InteractiveFly: GeneBrief

pico: Biological Overview | References

BIOLOGICAL OVERVIEW

MIG-10/RIAM/lamellipodin (MRL) proteins link activated Ras-GTPases with actin regulatory Ena/VASP proteins to induce local changes in cytoskeletal dynamics and cell motility. MRL proteins alter monomeric (G):filamentous (F) actin ratios, but the impact of these changes had not been fully appreciated. This study reports here that the Drosophila MRL ortholog, pico, is required for tissue and organismal growth. Reduction in pico levels resulted in reduced cell division rates, growth retardation, increased G:F actin ratios and lethality. Conversely, pico overexpression reduced G:F actin ratios and promoted tissue overgrowth in an epidermal growth factor (EGF) receptor (EGFR)-dependent manner. Consistently, in HeLa cells, lamellipodin was required for EGF-induced proliferation. pico and lamellipodin share the ability to activate serum response factor (SRF), a transcription factor that responds to reduced G:F-actin ratios via its co-factor Mal. Genetics data indicate that mal/SRF levels are important for pico-mediated tissue growth. It is proposed that MRL proteins link EGFR activation to mitogenic SRF signaling via changes in actin dynamics (Lyulcheva, 2008).

The construction of properly sized and functional tissues and organs during animal development requires tight control of cell growth, proliferation, differentiation, and death. Networks of intracellular signal transduction pathways that respond to various secreted ligands and cell surface proteins coordinate these processes. Elucidating the nature of the intracellular signaling networks that connect extracellular stimuli to basic cellular machinery controlling proliferation, growth, and morphology is not only critical for the understanding of tissue size regulation during normal development, but is also important for the identification of aberrant events underlying numerous disease processes, including cancer (Lyulcheva, 2008).

A number of pathways regulating cellular development are initiated by ligation of transmembrane receptor tyrosine kinases (RTKs), such as the epidermal growth factor (EGF) receptor (EGFR). One of the key mediators of RTK signaling is the Ras GTPase, capable of activating proteins harboring Ras association (RA) domains to initiate downstream signaling pathways, such as the mitogen-activated protein kinase (MAPK) cascade, and ultimately resulting in changes in gene transcription. The Ras/MAPK and other canonical RTK signaling pathways have been well characterized, yet they cannot account for all of the observed effects of their respective extracellular signals (Lyulcheva, 2008).

The MIG-10/Rap1-GTP-interacting adaptor molecule (RIAM)/lamellipodin (Lpd) (MRL) proteins are a family of recently identified molecular adaptors, harboring an RA, pleckstrin homology (PH), and several proline-rich domains (Krause, 2004; Lafuente, 2004). Several lines of evidence indicate that MRL proteins act downstream of Ras-like GTPases and transduce extracellular signals to changes in the actin cytoskeleton, cell motility, and adhesion. In particular, Lpd interacts with active Ras and RIAM with active Rap1. Consistent with this, only RIAM is required for Rap1-induced cell adhesion (Lafuente, 2004; Rodriguez-Viciana, 2004). Lpd also binds to PI(3,4)P2 via its PH domain, which is sufficient for membrane targeting after platelet-derived growth factor stimulation (Krause, 2004). Both Lpd and RIAM utilize their proline-rich motifs to directly interact with the Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) actin regulators, known to regulate lamellipodia formation and cell migration (Jenzora, 2005; Krause, 2004; Lafuente, 2004). In addition, Lpd knockdown impairs lamellipodia formation, whereas Lpd overexpression increases speed of lamellipodia protrusion in an Ena/VASP-dependent manner (Krause, 2004). Finally, both Lpd and RIAM have been shown to alter the cellular ratio between monomeric (G) and filamentous (F) actin (Krause, 2004; Lafuente, 2004), suggesting a wider role in regulating cell metabolism. Indeed, control of the G:F actin ratio is an essential way for cells to regulate gene transcription via the transcription factor serum response factor (SRF), and has been linked to changes in proliferation, migration, and differentiation (Lyulcheva, 2008).

