肌动蛋白解聚因-子/丝切蛋白:肌动蛋白重塑蛋白质家系

肌动蛋白解聚因子／丝切蛋白(actin depolymerizing factor／cofilin,ADF／cofilin)是肌动蛋白结合蛋白(actin—binding protein)家系。迄今为止,可以在所有的真核细胞中检测到ADF／cofilin。它们调节(纤)丝状肌动蛋白细胞骨架(F—actin eytoskeleton),影响细胞的各种生理功能。不同的生物有不同的ADF／cofilin,但其功能基本相似。ADF／cofilin可以使(纤)丝状肌动蛋白(F—actin)解聚合,而且这种解聚合活性是可逆的,ADF／cofilin切割F-actin并且能提高球状肌动蛋白(G—actin)离开纤维突出端(pointedend)的能力,其作用受很多因素调控。     

Actin Filament Bundling and Different Nucleating Effects of Mouse Diaphanous-Related Formin FH2 Domains on Actin/ADF and Actin/Cofilin Complexes

Mouse Diaphanous-related formins (mDias) are members of the formin protein family that nucleate actin polymerization and subsequently promote filamentous actin (F-actin) elongation by monomer addition to fast-growing barbed ends. It has been suggested that mDias preferentially recruit actin complexed to profilin due to their proline-rich FH1 domains. During filament elongation, dimeric mDias remain attached to the barbed ends by their FH2 domains, which form an anti-parallel ring-like structure enclosing the filament barbed ends. Dimer formation of mDia-FH2 domains is dependent on their N-terminal lasso and linker subdomains (connector). Here, we investigated the effect of isolated FH2 domains on actin polymerization using mDia1-FH2 domain plus connector, as well as core mDia1, mDia2, and mDia3 missing the connector, by cosedimentation and electron microscopy after negative staining. Analytical ultracentrifugation showed that core FH2 domains of mDia1 and mDia2 exhibited a low degree of dimer formation, whereas mDia3-FH2 minus connector and mDia1-FH2 plus connector readily dimerized. Only core mDia3-FH2 was able to nucleate actin polymerization. However, all tested core FH2 domains decorated and bundled F-actin, as demonstrated by electron microscopy after negative staining. Bundling activity was highest for mDia3-FH2, decreased for mDia2-FH2, and further decreased for mDia1-FH2. The mDia1-FH2 domain plus connector induced actin polymerization also in the absence of profilin, but failed to induce F-actin deformation and bundling. We also tested whether mDia1-FH2 was able to repolymerize actin in complex with different proteins that stabilize globular actin. The data obtained demonstrated that mDia1-FH2 induced actin repolymerization only from the actin/cofilin-1 complex, but not when complexed to actin depolymerizing factor, gelsolin segment 1, vitamin D binding protein, or deoxyribonuclease I.     

Slow Axonal Transport of Soluble Actin with Actin Depolymerizing Factor, Cofilin, and Profilin Suggests Actin Moves in an Unassembled Form

Abstract: We examined the axonal transport of actin and its monomer binding proteins, actin depolymerizing factor, cofilin, and profilin, in the chicken sciatic nerve following injection of [³⁵S]methionine into the lumbar spinal cord. At intervals up to 20 days after injection, nerves were cut into 1-cm segments and separated into Triton X-100-soluble and particulate fractions. Actin and its binding proteins were then isolated by affinity chromatography on DNase I-Sepharose and by one- and two-dimensional polyacrylamide gel electrophoresis. Fluorographic analysis showed that the specific activity of soluble actin was two to three times that of its particulate form and that soluble actin, cofilin, actin depolymerizing factor, and profilin were transported at similar rates in slow component b of axonal flow. Our data strongly support the view that the mobile form of actin in slow transport is soluble and that a substantial amount of this actin may travel as a complex with actin depolymerizing factor, cofilin, and profilin. Along labeled nerves the specific activity of the unphosphorylated form of actin depolymerizing factor, which binds actin, was not significantly different from that of its "inactive" phosphorylated form. This constancy in specific activity suggests that continuous inactivation and reactivation of actin depolymerizing factor occur during transport, which could contribute to the exchange of soluble actin with the filamentous actin pool.     

