Eileen L. Mallery

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.

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.

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.

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.

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.

The Endoplasmic Reticulum Is a Reservoir for WAVE/SCAR Regulatory Complex Signaling in the Arabidopsis Leaf

In Arabidopsis leaf pavement cells and trichomes, the ER is a reservoir for W/SRC signaling and may have a key role in the early steps of W/SRC activation. During plant cell morphogenesis, signal transduction and cytoskeletal dynamics interact to locally organize the cytoplasm and define the geometry of cell expansion. The WAVE/SCAR (for WASP family verprolin homologous/suppressor of cyclic AMP receptor) regulatory complex (W/SRC) is an evolutionarily conserved heteromeric protein complex. Within the plant kingdom W/SRC is a broadly used effector that converts Rho-of-Plants (ROP)/Rac small GTPase signals into Actin-Related Protein2/3 and actin-dependent growth responses. Although the components and biochemistry of the W/SRC pathway are well understood, a basic understanding of how cells partition W/SRC into active and inactive pools is lacking. In this paper, we report that the endoplasmic reticulum (ER) is an important organelle for W/SRC regulation. We determined that a large intracellular pool of the core W/SRC subunit NAP1, like the known positive regulator of W/SRC, the DOCK family guanine nucleotide-exchange factor SPIKE1 (SPK1), localizes to the surface of the ER. The ER-associated NAP1 is inactive because it displays little colocalization with the actin network, and ER localization requires neither activating signals from SPK1 nor a physical association with its W/SRC-binding partner, SRA1. Our results indicate that in Arabidopsis (Arabidopsis thaliana) leaf pavement cells and trichomes, the ER is a reservoir for W/SRC signaling and may have a key role in the early steps of W/SRC assembly and/or activation.