Ubiquitous among eukaryotes, the ADF/cofilins are essential proteins responsible for the high turnover rates of actin filaments in vivo. In vertebrates, ADF and cofilin are products of different genes. Both bind to F-actin cooperatively and induce a twist in the actin filament that results in the loss of the phalloidin-binding site. This conformational change may be responsible for the enhancement of the off rate of subunits at the minus end of ADF/cofilin-decorated filaments and for the weak filament-severing activity. Binding of ADF/cofilin is competitive with tropomyosin. Other regulatory mechanisms in animal cells include binding of phosphoinositides, phosphorylation by LIM kinases on a single serine, and changes in pH. Although vertebrate ADF/cofilins contain a nuclear localization sequence, they are usually concentrated in regions containing dynamic actin pools, such as the leading edge of migrating cells and neuronal growth cones. ADF/cofilins are essential for cytokinesis, phagocytosis, fluid phase endocytosis, and other cellular processes dependent upon actin dynamics.

Recent findings have significantly expanded our understanding of the regulation of actin depolymerizing factor (ADF)/cofilin proteins and the profound multifaceted impact that these well-established regulators of actin dynamics have on cell biology. In this review we discuss new aspects of previously documented regulation, such as phosphorylation, but also cover novel recently established modes of regulation and new functions of ADF (also known as destrin)/cofilin. We now understand that their activity responds to a vast array of inputs far greater than previously appreciated and that these proteins not only feed back to the crucially important dynamics of actin, but also to apoptosis cascades, phospholipid metabolism, and gene expression. We argue that this ability to respond to physiological changes by modulating those same changes makes the ADF/cofilin protein family a homeostatic regulator or ‘functional node’ in cell biology.

Inclusions containing actin-depolymerizing factor (ADF) and cofilin, abundant proteins in adult human brain, are prominent in hippocampal and cortical neurites of the post-mortem brains of Alzheimer's patients, especially in neurites contacting amyloid deposits. The origin and role of these inclusions in neurodegeneration are, however, unknown. Here we show that mediators of neurodegeneration induce the rapid formation of transient or persistent rod-like inclusions containing ADF/cofilin and actin in axons and dendrites of cultured hippocampal neurons. Rods form spontaneously within neurons overexpressing active ADF/cofilin, suggesting that the activation (by dephosphorylation) of ADF/cofilin that occurs in response to neurodegenerative stimuli is sufficient to induce rod formation. Persistent rods that span the diameter of the neurite disrupt microtubules and cause degeneration of the distal neurite without killing the neuron. These findings suggest a common pathway that can lead to loss of synapses.

The specific functions of greater than 40 vertebrate nonmuscle tropomyosins (Tms) are poorly understood. In this article we have tested the ability of two Tm isoforms, TmBr3 and the human homologue of Tm5 (hTM5 NM1 ), to regulate actin filament function. We found that these Tms can differentially alter actin filament organization, cell size, and shape. hTm5 NM1 was able to recruit myosin II into stress fibers, which resulted in decreased lamellipodia and cellular migration. In contrast, TmBr3 transfection induced lamellipodial formation, increased cellular migration, and reduced stress fibers. Based on coimmunoprecipitation and colocalization studies, TmBr3 appeared to be associated with actin-depolymerizing factor/cofilin (ADF)-bound actin filaments. Additionally, the Tms can specifically regulate the incorporation of other Tms into actin filaments, suggesting that selective dimerization may also be involved in the control of actin filament organization. We conclude that Tm isoforms can be used to specify the functional properties and molecular composition of actin filaments and that spatial segregation of isoforms may lead to localized specialization of actin filament function. INTRODUCTIONThe actin microfilament network is a primary cytoskeletal system involved in the development and maintenance of morphology within cells. The dynamic nature of the actinbased system and its organization is thought to regulate specific structural changes within different cellular regions (Gunning et al., 1998b). The function and form of the actin cytoskeleton is largely determined by actin-binding proteins that are associated with the polymeric structure. Tropomyosins (Tms), along with actin, are integral components of the microfilament cytoskeleton, although not all actin filaments have Tms bound to them (Bamburg, 1999). Tms bind largely by electrostatic charge to the helical groove of the actin filament and the Ͼ40 isoforms are obtained by alternative splicing from four genes, of which almost all are nonmuscle variants (Lees-Miller et al., 1990;Goodwin et al., 1991;Beisel and Kennedy, 1994;Dufour et al., 1998;Cooley and Bergtrom, 2001). Although a considerable amount of information exists as to the biochemical regulation of microfilament dynamics, little is known about the function of this large family of proteins in vertebrate nonmuscle cells.In vitro studies have shown that nonmuscle Tms are able to differentially protect actin from the severing action of gelsolin (Ishikawa et al., 1989) and can regulate the MgATPase activity of myosins to varying degrees (Fanning et al., 1994). The different binding strengths to actin are thought to impart a range of stability to the filaments (Matsumura and Yamashiro-Matsumura, 1985;Hitchcock-DeGregori et al., 1988;Pittenger et al., 1995). The impact of Tms on vertebrate cell morphology is poorly understood even though studies suggest the importance of Tm isoforms in regulating Article published online ahead of print. Mol. Biol. Cell 10.1091/ mbc.E02-04 -0244. Article and publication dat...

