BIOLOGICAL OVERVIEW

Cortactin is a Src substrate that interacts with F-actin and can stimulate actin polymerization by direct interaction with the Arp2/3 complex. Complete loss-of-function mutants of the single Drosophila Cortactin gene have been isolated. Mutants are viable and fertile, showing that Cortactin is not an essential gene. However, Cortactin mutants show distinct defects during oogenesis. During oogenesis, Cortactin protein is enriched at the F-actin rich ring canals in the germ line, and in migrating border cells. In Cortactin mutants, the ring canals are smaller than normal. A similar phenotype has been observed in Src64 mutants and in mutants for genes encoding Arp2/3 complex components, supporting that these protein products act together to control specific processes in vivo. Cortactin mutants also show impaired border cell migration. This invasive cell migration is guided by Drosophila EGFR and PDGF/VEGF receptor (PVR). Accumulation of Cortactin protein is positively regulated by PVR. Also, overexpression of Cortactin can by itself induce F-actin accumulation and ectopic filopodia formation in epithelial cells. Evidence is presented that Cortactin is one of the factors acting downstream of PVR and Src to stimulate F-actin accumulation. Cortactin is a minor contributor in this regulation, consistent with the Cortactin gene not being essential for development (Somogyi, 2004).

Cortactin was originally identified as a major substrate of the Src tyrosine kinase (Wu, 1991). The name Cortactin reflects that the protein binds to F-actin and that it localizes to the cell cortex, including membrane ruffles and lamellipodia (Wu, 1993). These features, plus the presence of an SH3 domain and proline-rich regions in the Cortactin protein, suggest that Cortactin might link signaling events to the actin cytoskeleton. Phosphorylation of Cortactin stimulated by Src modulates its activity in vivo (Huang, 1997). Cortactin phosphorylation and subcellular localization are also affected by receptor tyrosine kinases (RTKs) (Maa, 1992 and Zhan, 1993). The cortactin gene was also identified as EMS1, a putative oncogene encoding one of the transcripts amplified in certain human carcinomas (Schuuring, 1993). Directed overexpression of EMS1/cortactin has been shown to increase the motility of and invasion of fibroblasts (Patel, 1998) and the metastatic potential of breast cancer cells (Li, 2001). Cortactin is also enriched in 'invadopodia' from invasive tumor cells, cellular protrusions associated with degradation of extracellular matrix (Bowden, 1999). Together, these studies suggest that Cortactin may play a role in promoting cell motility of cancer cells, as well as of normal cells in response to growth factor stimulation (Somogyi, 2004 and references therein).

Recent studies have shown that Cortactin can directly influence actin polymerization. The Arp2/3 complex is an important nucleator of actin polymerization. It stimulates actin polymerization while binding to actin filaments and can thereby induce formation of a branched actin network. The Arp2/3 complex consists of multiple proteins and is very conserved through evolution, from yeast to man. Cortactin can bind to and activate the Arp2/3 complex as well as stabilize the resulting F-actin network (Uruno, 2001 and Weaver, 2001). Cortactin may act synergistically with proteins of the WASP family, that are potent regulators of Arp2/3 activity (Weaver, 2002). In addition to WASP and N-WASP, the WASP family includes the SCAR/WAVE proteins. The WASP family of proteins is regulated by GTPases of the Rho subfamily, namely CDC42 and Rac, WASP by direct binding (Higgs, 2001), and SCAR/WAVE by dissociation of an inhibitory complex (Eden, 2002). Cortactin can also associate with the adaptor protein WIP (WASP interacting protein), which binds monomeric actin and thus may aid Cortactin in actin nucleation (Kinley, 2003). Arp2/3 and its regulators are required for extensions of lamellipodia and other actin-rich structures, but also for intracellular trafficking events such as endocytosis. Cortactin has been suggested to link endocytosis to Arp2/3 mediated actin polymerization in mammalian cells. Cortactin is associated with dynamin2 and with vesicles (McNiven, 2000). Consistent with this type of function, inhibition of Cortactin inhibits receptor mediated endocytosis (Cao, 2003) (Somogyi, 2004 and references therein).

