BRICK1/HSPC300 functions with SCAR and the ARP2/3 complex to regulate epidermal cell shape in Arabidopsis

The Arp2/3 complex, a highly conserved nucleator of F-actin polymerization, is essential for a variety of eukaryotic cellular processes, including epidermal cell morphogenesis in Arabidopsis thaliana. Efficient nucleation of actin filaments by the Arp2/3 complex requires the presence of an activator such as a member of the Scar/WAVE family. In mammalian cells, a multiprotein complex consisting of WAVE, PIR121/Sra-1, Nap1, Abi-2 and HSPC300 mediates responsiveness of WAVE to upstream regulators such as Rac. Essential roles in WAVE complex assembly or function have been demonstrated for PIR121/Sra-1, Nap1 and Abi-2, but the significance of HSPC300 in this complex is unclear. Plant homologs of all mammalian WAVE complex components have been identified, including HSPC300, the mammalian homolog of maize BRICK1 (BRK1). We show that, like mutations disrupting the Arabidopsis homologs of PIR121/Sra-1, Nap1 and Scar/WAVE, mutations in the Arabidopsis BRK1 gene result in trichome and pavement cell morphology defects (and associated alterations in the F-actin cytoskeleton of expanding cells) similar to those caused by mutations disrupting the ARP2/3 complex itself. Analysis of double mutants provides genetic evidence that BRK1 functions in a pathway with the ARP2/3 complex. BRK1 is required for accumulation of SCAR1 protein in vivo, potentially explaining the apparently essential role of BRK1 in ARP2/3 complex function.     

The ROP2 GTPase controls the formation of cortical fine F-actin and the early phase of directional cell expansion during Arabidopsis organogenesis

Polar cell expansion in differentiating tissues is critical for the development and morphogenesis of plant organs and is modulated by hormonal and developmental signals, yet little is known about signaling in this fundamental process in plants. In contrast to tip-growing cells, such as pollen tubes and root hairs, cells in developing tissues are thought to expand by diffuse growth. In this study, we provide evidence that these cells expand in two phases with distinct mechanisms. In the early phase, cell expansion can occur in both longitudinal and radial or lateral directions and is mediated by Rop GTPase signaling, a mechanism known to control tip growth. The expression of a dominant-negative mutant for ROP2 (DN-rop2) inhibited polar cell expansion, whereas the expression of a constitutively active mutant (CA-rop2) caused isotropic expansion in the early phase. In the late phase, expansion occurs only in the longitudinal direction and is not affected by DN-rop2 or CA-rop2 expression. The transition from the early to the late phase coincides with the reorientation of cortical microtubules from random to transverse arrangements. Thus, cell expansion in the late phase is consistent with polar diffuse growth, in which polarity probably is defined by transverse cortical microtubules. We show that the direction of cell expansion in the early phase is associated with the localization of diffuse fine cortical F-actin in leaf epidermal cells. DN-rop2 expression specifically inhibited the formation of this F-actin, but not actin cables, whereas CA-rop2 expression caused delocalized distribution of this fine F-actin throughout the cell cortex. Furthermore, green fluorescent protein-ROP2 was localized preferentially to the cortical region of the cell, where expansion apparently occurs. These observations suggest that ROP2 control of the polar expansion of cells within tissues is analogous to the Rop control of tip growth and of tip-localized F-actin in pollen tubes and root hairs and that the tip growth mechanism also may modulate polar cell expansion in differentiating tissues.     

The putative Arabidopsis arp2/3 complex controls leaf cell morphogenesis

The evolutionarily conserved Arp2/3 complex has been shown to activate actin nucleation and branching in several eukaryotes, but its biological functions are not well understood in multicellular organisms. The model plant Arabidopsis provides many advantages for genetic dissection of the function of this conserved actin-nucleating machinery, yet the existence of this complex in plants has not been determined. We have identified Arabidopsis genes encoding homologs of all of the seven Arp2/3 subunits. The function of the putative Arabidopsis Arp2/3 complex has been studied using four homozygous T-DNA insertion mutants for ARP2, ARP3, and ARPC5/p16. All four mutants display identical defects in the development of jigsaw-shaped epidermal pavement cells and branched trichomes in the leaf. These loss-of-function mutations cause mislocalization of diffuse cortical F-actin to the neck region and inhibit lobe extension in pavement cells. The mutant trichomes resemble those treated with the actin-depolymerizing drug cytochalasin D, exhibiting stunted branches but dramatically enlarged stalks due to depolarized growth suggesting defects in the formation of a fine actin network. Our data demonstrate that the putative Arabidopsis Arp2/3 complex controls cell morphogenesis through its roles in cell polarity establishment and polar cell expansion. Furthermore, our data suggest a novel function for the putative Arp2/3 complex in the modulation of the spatial distribution of cortical F-actin and provide evidence that the putative Arp2/3 complex may activate the polymerization of some types of actin filaments in specific cell types.     

