To determine the molecular basis for genetic interactions between Src42A, shg and arm, whether or not protein products of these genes come together to form complexes within cells was examined using fractionated embryonic extracts. Membrane and cytosolic fractions were prepared from wild-type embryos and embryos with pnr-GAL4-dependent forced expression of either UAS-Src42A[WT], [KR] (dominant negative) or [YF] (activated). This section discusses endogenous interactions in wild-type embryos, whereas physical interactions in embryos with forced Src expression are described in the subsequent section (Takahashi, 2005).
Membrane and cytosolic fractions of wild-type embryos were treated with anti-Arm or anti-E-cad antibodies and the resultant immunoprecipitates were analyzed by SDS-PAGE and subsequent Western blotting. Any appreciable Src42A/Arm signals were detected in the E-cad immunoprecipitates of untransfected S2 cells, which express only a low level of E-cad (Takahashi, 2005).
Not only Arm but also Src42A and E-cad signals were detected in anti-Arm antibody immunoprecipitates obtained from the membrane fraction. Membranous Src42A signals were also coprecipitated by anti-E-cad antibody treatment. By contrast, there were no appreciable signals of E-cad on treating the cytosolic fraction with anti-Arm antibody. In the cytosolic fraction, Src42A signals co-precipitated with Arm appeared much less prominent than those in the membrane fraction. Accordingly, a significant fraction of membranous Arm, a core component of the putative adherens junction, may be considered to form a complex directly or indirectly with E-cad and Src42A as well (Takahashi, 2005).
Arm protein possesses 13 repeats referred to as Arm repeats, which provide binding sites for many Arm/ß-catenin-binding proteins. To determine whether Src42A binds to Arm directly and if so which part of Src42A is responsible for the Src-Arm interaction, a pull-down assay was carried out. Src42A was thus shown to bind to Arm through interaction of the 14 amino acid kinase domain peptide with Arm repeats (Takahashi, 2005).
In vertebrates, tyrosine phosphorylation has been shown to cause the binding of ß-catenin to E-cad to diminish significantly. Interactions between ß- and alpha-catenin may also be negatively regulated by tyrosine phosphorylation. Therefore studies were performed to see whether Arm phosphorylation requires Src activity. Arm was overexpressed in Drosophila S2 cells and RNAi was carried out to clarify any Src42A and/or Src64 involvement in Arm tyrosine-phosphorylation. The dsRNAs used specifically abolished protein expression of the corresponding target genes. Change in the degree of Arm tyrosine residue phosphorylation was monitored with Arm western blotting of anti-pTyr antibody immunoprecipitates of whole or E-cad-free cell extracts. The levels of Tyr-phosphorylated Arm were reduced by transfection with Src64 and/or Src42A dsRNAs, indicating that redundant Src function is required for Arm phosphorylation (Takahashi, 2005).
Consistent with the present findings on cytological accumulation of cytoplasmic E-cad and Arm signals in cells overexpressing wild type Src42A, increased signals of Arm and E-cad were noted in cytosolic fractions obtained from cells with forced expression of wild type or activated Src42A when using alpha-tubulin or Src42A as the control. Similar Src42A activity-dependent increase in cytosolic Arm signals was observed in anti-Arm precipitates and the supernatant fluid of anti-Src42A precipitates. Such increase may possibly be due to Arm tyrosine residue phosphorylation. However, it is considered that cytosolic Arm accumulation in cells with forced expression of wild type or activated Src42A may not necessarily arise from the tyrosine phosphorylation of Arm. Indeed, any Src42A-activity-dependent tyrosine phosphorylation appeared absent from Src42A-free Arm in the cytosolic fraction. Possibly, Src42A-dependent tyrosine phosphorylation activates some unknown factor responsible for cytosolic Arm stabilization, but not Arm itself (Takahashi, 2005).
The construction and maintenance of normal epithelia relies on local signals that guide cells into their proper niches and remove unwanted cells. Failure to execute this process properly may result in aberrant development or diseases, including cancer and associated metastasis. This study shows that local environment influences the behavior of dCsk-deficient cells. Broad loss of dCsk leads to enlarged and mispatterned tissues due to overproliferation, a block in apoptosis, and decreased cadherin-mediated adhesion. Loss of dCsk in discrete patches leads to a different outcome: epithelial exclusion, invasive migration, and apoptotic death. These latter phenotypes required sharp differences in dCsk activity between neighbors; dE-cadherin, P120-catenin, Rho1, JNK, and MMP2 mediate this signal. Together, these data demonstrate how the cellular microenvironment plays a central role in determining the outcome of altered dCsk activity, and reveal a role for P120-catenin in a mechanism that protects epithelial integrity by removing abnormal cells (Vidal, 2006).