A Cortactin protein has been identified in Drosophila (Katsube, 1998), but not in yeast. It is possible that other proteins substitute for Cortactin in yeast. It is also possible that the function of Cortactin is specific to higher eukaryotes that, contrary to yeast, use tyrosine kinases for cell-cell communication and cell regulation. There are multiple RTKs in Drosophila that control cell growth, differentiation and morphogenesis including EGFR, insulin receptor and PDGF/VEGF receptor. The Drosophila genome also encodes a number of non-receptor tyrosine kinases such as Src. There are two Src genes, Src42 and Src64. Src42 may act downstream of RTKs to modify their signaling. Src64 is specifically required for proper morphogenesis of actin-rich structures in the female germ line, called ring canals. Drosophila Cortactin protein is localized to the cell cortex and shows protein-protein interactions similar to that of mammalian Cortactin (Katsube, 1998). To understand the function of Cortactin in vivo, complete loss-of-function mutants of Drosophila Cortactin have been generated. Cortactin mutants are fully viable and are fertile, but have subtle defects during oogenesis. This analysis suggests that Cortactin acts downstream of RTKs and Src in vivo. The analysis also indicates that Cortactin is only a minor mediator of the effects of tyrosine kinases on the actin cytoskeleton (Somogyi, 2004 and references therein).

Cortactin was originally isolated as a substrate of the Src kinase. Further biochemical characterization of mammalian Cortactin indicates that it could directly affect actin polymerization by stimulating activity of the Arp2/3 complex in addition to stabilizing the F-actin network. Analysis of mammalian cells has also indicated that phosphorylation by Src is critical for Cortactin activity in affecting cell behavior such as cell migration. In fact, a non-phosphorylatable form can act as a dominant negative protein (Huang, 1998). Phosphorylation by Src can also affect the ability of Cortactin to crosslink F-actin (Huang, 1997), but phosphorylation may not directly affect Arp2/3 interaction (Weaver, 2001 and Uruno, 2001). Genetic analyses in Drosophila indicate that Src, Cortactin and Arp2/3 components control at least some of the same processes in vivo, rather than Src and Arp2/3 being associated with different aspects of Cortactin function. For example, in the germ line of the ovary, mutations in Src64, Cortactin or components of the Arp2/3 complex all affect the actin-rich structures called ring canals in a similar way. This supports the notion that Src, Cortactin and Arp2/3 act together in vivo. However, the interactions may be quite indirect. Perhaps Src induced phosphorylation of Cortactin influence its localization or stability in vivo, which in turn affects the ability of Cortactin to stimulate Arp2/3 (Somogyi, 2004).

The effects of Cortactin in border cells and other follicle cells allow its relationship to a specific RTK, namely PVR, to be investiged. This is of some interest as Cortactin has been implicated both in stimulation of actin polymerization per se, and in endocytosis. The two functions could reflect the same actin-regulatory biochemical function of Cortactin. PVR signaling stimulates actin polymerization in follicle cells and down-regulation of PVR via endocytosis might limit this effect. Genetic analysis indicates that Cortactin plays a positive role downstream of PVR in stimulating actin polymerization, rather than a negative role by limiting receptor signaling. That Cortactin also contributes in a minor way to stimulating receptor endocytosis, in these cells or in other cells, cannot be ruled out. However, genes encoding other proteins that directly control endocytosis such as alpha-adaptin or Hepatocyte growth factor regulated tyrosine kinase substrate are essential in Drosophila. Some mutants have phenotypes that reflect upregulation of specific signaling pathways indicating that endocytosis is important for limiting activity of these signaling pathways in vivo. One of the useful features of Drosophila as a model system is that distinctive defects are produced upon mis-regulation of different signaling pathways. However, no such visible phenotypes were seen in the Cortactin mutants (Somogyi, 2004).

