Arabidopsis Actin-Depolymerizing Factor AtADF4 Mediates Defense Signal Transduction Triggered by the Pseudomonas syringae Effector AvrPphB

The actin cytoskeleton has been implicated in plant defenses against pathogenic fungi and oomycetes with limited, indirect evidence. To date, there are no reports linking actin with resistance against phytopathogenic bacteria. The dynamic behavior of actin filaments is regulated by a diverse array of actin-binding proteins, among which is the Actin-Depolymerizing Factor (ADF) family of proteins. Here, we demonstrate that actin dynamics play a role in the activation of gene-for-gene resistance in Arabidopsis (Arabidopsis thaliana) following inoculation with the phytopathogenic bacterium Pseudomonas syringae pv tomato. Using a reverse genetics approach, we explored the roles of Arabidopsis ADFs in plant defenses. AtADF4 was identified as being specifically required for resistance triggered by the effector AvrPphB but not AvrRpt2 or AvrB. Recombinant AtADF4 bound to monomeric actin (G-actin) with a marked preference for the ADP-loaded form and inhibited the rate of nucleotide exchange on G-actin, indicating that AtADF4 is a bona fide actin-depolymerizing factor. Exogenous application of the actin-disrupting agent cytochalasin D partially rescued the Atadf4 mutant in the AvrPphB-mediated hypersensitive response, demonstrating that AtADF4 mediates defense signaling through modification of the actin cytoskeleton. Unlike the mechanism by which the actin cytoskeleton confers resistance against fungi and oomycetes, AtADF4 is not involved in resistance against pathogen entry. Collectively, this study identifies AtADF4 as a novel component of the plant defense signaling pathway and provides strong evidence for actin dynamics as a primary component that orchestrates plant defenses against P. syringae.The actin cytoskeleton has been implicated in plant defenses against pathogenic fungi and oomycetes (Hardham et al., 2007). Evidence largely comes from studies using actin cytoskeleton-disrupting agents, such as cytochalasins. Treatments with a variety of cytochalasins were shown to increase the penetration rate of both adapted and nonadapted pathogens in multiple plant-pathogen systems, thereby implicating the actin cytoskeleton as having a role in basal defenses and nonhost resistance (Kobayashi et al., 1997; Yun et al., 2003; Shimada et al., 2006; Miklis et al., 2007). The actin cytoskeleton may also play a role in race-specific resistance (Skalamera and Heath, 1998). To date, no reports linking actin dynamics with resistance against phytopathogenic bacteria have been published.While the actin cytoskeleton as a virulence target of plant pathogens has not been documented, it was well characterized in mammalian pathosystems, particularly in studies investigating macrophage interactions with the pathogenic bacterium Yersinia pestis (Mattoo et al., 2007). Yersinia species deliver a suite of effectors into the target host cell, and at least four of them (YopE, YpkA/YopO, YopT, and YopH) are involved in rearrangement of the actin cytoskeleton (Aepfelbacher and Heesemann, 2001). YopT, a Cys protease, targets a plasma membrane-localized Rho GTPase in affected phagocytes (Aepfelbacher and Heesemann, 2001). Cleavage of the GTPase by YopT releases the prenylated protein from the plasma membrane and disrupts the actin cytoskeleton, effectively shutting down phagocytosis, preventing elimination of the pathogen (Iriarte and Cornelis, 1998; Shao et al., 2002). Similarly, microbial pathogens also usurp host processes for the benefit of infection, disease, and death. Listeria species hijack the host''s cytoskeleton to move around inside the infected cell through the induction of directed polymerization of actin (Pistor et al., 1994). Salmonella injects into host cells two actin-binding proteins (SipA and SipC) as well as other regulators of actin dynamics to enhance phagocytic uptake and intracellular propagation (Galan and Zhou, 2000). In short, either by preventing polymerization or by promoting it, pathogens have evolved strategies to modify the host actin cytoskeleton for purposes of evading detection or eliciting disease and death.Dynamic actin cytoskeleton rearrangements are regulated by a pool of actin-binding proteins, which sense environmental changes and modulate the cytoskeleton through various biochemical activities (Hussey et al., 2006; Staiger and Blanchoin, 2006). Among the proteins that regulate these dynamic processes are the Actin-Depolymerizing Factor (ADF) family of proteins (Maciver and Hussey, 2002). In general, ADFs bind both monomeric (G-) and filamentous (F-) actin to increase actin dynamics. They function by severing F-actin to generate more ends for polymerization and by increasing the dissociation rate of actin monomers from the pointed ends (Maciver, 1998; Maciver and Hussey, 2002). Plant ADFs play roles in pollen tube growth (Chen et al., 2003), root formation (Thomas and Schiefelbein, 2002), and cold acclimation (Ouellet et al., 2001). There is also one report linking ADFs with plant defenses (Miklis et al., 2007). In that study, ectopic expression of barley (Hordeum vulgare) HvADF3 and several isovariants of Arabidopsis (Arabidopsis thaliana) ADFs in barley epidermal cells was shown to compromise penetration resistance to powdery mildew fungi (Miklis et al., 2007).The Arabidopsis-Pseudomonas syringae interaction provides an ideal model plant-pathogen system to study plant defense signaling. Like Yersinia species, P. syringae delivers effector proteins into the host cells via the type III secretion system and relies on these proteins for pathogenesis (Alfano and Collmer, 2004). However, once these proteins (Avr) are recognized either directly or indirectly by plant resistance (R) proteins, plant immune responses are activated (Jones and Dangl, 2006). Exciting progress has been made toward understanding the indirect recognition of several pairs of Avr-R proteins; the best examples include AvrB/AvrRPM1-RPM1, AvrRpt2-RPS2, and AvrPphB-RPS5. During activation of defense mediated by AvrB/AvrRPM1-RPM1 and AvrRpt2-RPS2, the phosphorylation or elimination of a third protein, RIN4, is essential (Mackey et al., 2002; Axtell and Staskawicz, 2003). In the case of AvrPphB-RPS5 recognition, the AvrPphB Cys protease of the same family as YopT (Shao et al., 2002) cleaves the plant protein kinase PBS1, inducing a conformational change in RPS5, which in turn leads to the activation of resistance (Ade et al., 2007). Although these studies have greatly enhanced our understanding of how pathogen effectors initiate plant defense responses, the ultimate signaling processes associated with the activation of resistance remain largely unknown, due to the limited number of genetic loci identified in these pathways. In this work, we hypothesize that actin-binding proteins play a role during plant-bacteria interactions based on the functional and structural similarity between AvrPphB and YopT.There are 11 ADFs in the Arabidopsis genome (Ruzicka et al., 2007). We utilized a reverse genetics approach to identify the putative roles these proteins play in plant resistance against the bacterial pathogen P. syringae pv tomato (Pst). AtADF4 was identified as a novel signaling component in the AvrPphB-RPS5-mediated defense signal transduction pathway. Loss of AtADF4 confers on Arabidopsis enhanced susceptibility to P. syringae expressing AvrPphB. Further subcellular localization and biochemical analyses, as well as pharmacological studies, suggest that AtADF4 functions as a bona fide actin-depolymerizing factor through modifying the actin cytoskeleton. Unlike the documented mechanism by which the actin cytoskeleton plays roles in resistance against fungi and oomycetes, the resistance against P. syringae mediated by AtADF4 is not involved in hindering pathogen entry.     