Actin Depolymerizing Factor (ADF/Cofilin) Enhances the Rate of Filament Turnover: Implication in Actin-based Motility

Actin-binding proteins of the actin depolymerizing factor (ADF)/cofilin family are thought to control actin-based motile processes. ADF1 from Arabidopsis thaliana appears to be a good model that is functionally similar to other members of the family. The function of ADF in actin dynamics has been examined using a combination of physical–chemical methods and actin-based motility assays, under physiological ionic conditions and at pH 7.8. ADF binds the ADPbound forms of G- or F-actin with an affinity two orders of magnitude higher than the ATP- or ADP-Pi– bound forms. A major property of ADF is its ability to enhance the in vitro turnover rate (treadmilling) of actin filaments to a value comparable to that observed in vivo in motile lamellipodia. ADF increases the rate of propulsion of Listeria monocytogenes in highly diluted, ADF-limited platelet extracts and shortens the actin tails. These effects are mediated by the participation of ADF in actin filament assembly, which results in a change in the kinetic parameters at the two ends of the actin filament. The kinetic effects of ADF are end specific and cannot be accounted for by filament severing. The main functionally relevant effect is a 25-fold increase in the rate of actin dissociation from the pointed ends, while the rate of dissociation from the barbed ends is unchanged. This large increase in the rate-limiting step of the monomer-polymer cycle at steady state is responsible for the increase in the rate of actin-based motile processes. In conclusion, the function of ADF is not to sequester G-actin. ADF uses ATP hydrolysis in actin assembly to enhance filament dynamics.     

ADF/Cofilin Is Not Essential but Is Critically Important for Actin Activities during Phagocytosis in Tetrahymena thermophila

ADF/cofilin is a highly conserved actin-modulating protein. Reorganization of the actin cytoskeleton in vivo through severing and depolymerizing of F-actin by this protein is essential for various cellular events, such as endocytosis, phagocytosis, cytokinesis, and cell migration. We show that in the ciliate Tetrahymena thermophila, the ADF/cofilin homologue Adf73p associates with actin on nascent food vacuoles. Overexpression of Adf73p disrupted the proper localization of actin and inhibited the formation of food vacuoles. In vitro, recombinant Adf73p promoted the depolymerization of filaments made of T. thermophila actin (Act1p). Knockout cells lacking the ADF73 gene are viable but grow extremely slowly and have a severely decreased rate of food vacuole formation. Knockout cells have abnormal aggregates of actin in the cytoplasm. Surprisingly, unlike the case in animals and yeasts, in Tetrahymena, ADF/cofilin is not required for cytokinesis. Thus, the Tetrahymena model shows promise for future studies of the role of ADF/cofilin in vivo.     

ATP和钾离子对肌动蛋白与前纤维蛋白结合的影响及其在植物肌动蛋白纯化中的应用

根据ATP和钾离子影响肌动蛋白聚合及肌动蛋白及肌动蛋白与前纤维蛋白结合的实验结果,通过对以往报道的利用多聚脯氨酸-琼脂糖亲和柱层析法纯化植物肌动蛋白过程中ATP及钾离子浓度的改变,不需要加源前纤维蛋白。可得到较大量高纯度具活性的花粉肌动蛋白。SDS凝胶电泳、免疫印迹、电镜负染及紫外检测表明,所得肌动蛋白在纯度、聚合特性等方面均与原方法所得结果相同,肌动蛋白产率为原方法的73.5%,而整个肌动蛋白提纯过程所需时间缩短了1/3,节省了纯化重组前纤维蛋白所需的费用,消除了外源前纤维蛋白的使用对一些实验室的限制,而且同时得到了大量纯化的花粉前纤维蛋白。     

Effects of ATP and K+ on Actin Binding to Profilin and the Application on Purification of Actin from Maize Pollen

