Heterodimeric capping protein from Arabidopsis is regulated by phosphatidic acid

The cytoskeleton is a key regulator of morphogenesis, sexual reproduction, and cellular responses to extracellular stimuli. Changes in the cellular architecture are often assumed to require actin-binding proteins as stimulus-response modulators, because many of these proteins are regulated directly by binding to intracellular second messengers or signaling phospholipids. Phosphatidic acid (PA) is gaining widespread acceptance as a major, abundant phospholipid in plants that is required for pollen tube tip growth and mediates responses to osmotic stress, wounding, and phytohormones; however, the number of identified effectors of PA is rather limited. Here we demonstrate that exogenous PA application leads to significant increases in filamentous actin levels in Arabidopsis suspension cells and poppy pollen grains. To investigate further these lipid-induced changes in polymer levels, we analyzed the properties of a key regulator of actin filament polymerization, the heterodimeric capping protein from Arabidopsis thaliana (AtCP). AtCP binds to PA with a K(d) value of 17 muM and stoichiometry of approximately 1:2. It also binds well to PtdIns(4,5)P(2), but not to several other phosphoinositide or acidic phospholipids. The interaction with PA inhibited the actin-binding activity of CP. In the presence of PA, CP is unable to block the barbed or rapidly growing and shrinking end of actin filaments. Precapped filament barbed ends can also be uncapped by addition of PA, allowing rapid filament assembly from an actin monomer pool that is buffered with profilin. The findings support a model in which the inhibition of CP activity in cells by elevated PA results in the stimulation of actin polymerization from a large pool of profilin-actin. Such regulation may be important for the response of plant cells to extracellular stimuli as well as for the normal process of pollen tube tip growth.     

Structural Insights into the Inhibition of Actin-Capping Protein by Interactions with Phosphatidic Acid and Phosphatidylinositol (4,5)-Bisphosphate

The actin cytoskeleton is a dynamic structure that coordinates numerous fundamental processes in eukaryotic cells. Dozens of actin-binding proteins are known to be involved in the regulation of actin filament organization or turnover and many of these are stimulus-response regulators of phospholipid signaling. One of these proteins is the heterodimeric actin-capping protein (CP) which binds the barbed end of actin filaments with high affinity and inhibits both addition and loss of actin monomers at this end. The ability of CP to bind filaments is regulated by signaling phospholipids, which inhibit the activity of CP; however, the exact mechanism of this regulation and the residues on CP responsible for lipid interactions is not fully resolved. Here, we focus on the interaction of CP with two signaling phospholipids, phosphatidic acid (PA) and phosphatidylinositol (4,5)-bisphosphate (PIP₂). Using different methods of computational biology such as homology modeling, molecular docking and coarse-grained molecular dynamics, we uncovered specific modes of high affinity interaction between membranes containing PA/phosphatidylcholine (PC) and plant CP, as well as between PIP₂/PC and animal CP. In particular, we identified differences in the binding of membrane lipids by animal and plant CP, explaining previously published experimental results. Furthermore, we pinpoint the critical importance of the C-terminal part of plant CPα subunit for CP–membrane interactions. We prepared a GST-fusion protein for the C-terminal domain of plant α subunit and verified this hypothesis with lipid-binding assays in vitro.     

Arabidopsis actin depolymerizing factor4 modulates the stochastic dynamic behavior of actin filaments in the cortical array of epidermal cells

Actin filament arrays are constantly remodeled as the needs of cells change as well as during responses to biotic and abiotic stimuli. Previous studies demonstrate that many single actin filaments in the cortical array of living Arabidopsis thaliana epidermal cells undergo stochastic dynamics, a combination of rapid growth balanced by disassembly from prolific severing activity. Filament turnover and dynamics are well understood from in vitro biochemical analyses and simple reconstituted systems. However, the identification in living cells of the molecular players involved in controlling actin dynamics awaits the use of model systems, especially ones where the power of genetics can be combined with imaging of individual actin filaments at high spatial and temporal resolution. Here, we test the hypothesis that actin depolymerizing factor (ADF)/cofilin contributes to stochastic filament severing and facilitates actin turnover. A knockout mutant for Arabidopsis ADF4 has longer hypocotyls and epidermal cells when compared with wild-type seedlings. This correlates with a change in actin filament architecture; cytoskeletal arrays in adf4 cells are significantly more bundled and less dense than in wild-type cells. Several parameters of single actin filament turnover are also altered. Notably, adf4 mutant cells have a 2.5-fold reduced severing frequency as well as significantly increased actin filament lengths and lifetimes. Thus, we provide evidence that ADF4 contributes to the stochastic dynamic turnover of actin filaments in plant cells.     

