Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.     

A new model for the interaction of dystrophin with F-actin

The F-actin binding and cross-linking properties of skeletal muscle dystrophin-glycoprotein complex were examined using high and low speed cosedimentation assays, microcapillary falling ball viscometry, and electron microscopy. Dystrophin-glycoprotein complex binding to F-actin saturated near 0.042 +/- 0.005 mol/ mol, which corresponds to one dystrophin per 24 actin monomers. Dystrophin-glycoprotein complex bound to F-actin with an average apparent Kd for dystrophin of 0.5 microM. These results demonstrate that native, full-length dystrophin in the glycoprotein complex binds F-actin with some properties similar to those measured for several members of the actin cross-linking super- family of proteins. However, we failed to observe dystrophin- glycoprotein complex-induced cross-linking of F-actin by three different methods, each positively controlled with alpha-actinin. Furthermore, high speed cosedimentation analysis of dystrophin- glycoprotein complex digested with calpain revealed a novel F-actin binding site located near the middle of the dystrophin rod domain. Recombinant dystrophin fragments corresponding to the novel actin binding site and the first 246 amino acids of dystrophin both bound F- actin but with significantly lower affinity and higher capacity than was observed with purified dystrophin-glycoprotein complex. Finally, dystrophin-glycoprotein complex was observed to significantly slow the depolymerization of F-actin, Suggesting that dystrophin may lie along side an actin filament through interaction with multiple actin monomers. These data suggest that although dystrophin is most closely related to the actin cross-linking superfamily based on sequence homology, dystrophin binds F-actin in a manner more analogous to actin side-binding proteins.     

Actin interacting protein1 and actin depolymerizing factor drive rapid actin dynamics in Physcomitrella patens

The remodeling of actin networks is required for a variety of cellular processes in eukaryotes. In plants, several actin binding proteins have been implicated in remodeling cortical actin filaments (F-actin). However, the extent to which these proteins support F-actin dynamics in planta has not been tested. Using reverse genetics, complementation analyses, and cell biological approaches, we assessed the in vivo function of two actin turnover proteins: actin interacting protein1 (AIP1) and actin depolymerizing factor (ADF). We report that AIP1 is a single-copy gene in the moss Physcomitrella patens. AIP1 knockout plants are viable but have reduced expansion of tip-growing cells. AIP1 is diffusely cytosolic and functions in a common genetic pathway with ADF to promote tip growth. Specifically, ADF can partially compensate for loss of AIP1, and AIP1 requires ADF for function. Consistent with a role in actin remodeling, AIP1 knockout lines accumulate F-actin bundles, have fewer dynamic ends, and have reduced severing frequency. Importantly, we demonstrate that AIP1 promotes and ADF is essential for cortical F-actin dynamics.     

Functional synergy of actin filament cross-linking proteins

The organization of filamentous actin (F-actin) in resilient networks is coordinated by various F-actin cross-linking proteins. The relative tolerance of cells to null mutations of genes that code for a single actin cross-linking protein suggests that the functions of those proteins are highly redundant. This apparent functional redundancy may, however, reflect the limited resolution of available assays in assessing the mechanical role of F-actin cross-linking/bundling proteins. Using reconstituted F-actin networks and rheological methods, we demonstrate how alpha-actinin and fascin, two F-actin cross-linking/bundling proteins that co-localize along stress fibers and in lamellipodia, could synergistically enhance the resilience of F-actin networks in vitro. These two proteins can generate microfilament arrays that "yield" at a strain amplitude that is much larger than each one of the proteins separately. F-actin/alpha-actinin/fascin networks display strain-induced hardening, whereby the network "stiffens" under shear deformations, a phenomenon that is non-existent in F-actin/fascin networks and much weaker in F-actin/alpha-actinin networks. Strain-hardening is further enhanced at high rates of deformation and high concentrations of actin cross-linking proteins. A simplified model suggests that the optimum results of the competition between the increased stiffness of bundles and their decreased density of cross-links. Our studies support a re-evaluation of the notion of functional redundancy among cytoskeletal regulatory proteins.     

Cooperation between actin-binding proteins of invasive Salmonella: SipA potentiates SipC nucleation and bundling of actin

Pathogen-induced remodelling of the host cell actin cytoskeleton drives internalization of invasive Salmonella by non-phagocytic intestinal epithelial cells. Two Salmonella actin-binding proteins are involved in internalization: SipC is essential for the process, while SipA enhances its efficiency. Using purified SipC and SipA proteins in in vitro assays of actin dynamics and F-actin bundling, we demonstrate that SipA stimulates substantially SipC-mediated nucleation of actin polymerization. SipA additionally enhances SipC-mediated F-actin bundling, and SipC-SipA collaboration generates stable networks of F-actin bundles. The data show that bacterial SipC and SipA cooperate to direct efficient modulation of actin dynamics, independently of host cell proteins. The ability of SipA to enhance SipC-induced reorganization of the actin cytoskeleton in vivo was confirmed using semi-permeabilized cultured mammalian cells.     