Arabidopsis ACTIN-DEPOLYMERIZING FACTOR7 Severs Actin Filaments and Regulates Actin Cable Turnover to Promote Normal Pollen Tube Growth

Actin filaments are often arranged into higher-order structures, such as the longitudinal actin cables that generate the reverse fountain cytoplasmic streaming pattern present in pollen tubes. While several actin binding proteins have been implicated in the generation of these cables, the mechanisms that regulate their dynamic turnover remain largely unknown. Here, we show that Arabidopsis thaliana ACTIN-DEPOLYMERIZING FACTOR7 (ADF7) is required for turnover of longitudinal actin cables. In vitro biochemical analyses revealed that ADF7 is a typical ADF that prefers ADP-G-actin over ATP-G-actin. ADF7 inhibits nucleotide exchange on actin and severs filaments, but its filament severing and depolymerizing activities are less potent than those of the vegetative ADF1. ADF7 primarily decorates longitudinal actin cables in the shanks of pollen tubes. Consistent with this localization pattern, the severing frequency and depolymerization rate of filaments significantly decreased, while their maximum lifetime significantly increased, in adf7 pollen tube shanks. Furthermore, an ADF7–enhanced green fluorescent protein fusion with defective severing activity but normal G-actin binding activity could not complement adf7, providing compelling evidence that the severing activity of ADF7 is vital for its in vivo functions. These observations suggest that ADF7 evolved to promote turnover of longitudinal actin cables by severing actin filaments in pollen tubes.     

