ELMOD2 is an Arl2 GTPase-activating protein that also acts on Arfs

Regulatory GTPases in the Ras superfamily employ a cycle of alternating GTP binding and hydrolysis, controlled by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs), as essential features of their actions in cells. Studies of these GAPs and guanine nucleotide exchange factors have provided important insights into our understanding of GTPase signaling and biology. Within the Ras superfamily, the Arf family is composed of 30 members in mammals, including 22 Arf-like (Arl) proteins. Much less is known about the mechanisms of cell regulation by Arls than by Arfs. We report the purification from bovine testis of an Arl2 GAP and its identity as ELMOD2, a protein with no previously described function. ELMOD2 is one of six human proteins that contain an ELMO domain, and a second member, ELMOD1, was also found to have Arl2 GAP activity. Surprisingly, ELMOD2 also exhibited GAP activity against Arf proteins even though it does not contain the canonical Arf GAP sequence signature. The broader specificity of ELMOD2, as well as the previously described role for ELMO1 and ELMO2 in linking Arf6 and Rac1 signaling, suggests that ELMO family members may play a more general role in integrating signaling pathways controlled by Arls and other GTPases.     

Mutations of the mouse ELMO domain containing 1 gene (Elmod1) link small GTPase signaling to actin cytoskeleton dynamics in hair cell stereocilia

Stereocilia, the modified microvilli projecting from the apical surfaces of the sensory hair cells of the inner ear, are essential to the mechanoelectrical transduction process underlying hearing and balance. The actin-filled stereocilia on each hair cell are tethered together by fibrous links to form a highly patterned hair bundle. Although many structural components of hair bundles have been identified, little is known about the signaling mechanisms that regulate their development, morphology, and maintenance. Here, we describe two naturally occurring, allelic mutations that result in hearing and balance deficits in mice, named roundabout (rda) and roundabout-2J (rda(2J)). Positional cloning identified both as mutations of the mouse ELMO domain containing 1 gene (Elmod1), a poorly characterized gene with no previously reported mutant phenotypes. The rda mutation is a 138 kb deletion that includes exons 1-5 of Elmod1, and rda(2J) is an intragenic duplication of exons 3-8 of Elmod1. The deafness associated with these mutations is caused by cochlear hair cell dysfunction, as indicated by conspicuous elongations and fusions of inner hair cell stereocilia and progressive degeneration of outer hair cell stereocilia. Mammalian ELMO-family proteins are known to be involved in complexes that activate small GTPases to regulate the actin cytoskeleton during phagocytosis and cell migration. ELMOD1 and ELMOD2 recently were shown to function as GTPase-activating proteins (GAPs) for the Arf family of small G proteins. Our finding connecting ELMOD1 deficiencies with stereocilia dysmorphologies thus establishes a link between the Ras superfamily of small regulatory GTPases and the actin cytoskeleton dynamics of hair cell stereocilia.     

ELMO Domains,Evolutionary and Functional Characterization of a Novel GTPase-activating Protein (GAP) Domain for Arf Protein Family GTPases

The human family of ELMO domain-containing proteins (ELMODs) consists of six members and is defined by the presence of the ELMO domain. Within this family are two subclassifications of proteins, based on primary sequence conservation, protein size, and domain architecture, deemed ELMOD and ELMO. In this study, we used homology searching and phylogenetics to identify ELMOD family homologs in genomes from across eukaryotic diversity. This demonstrated not only that the protein family is ancient but also that ELMOs are potentially restricted to the supergroup Opisthokonta (Metazoa and Fungi), whereas proteins with the ELMOD organization are found in diverse eukaryotes and thus were likely the form present in the last eukaryotic common ancestor. The segregation of the ELMO clade from the larger ELMOD group is consistent with their contrasting functions as unconventional Rac1 guanine nucleotide exchange factors and the Arf family GTPase-activating proteins, respectively. We used unbiased, phylogenetic sorting and sequence alignments to identify the most highly conserved residues within the ELMO domain to identify a putative GAP domain within the ELMODs. Three independent but complementary assays were used to provide an initial characterization of this domain. We identified a highly conserved arginine residue critical for both the biochemical and cellular GAP activity of ELMODs. We also provide initial evidence of the function of human ELMOD1 as an Arf family GAP at the Golgi. These findings provide the basis for the future study of the ELMOD family of proteins and a new avenue for the study of Arf family GTPases.     

