Tandem C2 domains mediate dynamic organelle targeting of a DOCK family guanine nucleotide exchange factor.

Multicellular organisms use dedicator of cytokinesis (DOCK) family guanine nucleotide exchange factors (GEFs) to activate Rac/Rho-of-plants small GTPases and coordinate cell shape change. In developing tissues, DOCK signals integrate cell-cell interactions with cytoskeleton remodeling, and the GEFs cluster reversibly at specific organelle surfaces to orchestrate cytoskeletal reorganization. The domain organizations among DOCK orthologs are diverse, and the mechanisms of localization control are poorly understood. Here, we use combinations of transgene complementation and live-cell imaging assays to uncover an evolutionarily conserved and essential localization determinant in the DOCK-GEF named SPIKE1. The SPIKE1-DHR3 domain is sufficient for organelle association in vivo, and displays a complicated lipid-binding selectivity for both phospholipid head groups and fatty acid chain saturation. SPIKE1-DHR3 is predicted to adopt a C2-domain structure and functions as part of a tandem C2 array that enables reversible clustering at the cell apex. This work provides mechanistic insight into how DOCK GEFs sense compositional and biophysical membrane properties at the interface of two organelle systems.

Spatial and temporal localization of SPIRRIG and WAVE/SCAR reveal roles for these proteins in actin-mediated root hair development.

Root hairs are single-cell protrusions that enable roots to optimize nutrient and water acquisition. These structures attain their tubular shapes by confining growth to the cell apex, a process called tip growth. The actin cytoskeleton and endomembrane systems are essential for tip growth; however, little is known about how these cellular components coordinate their activities during this process. Here, we show that SPIRRIG (SPI), a beige and Chediak Higashi domain-containing protein involved in membrane trafficking, and BRK1 and SCAR2, subunits of the WAVE/SCAR (W/SC) actin nucleating promoting complex, display polarized localizations in Arabidopsis thaliana root hairs during distinct developmental stages. SPI accumulates at the root hair apex via post-Golgi compartments and positively regulates tip growth by maintaining tip-focused vesicle secretion and filamentous-actin integrity. BRK1 and SCAR2 on the other hand, mark the root hair initiation domain to specify the position of root hair emergence. Consistent with the localization data, tip growth was reduced in spi and the position of root hair emergence was disrupted in brk1 and scar1234. BRK1 depletion coincided with SPI accumulation as root hairs transitioned from initiation to tip growth. Taken together, our work uncovers a role for SPI in facilitating actin-dependent root hair development in Arabidopsis through pathways that might intersect with W/SC.

A SPIKE1 signaling complex controls actin-dependent cell morphogenesis through the heteromeric WAVE and ARP2/3 complexes.

During morphogenesis, the actin cytoskeleton mediates cell-shape change in response to growth signals. In plants, actin filaments organize the cytoplasm in regions of polarized growth, and the filamentous arrays can be highly dynamic. Small GTPase signaling proteins termed Rho of plants (ROP)/RAC control actin polymerization. ROPs cycle between inactive GDP-bound and active GTP-bound forms, and it is the active form that interacts with effector proteins to mediate cytoskeletal rearrangement and cell-shape change. A class of proteins termed guanine nucleotide exchange factors (GEFs) generate GTP-ROP and positively regulate ROP signaling. However, in almost all experimental systems, it has proven difficult to unravel the complex signaling pathways from GEFs to the proteins that nucleate actin filaments. In this article, we show that the DOCK family protein SPIKE1 (SPK1) is a GEF, and that one function of SPK1 is to control actin polymerization via two heteromeric complexes termed WAVE and actin-related protein (ARP) 2/3. The genetic pathway was constructed by using a combination of highly informative spk1 alleles and detailed analyses of spk1, wave, and arp2/3 single and double mutants. Remarkably, we find that in addition to providing GEF activity, SPK1 associates with WAVE complex proteins and may spatially organize signaling. Our results describe a unique regulatory scheme for ARP2/3 regulation in cells, one that can be tested for widespread use in other multicellular organisms.

The association of the Arabidopsis actin-related protein2/3 complex with cell membranes is linked to its assembly status but not its activation.