Arabidopsis CDPK6 phosphorylates ADF1 at N-terminal serine 6 predominantly

Key message

We found that Arabidopsis AtADF1 was phosphorylated by AtCDPK6 at serine 6 predominantly and the phosphoregulation plays a key role in the regulation of ADF1-mediated depolymerization of actin filaments.

Abstract

Since actin-depolymerizing factor (ADF) is highly conserved among eukaryotes, it is one of the key modulators for actin organization. In plants, ADF is directly involved in the depolymerization of actin filaments, and therefore important for F-actin-dependent cellular activities. The activity of ADF is tightly controlled through a number of molecular mechanisms, including phosphorylation-mediated inactivation of ADF. To investigate Arabidopsis ADF1 phosphoregulation, we generated AtADF1 phosphorylation site-specific mutants. Using transient expression and stable transgenic approaches, we analyzed the ADF1 phosphorylation mutants in the regulation of actin filament organizations in plant cells. By in vitro phosphorylation assay, we showed that AtADF1 is phosphorylated by AtCDPK6 at serine 6 predominantly. Chemically induced expression of AtCDPK6 can negatively regulate the wild-type AtADF1 in depolymerizing actin filaments, but not those of the mutants AtADF1(S6A) and AtADF1(S6D). These results demonstrate a regulatory function of Arabidopsis CDPK6 in the N-terminal phosphorylation of AtADF1.     

A Yersinia effector and a Pseudomonas avirulence protein define a family of cysteine proteases functioning in bacterial pathogenesis

A Yersinia effector known as YopT and a Pseudomonas avirulence protein known as AvrPphB define a family of 19 proteins involved in bacterial pathogenesis. We show that both YopT and AvrPphB are cysteine proteases, and their proteolytic activities are dependent upon the invariant C/H/D residues conserved in the entire YopT family. YopT cleaves the posttranslationally modified Rho GTPases near their carboxyl termini, releasing them from the membrane. This leads to the disruption of actin cytoskeleton in host cells. The proteolytic activity of AvrPphB is essential for autoproteolytic cleavage of an AvrPphB precursor as well as for eliciting the hypersensitive response in plants. These findings provide new insights into mechanisms of animal and plant pathogenesis.     

A novel viral strategy for host factor recruitment: The co-opted proteasomal Rpn11 protein interaction hub in cooperation with subverted actin filaments are targeted to deliver cytosolic host factors for viral replication

Positive-strand (+)RNA viruses take advantage of the host cells by subverting a long list of host protein factors and transport vesicles and cellular organelles to build membranous viral replication organelles (VROs) that support robust RNA replication. How RNA viruses accomplish major recruitment tasks of a large number of cellular proteins are intensively studied. In case of tomato bushy stunt virus (TBSV), a single viral replication protein, named p33, carries out most of the recruitment duties. Yet, it is currently unknown how the viral p33 replication protein, which is membrane associated, is capable of the rapid and efficient recruitment of numerous cytosolic host proteins to facilitate the formation of large VROs. In this paper, we show that, TBSV p33 molecules do not recruit each cytosolic host factor one-by-one into VROs, but p33 targets a cytosolic protein interaction hub, namely Rpn11, which interacts with numerous other cytosolic proteins. The highly conserved Rpn11, called POH1 in humans, is the metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates. However, TBSV takes advantage of a noncanonical function of Rpn11 by exploiting Rpn11’s interaction with highly abundant cytosolic proteins and the actin network. We provide supporting evidence that the co-opted Rpn11 in coordination with the subverted actin network is used for delivering cytosolic proteins, such as glycolytic and fermentation enzymes, which are readily subverted into VROs to produce ATP locally in support of VRO formation, viral replicase complex assembly and viral RNA replication. Using several approaches, including knockdown of Rpn11 level, sequestering Rpn11 from the cytosol into the nucleus in plants or temperature-sensitive mutation in Rpn11 in yeast, we show the inhibition of recruitment of glycolytic and fermentation enzymes into VROs. The Rpn11-assisted recruitment of the cytosolic enzymes by p33, however, also requires the combined and coordinated role of the subverted actin network. Accordingly, stabilization of the actin filaments by expression of the Legionella VipA effector in yeast and plant, or via a mutation of ACT1 in yeast resulted in more efficient and rapid recruitment of Rpn11 and the selected glycolytic and fermentation enzymes into VROs. On the contrary, destruction of the actin filaments via expression of the Legionella RavK effector led to poor recruitment of Rpn11 and glycolytic and fermentation enzymes. Finally, we confirmed the key roles of Rpn11 and the actin filaments in situ ATP production within TBSV VROs via using a FRET-based ATP-biosensor. The novel emerging theme is that TBSV targets Rpn11 cytosolic protein interaction hub driven by the p33 replication protein and aided by the subverted actin filaments to deliver several co-opted cytosolic pro-viral factors for robust replication within VROs.     