Actin depolymerizing factor (ADF) occurs naturally in two forms, one of which contains a phosphorylated Ser and does not bind G-actin or depolymerize F-actin. Removal of this phosphate in vitro by alkaline phosphatase restores full F-actin depolymerizing activity. To identify the phosphorylation site, [32P]pADF was purified and digested with endoproteinase Lys-C. The digest contained only one 32P-labeled peptide. Further digestion with endoproteinase Asp-N and mass spectrometric analysis showed that this peptide came from the N terminus of ADF. Alkaline phosphatase treatment of one Asp-N peptide (mass 753) converted it to a peptide of mass 673, demonstrating that this peptide contains the phosphate group. Tandem mass spectrometric sequence analysis of this peptide identified the phosphorylated Ser as the encoded Ser3 (Ser2 in the processed protein). HeLa cells, transfected with either chick wild-type ADF cDNA or a cDNA mutated to code for Ala in place of Ser24 or Thr25, express and phosphorylate the exogenous ADF. Cells also expressed high levels of mutant ADF when Ser3 was deleted or converted to either Ala or Glu. However, none of these mutants was phosphorylated, confirming that Ser3 in the encoded ADF is the single in vivo regulatory site.

Slingshot (SSH) phosphatases and LIM kinases (LIMK)regulate actin dynamics via a reversible phosphorylation (inactivation) of serine 3 in actin-depolymerizing factor (ADF) and cofilin. Here we demonstrate that a multi-protein complex consisting of SSH-1L, LIMK1, actin, and the scaffolding protein, 14-3-3f, is involved, along with the kinase, PAK4, in the regulation of ADF/cofilin activity. Endogenous LIMK1 and SSH-1L interact in vitro and co-localize in vivo, and this interaction results in dephosphorylation and downregulation of LIMK1 activity. We also show that the phosphatase activity of purified SSH-1L is F-actin dependent and is negatively regulated via phosphorylation by PAK4. 14-3-3f binds to phosphorylated slingshot, decreases the amount of slingshot that co-sediments with F-actin, but does not alter slingshot activity. Here we define a novel ADF/cofilin phosphoregulatory complex and suggest a new mechanism for the regulation of ADF/cofilin activity in mediating changes to the actin cytoskeleton.

Summary Dendritic spines undergo actin-based growth and shrinkage during synaptic plasticity. The actin depolymerizing factor (ADF)/cofilin family of actin-associated proteins plays important roles in spine plasticity. Elevated ADF/cofilin activities often lead to reduced spine size and immature spine morphology, but can enhance synaptic potentiation in some cases. Therefore ADF/cofilin may exert distinct effects on postsynaptic structure and function. Here we report that ADF/cofilin-mediated actin dynamics regulate AMPA receptor (AMPAR) trafficking during synaptic potentiation, which is distinct from actin's structural role in spine morphology. We find that elevated ADF/cofilin activity markedly enhances surface addition of AMPARs after chemically-induced LTP (cLTP), whereas inhibition of ADF/cofilin abolishes AMPAR addition. Our data further show that cLTP elicits a temporal sequence of ADF/cofilin dephosphorylation and phosphorylation that underlies AMPAR trafficking and spine enlargement. These findings suggest a novel role for temporally-regulated ADF/cofilin activities in postsynaptic modifications of receptor number and spine size during synaptic plasticity.

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