dreadlocks

Protein Interactions

The yeast two-hybrid system was employed to identify proteins that interact with dPTP61F, a transmembrane protein tyrosine phosphatase. The protein Dreadlocks (Dock) was identified in this procedure. To further define the nature of the interaction between dPTP61F and Dock, in vitro binding assays were performed. GST fused to Dock was expressed in E. coli. GST-Dock was immobilized on glutathione-agarose and allowed to bind bacterially expressed full-length dPTP61Fn protein. Also tested for binding was the C-terminal half of dPTP61Fn containing the proline-rich domain (amino acids 298-535). Bound dPTP61Fn was detected by Western analysis using an affinity-purified polyclonal antibody. Immunoreactive proteins corresponding to the full-length and truncated version of dPTP61Fn are retained on the GST-Dock column. As expected for interactions mediated by the Dock SH3 domains and the PXXP motifs of dPTP61F, the C-terminal domain by itself is sufficient for binding to Dock (Clemens, 1996).

In order to demonstrate that Dock and dPTP61F interact in vivo, polyclonal antibodies recognizing Dock and dPTP61F were produced and affinity-purified by Western analysis. These antibodies detect a 50-kDa Dock protein and a 72-kDa dPTP61F protein present in Schneider II and Drosophila embryonic protein extracts. Both affinity-purified antibodies were used separately to immune precipitate Dock or dPTP61F from Schneider II cell and Drosophila embryonic soluble protein extracts. Proteins present in immune complexes were analyzed by immunoblotting with antibodies recognizing dPTP61F or phosphotyrosine. dPTP61F is detected when precipitated by anti-dPTP61F and is co-precipitated with Dock by anti-Dock. These results were observed in immune complexes from both Schneider II cell and Drosophila embryo extracts and provide conclusive evidence that dPTP61F and Dock associate in vivo. Precipitated proteins were also analyzed by immunoblotting with anti-phosphotyrosine. No tyrosine-phosphorylated bands corresponding to the molecular weights of Dock or dPTP61F were observed. However, two tyrosine-phosphorylated proteins of 190 kDa (p190) and 145 kDa (p145) co-precipitate with Dock (Clemens, 1996).

Dock was also employed in a two-hybrid screen to identify interacting proteins. Plasmid DNA was isolated from 37 positive colonies, and their insert cDNAs were sequenced. Translation of the sequences indicates that 29 of these clones are rich in proline. Most of these encode known proteins, which when translated in altered reading frames result in artificial proteins that are rich in proline. Notably, three of the eight non-proline-rich clones encode dPTP61F. Two of the three clones encode the full-length dPTP61F PTP while one encodes the C-terminal half only (amino acids 269-535). The latter result indicates that sequences in the C-terminal half of the PTP are sufficient for interaction with Dock (Clemens, 1996).

It is unlikely that Dock is a substrate of dPTP61F since immune precipitated Dock is not tyrosine-phosphorylated. The possibility that the Dock SH2 domain binds to the PTP is ruled out by the absence of tyrosine phosphorylation of dPTP61F. The interaction between Dock and dPTP61F is likely mediated by one or more SH3 domain/PXXP motif binding events as demonstrated by two-hybrid and in vitro binding data. Since every Dock two-hybrid positive contained all three SH3 domains, high affinity binding may be dependent on interactions involving more than one SH3 domain. This hypothesis is attractive since none of the two-hybrid screens with dPTP61F identified Drosophila proteins with two or fewer SH3 domains (i.e. Src, Drk, and SOS). The association of dPTP61F with Dock and its interaction with p190 and p145 provide initial insights into the complex of proteins likely to play a significant role in the Drosophila nervous system guidance mechanism (Clemens, 1996).

The expression of Dock interacting protein DPTP61F has been examined in detail. DPTP61F is expressed during Drosophila oogenesis and embryogenesis. DPTP61F transcripts are alternatively spliced to produce two isoforms of the protein that are targeted to different subcellular locations. DPTP61Fn accumulates in the nucleus, and DPTP61Fm associates with the membranes of the reticular network and the mitochondria. The spatial and temporal expression of the two alternative transcripts of dptp61F during Drosophila embryogenesis were also examined. The two isoforms are expressed in distinct patterns. The DPTP61Fn transcript is expressed in the mesoderm and neuroblast layer during germband extension and later in the gut epithelia. In comparison, the transcript encoding DPTP61Fm accumulates in 16 segmentally repeated stripes in the ectoderm during germband extension. These stripes are flanked by, and adjacent to, the domains of engrailed and wingless gene expression in the anterior/posterior axis. In stage 10 embryos, the domains of DPTP61Fm transcript accumulation are wedge shaped and roughly coincide with the area lateral to the denticle belts that will give rise to naked cuticle. The DPTP61Fm transcript is also expressed later in embryogenesis in the central nervous system. The segmental modulation of DPTP61Fm transcript accumulation in the A/P axis of the germband is regulated by the pair-rule genes, and the intrasegmental pattern of transcript accumulation is regulated by the segment polarity genes (Ursuliak, 1997).