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.     

Disassembly activity of actin-depolymerizing factor (ADF) is associated with distinct cellular processes in apicomplexan parasites

Proteins of the actin-depolymerizing factor (ADF)/cofilin family have been shown to be crucial for the motility and survival of apicomplexan parasites. However, the mechanisms by which ADF proteins fulfill their function remain poorly understood. In this study, we investigate the comparative activities of ADF proteins from Toxoplasma gondii and Plasmodium falciparum, the human malaria parasite, using a conditional T. gondii ADF-knockout line complemented with ADF variants from either species. We show that P. falciparum ADF1 can fully restore native TgADF activity, demonstrating functional conservation between parasites. Strikingly, mutation of a key basic residue (Lys-72), previously implicated in disassembly in PfADF1, had no detectable phenotypic effect on parasite growth, motility, or development. In contrast, organelle segregation was severely impaired when complementing with a TgADF mutant lacking the corresponding residue (Lys-68). Biochemical analyses of each ADF protein confirmed the reduced ability of lysine mutants to mediate actin depolymerization via filament disassembly although not severing, in contrast to previous reports. These data suggest that actin filament disassembly is essential for apicomplexan parasite development but not for motility, as well as pointing to genus-specific coevolution between ADF proteins and their native actin.     

Toxoplasma gondii Actin Depolymerizing Factor Acts Primarily to Sequester G-actin

Toxoplasma gondii is a protozoan parasite belonging to the phylum Apicomplexa. Parasites in this phylum utilize a unique process of motility termed gliding, which is dependent on parasite actin filaments. Surprisingly, 98% of parasite actin is maintained as G-actin, suggesting that filaments are rapidly assembled and turned over. Little is known about the regulated disassembly of filaments in the Apicomplexa. In higher eukaryotes, the related actin depolymerizing factor (ADF) and cofilin proteins are essential regulators of actin filament turnover. ADF is one of the few actin-binding proteins conserved in apicomplexan parasites. In this study we examined the mechanism by which T. gondii ADF (TgADF) regulates actin filament turnover. Unlike other members of the ADF/cofilin (AC) family, apicomplexan ADFs lack key F-actin binding sites. Surprisingly, this promotes their enhanced disassembly of actin filaments. Restoration of the C-terminal F-actin binding site to TgADF stabilized its interaction with filaments but reduced its net filament disassembly activity. Analysis of severing activity revealed that TgADF is a weak severing protein, requiring much higher concentrations than typical AC proteins. Investigation of TgADF interaction with T. gondii actin (TgACT) revealed that TgADF disassembled short TgACT oligomers. Kinetic and steady-state polymerization assays demonstrated that TgADF has strong monomer-sequestering activity, inhibiting TgACT polymerization at very low concentrations. Collectively these data indicate that TgADF promoted the efficient turnover of actin filaments via weak severing of filaments and strong sequestering of monomers. This suggests a dual role for TgADF in maintaining high G-actin concentrations and effecting rapid filament turnover.     

