A SPIKE1 Signaling Complex Controls Actin-Dependent Cell Morphogenesis Through the Heteromeric WAVE and ARP2/3 Complexes

A SPIKE1 signaling complex controls actin-dependent cell morphogenesis through the heteromeric WAVE and ARP2/3 complexes Dipanwita Basu*, Jie Le†, Taya Zakharova, Eileen L. Mallery, and Daniel B. Szymanski‡ Department of Agronomy, Purdue University, Lilly Hall, 915 W. State Street, West Lafayette, IN 47907-2054 Edited by Brian A. Larkins, University of Arizona, Tucson, AZ, and approved January 10, 2008 (received for review October 30, 2007) During morphogenesis, the actin cytoskeleton mediates cell-shape a conserved protein in both plants and animals and is a WAVE change in response to growth signals. In plants, actin filaments complex protein that directly interacts with active ROP/Rac (5, organize the cytoplasm in regions of polarized growth, and the 8). In cultured mammalian cells, Sra1 mediates the Rac- filamentous arrays can be highly dynamic. Small GTPase signaling dependent relocalization of the WAVE complex to leading edges proteins termed Rho of plants (ROP)/RAC control actin polymer- of lamellipodia (9), and in Arabidopsis, SRA1 and the WAVE ization. ROPs cycle between inactive GDP-bound and active GTP- complex positively regulate an actin filament-nucleating ma- bound forms, and it is the active form that interacts with effector chine, named the ARP2/3 complex (10). Plant and animal proteins to mediate cytoskeletal rearrangement and cell-shape WAVE complexes contain SCAR, a potent activator of ARP2/ change. A class of proteins termed guanine nucleotide exchange 3-dependent actin filament nucleation (8, 11). factors (GEFs) generate GTP–ROP and positively regulate ROP In both plant and animal systems, the proteins that generate signaling. However, in almost all experimental systems, it has the ROP/Rac signals and activate the WAVE–ARP2/3 pathway proven difficult to unravel the complex signaling pathways from are not known, and in general it has proven difficult to assign GEFs to the proteins that nucleate actin filaments. In this article, we clear functional linkages between GEFs and the proteins that show that the DOCK family protein SPIKE1 (SPK1) is a GEF, and that directly regulate the cytoskeleton. To our knowledge, a genetic one function of SPK1 is to control actin polymerization via two pathway linking a specific GEF to an actin filament nucleator has heteromeric complexes termed WAVE and actin-related protein not been described in any multicellular organism. Progress is (ARP) 2/3. The genetic pathway was constructed by using a com- hindered by genetic redundancy. For example, vertebrate ge- bination of highly informative spk1 alleles and detailed analyses of nomes encode large gene families of Dbl-homology domain and spk1, wave, and arp2/3 single and double mutants. Remarkably, DOCK family RhoGEFs (3, 4). In other cases, genetic studies of we find that in addition to providing GEF activity, SPK1 associates nonredundant GEFs (12) or actin filament nucleators (13) are with WAVE complex proteins and may spatially organize signaling. complicated by extreme pleiotropy and/or embryonic lethality. Our results describe a unique regulatory scheme for ARP2/3 reg- Genetic studies in Arabidopsis are not immune to such technical ulation in cells, one that can be tested for widespread use in other barriers. Although plants appear to lack the Dbl-homology multicellular organisms. domain-containing RhoGEFs that are widespread in animals, the Arabidopsis genome encodes 14 PRONE domain GEFs (14) ͉ ͉ ͉ DOCK cytoskeleton guanine nucleotide exchange factor that can affect the actin cytoskeleton and the tip growth of pollen ͉ Rho GTPase ROP tubes (15, 16). Arabidopsis has only a single DOCK family protein, SPIKE1 (SPK1). SPK1 (17), like all known WAVE and ell morphogenesis occurs in the context of tissue and organ ARP2/3 complex subunit genes (11), is ubiquitously expressed Cdevelopment. In the leaf epidermis, populations of cells and, based on the presence of the conserved DOCK homology regulate the location and extent of cell expansion, primarily region 2 (DHR2) domain, SPK1 originally was proposed to through the spatial control of the cytoskeleton and endomem- positively regulate ROP (17). Although the phenotype of spk1 brane systems. Plant cells (1, 2), like those of other eukaryotes plants is quite pleiotropic, the viability of the mutant and its (3, 4), use Rho family small GTPases as critical signaling proteins highly diagnostic trichome-swelling phenotype can be used to to dictate cell-shape change. Rho of plants (ROP) cycles be- discover novel morphogenesis pathways. tween GTP-bound active forms and GDP-bound inactive forms, In this article, we exploit a set of Arabidopsis epidermal and the nucleotide status of ROP is subject to complex regula- morphology mutants termed the ‘‘distorted group’’ to define a tion. One class of proteins termed guanine nucleotide exchange morphogenesis pathway from the DOCK family GEF SPK1 to factors (GEFs) catalyzes the exchange of bound GDP with