Arabidopsis GNARLED encodes a NAP125 homolog that positively regulates ARP2/3

In migrating cells, the actin filament nucleation activity of ARP2/3 is an essential component of dynamic cell shape change and motility. In response to signals from the small GTPase Rac1, alterations in the composition and/or subcellular localization of the WAVE complex lead to ARP2/3 activation. The human WAVE complex subunit, WAVE1/SCAR1, was first identified in Dictyostelium and is a direct ARP2/3 activator. In the absence of an intact WAVE complex, SCAR/WAVE protein is destabilized. Although the composition of the five-subunit WAVE complex is well characterized, the means by which individual subunits and fully assembled WAVE complexes regulate ARP2/3 in vivo are unclear. The molecular genetics of trichome distortion in Arabidopsis is a powerful system to understand how signaling pathways and ARP2/3 control multicellular development. In this paper we prove that the GNARLED gene encodes a homolog of the WAVE subunit NAP125. Despite the moderate level of amino acid identity between Arabidopsis and human NAP125, both homologs were functionally interchangeable in vivo and interacted physically with the putative Arabidopsis WAVE subunit ATSRA1. gnarled trichomes had nearly identical cell shape and actin cytoskeleton phenotypes when compared to ARP2/3 subunit mutants, suggesting that GRL positively regulates ARP2/3.     

The ARP2/3 complex mediates guard cell actin reorganization and stomatal movement in Arabidopsis

Guard cell actin reorganization has been observed in stomatal responses to a wide array of stimuli. However, how the guard cell signaling machinery regulates actin dynamics is poorly understood. Here, we report the identification of an allele of the Arabidopsis thaliana ACTIN-RELATED PROTEIN C2/DISTORTED TRICHOMES2 (ARPC2) locus (encoding the ARPC2 subunit of the ARP2/3 complex) designated high sugar response3 (hsr3). The hsr3 mutant showed increased transpirational water loss that was mainly due to a lesion in stomatal regulation. Stomatal bioassay analyses revealed that guard cell sensitivity to external stimuli, such as abscisic acid (ABA), CaCl(2), and light/dark transition, was reduced or abolished in hsr3. Analysis of a nonallelic mutant of the ARP2/3 complex suggested no pleiotropic effect of ARPC2 beyond its function in the complex in regard to stomatal regulation. When treated with ABA, guard cell actin filaments underwent fast disruption in wild-type plants, whereas those in hsr3 remained largely bundled. The ABA insensitivity phenotype of hsr3 was rescued by cytochalasin D treatment, suggesting that the aberrant stomatal response was a consequence of bundled actin filaments. Our work indicates that regulation of actin reassembly through ARP2/3 complex activity is crucial for stomatal regulation.     

The web and the rock: cell adhesion and the ARP2/3 complex

Cell locomotion entails functional and structural cooperation between cell surface adhesion and the actin cytoskeleton. A new paper by DeMali et al. provides new insights into the link between actin assembly and integrin adhesion at the leading edges of migrating cells.     

The role of Arabidopsis SCAR genes in ARP2-ARP3-dependent cell morphogenesis

The actin-nucleating ARP2-ARP3 complex controls cell shape in plants in many different cell types. Its activity is controlled by a multimeric complex containing BRK1 (also known as HSPC300), NAP1, SRA1, ABI and SCAR/WAVE. In this study, we focus on the function of the five putative SCAR homologues in Arabidopsis and we provide biochemical evidence that AtSCAR2 can activate the ARP2-ARP3 complex in vitro. Among the single mutants, mutations in only AtSCAR2 result in a subtle or weak phenotype similar to ARP2, ARP3 and other ;distorted' mutants. Double-mutant analysis revealed a redundancy with AtSCAR4. Systematic application of the yeast two-hybrid system and Bimolecular Fluorescence Complementation (BiFC) revealed a complex protein-interaction network between the ARP2-ARP3 complex and its genetically defined regulators. In addition to protein interactions known in other systems, we identified several new interactions, suggesting that SPIKE1 may be an integral component of the SCAR/WAVE complex and that SCAR proteins in plants might act as direct effectors of ROP GTPases.     