The method for purifying actin from maize pollen by poly-L-proline-sepharose affinity chromatography was modified by improving some of the experimental conditions (concentration of ATP and K+) and eliminating the use of recombinant human platelet profilin. SDS-PAGE, Western blotting, electron microscopy and ultraviolet absorption analysis demonstrated that the quality of the purified actin was similar to that obtained with the previous method. The yield was 73.5% of that of the previous method. However, the time required for preparation was 2/3 of that in the previous method and the cost of purifying recombinant profilin was saved. Furthermore, a large amount of purified pollen profilin could be obtained at the same time.     

Cofilin/ADF is required for retinal elongation and morphogenesis of the Drosophila rhabdomere

Drosophila photoreceptors undergo marked changes in their morphology during pupal development. These changes include a five-fold elongation of the retinal cell body and the morphogenesis of the rhabdomere, the light sensing structure of the cell. Here we show that twinstar (tsr), which encodes Drosophila cofilin/ADF (actin-depolymerizing factor), is required for both of these processes. In tsr mutants, the retina is shorter than normal, the result of a lack of retinal elongation. In addition, in a strong tsr mutant, the rhabdomere structure is disorganized and the microvilli are short and occasionally unraveled. In an intermediate tsr mutant, the rhabdomeres are not disorganized but have a wider than normal structure. The adherens junctions connecting photoreceptor cells to each other are also found to be wider than normal. We propose, and provide data supporting, that these wide rhabdomeres and adherens junctions are secondary events caused by the inhibition of retinal elongation. These results provide insight into the functions of the actin cytoskeleton during morphogenesis of the Drosophila eye.     

Cofilin/ADF is required for cell motility during Drosophila ovary development and oogenesis

The driving force behind cell motility is the actin cytoskeleton. Filopodia and lamellipodia are formed by the polymerization and extension of actin filaments towards the cell membrane. This polymerization at the barbed end of the filament is balanced by depolymerization at the pointed end, recycling the actin in a 'treadmilling' process. One protein involved in this process is cofilin/actin-depolymerizing factor (ADF), which can depolymerize actin filaments, allowing treadmilling to occur at an accelerated rate. Cofilin/ADF is an actin-binding protein that is required for actin-filament disassembly, cytokinesis and the organization of muscle actin filaments. There is also evidence that cofilin/ADF enhances cell motility, although a direct requirement in vivo has not yet been shown. Here we show that Drosophila cofilin/ADF, which is encoded by the twinstar (tsr) gene, promotes cell movements during ovary development and oogenesis. During larval development, cofilin/ADF is required for the cell rearrangement needed for formation of terminal filaments, stacks of somatic cells that are important for the initiation of ovarioles. It is also required for the migration of border cells during oogenesis. These results show that cofilin/ADF is an important regulator of actin-based cell motility during Drosophila development.     

Arp2/3 Complex from Acanthamoeba Binds Profilin and Cross-links Actin Filaments

The Arp2/3 complex was first purified from Acanthamoeba castellanii by profilin affinity chromatography. The mechanism of interaction with profilin was unknown but was hypothesized to be mediated by either Arp2 or Arp3. Here we show that the Arp2 subunit of the complex can be chemically cross-linked to the actin-binding site of profilin. By analytical ultracentrifugation, rhodamine-labeled profilin binds Arp2/3 complex with a K_d of 7 μM, an affinity intermediate between the low affinity of profilin for barbed ends of actin filaments and its high affinity for actin monomers. These data suggest the barbed end of Arp2 is exposed, but Arp2 and Arp3 are not packed together in the complex exactly like two actin monomers in a filament. Arp2/3 complex also cross-links actin filaments into small bundles and isotropic networks, which are mechanically stiffer than solutions of actin filaments alone. Arp2/3 complex is concentrated at the leading edge of motile Acanthamoeba, and its localization is distinct from that of α-actinin, another filament cross-linking protein. Based on localization and actin filament nucleation and cross-linking activities, we propose a role for Arp2/3 in determining the structure of the actin filament network at the leading edge of motile cells.     