The mechanisms that regulate organ size and shape are not well understood, but recent studies have pointed to the importance of local interactions between neighboring cells. For example, in the process known as 'cell competition', cells with relatively higher proliferative rates actively eliminate their neighbors by programmed cell death. Conversely, apoptotic cells send proliferative signals to their neighbors to compensate for their loss. In this way, normal tissue size is achieved. The misregulation of such mechanisms may contribute to the development of cancer, since most solid tumors arise from intact epithelia and are resistant to size-control signals. Tumors are particularly dangerous when linked to metastasis, a process in which cells leave the primary tumor and invade distant tissues. These processes are best understood within the context of an intact epithelium, in which the full range of cell interactions is retained. Work in Drosophila has provided an important in situ view of the action of oncogenes within epithelia (Vidal, 2006).
Src family kinases (SFKs) are active in a broad range of cancer types, including tumors of the breast, colon, and hematopoietic systems. SFK activity typically increases as tumorigenesis progresses and is associated with metastatic behavior. The major inhibitor of SFK activity is C-terminal Src kinase (Csk) and its paralog Chk; these may act as tumor suppressors in, e.g., breast cancer, presumably through their ability to inhibit Src activity and perhaps other pathways. Drosophila Csk acts primarily or exclusively through Src pathway regulation, and the reduction of dCsk activity by itself led to increased organ size, organismal lethality, and increased cell proliferation due to a failure to exit the cell cycle. However, neither Csk loss nor Src activation has been clearly linked to early events in tumorigenesis, bringing into question the role of Csk/Src in proliferation in vivo. Instead, Src is currently thought to be a major player in the metastatic events that occur later in oncogenesis. How Csk or Src promotes the metastatic behavior of cells in situ remains largely unknown (Vidal, 2006).
This study analyzed the phenotypes of dCsk in the context of developing epithelia. The outcome of a cell's loss of dCsk is linked to its cellular microenvironment. When dCsk activity is reduced broadly in the developing eye or wing, the result is overproliferation, inhibition of apoptosis, and decreased cell adhesion. Tissue integrity is retained, but dCsk cells become inappropriately mobile and fail to maintain their appropriate contacts. The outcome of these effects is an overgrown and mispatterned adult tissue. By contrast, loss of dCsk in discrete patches results in epithelial exclusion, invasive migration through the basal extracellular matrix, and eventual apoptotic death; these events occur exclusively at the boundary between dCsk and wild-type cells. Further emphasizing the unique nature of cells at this boundary, a specific requirement was found for a signal that includes Drosophila orthologs of E-cadherin, P120-catenin, RhoA, JNK, and the metalloprotease MMP2. Hence, this study explores the mechanisms by which the cellular microenvironment can direct different behaviors of cells, both in the regulation of apoptosis and epithelial integrity. It also uncovers a mechanism for the removal of abnormal cells from a normal epithelium (Vidal, 2006).
This study shows that reducing dCskactivity results in a blockade of apoptosis and downregulation of cellular adhesion. The work is consistent with the view that Csk is a tumor suppressor that acts at multiple steps. Mutations in the locus encoding the Csk paralog Chk have been described in breast tumors, and, in this study, it has been observed that human Chk can functionally replace dCsk. Therefore the experimental advantages of developing Drosophila imaginal epithelia were used to explore specific aspects of dCsk function that are relevant to the behavior of tumor cells (Vidal, 2006).
Visualization studies suggest that a reduction in dCsk activity leads to a failure of cells to stably retain associations with their neighbors, resulting in prolonged cell movement as cells slide across each other in a manner not observed in wild-type tissue. This may reflect a failure to establish stable junctions, excess cell motility, or both. Recent work has demonstrated a critical and dynamic role for the cadherin-based apical junctions in patterning the Drosophila retina. Misexpressing dE-cadherin prevents patterning defects in GMR>dCsk-IR retinas, suggesting that dCsk cells have reduced dE-cadherin function. Links between Csk, Src, cadherins, and junctional integrity have been reported in mammalian cell culture, and an association has been observed between Drosophila Src42A and dE-cadherin during embryonic development (Takahashi, 2005). The data are consistent with this view: misexpression of a kinase-dead form of Src42A leads to a disruption in the localization of the dE-cadherin-associated protein Armadillo; also, reduced Armadillo levels observed in dCsk retinas is rescued by dE-cadherin misexpression. Together, these data suggest that altering dCsk/Src activity affects cell movements by decreasing dE-cadherin adhesion (Vidal, 2006).
