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

Abstract

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 GTPbound 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.DOCK ͉ cytoskeleton ͉ guanine nucleotide exchange factor ͉ Rho GTPase ͉ ROP

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

“…Interestingly, the ER localization of NAP1 in pavement cells is not affected either in spk1, in which W/SRC is inactive (Basu et al, 2008), or in sra1 cells, in which W/SRC is incompletely assembled and has little or no activity Brembu et al, 2004;Saedler et al, 2004). These genetic data clearly demonstrate that inactive NAP1 associates with ER membranes and that its binding partner, SRA1, is not necessary for either the stability of the NAP1 protein or its association with the ER.…”

“…In angiosperms, ARP2/3 activation pathways are relatively simple compared with other organisms, because the ROP effector W/SRC complex acts as the sole source of activating signals (Szymanski, 2005;Zhang et al, 2008). In Arabidopsis, multiple activities, including the assembly of WAVE/SCAR proteins into a functional W/SRC through physical interactions with BRK1 (Frank et al, 2004;Zhang et al, 2005) and Ablinteractor-like proteins (Basu et al, 2005;Jörgens et al, 2010), and positive regulation by the ROPGEF SPK1 (Basu et al, 2008) are required to properly activate ARP2/3. However, the subcellular localization of pathway components is poorly understood, and a major challenge in the field is to determine the cellular mechanisms that partition W/SRC between inactive and fully activated pools.…”

“…There was a strong association between inducible expression of the toxins and the appearance of trichomes with severe trichome swelling and reduced branch number phenotypes (Singh et al, 2012). Although the exact mechanism of ROP-dependent control of W/SRC remains to be determined, the results described above in combination with the detection of direct interactions between the ROPGEF SPK1, active forms of ROP, and W/SRC subunits (Basu et al, , 2008Uhrig et al, 2007) strongly suggest that W/SRC is a ROP effector complex.…”

“…9C). SPK1 is an ER-localized ROPGEF (Zhang et al, 2010) and a known positive regulator of the W/SRC pathway (Basu et al, 2008;Zhang et al, 2010). In spk1 pavement cells, the ER has a reduced amount of ER tubulation and a more diffuse appearance compared with the wild type (Zhang et al, 2010), but it still contained normal GFP-HDEL-positive ER bodies (Fig.…”

“…The endoplasmic reticulum (ER) may also be involved in W/SRC regulation. The ER-localized DOCK family ROPGEF SPK1 (Zhang et al, 2010) physically associates with multiple W/SRC proteins (Uhrig et al, 2007;Basu et al, 2008) and, based on genetic criteria, is an upstream, positive regulator of the W/SRC-ARP2/3 pathway (Basu et al, 2008). In the leaf, one function of SPK1 is to promote normal trafficking between the ER and Golgi; however, arp2/3 mutants do not share ER-stress phenotypes with spk1 (Zhang et al, 2010), making it unclear if SPK1 and the ER are directly involved in W/SRC signaling.…”