This study reports the characterization of the Drosophila MRL ortholog, which has been named pico on the basis of the retarded growth phenotype resulting from pico knockdown or loss-of-function mutant. Reduction in pico levels results in reduced rates of cell growth and proliferation, whereas ectopic expression of pico promotes coordinated cell growth and proliferation, leading to tissue overgrowth. pico's effect on cell proliferation is conserved in its mammalian ortholog, Lpd. Evidence is presented that pico and Lpd link extracellular signaling to tissue growth via changes in actin dynamics and SRF activation. This is the first time that MRL proteins have been implicated in controlling cell proliferation and tissue growth (Lyulcheva, 2008).

Phylogenetic analysis has shown that pico (CG11940) encodes the only member of the MRL family of proteins in Drosophila. Two transcripts that were identified are generated from alternative transcription start sites of the pico transcription unit: pico and pico-L. pico-L encodes a 1159 amino acid protein that is identical to the protein encoded by pico, except for the presence of an additional 128 N-terminal residues. Both pico proteins contain RA and PH domains and proline-rich Ena/VASP binding sites characteristic of the MRL proteins (Lyulcheva, 2008).

This study shows that pico, which encodes the only Drosophila member of the MRL family of proteins, and its mammalian ortholog, Lpd, have a conserved role in the regulation of cellular proliferation. Reduced pico or Lpd levels result in reduced rates of cellular proliferation, but do not impair cell survival. Too much pico promotes coordinated growth and proliferation, leading to larger tissues with more normal-sized cells. In this respect, the effect of pico is distinct from that of many known Drosophila growth drivers. Growth regulators, such as Drosophila S6K, cause cells to accumulate mass faster than they can divide, primarily due to effects on translation, leading to cellular hypertrophy. Other regulators, such as E2F, can drive cell division without stimulating cell growth, leading to hyperplastic cellular hypotrophy and/or apoptosis (Lyulcheva, 2008).

Attenuating EGFR signaling abrogates the effect of ectopic pico on both F-actin accumulation and tissue growth. pico acts cell autonomously and is therefore unlikely to act upstream of Egfr by affecting the level of EGFR ligands. To rule out that pico regulates levels of EGFR, receptor levels and distribution was examined in wing imaginal discs overexpressing pico or pico^IR. EGFR levels and distribution in these genetic backgrounds resembled wild-type. Another possibility is that pico regulates EGFR activity. Although suitable reagents were not available to directly monitor EGFR activity levels in wing discs, effects on extracellular signal-regulated kinase (ERK) activation, which provides a molecular readout for EGFR/Ras/Raf signaling, were measured. Diphosphorylated (dp) ERK levels were not affected by ectopic pico. These data suggest that, rather than being upstream of EGFR, Pico needs to be activated by EGFR or a downstream component of EGFR signaling, such as activated Ras. Consistently, both Lpd and Pico bind to activated, but not wild-type, Ras. Furthermore, pico knockdown partially suppresses the effects of ectopic Egfr and activated Ras; in addition, Lpd knockdown impairs the EGF-induced increase in proliferation. Taken together, these data suggest that pico and Lpd are downstream effectors of EGFR (Lyulcheva, 2008).

Ena/VASP has been reported to act downstream of MRL proteins. Correspondingly, it was found that pico-mediated wing overgrowth and F-actin accumulation are sensitive to the levels of ena. Importantly, ena is also sufficient to cause overgrowth and F-actin accumulation when overexpressed. Changes in actin dynamics induced by Ena/VASP proteins can activate SRF-dependent gene expression in mammalian cells. Similarly, it was found that Pico and Lpd can activate SRF activity. Like pico, ectopic mal or bs/SRF in flies are sufficient to cause wing overgrowth. Pico-mediated overgrowth is sensitive to the levels of bs/SRF, but mal-induced overgrowth could not be suppressed by pico knockdown, suggesting that Mal/SRF may act downstream of pico in flies. Collectively, these data suggest that MRL proteins may exert their mitogenic effects by specifically interacting with Ena/VASP proteins and inducing SRF-responsive transcription. Interactions between EGFR, MRL proteins, Ena/VASP, and Mal may provide a mechanism linking growth factor signaling and Mal-mediated SRF activation (Lyulcheva, 2008).