Conserved interactions with cytoskeletal but not signaling elements are an essential aspect of Drosophila WASp function

Wiskott-Aldrich Syndrome proteins (WASp) serve as important regulators of cytoskeletal organization and function. These modular proteins, which are well-conserved among eukaryotic species, act to promote actin filament assembly in response to cues from various signal transduction pathways. Genetic analysis has revealed a requirement for the single Drosophila homolog, Wasp (Wsp), in cell-fate decisions governing specific neuronal lineages. We have used this unique developmental context to assess the contributions of established signaling and cytoskeletal partners of WASp. We present biochemical and genetic evidence that, as expected, Drosophila Wsp performs its developmental role via the Arp2/3 complex, indicating conservation of the cytoskeletal aspect of Wsp function in vivo. In contrast, we find that association with the key signaling molecules CDC42 and PIP2 is not an essential requirement, implying that activation of Wsp function in vivo depends on additional or alternative signaling pathways.     

Doing (F/L)PPPPs: EVH1 domains and their proline-rich partners in cell polarity and migration.

Actin filament assembly is a tightly regulated process that functions in many aspects of cell physiology. Members of the Ena/VASP (Drosophila Enabled/vasodilator-stimulated phosphoprotein) family are key players in regulating actin filament assembly, in many cases through their association with binding partners that display a particular proline-rich motif, FPPPP. Ena/VASP proteins interact with these partners via the highly conserved Ena/VASP homology 1 (EVH1) domain. The diverse array of binding partners for EVH1 domains, including cytoskeletal proteins such as zyxin, transmembrane guidance receptors such as Roundabout, and the T-cell signaling protein Fyb/SLAP, shows that these interactions are likely to be important in a number of cellular processes that require regulated actin filament assembly.     

A gelsolin-like protein from Papaver rhoeas pollen (PrABP80) stimulates calcium-regulated severing and depolymerization of actin filaments

The cytoskeleton is a key regulator of plant morphogenesis, sexual reproduction, and cellular responses to extracellular stimuli. During the self-incompatibility response of Papaver rhoeas L. (field poppy) pollen, the actin filament network is rapidly depolymerized by a flood of cytosolic free Ca2+ that results in cessation of tip growth and prevention of fertilization. Attempts to model this dramatic cytoskeletal response with known pollen actin-binding proteins (ABPs) revealed that the major G-actin-binding protein profilin can account for only a small percentage of the measured depolymerization. We have identified an 80-kDa, Ca(2+)-regulated ABP from poppy pollen (PrABP80) and characterized its biochemical properties in vitro. Sequence determination by mass spectrometry revealed that PrABP80 is related to gelsolin and villin. The molecular weight, lack of filament cross-linking activity, and a potent severing activity are all consistent with PrABP80 being a plant gelsolin. Kinetic analysis of actin assembly/disassembly reactions revealed that substoichiometric amounts of PrABP80 can nucleate actin polymerization from monomers, block the assembly of profilin-actin complex onto actin filament ends, and enhance profilin-mediated actin depolymerization. Fluorescence microscopy of individual actin filaments provided compelling, direct evidence for filament severing and confirmed the actin nucleation and barbed end capping properties. This is the first direct evidence for a plant gelsolin and the first example of efficient severing by a plant ABP. We propose that PrABP80 functions at the center of the self-incompatibility response by creating new filament pointed ends for disassembly and by blocking barbed ends from profilin-actin assembly.     

