Michael J. Deeks

Formins: intermediates in signal-transduction cascades that affect cytoskeletal reorganization

The control of cell growth and polarity depends on a dynamic actin cytoskeleton that has the ability to reorganize in response to developmental and environmental stimuli. In animals and fungi, formins are just one of the four major classes of poly-L-proline-containing (PLP) proteins that form part of the signal-transduction cascade that leads to rearrangement of the actin cytoskeleton. Analysis of the Arabidopsis genome sequence indicates that, unlike animals and fungi, formins are the only class of conserved profilin-binding PLP proteins in plants. Moreover, plant formins show significant structural differences compared with their animal and fungal counterparts, raising the possibility that plant formins are subject to novel mechanisms of control or perform unique roles in plants.

The plant formin AtFH4 interacts with both actin and microtubules, and contains a newly identified microtubule-binding domain

The dynamic behaviour of the actin cytoskeleton in plants relies on the coordinated action of several classes of actin-binding proteins (ABPs). These ABPs include the plant-specific subfamilies of actin-nucleating formin proteins. The model plant species Arabidopsis thaliana has over 20 formin proteins, all of which contain plant-specific regions in place of the GTPase-binding domain, formin homology (FH)3 domain, and DAD and DID motifs found in many fungal and animal formins. We have identified for the first time a plant-specific region of the membrane-integrated formin AtFH4 that mediates an association with the microtubule cytoskeleton. In vitro analysis shows that this region (named the GOE domain) binds directly to microtubules. Overexpressed AtFH4 accumulates at the endoplasmic reticulum membrane and co-aligns the endoplasmic reticulum with microtubules. The FH1 and FH2 domains of formins are conserved in plants, and we show that these domains of AtFH4 nucleate F-actin. Together, these data suggest that the combination of plant-specific and conserved domains enables AtFH4 to function as an interface between membranes and both major cytoskeletal networks.

Strategies of actin reorganisation in plant cells.

Spatial-temporal flexibility of the actin filament network (F-actin) is essential for all basic cellular functions and is governed by a stochastic dynamic model. In this model, actin filaments that randomly polymerise from a pool of free actin are bundled with other filaments and severed by ADF/cofilin. The fate of the severed fragments is not known. It has been proposed that the fragments are disassembled and the monomeric actin recycled for the polymerisation of new filaments. Here, we have generated tobacco cell lines and Arabidopsis plants expressing the actin marker Lifeact to address the mechanisms of F-actin reorganisation in vivo. We found that F-actin is more dynamic in isotropically expanding cells and that the density of the network changes with a periodicity of 70 seconds. The depolymerisation rate, but not the polymerisation rate, of F-actin increases when microtubules are destabilised. New filaments can be assembled from shorter free cytoplasmic fragments, from the products of F-actin severing and by polymerisation from the ends of extant filaments. Thus, remodelling of F-actin might not require bulk depolymerisation of the entire network, but could occur via severing and end-joining of existing polymers.

Arabidopsis NAP1 Is Essential for Arp2/3-Dependent Trichome Morphogenesis

The dynamic nature of the eukaryotic actin cytoskeleton is essential for the locomotion of animal cells and the morphogenesis of plant and fungal cells. The F-actin nucleating/branching activity of the Arp2/3 complex is a key function for all of these processes. The SCAR/WAVE family represents a group of Arp2/3 activators that are associated with lamellipodia formation. A protein complex of PIR121, NAP1, ABI, and HSPC300 is required for SCAR regulation by cell signaling pathways, but the exact nature of this interaction is controversial and represents a continually evolving model. The mechanism originally proposed was of a SCAR trans repressing complex supported by evidence from in vitro experiments. This model was reinforced by genetic studies in the Drosophila central nervous system and Dictyostelium, where the knockout of certain SCAR-complex components leads to excessive SCAR-mediated actin polymerization. Conflicting data have steadily accumulated from animal tissue culture experiments suggesting that the complex activates rather than represses in vivo SCAR activity. Recent biochemical evidence supports the SCAR-complex activator model. Here, we show that genetic observations in Arabidopsis are compatible with an activation model and provide one potential mechanism for the regulation of the newly identified Arabidopsis Arp2/3 complex.

The plant cytoskeleton, NET3C, and VAP27 mediate the link between the plasma membrane and endoplasmic reticulum

The cortical endoplasmic reticulum (ER) network in plants is a highly dynamic structure, and it contacts the plasma membrane (PM) at ER-PM anchor/contact sites. These sites are known to be essential for communication between the ER and PM for lipid transport, calcium influx, and ER morphology in mammalian and fungal cells. The nature of these contact sites is unknown in plants, and here, we have identified a complex that forms this bridge. This complex includes (1) NET3C, which belongs to a plant-specific superfamily (NET) of actin-binding proteins, (2) VAP27, a plant homolog of the yeast Scs2 ER-PM contact site protein, and (3) the actin and microtubule networks. We demonstrate that NET3C and VAP27 localize to puncta at the PM and that NET3C and VAP27 form homodimers/oligomers and together form complexes with actin and microtubules. We show that F-actin modulates the turnover of NET3C at these puncta and microtubules regulate the exchange of VAP27 at the same sites. Based on these data, we propose a model for the structure of the plant ER-PM contact sites.