Dock and Drosophila PAK-kinase have been shown to physically interact as do mammalian NIK and PAK. If Dock and Pak function in the same signaling pathway in developing R cells to regulate growth cone motility, they would be expected to colocalize in these cells. R cells extend their axons into the optic lobe during the third instar of larval development. Dock staining is markedly enriched in the lamina and medulla neuropils, consistent with its localization to R cell axons and growth cones (Garrity, 1996). In contrast, Dock is only expressed at low levels in the cell bodies of developing R cells as well as in neuronal cell bodies in the cortical regions in the lamina and medulla. Strong staining is seen, however, in the neuropils of the optic disc. The lamina and medulla neuropils contain no neuronal cell bodies and comprise axonal processes and growth cones. Both anti-Dock and anti-Pak antibodies stain the medulla neuropil uniformly, indicating that these proteins are expressed on many visual system fibers. Since the R7 and R8 axons only contribute a small fraction of the total number of fibers in the medulla, it is not possible to assess whether Pak and Dock are coexpressed in these axons. In contrast, at this stage in development, the vast majority of the processes in the lamina neuropil belong to R cells, including the expanded R1-R6 growth cones and axons of R7 and R8. Hence, Pak, like Dock, preferentially localizes to axons and growth cones (Hing, 1999).

To assess whether Pak is required for growth cone guidance, mutations disrupting its function were identified. It was assumed that, like dock, null mutations in Pak would cause recessive lethality. Accordingly, lethal mutations were identified in a small region of the chromosome within which Pak maps. From 9440 mutant lines containing randomly mutagenized third chromosomes, 238 lethal mutations mapping to a deficiency that deletes Pak were isolated. These were then tested against the same deficiency chromosome bearing a Pak-containing cosmid. The cosmid rescued 21 mutations. These fell into two groups based on complementation tests. A Pak cDNA expressed under the control of the heat shock promoter rescued the lethality associated with one complementation group, indicating that these mutations disrupt Pak function. These alleles are designated Pak¹ to Pak¹³ (Hing, 1999).

To assess R cell projections in Pak mutants, eye-brain complexes from transheterozygous larvae were stained with the R cell-specific antibody mAb24B10. In wild type, R cell axons grow from the eye disc, through the optic stalk, and into the optic ganglia during the third instar of larval development. The eight R cell axons from each ommatidium form a single bundle. These bundles spread out upon entering the optic lobe and form a smooth topographic map that reflects the arrangement of ommatidia in the eye. Growth cones of R1-R6 are seen as a band of immunoreactivity. In contrast, individual R8 growth cones are readily observed in the medulla neuropil. They are evenly spaced and exhibit a characteristic expanded morphology. At this stage of development, few of the R7 axons stain with mAb24B10. In Pak strong loss-of-function mutants, R cell axons extend into the brain normally. However, these fibers do not spread evenly within the lamina and medulla. As a result, some regions are hyperinnervated while others lack innervation. In the medulla neuropil, R cell axons fail to find their proper targets but instead, terminate as thick, blunt-ended fascicles. Hence, in contrast to wild type, Pak mutant R cells do not elaborate a smooth topographic map in the lamina and medulla neuropils. A small fraction of the R2-R5 neurons project through the lamina and into the medulla, indicating a modest disruption in ganglion target specificity. Eye-specific expression of a wild-type Pak cDNA under the control of the GMR promoter rescues the mutant phenotype. These data indicate that Pak is required for axon targeting but that it is not required for axon outgrowth because in these mutants R cell axons extend in the correct direction and into the target region. The Pak phenotypes are essentially indistinguishable from those previously described (Garrity, 1996) in dock mutants. While Pak has a profound effect on R cell projections, it does not disrupt R cell fate determination or differentiation (Hing, 1999).