The mechanism by which dCsk alters dE-cadherin function is not clear, but it is relevant to note that Src activation can shift cadherin-based cell adhesion from a 'strong' to a 'weak' adhesive state in mammalian cultured cells. Phosphorylation of cadherins and catenins may mediate 'inside-out' signaling that can alter the adhesive strength of the homophilic bond between cells (reviewed in Gumbiner, 2005). Evidence for such a mechanism has been provided for integrin-mediated focal adhesions (reviewed in Hynes, 2002), and Src activity can alter focal adhesions (Yeatman, 2004). However, normal basal membrane architecture was observed in dCsk cells, as assessed both by anti-integrin staining and by transmission electron microscopy, indicating that at least the gross structure is not affected (Vidal, 2006).
The ability of dCsk to influence cell proliferation, apoptosis, and cell adhesion is consistent with its ability to direct tissue overgrowth: reducing dCsk activity throughout a tissue (or the entire organism) leads to significantly enlarged tissues. This ability demonstrates that dCsk can participate in the mechanisms that set tissue size. A small number of other proteins have been implicated in this process, including Salvador, Hippo, and Lats/Warts, which show phenotypes that are strikingly similar to dCsk. Furthermore, dCsk can directly phosphorylate Lats/Warts (Stewart, 2003) in vitro (Vidal, 2006).
However, reduction of dCsk activity shows some important differences. Mutations in salvador, hippo, or lats/warts lead to an increase in Diap1 levels, which, in turn, blocks apoptotic cell death. By contrast, reductions in dCsk does not significantly alter Diap1 protein levels. Furthermore, although both Hippo and dCsk are required to exit the cell cycle, the cell cycle profile from hippo mutant cells is normal, while dCsk cells contain a significant shift toward G2/M (Read, 2004; Stewart, 2003). Perhaps the most striking difference is the effects of these factors on discrete mutant patches. While broad loss of dCsk activity leads to expanded tissues, surprisingly discrete patches of dCsk tissue are eliminated by neighboring cells. Unlike salvador, hippo, or lats/warts, clonal patches of dCsk cells fail to survive to adulthood. The effects of dCsk reduction are more similar to those reported for the tumor suppressor gene scribble. The scribble locus encodes a component of the septate junction that regulates cell polarity and proliferation; mutant cells display neoplastic overgrowth in a homotypic environment, but are removed by JNK-dependent apoptosis in discrete clonal patches abutting wild-type tissue (Vidal, 2006).
This work provides evidence that neighboring wild-type tissue provides a locally nonautonomous signal that leads to the removal of dCsk mutant cells. For example, FRT-derived clones of dCsk cells were out-competed by neighbors with normal levels of dCsk: this was most easily seen by the clonally related 'twin spot' of wild-type tissue that was consistently larger than the few surviving dCsk clones. In contrast, FRT-mediated dCsk clones that encompassed the entire eye survived and overproliferated. In the developing wing, cells at the periphery of sd>dCsk-IR or ptc>dCsk-IR expression domains were preferentially removed by apoptosis. This death is dependent not on absolute dCsk activity, but on the juxtaposition of cells that are starkly different in their levels of dCsk. Small differences, for example across the ptc>dCsk-IR or omb>dCsk-IR graded expression domains, did not trigger cell death (Vidal, 2006).
This translocation and death of dCsk-IR cells at the patched/wild-type boundary requires at least two steps. At boundaries with wild-type tissue, dCsk cells initially lose their apical profile, shift downward, and eventually become basally excluded from the epithelium. Such excluded cells then migrate away from the boundaries in both directions and eventually die by apoptosis. These events are strikingly reminiscent of those described for tumor cells undergoing metastasis. Altered activity of both Csk and Src has been implicated in a broad variety of tumors. Typically, however, increased Src activity is associated with later events in tumorigenesis, particularly metastasis. Although the connections between high Src activity and metastases are not understood, they likely include Src's ability to break cell-cell junctions and increase cell motility. Another hallmark of metastatic behavior is the ability to degrade basal extracellular matrix: this study also demonstrate a functional requirement for MMP2 activity during the translocation of mutant cells out of the wing epithelium (Vidal, 2006).