Arp2/3 and SCAR: plants move to the fore

The actin-nucleating Arp2/3 complex is essential for life in yeast and animals, but not in plants, in which mutants of Arp2/3 complex components show relatively minor developmental abnormalities. Animal cells control the activity of the Arp2/3 complex through the suppressor of cyclic AMP receptor (SCAR) complex to achieve cell motility. Amazingly, plants have also retained the SCAR cell-motility pathway, and now provide a unique model for the study of new aspects of SCAR function in the absence of cell motility.

A Superfamily of Actin-Binding Proteins at the Actin-Membrane Nexus of Higher Plants

Complex animals use a wide variety of adaptor proteins to produce specialized sites of interaction between actin and membranes. Plants do not have these protein families, yet actin-membrane interactions within plant cells are critical for the positioning of subcellular compartments, for coordinating intercellular communication, and for membrane deformation. Novel factors are therefore likely to provide interfaces at actin-membrane contacts in plants, but their identity has remained obscure. Here we identify the plant-specific Networked (NET) superfamily of actin-binding proteins, members of which localize to the actin cytoskeleton and specify different membrane compartments. The founding member of the NET superfamily, NET1A, is anchored at the plasma membrane and predominates at cell junctions, the plasmodesmata. NET1A binds directly to actin filaments via a novel actin-binding domain that defines a superfamily of thirteen Arabidopsis proteins divided into four distinct phylogenetic clades. Members of other clades identify interactions at the tonoplast, nuclear membrane, and pollen tube plasma membrane, emphasizing the role of this superfamily in mediating actin-membrane interactions.

Arabidopsis CAP1 – a key regulator of actin organisation and development

Maintenance of F-actin turnover is essential for plant cell morphogenesis. Actin-binding protein mutants reveal that plants place emphasis on particular aspects of actin biochemistry distinct from animals and fungi. Here we show that mutants in CAP1, an A. thaliana member of the cyclase-associated protein family, display a phenotype that establishes CAP1 as a fundamental facilitator of actin dynamics over a wide range of plant tissues. Plants homozygous for cap1 alleles show a reduction in stature and morphogenetic disruption of multiple cell types. Pollen grains exhibit reduced germination efficiency, and cap1 pollen tubes and root hairs grow at a decreased rate and to a reduced length. Live cell imaging of growing root hairs reveals actin filament disruption and cytoplasmic disorganisation in the tip growth zone. Mutant cap1 alleles also show synthetic phenotypes when combined with mutants of the Arp2/3 complex pathway, which further suggests a contribution of CAP1 to in planta actin dynamics. In yeast, CAP interacts with adenylate cyclase in a Ras signalling cascade; but plants do not have Ras. Surprisingly, cap1 plants show disruption in plant signalling pathways required for co-ordinated organ expansion suggesting that plant CAP has evolved to attain plant-specific signalling functions.

The ARP2/3 Complex Mediates Guard Cell Actin Reorganization and Stomatal Movement in Arabidopsis

This study reports the isolation of hsr3, an allele of the Arabidopsis ARPC2 locus encoding a subunit of the ARP2/3 complex involved in the regulation of the actin cytoskeleton. In hsr3, reduced abscisic acid–induced stomatal closure is associated with failure of actin reassembly, indicating that modulation of the cytoskeleton through the ARP2/3 complex is crucial for stomatal regulation. 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), CaCl2, 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.

Prieurianin/endosidin 1 is an actin‐stabilizing small molecule identified from a chemical genetic screen for circadian clock effectors in Arabidopsis thaliana

Chemical modulators are powerful tools to investigate biological processes. To identify circadian clock effectors, we screened a natural product library in the model plant Arabidopsis thaliana. Two compounds, prieurianin (Pri) and prieurianin acetate, were identified as causing a shorter circadian period. Recently, Pri was independently identified as a vesicle trafficking inhibitor and re-named endosidin 1 (ES1). Here we show that Pri primarily affects actin filament flexibility in vivo, later resulting in reduced severing and filament depolymerization. This stabilization of the actin cytoskeleton subsequently causes changes in vesicle trafficking. Pri also affected microfilaments in mammalian cells, indicating that its target is highly conserved; however, it did not alter actin dynamics in vitro, suggesting that its activity requires the presence of actin-associated proteins. Furthermore, well-characterized actin inhibitors shortened the period length of the Arabidopsis clock in a similar way to Pri, supporting the idea that Pri affects rhythms by altering the actin network. We conclude that actin-associated processes influence the circadian system in a light-dependent manner, but their disruption does not abolish rhythmicity. In summary, we propose that the primary effect of Pri is to stabilize the actin cytoskeleton system, thereby affecting endosome trafficking. Pri appears to stabilize actin filaments by a different mechanism from previously described inhibitors, and will be a useful tool to study actin-related cellular processes.