Are MRL proteins uniquely able to stimulate Mal/SRF-mediated tissue growth? Although other actin regulators are known to activate Mal/SRF (Posern, 2006), there is currently little data to indicate that they play a role in proliferation control. This might be explained if different transcriptional responses occur at different Mal-dependent SRF activation thresholds, leading to diverse cellular outcomes. Alternatively, other actin regulators might influence processes that limit net tissue growth. For instance, Rho activates Mal/SRF in mammalian cells, but increased Rho activity in flies is associated with loss of epithelial integrity and cell extrusion, which may negate any potential mal-mediated growth-promoting effects. These issues warrant further study in both flies and mammals. Future studies are also needed to characterize transcriptional targets of Drosophila SRF and resolve the contribution of SRF targets to MRL-mediated growth and proliferation (Lyulcheva, 2008).

Lpd expression appears to be differentially regulated in cancer compared to normal tissues (Dahl, 2005; Eppert, 2005; Ginestier, 2006). The data, showing a conserved role for MRL proteins in proliferation control, may provide a potential mechanistic explanation for these observations. In this regard, it is interesting that loss of pico or Lpd can abrogate the effects of EGFR/Erb signaling, deregulation of which has also been implicated in cancer progression. Collectively, these data suggest that MRL proteins might play a role in the pathogenesis of certain cancers and may therefore represent novel molecular targets for therapeutic intervention (Lyulcheva, 2008).

Lamellipodin and the Scar/WAVE complex cooperate to promote cell migration in vivo

Cell migration is essential for development, but its deregulation causes metastasis. The Scar/WAVE complex is absolutely required for lamellipodia and is a key effector in cell migration, but its regulation in vivo is enigmatic. Lamellipodin (Lpd) controls lamellipodium formation through an unknown mechanism. This study reports that Lpd directly binds active Rac, which regulates a direct interaction between Lpd and the Scar/WAVE complex via Abi. Consequently, Lpd controls lamellipodium size, cell migration speed, and persistence via Scar/WAVE in vitro. Moreover, Lpd knockout mice display defective pigmentation because fewer migrating neural crest-derived melanoblasts reach their target during development. Consistently, Lpd regulates mesenchymal neural crest cell migration cell autonomously in Xenopus laevis via the Scar/WAVE complex. Further, Lpd's Drosophila melanogaster orthologue Pico binds Scar, and both regulate collective epithelial border cell migration. Pico also controls directed cell protrusions of border cell clusters in a Scar-dependent manner. Taken together, Lpd is an essential, evolutionary conserved regulator of the Scar/WAVE complex during cell migration in vivo (Law, 2013).

This study reveals that Lpd colocalizes with the Scar/WAVE complex at the very edge of lamellipodia and directly interacts with this complex by binding to the Abi-SH3 domain. Active Rac directly binds Lpd, thereby regulating the interaction between Lpd and the Scar/WAVE complex. It is therefore postulated that Lpd acts as a platform to link active Rac and the Scar/WAVE complex at the leading edge of cells to regulate Scar/WAVE-Arp2/3 activity and thereby lamellipodium formation and cell migration (Law, 2013).