RhoE Regulates Actin Cytoskeleton Organization and Cell Migration

The actin cytoskeleton is regulated by Rho family proteins: in fibroblasts, Rho mediates the formation of actin stress fibers, whereas Rac regulates lamellipodium formation and Cdc42 controls filopodium formation. We have cloned the mouse RhoE gene, whose product is a member of the Rho family that shares (except in one amino acid) the conserved effector domain of RhoA, RhoB, and RhoC. RhoE is able to bind GTP but does not detectably bind GDP and has low intrinsic GTPase activity compared with Rac. The role of RhoE in regulating actin organization was investigated by microinjection in Bac1.2F5 macrophages and MDCK cells. In macrophages, RhoE induced actin reorganization, leading to the formation of extensions resembling filopodia and pseudopodia. In MDCK cells, RhoE induced the complete disappearance of stress fibers, together with cell spreading. However, RhoE did not detectably affect the actin bundles that run parallel to the outer membranes of cells at the periphery of colonies, which are known to be dependent on RhoA. In addition, RhoE induced an increase in the speed of migration of hepatocyte growth factor/scatter factor-stimulated MDCK cells, in contrast to the previously reported inhibition produced by activated RhoA. The subcellular localization of RhoE at the lateral membranes of MDCK cells suggests a role in cell-cell adhesion, as has been shown for RhoA. These results suggest that RhoE may act to inhibit signalling downstream of RhoA, altering some RhoA-regulated responses, such as stress fiber formation, but not affecting others, such as peripheral actin bundle formation.     

Swf1p, a member of the DHHC-CRD family of palmitoyltransferases, regulates the actin cytoskeleton and polarized secretion independently of its DHHC motif

Rho and Rab family GTPases play a key role in cytoskeletal organization and vesicular trafficking, but the exact mechanisms by which these GTPases regulate polarized cell growth are incompletely understood. A previous screen for genes that interact with CDC42, which encodes a Rho GTPase, found SWF1/PSL10. Here, we show Swf1p, a member of the DHHC-CRD family of palmitoyltransferases, localizes to actin cables and cortical actin patches in Saccharomyces cerevisiae. Deletion of SWF1 results in misorganization of the actin cytoskeleton and decreased stability of actin filaments in vivo. Cdc42p localization depends upon Swf1p primarily after bud emergence. Importantly, we revealed that the actin regulating activity of Swf1p is independent of its DHHC motif. A swf1 mutant, in which alanine substituted for the cysteine required for the palmitoylation activity of DHHC-CRD proteins, displayed wild-type actin organization and Cdc42p localization. Bgl2p-marked exocytosis was found wild type in this mutant, although invertase secretion was impaired. These data indicate Swf1p has at least two distinct functions, one of which regulates actin organization and Bgl2p-marked secretion. This report is the first to link the function of a DHHC-CRD protein to Cdc42p and the regulation of the actin cytoskeleton.     

The Arf family GTPase Arl4A complexes with ELMO proteins to promote actin cytoskeleton remodeling and reveals a versatile Ras-binding domain in the ELMO proteins family

The prototypical DOCK protein, DOCK180, is an evolutionarily conserved Rac regulator and is indispensable during processes such as cell migration and myoblast fusion. The biological activity of DOCK180 is tightly linked to its binding partner ELMO. We previously reported that autoinhibited ELMO proteins regulate signaling from this pathway. One mechanism to activate the ELMO-DOCK180 complex appears to be the recruitment of this complex to the membrane via the Ras-binding domain (RBD) of ELMO. In the present study, we aimed to identify novel ELMO-interacting proteins to further define the molecular events capable of controlling ELMO recruitment to the membrane. To do so, we performed two independent interaction screens: one specifically interrogated an active GTPase library while the other probed a brain cDNA library. Both methods converged on Arl4A, an Arf-related GTPase, as a specific ELMO interactor. Biochemically, Arl4A is constitutively GTP-loaded, and our binding assays confirm that both wild-type and constitutively active forms of the GTPase associate with ELMO. Mechanistically, we report that Arl4A binds the ELMO RBD and acts as a membrane localization signal for ELMO. In addition, we report that membrane targeting of ELMO via Arl4A promotes cytoskeletal reorganization including membrane ruffling and stress fiber disassembly via an ELMO-DOCK1800-Rac signaling pathway. We conclude that ELMO is capable of interacting with GTPases from Rho and Arf families, leading to the conclusion that ELMO contains a versatile RBD. Furthermore, via binding of an Arf family GTPase, the ELMO-DOCK180 is uniquely positioned at the membrane to activate Rac signaling and remodel the actin cytoskeleton.     