While evidence supports the view that the activity of Csk -- and presumably Src and perhaps other effectors -- can regulate metastatic behavior, it alone is not sufficient. First, reducing dCsk activity by itself is not sufficient to allow migrating cells to survive; the data suggest that most or all eventually die. This is consistent with previous work highlighting the importance of a 'two-hit' model to allow for stable tumor overgrowth and metastasis. A second mutation that prevents apoptotic cell death would be minimally required. Second, all cells within a discrete dCsk patch are not equivalent: cells at the boundary of the clone that border cells of strongly differing dCsk levels are exclusively prone to release from the epithelium. This work predicts that cells at the borders of some human tumors are especially prone toward metastatic behavior. Metastasis is often the most serious aspect of a tumor, and approaches that address the metastatic behavior of cells may need to take into account the properties of cells at the periphery. Understanding whether and how these cells are unique may help to more effectively target therapeutic intervention (Vidal, 2006).
In addition to enabling a detailed examination of dCsk cells and their behavior within an epithelium, this model system permitted identification of signaling components that are necessary to execute the aberrant cell mobility and cell death. The results indicate important roles for dE-cadherin, dP120ctn, Rho1, dJnk, and MMP2 (Vidal, 2006).
JNK-dependent apoptosis is required for a broad palette of related mechanisms such as cell competition in developing tissues and the removal of scribble mutant cells. JNK signaling is also associated with the movement of cells within epithelia, including dorsal closure in Drosophila and in mammals. Interestingly, JNK activity is required for the synthesis of MMP2 by v-Src-transformed mammalian cells (Vidal, 2006).
JNK activity can be triggered by several upstream signaling factors, including the small GTPases of the Rho family, and genetic data provide a link between dCsk, dJnk, and Rho1. Rho family proteins are key regulators of cell shape and motility. They also promote the cytoskeletal rearrangements required for epithelial-to-mesenchymal transitions (EMTs), and it is noted that dCsk boundary cells show a number of features that are reminiscent of EMTs. In Drosophila, Rho1 was found to induce an 'invasive' phenotype in wing disc cells, but, in this study, it was demonstrated that, similar to dCsk boundary cells, ptc>Rho1 misexpressing cells also undergo apoptotic death. Most importantly, halving the genetic dose of Rho1 strongly suppresses discrete loss of dCsk, but does not appreciably affect broad loss. Thus, Rho1 activity is linked to dCsk, and activation of Rho1 is sufficient to phenocopy both the apoptotic and migratory phenotypes of dCsk cells located near wild-type tissue (Vidal, 2006).
Previous work in mammalian cell culture has provided direct links between Src and P120-catenin, between cadherins and P120-catenin, and between RhoA and P120-catenin; the latter two interactions have been reported in Drosophila tissue culture systems as well. This study further supports links between these factors in dCsk boundary cells. Interestingly, although normal levels of both dP120ctn and Rho1 were required for the efficient removal of dCsk boundary cells, they were not required for the phenotypes resulting from broad loss of dCsk. The requirement for p120ctn specifically in boundary cells may explain why, although it is the only ortholog present in Drosophila, dP120ctn (Drosophila p120-catenin) is not required for organism viability (Vidal, 2006).
Both Src and P120-catenins are known to directly interact with cadherins, and, in fact, a role was demonstrated for dE-cadherin/Shotgun in the removal of dCsk cells. A model is postulated in which the loss of dCsk results in the remodeling of the zonula adherens, presumably by the phosphorylation of catenins and dE-cadherin itself by Src. Src activation is known to switch cadherin from a strong adhesive state to a weak one, providing one potential explanation for why dCsk retinal cells displayed reduced cell adhesion in situ. One critical question regarding cadherins is whether they have signaling roles that are independent of their adhesive properties. Perhaps relevant to this point, it was surprising to find that reducing dE-cadherin function leads to a suppression of the effects of dCsk-IR at the boundary. A simple dCsk-IR-mediated reduction in dE-cadherin adhesion would be enhanced by further reducing dE-cadherin activity, suggesting that dE-cadherin may provide an active signal that promotes boundary cells' release from the epithelium. If such a signal does exist, neighboring wild-type cells must trigger it, either through their own endogenous dE-cadherin or through a separate, local signal. Why are multiple (3-4) rows affected? The results are consistent with the creation of a successive new boundary as the previous row of cells descends, although other longer-range signals cannot be ruled out (Vidal, 2006).