Biological role and structural mechanism of twinfilin-capping protein interaction

Twinfilin and capping protein (CP) are highly conserved actin-binding proteins that regulate cytoskeletal dynamics in organisms from yeast to mammals. Twinfilin binds actin monomer, while CP binds the barbed end of the actin filament. Remarkably, twinfilin and CP also bind directly to each other, but the mechanism and role of this interaction in actin dynamics are not defined. Here, we found that the binding of twinfilin to CP does not affect the binding of either protein to actin. Furthermore, site-directed mutagenesis studies revealed that the CP-binding site resides in the conserved C-terminal tail region of twinfilin. The solution structure of the twinfilin-CP complex supports these conclusions. In vivo, twinfilin's binding to both CP and actin monomer was found to be necessary for twinfilin's role in actin assembly dynamics, based on genetic studies with mutants that have defined biochemical functions. Our results support a novel model for how sequential interactions between actin monomers, twinfilin, CP, and actin filaments promote cytoskeletal dynamics.     

Actin organization, bristle morphology, and viability are affected by actin capping protein mutations in Drosophila

Regulation of actin filament length and orientation is important in many actin-based cellular processes. This regulation is postulated to occur through the action of actin-binding proteins. Many actin-binding proteins that modify actin in vitro have been identified, but in many cases, it is not known if this activity is physiologically relevant. Capping protein (CP) is an actin-binding protein that has been demonstrated to control filament length in vitro by binding to the barbed ends and preventing the addition or loss of actin monomers. To examine the in vivo role of CP, we have performed a molecular and genetic characterization of the beta subunit of capping protein from Drosophila melanogaster. We have identified mutations in the Drosophila beta subunit-these are the first CP mutations in a multicellular organism, and unlike CP mutations in yeast, they are lethal, causing death during the early larval stage. Adult files that are heterozygous for a pair of weak alleles have a defect in bristle morphology that is correlated to disorganized actin bundles in developing bristles. Our data demonstrate that CP has an essential function during development, and further suggest that CP is required to regulate actin assembly during the development of specialized structures that depend on actin for their morphology.     

Requirements for Arabidopsis ATARP2 and ATARP3 during epidermal development

Plant cells employ the actin cytoskeleton to stably position organelles, as tracks for long distance transport, and to reorganize the cytoplasm in response to developmental and environmental cues. While diverse classes of actin binding proteins have been implicated in growth control, the mechanisms of cytoskeletal reorganization and the cellular functions of specific actin filament arrays are unclear. Arabidopsis trichome morphogenesis includes distinct requirements for the microtubule and actin filament cytoskeletons. It also is a genetically tractable process that is providing new knowledge about cytoskeleton function in plants. The "distorted group" of mutants defines a class of at least eight genes that are required during the actin-dependent phase of trichome growth. Using map-based cloning and a candidate gene approach, we identified mutations in ARP3 (ATARP3) and ARP2 (ATARP2) genes as the cause of the distorted1 (dis1) and wurm (wrm) phenotypes, respectively. ARP2 and ARP3 are components of the evolutionarily conserved ARP2/3 complex that nucleates actin filament polymerization [3]. Mutations in DIS1 and WRM caused severe trichome growth defects but had relatively mild effects on shoot development. DIS1 rescued the phenotype of Deltaarp3 when overexpressed in S. cerevisiae. Developing dis1 trichomes had defects in cytoplasmic actin bundle organization and reduced relative amounts of cytoplasmic actin filaments in developing branches.     

Tyrosine kinase signaling and type III effectors orchestrating Shigella invasion

Upon epithelial cell contact, Shigella type III effectors activate complex signaling pathways that induce localized membrane ruffling, resulting in Shigella invasion. Bacterial induced membrane ruffles require a timely coordination of cytoskeletal processes, including actin polymerization, filament reorganization and depolymerization, orchestrated by Rho GTPases and tyrosine kinases. An emerging concept is that multiple Shigella effectors act in synergy to promote actin polymerization in membrane extensions at the site of bacterial entry. Recent advances point to the role of Abl/Arg and Src tyrosine kinases as key regulators of bacterial induced cytoskeletal dynamics.     