Development of a fluorescent F-actin blot overlay assay for detection of F-actin binding proteins

Interactions between cellular proteins and filamentous (F) actin are key to many cellular functions, e.g., cell motility, endocytosis, cell:cell adhesion, and cell:substrate adhesion. Previously, a functional assay using 125I-labeled F-actin to detect a subset of F-actin binding proteins by blot overlay was developed. We have modified this assay to use the fluorescent label, Alexa 488, in place of 125Iodine. The detection limit for Alexa 488-labeled actin using a Molecular Dynamics STORM 860 Fluorescence/PhosphorImager was as little as 100pg of labeled actin. The Alexa 488 F-actin assay detects the same proteins from Dictyostelium discoideum and with approximately the same sensitivity (approximately 10 microg/ml F-actin final concentration) as the analogous 125I-labeled F-actin blot overlay. The use of Alexa 488 F-actin for blot overlay assays requires no radioactive materials and generates no hazardous waste. Assays can be performed on the laboratory bench top and the blots imaged directly with a blue laser scanner, either wet or dry. In addition, the Alexa 488 fluorophore is highly resistant to photobleaching, does not decay, and may be stored frozen or lyophilized. Alexa 488 F-actin is a stable, cost-effective, nonhazardous probe used for rapid identification of a subset of F-actin binding proteins.     

Regulation of Actin Tension in Plant Cells by Kinases and Phosphatases

Changes in the organization and mechanical properties of the actin network within plant and animal cells are primary responses to cell signaling. These changes are suggested to be mediated through the regulation of G/F-actin equilibria, alterations in the amount and/or type of actin-binding proteins, the binding of myosin to F-actin, and the formation of myosin filaments associated with F-actin. In the present communication, the cell optical displacement assay was used to investigate the role of phosphatases and kinases in modifying the tension and organization within the actin network of soybean cells. The results from these biophysical measurements suggest that: (a) calcium-regulated kinases and phosphatases are involved in the regulation of tension, (b) calcium transients induce changes in the tension and organization of the actin network through the stimulation of proteins containing calmodulin-like domains or calcium/calmodulin-dependent regulatory proteins, (c) myosin and/or actin cross-linking proteins may be the principal regulator(s) of tension within the actin network, and (d) these actin cross-linking proteins may be the principal targets of calcium-regulated kinases and phosphatases.     

Regulation of the pollen-specific actin-depolymerizing factor LlADF1

Pollen tube growth is dependent on a dynamic actin cytoskeleton, suggesting that actin-regulating proteins are involved. We have examined the regulation of the lily pollen-specific actin-depolymerizing factor (ADF) LlADF1. Its actin binding and depolymerizing activity is pH sensitive, inhibited by certain phosphoinositides, but not controlled by phosphorylation. Compared with its F-actin binding properties, its low activity in depolymerization assays has been used to explain why pollen ADF decorates F-actin in pollen grains. This low activity is incompatible with a role in increasing actin dynamics necessary to promote pollen tube growth. We have identified a plant homolog of actin-interacting protein, AIP1, which enhances the depolymerization of F-actin in the presence of LlADF1 by approximately 60%. Both pollen ADF and pollen AIP1 bind F-actin in pollen grains but are mainly cytoplasmic in pollen tubes. Our results suggest that together these proteins remodel actin filaments as pollen grains enter and exit dormancy.     

Real-time monitoring of actin polymerization in living cells using split luciferase

Real-time monitoring of actin polymerization in living cells is beneficial for characterizing cellular activities such as migration, proliferation, and death. We developed new bioluminescence-based probe proteins that enable the monitoring of actin polymerization in living cells. Unlike other ordinary split luciferase probes, our probes were incorporated in endogenous actin filament that enabled it to measure the actin polymerization quantitatively. The probe proteins exhibited a dose-responsive decrease in photon emission intensity in response to the filamentous (F)-actin-disrupting agent latrunculin A. This technique has a high sensitivity with a high signal-to-noise ratio and is nontoxic compared with other methods of monitoring actin polymerization in living cells. Using this technique, we succeeded in monitoring the F-actin level in living cells during apoptosis progression induced by UV irradiation continuously for 12 h. F-actin was transiently upregulated after UV irradiation. Since UV-induced cell death was enhanced by treatment with latrunculin A during the period which F-actin is increased, transient upregulation of F-actin after UV is likely a protective reaction against UV-induced cell death. Our novel technique is an effective tool for investigating actin polymerization in living cells.     