It is noted that reducing dCsk activity by itself is not sufficient to direct stable tumor overgrowth, supporting the importance of a 'two-hit' model in Drosophila. Loss of the junction protein Scribble showed similar phenotypes to dCsk, including apoptosis, but was found to confer survival and metastatic-like behavior to cells in the presence of an activated Ras isoform. Interestingly, coexpression of dE-cadherin prevents this metastatic behavior (Vidal, 2006).
Finally, how can dP120ctn and Rho1 promote release of dCsk near wild-type boundaries but not act similarly with other dCsk cells? One source of information is the cadherins themselves: the boundary creates an interface of cadherins that have been exposed to different levels of Csk and, presumably, Src activity. This unusual interface may generate the needed dE-cadherin signal. Importantly, recent work has noted a change in the subcellular localization of P120-catenin and E-cadherin specifically at the border of human tumor tissues. At the time that ptc>dCsk-IR boundary cells lose their apical profiles, this study found that dP120ctn is relocalized to the cytoplasm. These results again emphasize the possibility that cells at tumor boundaries pose a special risk of undergoing epithelial-to-mesenchymal-like transitions and metastatic behavior. Metastasis is often the most serious complication of progressing tumors. Targeting therapies to this aspect of cancer may benefit from considering boundary cells and their potentially distinctive properties (Vidal, 2006).
The cellular machinery promoting phagocytosis of corpses of apoptotic cells is well conserved from worms to mammals. An important component is the Caenorhabditis elegans engulfment receptor CED-1 and its Drosophila orthologue, Draper. The CED-1/Draper signalling pathway is also essential for the phagocytosis of other types of 'modified self' including necrotic cells, developmentally pruned axons and dendrites, and axons undergoing Wallerian degeneration. This study shows that Drosophila Shark, a non-receptor tyrosine kinase similar to mammalian Syk and Zap-70 (Ferrante, 1995), binds Draper through an immunoreceptor tyrosine-based activation motif (ITAM) in the Draper intracellular domain. Shark activity is essential for Draper-mediated signalling events in vivo, including the recruitment of glial membranes to severed axons and the phagocytosis of axonal debris and neuronal cell corpses by glia. The Src family kinase (SFK) Src42A can markedly increase Draper phosphorylation and is essential for glial phagocytic activity. It is proposed that ligand-dependent Draper receptor activation initiates the Src42A-dependent tyrosine phosphorylation of Draper, the association of Shark and the activation of the Draper pathway. These Draper-Src42A-Shark interactions are strikingly similar to mammalian immunoreceptor-SFK-Syk signalling events in mammalian myeloid and lymphoid cells. Thus, Draper seems to be an ancient immunoreceptor with an extracellular domain tuned to modified self, and an intracellular domain promoting phagocytosis through an ITAM-domain-SFK-Syk-mediated signalling cascade (Ziegenfuss, 2008).
Developing tissues produce excessive numbers of cells and selectively destroy a subpopulation through programmed cell death to regulate growth. Rapid clearance of cell corpses is essential for maintaining tissue homeostasis and preventing the release of potentially cytotoxic or antigenic molecules from dying cells, and defects in cell corpse clearance are closely associated with autoimmune and inflammatory diseases. In C. elegans the CED-1 receptor is expressed in engulfing cells, where it acts to recognize cell corpses and drive their phagocytosis. CED-1 promotes engulfment through an intracellular NPXY motif, a binding site for proteins containing a phosphotyrosine-binding (PTB) domain, and a YXXL motif, a potential interaction site for proteins containing SH2 domains. The PTB domain adaptor protein CED-6 can bind the NPXY motif of CED-1, is required for cell corpse engulfment and acts in the same genetic pathway as CED-1. CED-1 ultimately mediates actin-dependent cytoskeletal reorganization through the Rac1 GTPase, and Dynamin modulates vesicle dynamics downstream of CED-1 during engulfment, but the molecular signalling cascade that allows CED-1 to execute phagocytic events remains poorly defined (Ziegenfuss, 2008).