It is perhaps surprising that Cortactin in not an essential gene. Many modulators of essential processes in the cell such as dynamics of the cytoskeleton, cell adhesion or cell signaling are very well conserved in higher eukaryotes. They may add to the robustness and fidelity of the regulation, but they may only be essential to the organism if their absence completely changes the behavior or fate of specific, important cells. In mammals, there are often multiple closely related genes and simple redundancy between these gene products may explain an absence of phenotypes in knockout mice. In Drosophila, this type of simple redundancy is less frequent. For example, there is no evidence for another Cortactin gene in the sequenced Drosophila genome. However, more distantly related genes may have overlapping functions. Subtle phenotypes may also reflect that one process can be regulated in multiple ways. Combining multiple mutations can then be used to genetically help define which genes and pathways overlap in function (Somogyi, 2004).

I-BAR protein antagonism of endocytosis mediates directional sensing during guided cell migration

Although directed cellular migration facilitates the coordinated movement of cells during development and repair, the mechanisms regulating such migration remain poorly understood. Missing-in-metastasis (MIM) is a defining member of the inverse Bin/Amphiphysin/Rvs domain (I-BAR) subfamily of lipid binding, cytoskeletal regulators whose levels are altered in a number of cancers. This study provides the first genetic evidence that an I-BAR protein regulates directed cell migration in vivo. Drosophila MIM (dmim, CG33558) is involved in Drosophila border cell migration, with loss of dmim function resulting in a lack of directional movement by the border cell cluster. In vivo endocytosis assays combined with genetic analyses demonstrate that the dmim product regulates directed cell movement by inhibiting endocytosis and antagonizing the activities of the CD2-associated protein/cortactin complex in these cells. These studies demonstrate that DMIM antagonizes pro-endocytic components to facilitate polarity and localized guidance cue sensing during directional cell migration (Quinones, 2010).

Directed cellular migration facilitates the coordinated movement of individual cells, cell clusters, and sheets of cells during development and regeneration. Directed cell migration is important for individual cells as well as groups of cells; there are key differences between these types of migrating cells. Individual cells migrating, either on a plastic dish or in a living tissue matrix, must correctly sense or respond to migratory cues. These cells then undergo changes in their cytoskeletal structure in order to project their cell bodies forward, put down new adhesion complexes, and remove older adhesion complexes at the trailing end of the cell. Although cells in a cluster or sheet must also respond to directional cues, they must also maintain the correct cell-cell adhesions and spatial awareness in order to maintain their structure. Directed cluster migration forms the foundation for organ morphogenesis and, when abnormal, has been implicated in disease states such as mental retardation, birth defects, and cancer. Insights into directed cell migration of groups of cells have come from the studies of relatively small clusters of cells, such as border cell migration during Drosophila oogenesis or the rearrangement of larger sheets, as is the case during vertebrate gastrulation. In Drosophila border cell migration, cell clusters initially become autonomously motile, elaborating nondirectional actin-based cellular extensions with little net cellular displacement. Border cell migration is mediated in response to local migratory cues emanating from the ovary via guidance receptors, the Drosophila epidermal growth factor receptor (DER) and the PDGF/VEGF-like (PVR) receptor, on the cluster surface. Signaling through the receptors allows border cell membranes to become polarized to form actin-based membrane extensions and migrate along the growth factor gradient. Genetic studies overexpressing DER in border cells indicate that it is not the total levels of receptor, but the location of activated receptors that determines directional migration. Despite its importance, understanding of signaling mechanisms downstream of the guidance receptors that operate in the context of developing organisms remains primitive (Quinones, 2010).