A Point Mutation in p190A RhoGAP Affects Ciliogenesis and Leads to Glomerulocystic Kidney Defects

Rho family GTPases act as molecular switches regulating actin cytoskeleton dynamics. Attenuation of their signaling capacity is provided by GTPase-activating proteins (GAPs), including p190A, that promote the intrinsic GTPase activity of Rho proteins. In the current study we have performed a small-scale ENU mutagenesis screen and identified a novel loss of function allele of the p190A gene Arhgap35, which introduces a Leu1396 to Gln substitution in the GAP domain. This results in decreased GAP activity for the prototypical Rho-family members, RhoA and Rac1, likely due to disrupted ordering of the Rho binding surface. Consequently, Arhgap35-deficient animals exhibit hypoplastic and glomerulocystic kidneys. Investigation into the cystic phenotype shows that p190A is required for appropriate primary cilium formation in renal nephrons. P190A specifically localizes to the base of the cilia to permit axoneme elongation, which requires a functional GAP domain. Pharmacological manipulations further reveal that inhibition of either Rho kinase (ROCK) or F-actin polymerization is able to rescue the ciliogenesis defects observed upon loss of p190A activity. We propose a model in which p190A acts as a modulator of Rho GTPases in a localized area around the cilia to permit the dynamic actin rearrangement required for cilia elongation. Together, our results establish an unexpected link between Rho GTPase regulation, ciliogenesis and glomerulocystic kidney disease.     

Allelic Mutations of KITLG,Encoding KIT Ligand,Cause Asymmetric and Unilateral Hearing Loss and Waardenburg Syndrome Type 2

Linkage analysis combined with whole-exome sequencing in a large family with congenital and stable non-syndromic unilateral and asymmetric hearing loss (NS-UHL/AHL) revealed a heterozygous truncating mutation, c.286_303delinsT (p.Ser96Ter), in KITLG. This mutation co-segregated with NS-UHL/AHL as a dominant trait with reduced penetrance. By screening a panel of probands with NS-UHL/AHL, we found an additional mutation, c.200_202del (p.His67_Cys68delinsArg). In vitro studies revealed that the p.His67_Cys68delinsArg transmembrane isoform of KITLG is not detectable at the cell membrane, supporting pathogenicity. KITLG encodes a ligand for the KIT receptor. Also, KITLG-KIT signaling and MITF are suggested to mutually interact in melanocyte development. Because mutations in MITF are causative of Waardenburg syndrome type 2 (WS2), we screened KITLG in suspected WS2-affected probands. A heterozygous missense mutation, c.310C>G (p.Leu104Val), that segregated with WS2 was identified in a small family. In vitro studies revealed that the p.Leu104Val transmembrane isoform of KITLG is located at the cell membrane, as is wild-type KITLG. However, in culture media of transfected cells, the p.Leu104Val soluble isoform of KITLG was reduced, and no soluble p.His67_Cys68delinsArg and p.Ser96Ter KITLG could be detected. These data suggest that mutations in KITLG associated with NS-UHL/AHL have a loss-of-function effect. We speculate that the mechanism of the mutation underlying WS2 and leading to membrane incorporation and reduced secretion of KITLG occurs via a dominant-negative or gain-of-function effect. Our study unveils different phenotypes associated with KITLG, previously associated with pigmentation abnormalities, and will thereby improve the genetic counseling given to individuals with KITLG variants.     

Characterization of a novel interaction between ELMO1 and ERM proteins

ERMs are closely related proteins involved in cell migration, cell adhesion, maintenance of cell shape, and formation of microvilli through their ability to cross-link the plasma membrane with the actin cytoskeleton. ELMO proteins are also known to regulate actin cytoskeleton reorganization through activation of the small GTPbinding protein Rac via the ELMO-Dock180 complex. Here we showed that ERM proteins associate directly with ELMO1 as purified recombinant proteins in vitro and at endogenous levels in intact cells. We mapped ERM binding on ELMO1 to the N-terminal 280 amino acids, which overlaps with the region required for binding to the GTPase RhoG, but is distinct from the C-terminal Dock180 binding region. Consistent with this, ELMO1 could simultaneously bind both radixin and Dock180, although radixin did not alter Rac activation via the Dock180-ELMO complex. Most interestingly, radixin binding did not affect ELMO binding to active RhoG and a trimeric complex of active RhoG-ELMO-radixin could be detected. Moreover, the three proteins colocalized at the plasma membrane. Finally, in contrast to most other ERM-binding proteins, ELMO1 binding occurred independently of the state of radixin C-terminal phosphorylation, suggesting an ELMO1 interaction with both the active and inactive forms of ERM proteins and implying a possible role of ELMO in localizing or retaining ERM proteins in certain cellular sites. Together these data suggest that ELMO1-mediated cytoskeletal changes may be coordinated with ERM protein crosslinking activity during dynamic cellular functions.     