Putative Arabidopsis homologues of metazoan coiled-coil cytoskeletal proteins

The Arabidopsis thaliana genome encodes about 386 proteins with coiled-coil domains of at least 50 amino acids in length. In mammalian systems, many coiled-coil proteins are part of various cytoskeletal networks including intermediate filament protein, actin-binding proteins and MAP (microtubule-associated proteins). Immunological evidence suggests that some of these cytoskeletal proteins, such as lamins, keratins and tropomyosins, may be conserved in Arabidopsis. However, coiled-coil proteins are of low complexity, and thus, traditional sequence comparison algorithms, such as BLAST may not detect homologies. Here, we use the PROPSEARCH algorithm to detect putative coiled-coil cytoskeletal protein homologues in Arabidopsis. This approach reveals putative intermediate filament protein homologues of filensin, lamin and keratin; putative actin-binding homologues of ERM (ezrin/radixin/moesin), periplakin, utrophin, tropomyosin and paramyosin, and putative MAP homologues of restin/CLIP-170 (cytoplasmic linker protein-170). We suggest that the AtFPP (Arabiopsis thaliana filament-like plant protein) and AtMAP70 (Arabidopsis microtubule-associated protein 70) families of coiled-coil proteins may, in fact, be related to lamins and function as intermediate filament proteins.     

Actin filament barbed end elongation with nonmuscle MgATP-actin and MgADP-actin in the presence of profilin

We have quantitated the in vitro interactions of profilin and the profilin-actin complex (PA) with the actin filament barbed end using profilin and nonmuscle beta,gamma-actin prepared from bovine spleen. Actin filament barbed end elongation was initiated from spectrin seeds in the presence of varying profilin concentrations and followed by light scattering. We find that profilin inhibits actin polymerization and that this effect is much more pronounced for beta,gamma-actin than for alpha-skeletal muscle actin. Profilin binds to beta,gamma-actin filament barbed ends with an equilibrium constant of 20 microM, decreases the filament elongation rate by blocking addition of actin monomers, and increases the dissociation rate of actin monomers from the filament end. PA containing bound MgADP supports elongation of the actin filament barbed end, indicating that ATP hydrolysis is not necessary for PA elongation of filaments. Initial analysis of the energetics for these reactions suggested an apparent greater negative free energy change for actin filament elongation from PA than elongation from monomeric actin. However, we calculate that the free energy changes for the two elongation pathways are equal if the profilin-induced weakening of nucleotide binding to actin is taken into consideration.     

Click chemistry and optogenetic approaches to visualize and manipulate phosphatidic acid signaling

The simple structure of phosphatidic acid (PA) belies its complex biological functions as both a key phospholipid biosynthetic intermediate and a potent signaling molecule. In the latter role, PA controls processes including vesicle trafficking, actin dynamics, cell growth, and migration. However, experimental methods to decode the pleiotropy of PA are sorely lacking. Because PA metabolism and trafficking are rapid, approaches to accurately visualize and manipulate its levels require high spatiotemporal precision. Here, we describe recent efforts to create a suite of chemical tools that enable imaging and perturbation of PA signaling. First, we describe techniques to visualize PA production by phospholipase D (PLD) enzymes, which are major producers of PA, called Imaging Phospholipase D Activity with Clickable Alcohols via Transphosphatidylation (IMPACT). IMPACT harnesses the ability of endogenous PLD enzymes to accept bioorthogonally tagged alcohols in transphosphatidylation reactions to generate functionalized reporter lipids that are subsequently fluorescently tagged via click chemistry. Second, we describe two light-controlled approaches for precisely manipulating PA signaling. Optogenetic PLDs use light-mediated heterodimerization to recruit a bacterial PLD to desired organelle membranes, and photoswitchable PA analogs contain azobenzene photoswitches in their acyl tails, enabling molecular shape and bioactivity to be controlled by light. We highlight select applications of these tools for studying GPCR–G_q signaling, discovering regulators of PLD signaling, tracking intracellular lipid transport pathways, and elucidating new oncogenic signaling roles for PA. We envision that these chemical tools hold promise for revealing many new insights into lipid signaling pathways.     

Clustering of VASP actively drives processive, WH2 domain-mediated actin filament elongation

Vasodilator-stimulated phosphoprotein (VASP) is a key regulator of dynamic actin structures like filopodia and lamellipodia, but its precise function in their formation is controversial. Using in vitro TIRF microscopy, we show for the first time that both human and Dictyostelium VASP are directly involved in accelerating filament elongation by delivering monomeric actin to the growing barbed end. In solution, DdVASP markedly accelerated actin filament elongation in a concentration-dependent manner but was inhibited by low concentrations of capping protein (CP). In striking contrast, VASP clustered on functionalized beads switched to processive filament elongation that became insensitive even to very high concentrations of CP. Supplemented with the in vivo analysis of VASP mutants and an EM structure of the protein, we propose a mechanism by which membrane-associated VASP oligomers use their WH2 domains to effect both the tethering of actin filaments and their processive elongation in sites of active actin assembly.     