Exo70 stimulates the arp2/3 complex for lamellipodia formation and directional cell migration

Directional cell migration requires the coordination of actin assembly and membrane remodeling. The exocyst is an octameric protein complex essential for exocytosis and plasma membrane remodeling [1, 2]. A component of the exocyst, Exo70, directly interacts with the Arp2/3 complex, a core nucleating factor for the generation of branched actin networks for cell morphogenesis and migration [3-9]. Using in?vitro actin polymerization assay and time-lapse total internal reflection fluorescence microscopy, we found that Exo70 functions as a kinetic activator of the Arp2/3 complex that promotes actin filament nucleation and branching. We further found that the effect of Exo70 on actin is mediated by promoting the interaction of the Arp2/3 complex with WAVE2, a member of the N-WASP/WAVE family of nucleation promoting factors. At the cellular level, the stimulatory effect of Exo70 on the Arp2/3 complex is required for lamellipodia formation and maintaining directional persistence of cell migration. Our findings provide a novel mechanism for regulating actin polymerization and branching for effective membrane protrusion during cell morphogenesis and migration.     

The ARP2/3 complex: an actin nucleator comes of age

The cellular functions of the actin cytoskeleton require precise regulation of both the initiation of actin polymerization and the organization of the resulting filaments. The actin-related protein-2/3 (ARP2/3) complex is a central player in this regulation. A decade of study has begun to shed light on the molecular mechanisms by which this powerful machine controls the polymerization, organization and recycling of actin-filament networks, both in vitro and in the living cell.     

Mutations in actin-related proteins 2 and 3 affect cell shape development in Arabidopsis

ACTIN-RELATED PROTEINS 2 and 3 form the major subunits of the ARP2/3 complex, which is known as an important regulator of actin organization in diverse organisms. Here, we report that two genes, WURM and DISTORTED1, which are important for cell shape control in Arabidopsis, encode the plant ARP2 and ARP3 orthologs, respectively. Mutations in these genes result in misdirected expansion of various cell types: trichome expansion is randomized, pavement cells fail to produce lobes, hypocotyl cells curl out of the normal epidermal plane, and root hairs are sinuous. At the subcellular level, cell shape changes are linked to severe filamentous actin aggregation and compromised vacuole fusion. Because all seven subunits of the ARP2/3 complex are present in plants, our data indicate that this complex may play a pivotal role during plant cell morphogenesis.     

Regulation of N-WASP and the Arp2/3 complex by Abp1 controls neuronal morphology

Polymerization and organization of actin filaments into complex superstructures is indispensable for structure and function of neuronal networks. We here report that knock down of the F-actin-binding protein Abp1, which is important for endocytosis and synaptic organization, results in changes in axon development virtually identical to Arp2/3 complex inhibition, i.e., a selective increase of axon length. Our in vitro and in vivo experiments demonstrate that Abp1 interacts directly with N-WASP, an activator of the Arp2/3 complex, and releases the autoinhibition of N-WASP in cooperation with Cdc42 and thereby promotes N-WASP-triggered Arp2/3 complex-mediated actin polymerization. In line with our mechanistical studies and the colocalization of Abp1, N-WASP and Arp2/3 at sites of actin polymerization in neurons, we reveal an essential role of Abp1 and its cooperativity with Cdc42 in N-WASP-induced rearrangements of the neuronal cytoskeleton. We furthermore show that introduction of N-WASP mutants lacking the ability to bind Abp1 or Cdc42, Arp2/3 complex inhibition, Abp1 knock down, N-WASP knock down and Arp3 knock down, all cause identical neuromorphological phenotypes. Our data thus strongly suggest that these proteins and their complex formation are important for cytoskeletal processes underlying neuronal network formation.     

Overexpression of profilin 3 affects cell elongation and F-actin organization in Arabidopsis thaliana

Key message

Reduced levels of profilin 3 do not have a noticeable phenotypic effect; however, elevated profilin 3 levels result in decreased hypocotyl length due to a reduction in cell elongation and F-actin reorganization.