Knockdown of Lpd expression or KO of Lpd highly impaired lamellipodium formation, phenocopying the effect of Scar/WAVE complex knockdown on lamellipodium formation. Conversely, it was observed that overexpression of Lpd increased lamellipodia size in Xenopus NC cells, and this was dependent on the interaction with Abi, linking it to the Scar/WAVE complex. Overexpression of Pico, the Lpd fly orthologue, aberrantly increased the number and frequency of cellular protrusions at the rear of border cell clusters in a Scar-dependent manner, which suggests that the regulation of Scar/WAVE by Lpd is evolutionary conserved. Collectively, these data suggest that Lpd functions to generate lamellipodia via the Scar/WAVE complex (Law, 2013).

Lpd or Pico knockdown or Lpd KO impaired cell migration in vitro and in vivo in Drosophila, Xenopus, and mice. Lpd KO or knockdown cells were unable to migrate via lamellipodia but instead migrated very slowly by extending filopodia. The same residual migration mode had been observed for Arp2/3 knockdown cells. Arp2/3 is activated by the Scar/WAVE complex to regulate cell migration. It was also observed that both Lpd and Abi knockdown impaired NC migration in vivo. Consistently, it was found that Lpd and Abi-Scar/WAVE are in the same pathway regulating cell migration. This is consistent with recent studies suggesting that the Lpd orthologue in C. elegans, mig-10, genetically interacts with abi-1 to regulate axon guidance, synaptic vesicle clustering, and excretory canal outgrowth in C. elegans (Stavoe, 2012; Xu, 2012; McShea, 2013). Collectively, these results suggest that Lpd functions in cell migration via the Scar/WAVE complex in mammalian cells, Xenopus NC cells, and Drosophila border cells (Law, 2013).

Lpd not only interacts with the Scar/WAVE complex but also directly binds to Ena/VASP proteins. Ena/VASP proteins regulate actin filament length by temporarily preventing capping of barbed ends and by recruiting profilin-actin to the growing end of actin filaments. In contrast, the Scar/WAVE-Arp2/3 complexes increase branching of actin filaments. Lamellipodia with a highly branched actin network protrude more slowly but are more persistent, whereas lamellipodia with longer, less branched actin filaments protrude faster but are less stable and quickly turn into ruffles. It was observed that Lpd overexpression increases cell migration in a Scar/WAVE- and not Ena/VASP-dependent manner. This is consistent with a predominant function of Scar/WAVE downstream of Lpd to regulate a highly branched actin network supporting persistent lamellipodia protrusion and cell migration. Other actin-dependent cell protrusions such as axon extension or dorsal ruffles of fibroblasts require Lpd-Ena/VASP-mediated F-actin structures (Law, 2013).

Collective cell migration describes a group of cells that moves together and affect each other, and various types of collective cell migration exists during development and cancer invasion. Xenopus NC cells migrate as loose streams, whereas Drosophila border cells migrate as a cluster of cells with close cell-cell contacts. This study found that Rac regulates Lpd and Scar/WAVE interaction and that both are required for Xenopus NC migration, which is consistent with previous work in which Rac activity mediates this type of migration. Similarly, NC-derived melanoblast migration in the mouse depends on Rac-Scar/WAVE-Arp2/3, and it was found that Lpd functions in this process as well (Law, 2013).

Drosophila border cell clusters migrate through the fly egg chamber in two phases: an early part characterized by large and persistent front extensions, which are regulated predominantly by PVR (the fly PDGF receptor); and a late part characterized by dynamic collective 'tumbling' behavior. Surprisingly, Pico overexpression resulted in the appearance of a higher proportion of rear facing extensions, a phenotype previously observed with dominant-negative PVR, causing premature tumbling of the border cell cluster. This suggests that Pico function is normally tightly controlled to stabilize specific extensions and functions also in guidance of collective cell migration. Because Lpd-Scar/WAVE control single cell migration as well as collective cell migration, this suggests that they function as general regulators of cell migration (Law, 2013).

Collectively, this study has identified a novel pathway in which Lpd functions as an essential, evolutionary conserved regulator of the Scar/WAVE complex during cell migration in vivo (Law, 2013).