Glia are the primary phagocytic cell type in the developing and mature brain. Glia rapidly engulf neuronal cell corpses produced during development, as well as neuronal debris generated during axon pruning or during Wallerian degeneration in the adult brain. In Drosophila, glial phagocytosis of these engulfment targets requires Draper, the fly orthologue of CED-1. Draper, like CED-1, contains 15 extracellular atypical epidermal growth factor (EGF) repeats, a single transmembrane domain, and NPXY and YXXL motifs in its intracellular domain. Drosophila Ced-6 is also required for the clearance of pruned axons, indicating possible conservation of the interaction between CED-1 and CED-6 in flies, but additional signalling molecules acting downstream of Draper have not been identified (Ziegenfuss, 2008).
Draper was identified in a yeast two-hybrid screen for molecules interacting with the regulatory region of Shark. When LexA-Shark, constitutively active Src kinase and AD-Draper are present, Shark and Draper interact physically. In the absence of Src kinase, Shark and Draper fail to interact, indicating that phosphorylation of Draper by Src may be essential for Shark-Draper interactions. The Draper intracellular domain contains an ITAM (YXXI/L-X6-12-YXXL), a key domain found in many mammalian immunoreceptors including Fc, T-cell and B-cell receptors. SFKs phosphorylate the tyrosines in ITAM domains, thereby allowing ITAM association with SH2-domain-containing signal transduction proteins including Syk and Zap-70. Y-->F substitutions of the tyrosine residues were generated within or near the Draper ITAM, and it was found that Tyr 949 and Tyr 934 are critical for robust Draper-Shark binding. These correspond to the consensus tyrosine residues in the predicted Draper ITAM. Plasmids were transfected with carboxy-terminally haemagglutinin-tagged Draper (Draper-HA) or with Draper-HA and Shark with an amino-terminal Myc tag (Myc-Shark) into Drosophila S2 cells, immunoprecipitated with anti-HA antibodies, and western blots were performed with anti-phosphotyrosine, anti-Myc and anti-HA antibodies. Myc-Shark was found to co-immunoprecipitate with Draper-HA, and that anti-phosphotyrosine antibodies label a band corresponding to the position of Draper-HA that is absent in empty vector controls. Further, it was found that a Y949F substitution markedly reduces Draper-Shark association. Taken together, these data indicate Draper and Shark can associate physically through the Draper ITAM domain (Ziegenfuss, 2008).
Attempts were made to determine whether Shark is required for glial phagocytic activity in vivo. Severing adult Drosophila olfactory receptor neurons (ORNs) initiates Wallerian degeneration of ORN axons. Antennal lobe glia surrounding these severed axons respond to this injury by extending membranes towards severed axons and engulfing degenerating axonal debris. These glia express high levels of Draper, and in draperδ5 null mutants, glia fail to respond morphologically to axon injury, and severed axons are not cleared from the central nervous system (CNS). Thus, both the extension of glial membranes to severed axons and the phagocytosis of degenerating axonal debris require Draper signalling (Ziegenfuss, 2008).
Whether Shark function in glia is essential for glial responses to axon injury was explored by driving a UAS-regulated double-stranded RNAi construct designed to target shark (sharkRNAi) with the glial-specific repo-Gal4 driver, severing ORN axons, and assaying the recruitment of Draper and green fluorescent protein (GFP)-labelled glial membranes to severed axons. Maxillary palp-derived ORN axons project to 6 of the roughly 50 glomeruli in the antennal lobe. Within hours after maxillary palps have been ablated in control animals, Draper immunoreactivity decorates severed axons projecting to and within maxillary palp ORN-innervated glomeruli, and GFP-labelled glial membranes are recruited to these severed axons. Strikingly, knocking down Shark in glia completely suppresses these events. Next, antennal ORN axons were severed; these axons project to about 44 of the 50 antennal lobe glomeruli. Antennal ablation therefore injures nearly all glomeruli in the antennal lobe and results in the majority of antennal lobe glia in control animals upregulating Draper and undergoing hypertrophy. It was found that knocking down Shark in glia also blocks this glial response to axon injury. Thus, Shark is essential for all axon-injury-induced changes in glial morphology and Draper expression (Ziegenfuss, 2008).