Abstract

The actin cytoskeleton is critical for a variety of cellular processes. The small actin monomer proteins, profilins (PRFs), are encoded by five highly conserved isoforms in Arabidopsis thaliana. PRF3, one of the vegetative isoforms, has 36 more N-terminal amino acid residues than the other four PRFs; however, the functions of PRF3 are mostly unknown. In this study, we demonstrated that PRF3 was strongly expressed in young seedlings, rosette leaves, and cauline leaves, but was weakly expressed in 14-day-old seedlings and flowers. Our data also showed that PRF3 could increase the critical concentration (Cc) of actin assembly in vitro. Overexpression of the full-length PRF3 cDNA resulted in a decrease in the lengths of roots and hypocotyls and delayed seed germination, but PRF3-ΔN36 transgenic plants and prf3 mutant plants showed normal growth when compared with wild-type plants. Microscopy observation revealed that cell elongation was inhibited in the hypocotyl and that F-actin was reorganized by destabilizing microfilaments. These results suggest that the dwarf phenotype of the PRF3 overexpression seedlings may be related to a reduction in cell length and F-actin rearrangement.     

Phosphorylation of the Arp2 subunit relieves auto-inhibitory interactions for Arp2/3 complex activation

Actin filament assembly by the actin-related protein (Arp) 2/3 complex is necessary to build many cellular structures, including lamellipodia at the leading edge of motile cells and phagocytic cups, and to move endosomes and intracellular pathogens. The crucial role of the Arp2/3 complex in cellular processes requires precise spatiotemporal regulation of its activity. While binding of nucleation-promoting factors (NPFs) has long been considered essential to Arp2/3 complex activity, we recently showed that phosphorylation of the Arp2 subunit is also necessary for Arp2/3 complex activation. Using molecular dynamics simulations and biochemical assays with recombinant Arp2/3 complex, we now show how phosphorylation of Arp2 induces conformational changes permitting activation. The simulations suggest that phosphorylation causes reorientation of Arp2 relative to Arp3 by destabilizing a network of salt-bridge interactions at the interface of the Arp2, Arp3, and ARPC4 subunits. Simulations also suggest a gain-of-function ARPC4 mutant that we show experimentally to have substantial activity in the absence of NPFs. We propose a model in which a network of auto-inhibitory salt-bridge interactions holds the Arp2 subunit in an inactive orientation. These auto-inhibitory interactions are destabilized upon phosphorylation of Arp2, allowing Arp2 to reorient to an activation-competent state.     

Pathway of actin filament branch formation by Arp2/3 complex

A spectroscopic assay using pyrene-labeled fission yeast Arp2/3 complex revealed that the complex binds to and dissociates from actin filaments extremely slowly with or without the nucleation-promoting factor fission yeast Wsp1-VCA. Wsp1-VCA binds both Arp2/3 complex and actin monomers with high affinity. These two ligands have only modest impacts on the interaction of the other ligand with VCA. Simulations of a mathematical model based on the kinetic parameters determined in this study and elsewhere account for the full time course of actin polymerization in the presence of Arp2/3 complex and Wsp1-VCA and show that an activation step, postulated to follow binding of a ternary complex of Arp2/3 complex, a bound nucleation-promoting factor, and an actin monomer to an actin filament, has a rate constant at least 0.15 s(-1). Kinetic parameters determined in this study constrain the process of actin filament branch formation during cellular motility to one main pathway.     

Dynamics between actin and the VE-cadherin/catenin complex: Novel aspects of the ARP2/3 complex in regulation of endothelial junctions

Endothelial adherens junctions are critical for physiological and pathological processes such as differentiation, maintenance of entire monolayer integrity, and the remodeling. The endothelial-specific VE-cadherin/catenin complex provides the backbone of adherens junctions and acts in close interaction with actin filaments and actin/myosin-mediated contractility to fulfill the junction demands. The functional connection between the cadherin/catenin complex and actin filaments might be either directly through α-catenins, or indirectly e.g., via linker proteins such as vinculin, p120ctn, α-actinin, or EPLIN. However, both junction integrity and dynamic remodeling have to be contemporarily coordinated. The actin-related protein complex ARP2/3 and its activating molecules, such as N-WASP and WAVE, have been shown to regulate the lammellipodia-mediated formation of cell junctions in both epithelium and endothelium. Recent reports now demonstrate a novel aspect of the ARP2/3 complex and the nucleating-promoting factors in the maintenance of endothelial barrier function and junction remodeling of established endothelial cell junctions. Those mechanisms open novel possibilities; not only in fulfilling physiological demands but obtained information may be of critical importance in pathologies such as wound healing, angiogenesis, inflammation, and cell diapedesis.     