Nedd4, a human ubiquitin ligase, affects actin cytoskeleton in yeast cells

Human Nedd4 ubiquitin ligase is involved in protein trafficking, signal transduction and oncogenesis. Nedd4 with an inactive WW4 domain is toxic to yeast cells. We report here that actin cytoskeleton is abnormal in yeast cells expressing the NEDD4 or NEDD4w4 gene and these cells are more sensitive to Latrunculin A, an actin-depolymerizing drug. These phenotypes are less pronounced when a mutation inactivating the catalytic domain of the ligase has been introduced. In contrast, overexpression of the LAS17 gene, encoding an activator of the Arp2/3 actin nucleating complex, is detrimental to NEDD4w4-expressing cells. The level of Las17p is increased in cells overproducing Nedd4w4 and this depends partially on its catalytic domain. Expression of genes encoding Nedd4 variants, like overexpression of LAS17, suppresses the growth defect of the arp2-1 strain. Our results suggest that human Nedd4 ligase inhibits yeast cell growth by disturbing the actin cytoskeleton, in part by increasing Las17p level, and that Nedd4 ubiquitination targets may include actin cytoskeleton-associated proteins conserved in evolution.     

Dynamics of the Rho-family small GTPases in actin regulation and motility

The p21 Rho-family of small GTPases are master regulators of actin cytoskeleton rearrangements. Their functions have been well characterized in terms of their effects toward various actin-modulating protein targets. However, more recent studies have shown that the dynamics of Rho GTPase activities are highly complex and tightly regulated in order to achieve their specific subcellular localization. Furthermore, these localized effects are highly dynamic, often spanning the time-scale of seconds, making the interpretation of traditional biochemical approaches inadequate to fully decipher these rapid mechanisms in vivo. Here, we provide an overview of Rho family GTPase biology, and introduce state-of-the-art approaches to study the dynamics of these important signaling proteins that ultimately coordinate the actin cytoskeleton rearrangements during cell migration.Key words: Rho, dynamics, live-cell imaging, FRET, migration, actin, biosensors     

Two closely spaced missense COL3A1 variants in cis cause vascular Ehlers‐Danlos syndrome in one large Chinese family

Vascular Ehlers‐Danlos syndrome (vEDS) is a rare and severe hereditary connective tissue disease arising from a mutation in the type III collagen alpha I chain (COL3A1) gene, with a poor prognosis due to exceptional vascular ruptures and premature death. Herein, starting from a 36‐year‐old Chinese male patient with a complaint of upper abdominal pain, we collected clinical data of and performed a genetic analysis of a total of 20 family members. We identified two closely spaced COL3A1 missense variants in cis, p.Leu734Phe (c.2199_2200TC>AT) and p.Gly741Ser (c.2221G>A), as the cause of vEDS in this family. p.Gly741Ser, a glycine substitution mutation, has been previously reported, whereas p.Leu734Phe, a non‐glycine substitution mutation, is novel. We analysed their independent and combined effects on the COL3A1 level in transfected skin fibroblast cells by means of Western blotting. We found that both variants independently led to a reduced COL3A1 level and, when combined, led to an even more reduced COL3A1 level compared to the wild type. Thus, each missense variant can be independently classified as a pathogenic variant, albeit with a synergetic effect when occurring together. Moreover, our genetic findings provide an explanation for four previous sudden deaths and identified two high‐risk carriers in the family.     

Control of T lymphocyte morphology by the GTPase Rho

Background  

Rho family GTPase regulation of the actin cytoskeleton governs a variety of cell responses. In this report, we have analyzed the role of the GTPase Rho in maintenance of the T lymphocyte actin cytoskeleton.     