In growing plant cells, the combined activities of the cytoskeleton, endomembrane, and cell wall biosynthetic systems organize the cytoplasm and define the architecture and growth properties of the cell. These biosynthetic machineries efficiently synthesize, deliver, and recycle the raw materials that support cell expansion. The precise roles of the actin cytoskeleton in these processes are unclear. Certainly, bundles of actin filaments position organelles and are a substrate for long-distance intracellular transport, but the functional linkages between dynamic actin filament arrays and the cell growth machinery are poorly understood. The Arabidopsis (Arabidopsis thaliana) "distorted group" mutants have defined protein complexes that appear to generate and convert small GTPase signals into an Actin-Related Protein2/3 (ARP2/3)-dependent actin filament nucleation response. However, direct biochemical knowledge about Arabidopsis ARP2/3 and its cellular distribution is lacking. In this paper, we provide biochemical evidence for a plant ARP2/3. The plant complex utilizes a conserved assembly mechanism. ARPC4 is the most critical core subunit that controls the assembly and steady-state levels of the complex. ARP2/3 in other systems is believed to be mostly a soluble complex that is locally recruited and activated. Unexpectedly, we find that Arabidopsis ARP2/3 interacts strongly with cell membranes. Membrane binding is linked to complex assembly status and not to the extent to which it is activated. Mutant analyses implicate ARP2 as an important subunit for membrane association.

Arabidopsis BRICK1/HSPC300 is an essential WAVE-complex subunit that selectively stabilizes the Arp2/3 activator SCAR2.

The actin cytoskeleton dynamically reorganizes the cytoplasm during cell morphogenesis. The actin-related protein (Arp)2/3 complex is a potent nucleator of actin filaments that controls a variety of endomembrane functions including the endocytic internalization of plasma membrane , vacuole biogenesis , plasma-membrane protrusion in crawling cells , and membrane trafficking from the Golgi . Therefore, Arp2/3 is an important signaling target during morphogenesis. The evolutionarily conserved Rac-WAVE-Arp2/3 pathway links actin filament nucleation to cell morphogenesis . WAVE translates Rac-GTP signals into Arp2/3 activation by regulating the stability and/or localization of the activator subunit Scar/WAVE . The WAVE complex includes Sra1/PIR121/CYFIP1, Nap1/NAP125, Abi-1/Abi-2, Brick1(Brk1)/HSPC300, and Scar/WAVE : Defining the in vivo function of each subunit is an important step toward understanding this complicated signaling pathway. Brk1/HSPC300 has been the most recalcitrant WAVE-complex protein and has no known function. In this paper, we report that Arabidopsis brick1 (brk1) is a member of the "distorted group" of trichome morphology mutants, a group that defines a WAVE-ARP2/3 morphogenesis pathway . In this paper we provide the first strong genetic and biochemical evidence that BRK1 is a critical WAVE-complex subunit that selectively stabilizes the Arp2/3 activator SCAR2.

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.

Patterning mechanisms of cytoskeletal and cell wall systems during leaf trichome morphogenesis.

The plant actin cytoskeleton is an unstable network of filaments that influences polarized growth through poorly understood mechanisms. Here, we used a combination of live cell imaging and finite element computational modelling of Arabidopsis trichome morphogenesis to determine how the actin and microtubule cytoskeletons cooperate to pattern the cell wall and growth. The actin-related protein (ARP)2/3 complex generates an actin meshwork that operates within a tip-localized, microtubule-depleted zone to modulate cell wall anisotropy locally. The actin meshwork also positions an actin bundle network that organizes organelle flow patterns. This activity is required to maintain cell wall thickness gradients that enable tip-biased diffuse growth. These newly discovered couplings between cytoskeletal patterns and wall textures provide important insights into the cellular mechanism of growth control in plants.

ARP2/3 localization in Arabidopsis leaf pavement cells: a diversity of intracellular pools and cytoskeletal interactions.

In plant cells the actin cytoskeleton adopts many configurations, but is best understood as an unstable, interconnected track that rearranges to define the patterns of long distance transport of organelles during growth. Actin filaments do not form spontaneously; instead filament nucleators, such as the evolutionarily conserved actin-related protein (ARP) 2/3 complex, can efficiently generate new actin filament networks when in a fully activated state. A growing number of genetic experiments have shown that ARP2/3 is necessary for morphogenesis in processes that range from tip growth during root nodule formation to the diffuse polarized growth of leaf trichomes and pavement cells. Although progress has been rapid in the identification of proteins that function in series to positively regulate ARP2/3, less has been learned about the actual function of ARP2/3 in cells. In this paper, we analyze the localization of ARP2/3 in Arabidopsis leaf pavement cells. We detect a pool of ARP2/3 in the nucleus, and also find that ARP2/3 is efficiently and specifically clustered on multiple organelle surfaces and associates with both the actin filament and microtubule cytoskeletons. Our mutant analyses and ARP2/3 and actin double labeling experiments indicate that the clustering of ARP2/3 on organelle surfaces and an association with actin bundles does not necessarily reflect an active pool of ARP2/3, and instead most of the complex appears to exist as a latent organelle-associated pool.