Mechanisms responsible for F-actin stabilization after lysis of polymorphonuclear leukocytes

While actin polymerization and depolymerization are both essential for cell movement, few studies have focused on actin depolymerization. In vivo, depolymerization can occur exceedingly rapidly and in a spatially defined manner: the F-actin in the lamellipodia depolymerizes in 30 s after chemoattractant removal (Cassimeris, L., H. McNeill, and S. H. Zigmond. 1990. J. Cell Biol. 110:1067-1075). To begin to understand the regulation of F-actin depolymerization, we have examined F-actin depolymerization in lysates of polymorphonuclear leukocytes (PMNs). Surprisingly, much of the cell F-actin, measured with a TRITC-phalloidin-binding assay, was stable after lysis in a physiological salt buffer (0.15 M KCl): approximately 50% of the F-actin did not depolymerize even after 18 h. This stable F-actin included lamellar F-actin which could still be visualized one hour after lysis by staining with TRITC-phalloidin and by EM. We investigated the basis for this stability. In lysates with cell concentrations greater than 10(7) cells/ml, sufficient globular actin (G-actin) was present to result in a net increase in F-actin. However, the F-actin stability was not solely because of the presence of free G-actin since addition of DNase I to the lysate did not increase the F-actin loss. Nor did it appear to be because of barbed end capping factors since cell lysates provided sites for barbed end polymerization of exogenous added actin. The stable F-actin existed in a macromolecular complex that pelleted at low gravitational forces. Increasing the salt concentration of the lysis buffer decreased the amount of F-actin that pelleted at low gravitational forces and increased the amount of F-actin that depolymerized. Various actin-binding and cross-linking proteins such as tropomyosin, alpha-actinin, and actin-binding protein pelleted with the stable F-actin. In addition, we found that alpha-actinin, a filament cross-linking protein, inhibited the rate of pyrenyl F-actin depolymerization. These results suggested that actin cross-linking proteins may contribute to the stability of cellular actin after lysis. The activity of crosslinkers may be regulated in vivo to allow rapid turnover of lamellipodia F-actin.     

The bimodal role of filamin in controlling the architecture and mechanics of F-actin networks

Reconstituted actin filament networks have been used extensively to understand the mechanics of the actin cortex and decipher the role of actin cross-linking proteins in the maintenance and deformation of cell shape. However, studies of the mechanical role of the F-actin cross-linking protein filamin have led to seemingly contradictory conclusions, in part due to the use of ill-defined mechanical assays. Using quantitative rheological methods that avoid the pitfalls of previous studies, we systematically tested the complex mechanical response of reconstituted actin filament networks containing a wide range of filamin concentrations and compared the mechanical function of filamin with that of the cross-linking/bundling proteins alpha-actinin and fascin. At steady state and within a well defined linear regime of small non-destructive deformations, F-actin solutions behave as highly dynamic networks (actin polymers are still sufficiently mobile to relax the stress) below the cross-linking-to-bundling threshold filamin concentration, and they behave as covalently cross-linked gels above that threshold. Under large deformations, F-actin networks soften at low filamin concentrations and strain-harden at high filamin concentrations. Filamin cross-links F-actin into networks that are more resilient, stiffer, more solid-like, and less dynamic than alpha-actinin and fascin. These results resolve the controversy by showing that F-actin/filamin networks can adopt diametrically opposed rheological behaviors depending on the concentration in cross-linking proteins.     

Cingulin interacts with F-actin in vitro.

Cingulin, a M(r) 140-160 kDa protein of the cytoplasmic plaque of epithelial tight junctions (TJ), interacts in vitro with TJ proteins and myosin. Here we investigated cingulin interaction with actin, using His-tagged, full-length Xenopus laevis cingulin expressed in insect cells, and glutathione S-transferase (GST) fusion proteins of fragments of cingulin expressed in bacteria. Purified full-length cingulin co-pelleted with F-actin after high speed centrifugation, and promoted the sedimentation of F-actin under low speed centrifugation, suggesting that cingulin is an actin-cross-linking protein. The actin interaction of GST fusion proteins containing fragments of Xenopus cingulin suggested that the F-actin binding site is between residues 101 and 294.     