To determine whether Shark is required for glial phagocytosis of severed axons, a subset of maxillary palp ORN axons were labelled with mCD8::GFP, Shark function was knocked down in glia, and the clearance of severed axons was assayed. In control animals severed GFP-labelled ORN axonal debris was cleared from the CNS within 5 days. In contrast, glial-specific sharkRNAi potently suppresses the clearance of degenerating axons, with severed axons lingering in the CNS for at least 5 days. Then whether mutations in the shark gene affected the glial clearance of degenerating axons was examined. The null allele of shark, shark1, is pupal lethal (Fernandez, 2000). Therefore glial responses to axon injury were assayed in shark1 heterozygous mutants, and dominant genetic interactions between draperδ5 and shark1 were tested. It was found that both draperδ5/+ and shark1/+ animals showed defects in glial phagocytic function: 5 days after injury, significant amounts of axonal debris remained within OR85e-innervated glomeruli. Moreover, shark1/+; draperδ5/+ animals showed a striking suppression of glial clearance of severed axons almost equivalent to that of draperδ5 mutants. Thus, shark mutations dominantly suppress the glial clearance of degenerating ORN axons, and this phenotype is strongly enhanced by removing one copy of draper. These data, taken together with sharkRNAi data, show that Shark is essential for the clearance of degenerating axons by glia (Ziegenfuss, 2008).
Is Shark required for the glial clearance of neuronal cell corpses? In embryonic stage 14-15 control animals, 24.4 cell corpses were found per hemisegment. In contrast, it was found that shark1 null mutants showed a marked increase in CNS cell corpses, with null mutants containing almost twice as many corpses per hemisegment. shark1/Df(2R)6063 mutants accumulate cell corpses at levels similar to those in shark1, indicating that this phenotype maps to shark. These cell corpse engulfment phenotypes are indistinguishable from that of draperδ5 mutants. It is concluded that Shark, like Draper, is also essential for the efficient clearance of embryonic neuronal cell corpses by glia (Ziegenfuss, 2008).
Because it was found that Shark binds Draper only in the presence of an active Src kinase in two-hybrid assays, Drosophila Src kinases were screened for roles in glial phagocytic activity. Interestingly, it was found that glia-specific knockdown of Src42A (src42ARNAi) potently suppress glial phagocytic activity: in src42ARNAi animals, Draper is not recruited to severed maxillary palp axons; glial hypertrophy and upregulation of Draper after antennal ablation was blocked; and GFP-labelled severed axons lingered in the CNS for 5 days. Knockdown of two other Drosophila Src kinases, Btk29A and Src64B, had no effect on the glial phagocytosis of severed axons. Thus, Src42A seems to be essential for all morphological responses of glia to axon injury and for the efficient clearance of degenerating axonal debris from the CNS (Ziegenfuss, 2008).
It was predicted that Draper phosphorylation status should be sensitive to the SFK inhibitor PP2. Indeed, addition of PP2 to S2 cultures led to a decrease in the phosphorylation of Draper and Draper-Shark association. Strikingly, co-transfection of Draper and Src42A led to a marked increase in Draper phosphorylation, which was PP2-sensitive and Draper-specific. Draper-Shark interactions are not dependent on Shark kinase activity because kinase-dead Shark (Shark K698R) associates with Draper. These data indicate that Src42A may phosphorylate the Draper intracellular domain, thereby increasing the association of Shark with Draper and the activation of downstream glial phagocytic signalling (Ziegenfuss, 2008).
This study has identified Shark and Src42A as novel components of the Draper pathway. One potential model for Draper-Shark-Src42A interactions is that Shark and Src42A drive the recruitment of Draper to engulfment targets. However, CED-1 has been shown to cluster around cell corpses even in the absence of its intracellular domain. Moreover, Zap-70 and Syk bind phosphorylated ITAM domains in mammalian immunoreceptors when ITAM domains are phosphorylated by Src after ligand-dependent receptor. Therefore a model is favoured in which the engagement of Draper with its ligand (presumably presented by engulfment targets) promotes receptor clustering, tyrosine phosphorylation of Draper by Src42A, association of Shark, and activation of downstream phagocytic signalling events. This work suggests that Draper is an ancient immunoreceptor in which the extracellular domain is tuned to recognize modified self and the intracellular domain signals through ITAM-Src-Syk-mediated mechanisms. This is the first identification of ITAM-Src-Syk signalling in invertebrates, and it suggests that a pathway similar to Draper-Ced-1 may ultimately have given rise to ITAM-based signalling cascades in mammalian myeloid and lymphoid cells, including those regulated by Fc, B-cell and T-cell receptors (Ziegenfuss, 2008).
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