Previous work points to a central role for guidance receptor endocytosis in interpreting local migratory cues to the underlying cytoskeleton. In cultured mammalian cells, localized receptor-mediated endocytosis and receptor recycling amplifies the guidance signal to focally activate key regulators of the cytoskeleton such as the GTPase Rac1. Similar mechanisms appear to control Drosophila border cell migration. Forward genetic screens for migration mutants have identified cue-specific components such as the nonreceptor tyrosine kinase Src, components of the endocytic machinery, and the CD2-associated protein (CD2AP)/cortactin complex. Each of these components has been shown to regulate endocytosis and cell migration, but little information exists about how they function to regulate directionality during migration (Quinones, 2010).

Increasing attention has been directed toward the Bin/Amphiphysin/Rvs (BAR) superfamily of proteins and their role in endocytosis and vesicle trafficking (Dawson, 2006; Frost, 2007). Each of the BAR domain subfamilies of curvature-dependent molecular scaffolds are thought to bring together effector complexes to distinct lipid surface in order to regulate actin cytoskeletal remodeling near vesicles. For example, the BAR protein endophilin is critical for early vesicle scission and EGF receptor signaling (Kaneko, 2005), and isoforms have been associated with both tumor suppression and oncogenesis. Endophilin is recruited in a receptor-dependent manner through the formation of complexes with the Cbl-associated proteins CIN85 and CD2AP. The endophilin/CD2AP complex in turn mediates vesicle scission through the recruitment of cortactin and the actin-polymerization machinery (Dikic, 2002; Lynch, 2003; Kaksonen, 2006; Quinones, 2010 and references therein).

The newest family of BAR domain proteins is the inverse or IMD BAR (I-BAR) family. IMD proteins are defined by the proteins Missing-In-Metastasis (MIM) and the insulin receptor substrate 53 (IRSp53) cytoskeletal regulators (Miki, 2000; Lee, 2002). MIM was originally identified as a gene whose expression is down-regulated in a variety of urogenital metastatic cancer, but other studies have also demonstrated elevated MIM levels in many hedgehog-dependent tumors and metastatic endodermal tumors such as hepatocellular carcinomas. Like many BAR family proteins, MIM contains several protein-protein interaction modules that suggest it functions to scaffold protein complexes at membranes. Crystal structure analysis indicates that the shape of the IMD dimer is the most convex of the family members thus far, suggesting that the I-BAR family senses a very distinct class of membranes. I-BAR family members have also been well studied as membrane-deforming proteins with the capacity to cause membrane tubulation and projections. Because each of the other BAR family members has roles in positively regulating endocytosis, the convex shape of the I-BAR proteins is proposed to be involved in antagonizing endocytosis (Quinones, 2010).

This study provides the first in vivo genetic evidence for the involvement of an I-BAR family member in regulating directional migration. MIM and cortactin antagonism is shown to underlie a novel molecular steering mechanism (Quinones, 2010).

This study shows, through genetic interaction and live-cell imaging, that migrating cells use a MIM-dependent steering mechanism to interpret local migratory signals. MIM’s role appears to be general, as both border cell and PGC migration are affected in dmim mutants and involve different cell types responding to different guidance cues. The data indicate that MIM inhibits guidance receptor endocytosis by competing directly with CD2AP for cortactin, resulting in dampened guidance receptor signaling. This study provides the first genetic and biochemical evidence for the function of a member of the I-BAR family of proteins in directed cell migration, and provides a mechanistic link between MIM and cell migration (Quinones, 2010).

Directional cell migration is a complex process requiring dynamic rearrangements of the cytoskeleton and precise directional sensing of local migratory cues. Live-cell imaging data suggest that DMIM is involved in directing cell migration through the inhibition of endocytosis. Although previous studies demonstrate that MIM is an actin cytoskeletal remodeling protein, the current imaging studies argue against a major, direct role for MIM in general actin polymerization. Consistent with this notion is the lack of apparent defects in adherens junctions, the actin cytoskeleton, or anteroposterior polarity in dmim mutant egg chambers. This is not to say that MIM does not affect the actin cytoskeleton in other cases of cell migration, just that in the case of Drosophila border cell migration the function of DMIM is not required for actin cytoskeletal dynamics. Previous studies have also implicated MIM in regulating Sonic Hedgehog signaling. Mutations in the hedgehog pathway component costal2 result in aberrant border cell numbers, but dmim mutants display a wild-type number of border cells. This discrepancy could be explained in part due to the observation that MIM associates with vertebrate Suppressor of Fused, which is redundant in flies. The data presented in this study uncover migratory defects in PGCs, border cells, and vertebrate cultured fibroblasts, all responding to different migratory cues. This suggests that although cells use different cues and receptors for migration in a variety of systems, the regulation of this process at the level of endocytosis appears to be shared (Quinones, 2010).