Structural insights into actin-binding,branching and bundling proteins

Structural advances in our understanding of the functions of the actin cytoskeleton have come from diverse sources. On the one hand, the determination of the structure of a bacterial actin-like protein MreB reveals the prokaryotic origins of the actin cytoskeleton, whereas on the other, cryo-electron microscopy and crystallography have yielded reconstructions of many actin crosslinking, regulatory and binding proteins in complex with F-actin. Not least, a high-resolution structure of the Arp2/3 complex and a reconstruction with F-actin provides considerable insight into the eukaryotic machinery, vital for the formation of new F-actin barbed ends, a prerequisite for rapid actin polymerisation involved in cell shape change and motility.     

Control of actin turnover by a salmonella invasion protein

Salmonella force their way into nonphagocytic host intestinal cells to initiate infection. Uptake is triggered by delivery into the target cell of bacterial effector proteins that stimulate cytoskeletal rearrangements and membrane ruffling. The Salmonella invasion protein A (SipA) effector is an actin binding protein that enhances uptake efficiency by promoting actin polymerization. SipA-bound actin filaments (F-actin) are also resistant to artificial disassembly in vitro. Using biochemical assays of actin dynamics and actin-based motility models, we demonstrate that SipA directly arrests cellular mechanisms of actin turnover. SipA inhibits ADF/cofilin-directed depolymerization both by preventing binding of ADF and cofilin and by displacing them from F-actin. SipA also protects F-actin from gelsolin-directed severing and reanneals gelsolin-severed F-actin fragments. These data suggest that SipA focuses host cytoskeletal reorganization by locally inhibiting both ADF/cofilin- and gelsolin-directed actin disassembly, while simultaneously stimulating pathogen-induced actin polymerization.     

The association of 14-3-3gamma and actin plays a role in cell division and apoptosis in astrocytes

The 14-3-3 protein family plays critical regulatory roles in signaling pathways in cell division and apoptosis. 14-3-3gamma is mainly expressed in brain. Using primary cultures of cerebral cortical astrocytes, we investigated the relationships between 14-3-3gamma proteins and actin in astrocytes in cell division and under ischemia. Our results showed that endogenous 14-3-3gamma proteins in immature astrocytes appeared filamentous and co-localized with filamentous actin (F-actin). During certain stages of mitosis, 14-3-3gamma proteins first aggregated and then formed a ring-like structure that surrounded the daughter nuclei and enclosed the F-actin. In 4-week-old cultures of astrocytes, 14-3-3gamma proteins appeared as punctate aggregates in the cytoplasm. Under ischemia, 14-3-3gamma proteins formed filamentous structures and were closely associated with F-actin in surviving astrocytes. However, in apoptotic astrocytes, the intensity of immunostaining of 14-3-3gamma proteins in the cytoplasm decreased. The proteins aggregated around the nucleus and dissociated from the actin filaments. Reciprocal co-immunoprecipitations demonstrated that endogenous 14-3-3gamma proteins bound to detergent-soluble actin and the level of binding increased after 4h of ischemia. As actin is a critical structural protein heavily involved in cell division and apoptotic death, our findings suggest that 14-3-3gamma proteins play a role in cytoskeletal function during the process of cell division and apoptosis in astrocytes in association with actin.     

Ena/VASP proteins enhance actin polymerization in the presence of barbed end capping proteins

Ena/VASP proteins influence the organization of actin filament networks within lamellipodia and filopodia of migrating cells and in actin comet tails. The molecular mechanisms by which Ena/VASP proteins control actin dynamics are unknown. We investigated how Ena/VASP proteins regulate actin polymerization at actin filament barbed ends in vitro in the presence and absence of barbed end capping proteins. Recombinant His-tagged VASP increased the rate of actin polymerization in the presence of the barbed end cappers, heterodimeric capping protein (CP), CapG, and gelsolin-actin complex. Profilin enhanced the ability of VASP to protect barbed ends from capping by CP, and this required interactions of profilin with G-actin and VASP. The VASP EVH2 domain was sufficient to protect barbed ends from capping, and the F-actin and G-actin binding motifs within EVH2 were required. Phosphorylation by protein kinase A at sites within the VASP EVH2 domain regulated anti-capping and F-actin bundling by VASP. We propose that Ena/VASP proteins associate at or near actin filament barbed ends, promote actin assembly, and restrict the access of barbed end capping proteins.     