the the actin filament nucleator ARP2/3. Our genetic and biochem- endogenous pool of GTP and positively regulates ROP signaling ical data indicate that ROP activation is the critical function for (2, 4). GTP-bound ROP physically interacts with diverse classes SPK1. We find that SPK1 not only generates signals that activate of effector proteins to control trafficking through the endomem- brane system and the organization of the cytoskeleton (5–7). This article defines a complete morphogenetic pathway from Author contributions: D.B. and J.L. designed research; D.B., J.L., and T.Z. performed ROP activation to actin-related protein (ARP) 2/3-dependent research; E.L.M. contributed new reagents/analytic tools; and D.B.S. wrote the paper. regulation of the actin cytoskeleton. The authors declare no conflict of interest. In the leaf epidermis, three different ROP effectors are This article is a PNAS Direct Submission. known. Interactor of constitutive active ROPs 1 (ICR1) is required *Present address: Department of Microbiology, University of Virginia School of Medicine, for the polarized growth and the normal lobing of pavement cells Charlottesville, VA 22908. and appears to link ROP signaling to the exocyst complex and †Present address: Department of Botany, University of British Columbia, Vancouver, BC, the secretory pathway (7). Two other genes, Rop-interactive Canada V6T 1Z4. CRIB motif 4 (RIC4) (6) and PIROGI/KLUNKER/PIRP/SRA1 ‡To whom correspondence should be addressed. E-mail: [email protected]. (referred to in this article as SRA1) (5), are thought to link ROP This article contains supporting information online at www.pnas.org/cgi/content/full/ signaling to actin-dependent polarized growth. The genetic and 0710294105/DC1. biochemical functions of SRA1 are well characterized. SRA1 is © 2008 by The National Academy of Sciences of the USA 4044–4049 ͉ PNAS ͉ March 11, 2008 ͉ vol. 105 ͉ no. 10 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0710294105 Downloaded by guest on September 25, 2021 We therefore tested the ability of endogenous SPK1 to associate with ROP. In coimmunoprecipitation (coIP) experiments using an antibody directed against SPK1, we found that endogenous SPK1 and ROP associated with each other (Fig. 2A). DHR2 affected the formation of SPK1–ROP complexes because we failed to detect ROP in coIP experiments using extracts prepared from spk1-3 plants (Fig. 2A). DHR2 also mediated the association of SPK1 with ROP in pull-down assays using purified GST-tagged ROPs. The preferential binding to a nucleotide-depleted Rho apoprotein is a hallmark of GEFs (4), and we found that endogenous SPK1 associated with GST– ROP2 in either the nucleotide-depleted or the GDP-bound forms (Fig. 2B). As expected, the association required an intact DHR2 domain (Fig. 2B). Furthermore, the interaction was specific to ROP, because SPK1 binding to human Rac was not detected (Fig. 2B). SPK1 and ROP directly interact because a purified C-terminal fragment of SPK1 (996-DHR2) efficiently sedimented inactive forms of ROP2 (Fig. 2C). We suspect that the previously reported failure to detect direct interactions between SPK1 and ROP (14) reflects a weakness of the yeast two-hybrid assay. The Arabidopsis ROP GTPase gene family consists of 11 members (20), many of which are expressed in the shoot and are candidate targets of SPK1 regulation. We therefore tested the SPK1-binding activity of several shoot-expressed prenylated ROPs (ROP2 to ROP5) and the S-acylated isoform, ROP10 (21). In triplicate pull-down assays that used full-length recombinant SPK1, there were no significant differences in the strength PLANT BIOLOGY of SPK1 binding to any of the different ROP isoforms (Fig. 2D). However, for each of the ROPs tested, SPK1-binding was highly selective for nucleotide-depleted or GDP-bound forms [Fig. 2D and supporting information (SI) Fig. 5]. The ability of SPK1 and 996-DHR2 to bind GDP–ROP is unique compared with other Fig. 1. The SPK1 Dock homology region 2 (DHR2) domain provides critical functions in vivo.(A) The domain organization of SPK1 protein. The locations of known DOCKs, such as Dock180 and Zizimin (18, 19, 22). the spk1-1 and spk1-3 mutations are indicated. The conserved regions of SPK1 are Full-length recombinant SPK1 has GEF activity. The intrinsic labeled. (B) Wild-type, spk1-1, and spk1-3 seedlings at 10 DAG. (C) Pavement rate of ROP2 nucleotide exchange was slow with an apparent Ϫ1 cell–cell adhesion defects of spk1 cotyledons and leaves. Wild-type, spk1-1, and off-rate of bound nucleotide (kobs) of 0.03 min , and SPK1 spk1-3 seedlings were stained with 0.1 ␮M FM1–43 (green) to visualize cell accelerated the nucleotide exchange reaction Ϸ7-fold (Fig. 2E), Ϫ1 boundaries. The autofluorescence from the chloroplasts of the underlying me- with kobs ϭ 0.21 min . Insect extracts containing an irrelevant sophyll cells is red and is clearly visible within pavement cell gaps. (D) SEM of stage expressed protein had no effect on the reaction kinetics (Fig.

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