Inhibitory Mechanism of FAT4 Gene Expression in Response to Actin Dynamics during Src-Induced Carcinogenesis

Oncogenic transformation is characterized by morphological changes resulting from alterations in actin dynamics and adhesive activities. Emerging evidence suggests that the protocadherin FAT4 acts as a tumor suppressor in humans, and reduced FAT4 gene expression has been reported in breast and lung cancers and melanoma. However, the mechanism controlling FAT4 gene expression is poorly understood. In this study, we show that transient activation of the Src oncoprotein represses FAT4 mRNA expression through actin depolymerization in the immortalized normal human mammary epithelial cell line MCF-10A. Src activation causes actin depolymerization via the MEK/Erk/Cofilin cascade. The MEK inhibitor U0126 blocks the inhibitory effect of Src on FAT4 mRNA expression and Src-induced actin depolymerization. To determine whether actin dynamics act on the regulation of FAT4 mRNA expression, we treated MCF-10A cells with the ROCK inhibitor Y-27632. Y-27632 treatment decreased FAT4 mRNA expression. This suppressive effect was blocked by siRNA-mediated knockdown of Cofilin1. Furthermore, simultaneous administration of Latrunculin A (an actin depolymerizing agent), Y-27632, and Cofilin1 siRNA to the cells resulted in a marked reduction of FAT4 mRNA expression. Intriguingly, we also found that FAT4 mRNA expression was reduced under both low cell density and low stiffness conditions, which suggests that mechanotransduction affects FAT4 mRNA expression. Additionally, we show that siRNA-mediated FAT4 knockdown induced the activity of the Hippo effector YAP/TAZ in MCF-10A cells. Taken together, our results reveal a novel inhibitory mechanism of FAT4 gene expression through actin depolymerization during Src-induced carcinogenesis in human breast cells.     

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.     

ADF2 is required for transformation of the ookinete and sporozoite in malaria parasite development

Malaria parasites undergo two rounding-up transformations in their life cycle: the ookinete-to-oocyst transformation in the mosquito midgut, and the sporozoite-to-EEF (exo-erythrocytic form) differentiation in the host hepatocyte. Both events are characterized by the loss of polarity, implying that cytoskeletal reorganization is involved. In other eukaryotes, regulation of the actin skeleton is fundamental to subcellular remodeling. Although filamentous actin is well known to be involved in the motility of apicomplexan parasites, its participation in their morphological regulation is still largely unexplored. Here we describe the fundamental role of Actin depolymerization factor 2 (ADF2), a vector-stage-specific ADF isoform, in morphological changes accompanying the parasite life cycle. Among protozoan parasites, Plasmodium is unique in having two actin and two ADF genes. Disruption of the ADF2 gene in the rodent malaria parasite P. berghei had no effect on ookinete development or its subsequent invasion of the midgut. However, both the ookinete-to-oocyst and sporozoite-to-EEF transformations showed significant defects. These results indicated that Plasmodium ADF2 plays a pivotal role in transformation in the malaria parasite life cycle.     

Defects in actin dynamics lead to an autoinflammatory condition through the upregulation of CXCL5

Background

Destrin (DSTN) is a member of the ADF/cofilin family of proteins and is an important regulator of actin dynamics. The primary function of destrin is to depolymerize filamentous actin into its monomeric form and promote filament severing. While progress has been made in understanding the biochemical functions of the ADF/cofilin proteins, the study of an animal model for cells deficient for DSTN provides an opportunity to investigate the physiological processes regulated by proper actin dynamics in vivo. A spontaneous mouse mutant, corneal disease 1(corn1), is deficient for DSTN, which causes epithelial hyperproliferation and neovascularization in the cornea. Dstn^corn1 mice exhibit an actin dynamics defect in the cornea as evidenced by the formation of actin stress fibers in the epithelial cells. Previously, we observed a significant infiltration of leukocytes into the cornea of Dstn^corn1 mice as well as the upregulation of proinflammatory molecules. In this study, we sought to characterize this inflammatory condition and explore the physiological mechanism through which a loss of Dstn function leads to inflammation.

Methodology/Principal Findings

Through immunofluorescent analyses, we observed a significant recruitment of neutrophils and macrophages to the Dstn^corn1 cornea, demonstrating that the innate immune system is spontaneously activated in this mutant. The inflammatory chemokine, CXCL5, was ectopically expressed in the corneal epithelial cells of Dstn^corn1 mice, and targeting of the receptor for this chemokine inhibited neutrophil recruitment. An inflammatory reaction was not observed in the cornea of allelic mutant strain, Dstn^corn1-2J, which has a milder defect in actin dynamics in the corneal epithelial cells.