Synaptic vesicle clustering requires a distinct MIG-10/Lamellipodin isoform and ABI-1 downstream from Netrin

The chemotrophic factor Netrin can simultaneously instruct different neurodevelopmental programs in individual neurons in vivo. How neurons correctly interpret the Netrin signal and undergo the appropriate neurodevelopmental response is not understood. This study identified MIG-10 isoforms as critical determinants of individual cellular responses to Netrin. Distinct MIG-10 isoforms, varying only in their N-terminal motifs, can localize to specific subcellular domains and are differentially required for discrete neurodevelopmental processes in vivo. MIG-10B was identified as an isoform uniquely capable of localizing to presynaptic regions and instructing synaptic vesicle clustering in response to Netrin. MIG-10B interacts with Abl-interacting protein-1 (ABI-1)/Abi1, a component of the WAVE complex, to organize the actin cytoskeleton at presynaptic sites and instruct vesicle clustering through SNN-1/Synapsin. A motif in the MIG-10B N-terminal domain was identified that is required for its function and localization to presynaptic sites. With this motif, a dominant-negative MIG-10B construct was engineered that disrupts vesicle clustering and animal thermotaxis behavior when expressed in a single neuron in vivo. These findings indicate that the unique N-terminal domains confer distinct MIG-10 isoforms with unique capabilities to localize to distinct subcellular compartments, organize the actin cytoskeleton at these sites, and instruct distinct Netrin-dependent neurodevelopmental programs (Stavoe, 2012).

Abelson interactor-1 (ABI-1) interacts with MRL adaptor protein MIG-10 and is required in guided cell migrations and process outgrowth in C. elegans

Directed cell migration and process outgrowth are vital to proper development of many metazoan tissues. These processes are dependent on reorganization of the actin cytoskeleton in response to external guidance cues. During development of the nervous system, the MIG-10/RIAM/Lamellipodin (MRL) signaling proteins are thought to transmit positional information from surface guidance cues to the actin polymerization machinery, and thus to promote polarized outgrowth of axons. In C. elegans, mutations in the MRL family member gene mig-10 result in animals that have defects in axon guidance, neuronal migration, and the outgrowth of the processes or 'canals' of the excretory cell, which is required for osmoregulation in the worm. In addition, mig-10 mutant animals have recently been shown to have defects in clustering of vesicles at the synapse. To determine additional molecular partners of MIG-10, a yeast two-hybrid screen was conducted using isoform MIG-10A as bait, and Abelson-interactor protein-1 (ABI-1) was isolated. ABI-1, a downstream target of Abl non-receptor tyrosine kinase, is a member of the WAVE regulatory complex (WRC) involved in the initiation of actin polymerization. Further analysis using a co-immunoprecipitation system confirmed the interaction of MIG-10 and ABI-1 and showed that it requires the SH3 domain of ABI-1. Single mutants for mig-10 and abi-1 displayed similar phenotypes of incomplete migration of the ALM neurons and truncated outgrowth of the excretory cell canals, suggesting that the ABI-1/MIG-10 interaction is relevant in vivo. Cell autonomous expression of MIG-10 isoforms rescued both the neuronal migration and the canal outgrowth defects, showing that MIG-10 functions autonomously in the ALM neurons and the excretory cell. These results suggest that MIG-10 and ABI-1 interact physically to promote cell migration and process outgrowth in vivo. In the excretory canal, ABI-1 is thought to act downstream of UNC-53/NAV2, linking this large scaffolding protein to actin polymerization during excretory canal outgrowth. abi-1^RNAi enhanced the excretory canal truncation observed in mig-10 mutants, while double mutant analysis between unc-53 and mig-10 showed no increased truncation of the posterior canal beyond that observed in mig-10 mutants. Morphological analysis of mig-10 and unc-53 mutants showed that these genes regulate canal diameter as well as its length, suggesting that defective lumen formation may be linked to the ability of the excretory canal to grow out longitudinally. Taken together, these results suggest that MIG-10, UNC-53, and ABI-1 act sequentially to mediate excretory cell process outgrowth (McShea, 2013).