These studies identify DMIM as novel I-BAR protein, and one of the first negative regulators of endocytosis with a role in guided cell migration. Genetic, cell biological, and biochemical data support the model that DMIM and CD2AP compete for cortactin in regulating receptor-mediated endocytosis. The observation that removing both MIM and cindr/cortactin results in wild-type migration suggests that MIM and cortactin constitute one of several redundant regulatory systems to control the directional migration of the border cells. Because removal of both proteins restores normal border cell migration but disruption of clathrin and dynamin function does not, it is speculated that other combinations of pro- and anti-endocytosis complexes downstream of dynamin must be operating to balance this process in migrating cells. Although this study has seen a trend in increased endocytosis with MIM/endophilin double knockdown, the lack of complete rescue further suggests endophilin possesses MIM-independent endocytosis functions. Current studies are ongoing to identify these additional signaling pathways in the sensitized dmim;dcortactin background. More importantly, the data show the dramatic effects on migration when components of the steering mechanism are missing or out of balance. Similar effects have also been seen with gross overexpression of cortactin and may explain the relatively high frequency of cortactin and MIM alterations in late-stage cancers (Quinones, 2010).

These results provide a new mechanistic understanding of BAR domain function by showing that directional sensing comes in part from protein complexes competing for common effector proteins during endocytosis. These data support the notion that MIM acts to dampen guidance receptor signaling at a variety of ligand concentrations by sequestering cortactin. Guidance cue binding assembles the N-BAR subfamily member endophilin and its adapter CD2AP, which binds cortactin, shifting it away from MIM sequestration. It is postulated that increased endocytosis and MIM's persistent binding of cortactin prevent the cell from improperly sensing guidance cues and misinterpreting directional differences. Previous studies suggest that phosphorylation of cortactin modulates its interaction with a number of proteins; however, no such alteration was detected using phosphospecific cortactin antibodies in this system. Consistent with this data are the lack of localized bulk MIM protein at the leading edge of cultured cells or rescued dmim; DMIM+ border cells. Altogether, these data suggest that there is a novel MIM-dependent steering mechanism that guides cell migration through interactions with other protein complexes. The importance for regulating both polarity and a localized response to external stimuli during the migration of the border cell cluster has been demonstrated. Previous studies focused on regulation of receptor tyrosine kinase signaling through the action of key proteins involved in the endocytosis of the receptor. This study focused on a negative regulator of endocytosis, which in the Drosophila border cell cluster regulates both polarity and a local response to guidance cues as a means of mediating directional migration (Quinones, 2010).

GENE STRUCTURE

cDNA clone length - 4067

Bases in 5' UTR - 732

Exons - 4

Bases in 3' UTR - 5420

PROTEIN STRUCTURE

Amino Acids - 559

Structural Domains

The gene coding for Drosophila Cortactin was cloned as a Polychaetoid interacting protein using the yeast two hybrid technique. Drosophila Cortactin is a 559-amino acid protein highly expressed in embryos, larvae, and pupae but relatively underexpressed in adult flies. An SH3 domain of approximately 60 amino acids dominates the C-terminal region of Cortactin; no other significant structural motif is found. The SH3 domain is known to bind to a PXXP motif often found in proline-rich regions (Katsube, 1998).

date revised: 25 September 2023

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