH2 domain shows it to be a "donut shaped" anti-parallel dimer, with a central hole created by two sets of dimerization interactions between subunits (5). Mutational analysis reveals that surfaceexposed residues on the interior of the donut are necessary for Bni1p-mediated actin assembly and protection from capping protein. This dimer is very stable, but flexible because of an extende...
Abstract. Satellite-derived soil moisture provides more spatially and temporally extensive data than in situ observations. However, satellites can only measure water in the top few centimeters of the soil. Root zone soil moisture is more important, particularly in vegetated regions. Therefore estimates of root zone soil moisture must be inferred from nearsurface soil moisture retrievals. The accuracy of this inference is contingent on the relationship between soil moisture in the near-surface and the soil moisture at greater depths. This study uses cross correlation analysis to quantify the association between near-surface and root zone soil moisture using in situ data from the United States Great Plains. Our analysis demonstrates that there is generally a strong relationship between near-surface (5-10 cm) and root zone (25-60 cm) soil moisture. An exponential decay filter is used to estimate root zone soil moisture using near-surface soil moisture derived from the Soil Moisture and Ocean Salinity (SMOS) satellite. Root zone soil moisture derived from SMOS surface retrievals is compared to in situ soil moisture observations in the United States Great Plains. The SMOSbased root zone soil moisture had a mean R 2 of 0.57 and a mean Nash-Sutcliffe score of 0.61 based on 33 stations in Oklahoma. In Nebraska, the SMOS-based root zone soil moisture had a mean R 2 of 0.24 and a mean Nash-Sutcliffe score of 0.22 based on 22 stations. Although the performance of the exponential filter method varies over space and time, we conclude that it is a useful approach for estimating root zone soil moisture from SMOS surface retrievals.
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