Phosphatidic acid regulation of PIPKI is critical for actin cytoskeletal reorganization

Type I phosphatidylinositol-4-phosphate 5-kinase (PIPKI) is the main enzyme generating the lipid second messenger phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], which has critical functions in many cellular processes, such as cytoskeletal reorganization, membrane trafficking, and signal transduction. All three members of the PIPKI family are activated by phosphatidic acid (PA). However, how PA regulates the activity and functions of PIPKI have not been fully elucidated. In this study, we identify a PA-binding site on PIPKIγ. Mutation of this site inhibited the PA-stimulated activity and membrane localization of PIPKIγ as well as the formation of actin comets and foci induced by PIPKIγ. We also demonstrate that phospholipase D (PLD) generates a pool of PA involved in PIPKIγ regulation by showing that PLD inhibitors blocked the membrane localization of PIPKIγ and its ability to induce actin cytoskeletal reorganization. Targeting the PIPKIγ PA-binding-deficient mutant to membranes by a membrane localization sequence failed to restore the actin reorganization activity of PIPKIγ, suggesting that PA binding is not only involved in recruiting PIPKIγ to membranes but also may induce a conformational change. Taken together, these results reveal a new molecular mechanism through which PA regulates PIPKI and provides direct evidence that PA is important for the localization and functions of PIPKI in intact cells.     

Actin Turnover Is Required for Myosin-Dependent Mitochondrial Movements in Arabidopsis Root Hairs

Background

Previous studies have shown that plant mitochondrial movements are myosin-based along actin filaments, which undergo continuous turnover by the exchange of actin subunits from existing filaments. Although earlier studies revealed that actin filament dynamics are essential for many functions of the actin cytoskeleton, there are little data connecting actin dynamics and mitochondrial movements.

Methodology/Principal Findings

We addressed the role of actin filament dynamics in the control of mitochondrial movements by treating cells with various pharmaceuticals that affect actin filament assembly and disassembly. Confocal microscopy of Arabidopsis thaliana root hairs expressing GFP-FABD2 as an actin filament reporter showed that mitochondrial distribution was in agreement with the arrangement of actin filaments in root hairs at different developmental stages. Analyses of mitochondrial trajectories and instantaneous velocities immediately following pharmacological perturbation of the cytoskeleton using variable-angle evanescent wave microscopy and/or spinning disk confocal microscopy revealed that mitochondrial velocities were regulated by myosin activity and actin filament dynamics. Furthermore, simultaneous visualization of mitochondria and actin filaments suggested that mitochondrial positioning might involve depolymerization of actin filaments on the surface of mitochondria.

Conclusions/Significance

Base on these results we propose a mechanism for the regulation of mitochondrial speed of movements, positioning, and direction of movements that combines the coordinated activity of myosin and the rate of actin turnover, together with microtubule dynamics, which directs the positioning of actin polymerization events.     

DISTORTED3/SCAR2 is a putative arabidopsis WAVE complex subunit that activates the Arp2/3 complex and is required for epidermal morphogenesis

In a plant cell, a subset of actin filaments function as a scaffold that positions the endomembrane system and acts as a substrate on which organelle motility occurs. Other actin filament arrays appear to be more dynamic and reorganize in response to growth signals and external cues. The distorted group of trichome morphology mutants provides powerful genetic tools to study the control of actin filament nucleation in the context of morphogenesis. In this article, we report that DISTORTED3 (DIS3) encodes a plant-specific SCAR/WAVE homolog. Null alleles of DIS3, like those of other Arabidopsis thaliana WAVE and Actin-Related Protein (ARP) 2/3 subunit genes, cause trichome distortion, defects in cell-cell adhesion, and reduced hypocotyl growth in etiolated seedlings. DIS3 efficiently activates the actin filament nucleation and branching activity of vertebrate Arp2/3 and functions within a WAVE-ARP2/3 pathway in vivo. DIS3 may assemble into a WAVE complex via a physical interaction with a highly diverged Arabidopsis Abi-1-like bridging protein. These results demonstrate the utility of the Arabidopsis trichome system to understand how the WAVE and ARP2/3 complexes translate signaling inputs into a coordinated morphogenetic response.