Mechanism of interaction of Acanthamoeba actophorin (ADF/Cofilin) with actin filaments.

We characterized the interaction of Acanthamoeba actophorin, a member of ADF/cofilin family, with filaments of amoeba and rabbit skeletal muscle actin. The affinity is about 10 times higher for muscle actin filaments (Kd = 0.5 microM) than amoeba actin filaments (Kd = 5 microM) even though the affinity for muscle and amoeba Mg-ADP-actin monomers (Kd = 0.1 microM) is the same (Blanchoin, L., and Pollard, T. D. (1998) J. Biol. Chem. 273, 25106-25111). Actophorin binds slowly (k+ = 0.03 microM-1 s-1) to and dissociates from amoeba actin filaments in a simple bimolecular reaction, but binding to muscle actin filaments is cooperative. Actophorin severs filaments in a concentration-dependent fashion. Phosphate or BeF3 bound to ADP-actin filaments inhibit actophorin binding. Actophorin increases the rate of phosphate release from actin filaments more than 10-fold. The time course of the interaction of actophorin with filaments measured by quenching of the fluorescence of pyrenyl-actin or fluorescence anisotropy of rhodamine-actophorin is complicated, because severing, depolymerization, and repolymerization follows binding. The 50-fold higher affinity of actophorin for Mg-ADP-actin monomers (Kd = 0.1 microM) than ADP-actin filaments provides the thermodynamic basis for driving disassembly of filaments that have hydrolyzed ATP and dissociated gamma-phosphate.     

Probing the Plant Actin Cytoskeleton during Cytokinesis and Interphase by Profilin Microinjection

We have examined the cytological effects of microinjecting recombinant birch profilin in dividing and interphase stamen hair cells of Tradescantia virginiana. Microinjection of profilin at anaphase and telophase led to a marked effect on cytokinesis; cell plate formation was often delayed, blocked, or completely inhibited. In addition, the initial appearance of the cell plate was wrinkled, thin, and sometimes fragmented. Injection of profilin at interphase caused a thinning or the collapse of cytoplasmic strands and a retardation or inhibition of cytoplasmic streaming in a dose-dependent manner. Confocal laser scanning microscopy of rhodamine-phalloidin staining in vivo revealed that high levels of microinjected profilin induced a degradation of the actin cytoskeleton in the phragmoplast, the perinuclear zone, and the cytoplasmic strands. However, some cortical actin filaments remained intact. The data demonstrate that profilin has the ability to act as a regulator of actin-dependent events and that centrally located actin filaments are more sensitive to microinjected profilin than are cortical actin filaments. These results add new evidence supporting the hypothesis that actin filaments play a crucial role in the formation of the cell plate and provide mechanical support for the cytoplasmic strands in interphase cells.     

Xenopus Actin Depolymerizing Factor/Cofilin (XAC) Is Responsible for the Turnover of Actin Filaments in Listeria monocytogenes Tails

In contrast to the slow rate of depolymerization of pure actin in vitro, populations of actin filaments in vivo turn over rapidly. Therefore, the rate of actin depolymerization must be accelerated by one or more factors in the cell. Since the actin dynamics in Listeria monocytogenes tails bear many similarities to those in the lamellipodia of moving cells, we have used Listeria as a model system to isolate factors required for regulating the rapid actin filament turnover involved in cell migration. Using a cell-free Xenopus egg extract system to reproduce the Listeria movement seen in a cell, we depleted candidate depolymerizing proteins and analyzed the effect that their removal had on the morphology of Listeria tails. Immunodepletion of Xenopus actin depolymerizing factor (ADF)/cofilin (XAC) from Xenopus egg extracts resulted in Listeria tails that were approximately five times longer than the tails from undepleted extracts. Depletion of XAC did not affect the tail assembly rate, suggesting that the increased tail length was caused by an inhibition of actin filament depolymerization. Immunodepletion of Xenopus gelsolin had no effect on either tail length or assembly rate. Addition of recombinant wild-type XAC or chick ADF protein to XAC-depleted extracts restored the tail length to that of control extracts, while addition of mutant ADF S3E that mimics the phosphorylated, inactive form of ADF did not reduce the tail length. Addition of excess wild-type XAC to Xenopus egg extracts reduced the length of Listeria tails to a limited extent. These observations show that XAC but not gelsolin is essential for depolymerizing actin filaments that rapidly turn over in Xenopus extracts. We also show that while the depolymerizing activities of XAC and Xenopus extract are effective at depolymerizing normal filaments containing ADP, they are unable to completely depolymerize actin filaments containing AMPPNP, a slowly hydrolyzible ATP analog. This observation suggests that the substrate for XAC is the ADP-bound subunit of actin and that the lifetime of a filament is controlled by its nucleotide content.     