The Nck-interacting kinase NIK increases Arp2/3 complex activity by phosphorylating the Arp2 subunit

The nucleating activity of the Arp2/3 complex promotes the assembly of branched actin filaments that drive plasma membrane protrusion in migrating cells. Arp2/3 complex binding to nucleation-promoting factors of the WASP and WAVE families was previously thought to be sufficient to increase nucleating activity. However, phosphorylation of the Arp2 subunit was recently shown to be necessary for Arp2/3 complex activity. We show in mammary carcinoma cells that mutant Arp2 lacking phosphorylation assembled with endogenous subunits and dominantly suppressed actin filament assembly and membrane protrusion. We also report that Nck-interacting kinase (NIK), a MAP4K4, binds and directly phosphorylates the Arp2 subunit, which increases the nucleating activity of the Arp2/3 complex. In cells, NIK kinase activity was necessary for increased Arp2 phosphorylation and plasma membrane protrusion in response to epidermal growth factor. NIK is the first kinase shown to phosphorylate and increase the activity of the Arp2/3 complex, and our findings suggest that it integrates growth factor regulation of actin filament dynamics.     

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.     

Arp2/3 complex inhibition radically alters lamellipodial actin architecture,suspended cell shape,and the cell spreading process

Recent studies have investigated the dendritic actin cytoskeleton of the cell edge''s lamellipodial (LP) region by experimentally decreasing the activity of the actin filament nucleator and branch former, the Arp2/3 complex. Here we extend these studies via pharmacological inhibition of the Arp2/3 complex in sea urchin coelomocytes, cells that possess an unusually broad LP region and display correspondingly exaggerated centripetal flow. Using light and electron microscopy, we demonstrate that Arp2/3 complex inhibition via the drug CK666 dramatically altered LP actin architecture, slowed centripetal flow, drove a lamellipodial-to-filopodial shape change in suspended cells, and induced a novel actin structural organization during cell spreading. A general feature of the CK666 phenotype in coelomocytes was transverse actin arcs, and arc generation was arrested by a formin inhibitor. We also demonstrate that CK666 treatment produces actin arcs in other cells with broad LP regions, namely fish keratocytes and Drosophila S2 cells. We hypothesize that the actin arcs made visible by Arp2/3 complex inhibition in coelomocytes may represent an exaggerated manifestation of the elongate mother filaments that could possibly serve as the scaffold for the production of the dendritic actin network.     

The N-terminal region and the mid-region complex of the integrin beta 2 subunit

In the primary sequence of the integrin beta subunit, the N-terminal region (NTR) and mid-region are separated by the I-like domain. To determine the spatial relationship and functional properties of the integrin beta(2) NTR and mid-region, we constructed beta(2)/beta(7) chimeras in which the NTR, I-like domain, and the mid-region of the beta(2) subunit were replaced by those of beta(7). Changing either the beta(2) NTR or mid-region, but not the I-like domain to that of beta(7) did not affect LFA-1 (alpha(L)beta(2)) formation and surface expression. Thus, the specificity of alpha(L)beta(2) pairing is conferred by the I-like domain but not the NTR or mid-region. Using these chimeras, the epitopes of six anti-beta(2) mAbs (H52, 7E4, AZN-L18, AZN-L27, KIM202, and MEM-148) were mapped. All except H52 require both the NTR and mid-region for epitope expression. Since these mAbs have distinct properties in terms of epitope expression and effect on LFA-1 binding to ICAM-1, we conclude that the beta(2) NTR and mid-region interact extensively. Although the I-like domain is located between the NTR and mid-region, its removal does not affect the folding of the beta(2) NTR/mid-region complex because this complex alone can be expressed as a soluble protein and precipitated by the appropriate mAbs. Finally, the mAbs H52 and 7E4, abrogated KIM185- but not Mg/EGTAinduced LFA-1/ICAM-1 binding and the epitope of MEM-148 is expressed on Mg/EGTA-activated but not resting LFA-1. These results suggest that the NTR/mid-region complex is involved in the regulation of LFA-1 function.