Arabidopsis SCARs function interchangeably to meet actin-related protein 2/3 activation thresholds during morphogenesis.

During polarized growth and tissue morphogenesis, cells must reorganize their cytoplasm and change shape in response to growth signals. Dynamic polymerization of actin filaments is one cellular component of polarized growth, and the actin-related protein 2/3 (ARP2/3) complex is an important actin filament nucleator in plants. ARP2/3 alone is inactive, and the Arabidopsis thaliana WAVE complex translates Rho-family small GTPase signals into an ARP2/3 activation response. The SCAR subunit of the WAVE complex is the primary activator of ARP2/3, and plant and vertebrate SCARs are encoded by a small gene family. However, it is unclear if SCAR isoforms function interchangeably or if they have unique properties that customize WAVE complex functions. We used the Arabidopsis distorted group mutants and an integrated analysis of SCAR gene and protein functions to address this question directly. Genetic results indicate that each of the four SCARs functions in the context of the WAVE-ARP2/3 pathway and together they define the lone mechanism for ARP2/3 activation. Genetic interactions among the scar mutants and transgene complementation studies show that the activators function interchangeably to meet the threshold for ARP2/3 activation in the cell. Interestingly, double, triple, and quadruple mutant analyses indicate that individual SCAR genes vary in their relative importance depending on the cell type, tissue, or organ that is analyzed. Differences among SCARs in mRNA levels and the biochemical efficiency of ARP2/3 activation may explain the functional contributions of individual genes.

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.

DISTORTED2 encodes an ARPC2 subunit of the putative Arabidopsis ARP2/3 complex.

Arabidopsis trichomes are unicellular, branched structures that have highly constrained requirements for the cytoskeleton. The 'distorted group' genes function downstream from microtubule-based branch initiation, and are required during the actin-dependent phase of polarized stalk and branch expansion. Of the eight known 'distorted group' genes, a subset encode homologs of ARP2/3 complex subunits. In eukaryotic cells, the seven-protein ARP2/3 complex nucleates actin filament networks that push on the plasma membrane and organelles. In plants cells, the existence and function of an ARP2/3 complex is unclear. In this paper, we report that DISTORTED2 (DIS2) encodes a paralogue of the ARP2/3 complex subunit ARPC2. DIS2 has ARPC2 activity, based on its ability to rescue the growth defects of arpc2 (arc35Delta) null yeast cells. Like known ARPC2s, DIS2 physically interacts with ARPC4. Mutations in DIS2 cause a distorted trichome phenotype, defects in cell-cell adhesion, and a modest reduction in shoot FW. The actin cytoskeleton in dis2 trichomes is extensive, but developing branches fail to generate and maintain highly organized cytoplasmic actin bundles.

Interchangeable functions of Arabidopsis PIROGI and the human WAVE complex subunit SRA1 during leaf epidermal development.

The WAVE complex is an essential regulator of actin-related protein (ARP) 2/3-dependent actin filament nucleation and cell shape change in migrating cells. Although the composition of the WAVE complex is well characterized, the cellular mechanisms that control its activity and localization are not well known. The 'distorted group' defines a set of Arabidopsis genes that are required to remodel the actin cytoskeleton and maintain the polarized elongation of branched, hair-like cells termed trichomes. Several loci within this group encode homologs of ARP2/3 subunits. In addition to trichome distortion, ARP2/3 subunit mutants have reduced shoot fresh weight and widespread defects in epidermal cell-cell adhesion. The precise cellular function of plant ARP2/3, and the means by which it is regulated, is not known. In this paper, we report that the 'distorted group' gene PIROGI encodes a homolog of the WAVE complex subunit SRA1. The similar cell shape and actin phenotypes of pir and ARP2/3 complex subunit mutants suggest that PIROGI positively regulates ARP2/3. PIROGI directly interacts with the small GTPase ATROP2 with isoform specificity and with selectivity for active forms of the protein. PIROGI shares only 30% amino acid identity with its human homolog. However, both WAVE subunit homologs are functionally interchangeable and display identical physical interactions with RHO family GTPases and the Arabidopsis homolog of the WAVE complex subunit NAP125. These results demonstrate the utility of the 'distorted group' mutants to study ARP2/3 complex functions from signaling input to cell shape output.