MAP2c, but not tau, binds and bundles F-actin via its microtubule binding domain

BACKGROUND: MAP2 and tau are abundant microtubule-associated proteins (MAPs) in neurons. The development of neuronal dendrites and axons requires a dynamic interaction between microtubules and actin filaments. MAPs represent good candidates to mediate such interactions. Although MAP2c and tau have similar, well-characterized microtubule binding activities, their actin interaction is poorly understood. RESULTS: Here, we show by using a cosedimentation assay that MAP2c binds F-actin. Upon actin binding, MAP2c organizes F-actin into closely packed actin bundles. Moreover, we show by using a deletion approach that MAP2c's microtubule binding domain (MTBD) is both necessary and sufficient for both F-actin binding and bundling activities. Surprisingly, even though the MAP2 and tau MTBDs share high sequence homology and possess similar microtubule binding activities, tau is unable to bind or bundle F-actin. Furthermore, experiments with chimeric proteins demonstrate that the actin binding activity fully correlates with the ability to promote neurite initiation in neuroblastoma cells. CONCLUSIONS: These results provide the first demonstration that the MAP2c and tau MTBD domains exhibit distinct properties, diverging in actin binding and neurite initiation activities. These results implicate a novel actin function for MAP2c in neuronal morphogenesis and furthermore suggest that actin interactions could contribute to functional differences between MAP2 and tau in neurons.     

Inorganic phosphate regulates the binding of cofilin to actin filaments

Inorganic phosphate (Pi) and cofilin/actin depolymerizing factor proteins have opposite effects on actin filament structure and dynamics. Pi stabilizes the subdomain 2 in F-actin and decreases the critical concentration for actin polymerization. Conversely, cofilin enhances disorder in subdomain 2, increases the critical concentration, and accelerates actin treadmilling. Here, we report that Pi inhibits the rate, but not the extent of cofilin binding to actin filaments. This inhibition is also significant at physiological concentrations of Pi, and more pronounced at low pH. Cofilin prevents conformational changes in F-actin induced by Pi, even at high Pi concentrations, probably because allosteric changes in the nucleotide cleft decrease the affinity of Pi to F-actin. Cofilin induced allosteric changes in the nucleotide cleft of F-actin are also indicated by an increase in fluorescence emission and a decrease in the accessibility of etheno-ADP to collisional quenchers. These changes transform the nucleotide cleft of F-actin to G-actin-like. Pi regulation of cofilin binding and the cofilin regulation of Pi binding to F-actin can be important aspects of actin based cell motility.     

Mutational analysis of an actin-binding site of cofilin and characterization of chimeric proteins between cofilin and destrin.

Cofilin and destrin are two related low molecular weight mammalian actin-binding proteins. Cofilin is an F-actin side-binding and pH-dependent actin-depolymerizing protein, and destrin is a pH-independent actin-depolymerizing protein. We have introduced a few point mutations within an actin-binding sequence of cofilin. Biochemical analyses of these mutant proteins have clearly shown that Lys112 and Lys114 of cofilin are crucially but differently involved in its interaction with actin and phosphatidylinositol 4,5-bisphosphate. This is the first example among actin-binding proteins whose point mutations inactivate their interaction with actin in vitro. We have also made and characterized a series of chimeric proteins between cofilin and destrin to identify the regions responsible for the pH dependence and the F-actin side binding activity of cofilin. Our results suggest that a central region consisting of 42 amino acid residues and a carboxyl-terminal quarter of cofilin are both involved in regulation of the pH-dependent actin depolymerizing activity and the activity to bind along F-actin.     

Cucumber Mosaic Virus Movement Protein Severs Actin Filaments to Increase the Plasmodesmal Size Exclusion Limit in Tobacco

Plant viral movement proteins (MPs) enable viruses to pass through cell walls by increasing the size exclusion limit (SEL) of plasmodesmata (PD). Here, we report that the ability of Cucumber mosaic virus (CMV) MP to increase the SEL of the PD could be inhibited by treatment with the actin filament (F-actin)–stabilizing agent phalloidin but not by treatment with the F-actin–destabilizing agent latrunculin A. In vitro studies showed that CMV MP bound globular and F-actin, inhibited actin polymerization, severed F-actin, and participated in plus end capping of F-actin. Analyses of two CMV MP mutants, one with and one without F-actin severing activities, demonstrated that the F-actin severing ability was required to increase the PD SEL. Furthermore, the Tobacco mosaic virus MP also exhibited F-actin severing activity, and its ability to increase the PD SEL was inhibited by treatment with phalloidin. Our data provide evidence to support the hypothesis that F-actin severing is required for MP-induced increase in the SEL of PD. This may have broad implications in the study of the mechanisms of actin dynamics that regulate cell-to-cell transport of viral and endogenous proteins.