Actin-binding Verprolin Is a Polarity Development Protein Required for the Morphogenesis and Function of the Yeast Actin Cytoskeleton

Yeast verprolin, encoded by VRP1, is implicated in cell growth, cytoskeletal organization, endocytosis and mitochondrial protein distribution and function. We show that verprolin is also required for bipolar bud-site selection. Previously we reported that additional actin suppresses the temperature-dependent growth defect caused by a mutation in VRP1. Here we show that additional actin suppresses all known defects caused by vrp1-1 and conclude that the defects relate to an abnormal cytoskeleton. Using the two-hybrid system, we show that verprolin binds actin. An actin-binding domain maps to the LKKAET hexapeptide located in the first 70 amino acids. A similar hexapeptide in other acting-binding proteins was previously shown to be necessary for actin-binding activity. The entire 70– amino acid motif is conserved in novel higher eukaryotic proteins that we predict to be actin-binding, and also in the actin-binding proteins, WASP and N-WASP. Verprolin-GFP in live cells has a cell cycle-dependent distribution similar to the actin cortical cytoskeleton. In fixed cells hemagglutinin-tagged Vrp1p often co-localizes with actin in cortical patches. However, disassembly of the actin cytoskeleton using Latrunculin-A does not alter verprolin's location, indicating that verprolin establishes and maintains its location independent of the actin cytoskeleton. Verprolin is a new member of the actin-binding protein family that serves as a polarity development protein, perhaps by anchoring actin. We speculate that the effects of verprolin upon the actin cytoskeleton might influence mitochondrial protein sorting/function via mRNA distribution.     

A Novel Alpha Cardiac Actin (ACTC1) Mutation Mapping to a Domain in Close Contact with Myosin Heavy Chain Leads to a Variety of Congenital Heart Defects,Arrhythmia and Possibly Midline Defects

Background

A Lebanese Maronite family presented with 13 relatives affected by various congenital heart defects (mainly atrial septal defects), conduction tissue anomalies and midline defects. No mutations were found in GATA4 and NKX2-5.

Methods and Results

A set of 399 poly(AC) markers was used to perform a linkage analysis which peaked at a 2.98 lod score on the long arm of chromosome 15. The haplotype analysis delineated a 7.7 meganucleotides genomic interval which included the alpha-cardiac actin gene (ACTC1) among 36 other protein coding genes. A heterozygous missense mutation was found (c.251T>C, p.(Met84Thr)) in the ACTC1 gene which changed a methionine residue conserved up to yeast. This mutation was absent from 1000 genomes and exome variant server database but segregated perfectly in this family with the affection status. This mutation and 2 other ACTC1 mutations (p.(Glu101Lys) and p.(Met125Val)) which result also in congenital heart defects are located in a region in close apposition to a myosin heavy chain head region by contrast to 3 other alpha-cardiac actin mutations (p.(Ala297Ser),p.(Asp313His) and p.(Arg314His)) which result in diverse cardiomyopathies and are located in a totally different interaction surface.

Conclusions

Alpha-cardiac actin mutations lead to congenital heart defects, cardiomyopathies and eventually midline defects. The consequence of an ACTC1 mutation may in part be dependent on the interaction surface between actin and myosin.     

Dynamics of the Rho-family small GTPases in actin regulation and motility

The p21 Rho-family of small GTPases are master regulators of actin cytoskeleton rearrangements. Their functions have been well characterized in terms of their effects toward various actin-modulating protein targets. However, more recent studies have shown that the dynamics of Rho GTPase activities are highly complex and tightly regulated in order to achieve their specific subcellular localization. Furthermore, these localized effects are highly dynamic, often spanning the time-scale of seconds, making the interpretation of traditional biochemical approaches inadequate to fully decipher these rapid mechanisms in vivo. Here, we provide an overview of Rho family GTPase biology, and introduce state-of-the-art approaches to study the dynamics of these important signaling proteins that ultimately coordinate the actin cytoskeleton rearrangements during cell migration.     

Genetic Analysis of the Saccharomyces Cerevisiae Rho3 Gene, Encoding a Rho-Type Small Gtpase, Provides Evidence for a Role in Bud Formation

RHO3 encodes a Rho-type small GTPase of the yeast Saccharomyces cerevisiae. We isolated temperature-sensitive alleles and a dominant active allele of RHO3. Ts(-) rho3 cells lost cell polarity during bud formation and grew more isotropically than wild-type cells at nonpermissive temperatures. In contrast, cells carrying a dominant active mutant RHO3 displayed cold sensitivity, and the cells became elongated and bent, often at the position where actin patches were concentrated. These phenotypes of the rho3 mutants strongly suggest that RHO3 is involved in directing the growing points during bud formation. In addition, we found that SRO6, previously isolated as a multicopy suppressor of rho3, is the same as SEC4. The sec4-2 mutation was synthetic lethal with temperature-sensitive rho3 mutations and suppressed the cold sensitivity caused by a dominant active mutant RHO3. The genetic interactions between RHO3 and SEC4, taken together with the fact that the Rab-type GTPase Sec4p is required to fuse secretory vesicles together with plasma membrane for exocytosis, support a model in which the Rho3p pathway modulates morphogenesis during bud growth via directing organization of the actin cytoskeleton and the position of the secretory machinery for exocytosis.     