Abl2/Abl-related Gene Stabilizes Actin Filaments,Stimulates Actin Branching by Actin-related Protein 2/3 Complex,and Promotes Actin Filament Severing by Cofilin

Both Arp2/3 complex and the Abl2/Arg nonreceptor tyrosine kinase are essential to form and maintain diverse actin-based structures in cells, including cell edge protrusions in fibroblasts and cancer cells and dendritic spines in neurons. The ability of Arg to promote cell edge protrusions in fibroblasts does not absolutely require kinase activity, raising the question of how Arg might modulate actin assembly and turnover in the absence of kinase function. Arg has two distinct actin-binding domains and interacts physically and functionally with cortactin, an activator of the Arp2/3 complex. However, it was not known whether and how Arg influences actin filament stability, actin branch formation, or cofilin-mediated actin severing or how cortactin influences these reactions of Arg with actin. Arg or cortactin bound to actin filaments stabilizes them from depolymerization. Low concentrations of Arg and cortactin cooperate to stabilize filaments by slowing depolymerization. Arg stimulates formation of actin filament branches by Arp2/3 complex and cortactin. An Arg mutant lacking the C-terminal calponin homology actin-binding domain stimulates actin branch formation by the Arp2/3 complex, indicative of autoinhibition. ArgΔCH can stimulate the Arp2/3 complex even in the absence of cortactin. Arg greatly potentiates cofilin severing of actin filaments, and cortactin attenuates this enhanced severing. The ability of Arg to stabilize filaments, promote branching, and increase severing requires the internal (I/L)WEQ actin-binding domain. These activities likely underlie important roles that Arg plays in the formation, dynamics, and stability of actin-based cellular structures.     

Arp2/3 and Mena/VASP Require Profilin 1 for Actin Network Assembly at the Leading Edge

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Assembly and Turnover of Short Actin Filaments by the Formin INF2 and Profilin

INF2 (inverted formin 2) is a formin protein with unique biochemical effects on actin. In addition to the common formin ability to accelerate actin nucleation and elongation, INF2 can also sever filaments and accelerate their depolymerization. Although we understand key attributes of INF2-mediated severing, we do not understand the mechanism by which INF2 accelerates depolymerization subsequent to severing. Here, we show that INF2 can create short filaments (<60 nm) that continuously turn over actin subunits through a combination of barbed end elongation, severing, and WH2 motif-mediated depolymerization. This pseudo-steady state condition occurs whether starting from actin filaments or monomers. The rate-limiting step of the cycle is nucleotide exchange of ADP for ATP on actin monomers after release from the INF2/actin complex. Profilin addition has two effects: 1) to accelerate filament turnover 6-fold by accelerating nucleotide exchange and 2) to shift the equilibrium toward polymerization, resulting in longer filaments. In sum, our findings show that the combination of multiple interactions of INF2 with actin can work in concert to increase the ATP turnover rate of actin. Depending on the ratio of INF2:actin, this increased flux can result in rapid filament depolymerization or maintenance of short filaments. We also show that high concentrations of cytochalasin D accelerate ATP turnover by actin but through a different mechanism from that of INF2.