Search PubMed for articles about Drosophila Pico

Dahl, E., et al. (2005). Systematic identification and molecular characterization of genes differentially expressed in breast and ovarian cancer. J. Pathol. 205: 21-28. PubMed ID: 15586368

Eppert, K., et al. (2005). Altered expression and deletion of RMO1 in osteosarcoma. Int. J. Cancer 114: 738-746. PubMed ID: 1560930

Ginestier, C., et al. (2006). Prognosis and gene expression profiling of 20q13-amplified breast cancers. Clin. Cancer Res. 12: 4533-4544. PubMed ID: 16899599

Jenzora, A., et al. (2005). PREL1 provides a link from Ras signalling to the actin cytoskeleton via Ena/VASP proteins. FEBS Lett. 579: 455-463. PubMed ID: 15642358

Krause, M., et al. (2004). Lamellipodin, an Ena/VASP ligand, is implicated in the regulation of lamellipodial dynamics. Dev. Cell 7: 571-583. PubMed ID: 15469845

Lafuente, et al. (2004). RIAM, an Ena/VASP and Profilin ligand, interacts with Rap1-GTP and mediates Rap1-induced adhesion. Dev. Cell 7: 585-595. PubMed ID: 15469846

Law, A. L., Vehlow, A., Kotini, M., Dodgson, L., Soong, D., Theveneau, E., Bodo, C., Taylor, E., Navarro, C., Perera, U., Michael, M., Dunn, G. A., Bennett, D., Mayor, R. and Krause, M. (2013). Lamellipodin and the Scar/WAVE complex cooperate to promote cell migration in vivo. J Cell Biol. 203(4): 673-89. PubMed ID: 24247431

Lyulcheva, E., Taylor, E., Michael, M., Vehlow, A., Tan, S., Fletcher, A., Krause, M. and Bennett, D. (2008). Drosophila pico and its mammalian ortholog lamellipodin activate serum response factor and promote cell proliferation. Dev. Cell 15(5): 680-90. PubMed ID: 19000833

McShea, M. A., Schmidt, K. L., Dubuke, M. L., Baldiga, C. E., Sullender, M. E., Reis, A. L., Zhang, S., O'Toole, S. M., Jeffers, M. C., Warden, R. M., Kenney, A. H., Gosselin, J., Kuhlwein, M., Hashmi, S. K., Stringham, E. G. and Ryder, E. F. (2013). Abelson interactor-1 (ABI-1) interacts with MRL adaptor protein MIG-10 and is required in guided cell migrations and process outgrowth in C. elegans. Dev Biol 373: 1-13. PubMed ID: 23022657

Posern, G. and Treisman, R. (2006). Actin' together: serum response factor, its cofactors and the link to signal transduction. Trends Cell Biol. 16: 588-596. PubMed ID: 17035020

Rodriguez-Viciana, P., Sabatier, C. and McCormick, F. (2004). Signaling specificity by Ras family GTPases is determined by the full spectrum of effectors they regulate. Mol. Cell. Biol. 24: 4943-4954. PubMed ID: 15143186

Stavoe, A. K., Nelson, J. C., Martinez-Velazquez, L. A., Klein, M., Samuel, A. D. and Colon-Ramos, D. A. (2012). Synaptic vesicle clustering requires a distinct MIG-10/Lamellipodin isoform and ABI-1 downstream from Netrin. Genes Dev 26: 2206-2221. PubMed ID: 23028145

Xu, Y. and Quinn, C. C. (2012). MIG-10 functions with ABI-1 to mediate the UNC-6 and SLT-1 axon guidance signaling pathways. PLoS Genet 8: e1003054. PubMed ID: 23209429

date revised: 10 February 2014

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