p19Arf-p53 tumor suppressor pathway regulates cell motility by suppression of phosphoinositide 3-kinase and Rac1 GTPase activities

The p19(Arf)-p53 tumor suppressor pathway plays a critical role in cell-cycle checkpoint control and apoptosis, whereas Rho family small GTPases are key regulators of actin structure and cell motility. By using primary mouse embryonic fibroblasts that lack Arf, p53, or both, we studied the involvement of the p19(Arf)-p53 pathway in the regulation of cell motility and its relationship with Rho GTPases. Deletion of Arf and/or p53 led to actin cytoskeleton reorganization and a significant increase in cell motility. The endogenous phosphoinositide (PI) 3- kinase and Rac1 activities were elevated in Arf(-/-) and p53(-/-) cells, and these activities are required for p19(Arf)- and p53-regulated migration. Reintroduction of the wild type Arf or p53 genes into Arf(-/-) or p53(-/-) cells reversed the PI 3-kinase and Rho GTPase activities as well as the migration phenotype. These results suggest a functional relationship between an established tumor suppressor pathway and a signaling module that controls actin structure and cell motility and show that p19(Arf) and p53 negatively regulate cell migration by suppression of PI 3-kinase and Rac1 activities.     

A New Member of the Rho Family, Rnd1, Promotes Disassembly of Actin Filament Structures and Loss of Cell Adhesion

Members of the Rho GTPase family regulate the organization of the actin cytoskeleton in response to extracellular growth factors. We have identified three proteins that form a distinct branch of the Rho family: Rnd1, expressed mostly in brain and liver; Rnd2, highly expressed in testis; and Rnd3/RhoE, showing a ubiquitous low expression. At the subcellular level, Rnd1 is concentrated at adherens junctions both in confluent fibroblasts and in epithelial cells. Rnd1 has a low affinity for GDP and spontaneously exchanges nucleotide rapidly in a physiological buffer. Furthermore, Rnd1 lacks intrinsic GTPase activity suggesting that in vivo, it might be constitutively in a GTP-bound form. Expression of Rnd1 or Rnd3/RhoE in fibroblasts inhibits the formation of actin stress fibers, membrane ruffles, and integrin-based focal adhesions and induces loss of cell–substrate adhesion leading to cell rounding (hence Rnd for “round”). We suggest that these proteins control rearrangements of the actin cytoskeleton and changes in cell adhesion.     

Fine-Tuning of the Actin Cytoskeleton and Cell Adhesion During Drosophila Development by the Unconventional Guanine Nucleotide Exchange Factors Myoblast City and Sponge

The evolutionarily conserved Dock proteins function as unconventional guanine nucleotide exchange factors (GEFs). Upon binding to engulfment and cell motility (ELMO) proteins, Dock–ELMO complexes activate the Rho family of small GTPases to mediate a diverse array of biological processes, including cell motility, apoptotic cell clearance, and axon guidance. Overlapping expression patterns and functional redundancy among the 11 vertebrate Dock family members, which are subdivided into four families (Dock A, B, C, and D), complicate genetic analysis. In both vertebrate and invertebrate systems, the actin dynamics regulator, Rac, is the target GTPase of the Dock-A subfamily. However, it remains unclear whether Rac or Rap1 are the in vivo downstream GTPases of the Dock-B subfamily. Drosophila melanogaster is an excellent genetic model organism for understanding Dock protein function as its genome encodes one ortholog per subfamily: Myoblast city (Mbc; Dock A) and Sponge (Spg; Dock B). Here we show that the roles of Spg and Mbc are not redundant in the Drosophila somatic muscle or the dorsal vessel. Moreover, we confirm the in vivo role of Mbc upstream of Rac and provide evidence that Spg functions in concert with Rap1, possibly to regulate aspects of cell adhesion. Together these data show that Mbc and Spg can have different downstream GTPase targets. Our findings predict that the ability to regulate downstream GTPases is dependent on cellular context and allows for the fine-tuning of actin cytoskeletal or cell adhesion events in biological processes that undergo cell morphogenesis.