Conclusions/Significance

This study shows that severe defects in actin dynamics lead to an autoinflammatory condition that is mediated by the expression of CXC chemokines.     

Arabidopsis AtADF1 is Functionally Affected by Mutations on Actin Binding Sites

The plant actin depolymerizing factor (ADF) binds to both monomeric and filamentous actin, and is directly involved in the depolymerization of actin filaments. To better understand the actin binding sites of the Arabidopsis thaliana L. AtADF1, we generated mutants of AtADF1 and investigated their functions in vitro and in vivo. Analysis of mutants harboring amino acid substitutions revealed that charged residues (Arg98 and Lys100) located at the α‐helix 3 and forming an actin binding site together with the N‐terminus are essential for both G‐ and F‐actin binding. The basic residues on the β‐strand 5 (K82/A) and the α‐helix 4 (R135/A, R137/A) form another actin binding site that is important for F‐actin binding. Using transient expression of CFP‐tagged AtADF1 mutant proteins in onion (Allium cepa) peel epidermal cells and transgenic Arabidopsis thaliana L. plants overexpressing these mutants, we analyzed how these mutant proteins regulate actin organization and affect seedling growth. Our results show that the ADF mutants with a lower affinity for actin filament binding can still be functional, unless the affinity for actin monomers is also affected. The G‐actin binding activity of the ADF plays an essential role in actin binding, depolymerization of actin polymers, and therefore in the control of actin organization.     

The Legionella pneumophila Effector VipA Is an Actin Nucleator That Alters Host Cell Organelle Trafficking

Legionella pneumophila, the causative agent of Legionnaires'' disease, invades and replicates within macrophages and protozoan cells inside a vacuole. The type IVB Icm/Dot secretion system is necessary for the translocation of effector proteins that modulate vesicle trafficking pathways in the host cell, thus avoiding phagosome-lysosome fusion. The Legionella VipA effector was previously identified by its ability to interfere with organelle trafficking in the Multivesicular Body (MVB) pathway when ectopically expressed in yeast. In this study, we show that VipA binds actin in vitro and directly polymerizes microfilaments without the requirement of additional proteins, displaying properties distinct from other bacterial actin nucleators. Microscopy studies revealed that fluorescently tagged VipA variants localize to puncta in eukaryotic cells. In yeast these puncta are associated with actin-rich regions and components of the Multivesicular Body pathway such as endosomes and the MVB-associated protein Bro1. During macrophage infection, native translocated VipA associated with actin patches and early endosomes. When ectopically expressed in mammalian cells, VipA-GFP displayed a similar distribution ruling out the requirement of additional effectors for binding to its eukaryotic targets. Interestingly, a mutant form of VipA, VipA-1, that does not interfere with organelle trafficking is also defective in actin binding as well as association with early endosomes and shows a homogeneous cytosolic localization. These results show that the ability of VipA to bind actin is related to its association with a specific subcellular location as well as its role in modulating organelle trafficking pathways. VipA constitutes a novel type of actin nucleator that may contribute to the intracellular lifestyle of Legionella by altering cytoskeleton dynamics to target host cell pathways.     

RNAi depleted Drosophila cell extracts to dissect signaling pathways leading to actin polymerization

Dissection of signal transduction pathways leading to actin polymerization has been performed in cytosolic extracts. In such assays, the implication of an effector molecule is demonstrated by the loss of actin polymerization upon its depletion and the restoration of actin polymerization upon its add-back. Two major limitations in the wide use of this approach have been the availability of immunodepleting antibodies and the functional redundancy for many classes of effector molecules encoded by vertebrate genomes. To circumvent these limitations, we developed extracts derived from S2 Drosophila cells, which are competent for actin polymerization. In this system, depleted extracts are simply obtained from cells cultured with long double stranded RNAs in the medium. We validated the method by showing that beads coated with the C-terminal domain of Wave2 were no longer able to trigger actin polymerization in an extract depleted of the Arp2/3 complex. We also examined the complete set of Drosophila small GTPases of the Rho family for their ability to polymerize actin in such extracts, and found that only dCdc42 was able to induce actin polymerization. Using RNAi depleted extract, we confirmed that dCdc42 triggers actin polymerization in a Wasp dependent manner.