If Pak functions downstream of Dock, then a constitutively active form of Pak should rescue Dock mutations. If a key step in Dock function is to recruit Pak to the membrane, then membrane-tethered Pak, GMR-Pak^myr, may rescue some aspects of the dock mutant phenotype. To test this, a single copy of the transgene was introduced into a dock null background. In dock mutants, R cell axons form large abnormal fascicles in the optic ganglia. This leads to hyperinnervated regions separated by areas lacking innervation in both the lamina and medulla. In addition to disrupting targeting, R cell axon terminals in the medulla are thick and blunt ended. The dock phenotype was shown to be substantially rescued by GMR-Pak^myr. Axon bundles between the lamina and medulla are thinner in rescued flies. Growth cones in the medulla are expanded and spread out more evenly, giving rise to an array of terminals. Quantification of the expanded growth cones in the medulla shows an increase from less than 2 in dock mutants to 64 in dock mutants carrying a copy of GMR-Pak^myr. This represents a restoration of about half the number of growth cones, compared with wild-type preparations of similar age. Rescue requires both myristylation and kinase activity; rescue is not observed with GMR-Pak^wt, kinase inactive or constitutively active soluble Pak. These data are consistent with models in which recruitment of Pak to the membrane by Dock is an essential regulatory step in R cell axon guidance (Hing, 1999).

Mammalian Paks bind Nck. The interaction sites have been mapped to the SH3-2 domain of Nck and the N-terminal-most PXXP site in Pak. Drosophila Pak contains an N-terminal PXXP site highly related to mammalian Paks, which bind to Nck (Harden, 1996). A test was performed to see whether Drosophila Pak interacts with Dock through these sites in a yeast two-hybrid assay. Indeed, Pak and Dock do interact. The DNA-binding domain of Lex A fused to full-length Dock (LexA-Dock) interacts strongly with a fusion protein containing the Gal4 activation domain and full-length Pak (GAD-Pak). The SH3-2 domain of Dock is necessary for interaction with Pak. LexA-Dock fusion proteins carrying mutations in the SH3-1 and SH3-3 domains, designed to disrupt interactions with proline-rich sequences, exhibit a similar level of interaction to that seen for LexA-Dock. Conversely, the same mutations introduced into the SH3-2 domain abolish this interaction. It has been demonstrated that the SH3-2 domain is not only necessary, but it is sufficient for binding to Pak. While Lex-SH3-2 interacts strongly, neither LexA-SH3-1 nor LexA-SH3-3 interacts with GAD-Pak (Hing, 1999).

The requirement for the N-terminal PXXP site of Pak for these interactions was demonstrated in a separate series of experiments in which LexA-Dock was tested for interactions with three different forms of the N-terminal region of Pak fused to GAD. The N-terminal region of Pak interacts strongly with LexA-Dock. Conversely, LexA-Dock does not interact with Pak N-terminal fragments containing point mutations in which arginine 14 is changed to methionine (R14M) or proline 9 is changed to leucine (P9L) (Hing, 1999).

To assess whether these proteins can associate in vivo, immunoprecipitation experiments were carried out in Drosophila S2 cells. Both Dock and Pak are endogenously expressed in these cells. S2 cell lysates were incubated with either anti-Dock antibody or a control antiserum and precipitated with protein A beads. The immunoprecipitates were analyzed on Western blots with anti-Pak antibodies. Pak was found in the anti-Dock immunoprecipates, but not in the controls (Hing, 1999).

Drosophila PAK-kinase binds Rac and Cdc42 proteins (Harden, 1996). Pak kinase activity is stimulated by Rac or Cdc42. This occurs through direct binding of Rac/Cdc42 in the GTP-bound form to the CRIB domain. Previous studies have demonstrated that substituting leucines for two conserved histidines in the CRIB site of mammalian Pak prevents this interaction. Similarly, it has been demonstrated in the yeast two-hybrid assay that this mutation prevents binding of Drosophila Pak to Cdc42. A Pak transgene carrying these mutations fails to rescue the Pak mutant phenotype, indicating that interaction between Pak and Cdc42/Rac is necessary for R cell growth cone guidance (Hing, 1999).

DNA sequence analysis reveals a missense mutation that changes a proline within the conserved Dock-binding site to a leucine (P9L) in Pak⁴. This substitution abolishes the interaction between Dock and Pak in the yeast two-hybrid assay. Pak⁴ protein is expressed at wild-type levels in eye-brain complexes and is localized to the optic lobe neuropil as in wild type. R cell axon guidance defects in Pak⁴/Pak¹¹ are indistinguishable from the strong loss-of-function phenotypes associated with Pak⁶/Pak¹¹ and dock null alleles. Hence, the Dock-binding site in Pak is essential for its function. This finding and the similarities between the dock and Pak mutant phenotypes provide strong evidence that direct interaction between Dock and Pak is essential for R cell axon guidance (Hing, 1999).

Recent studies suggest that the SH2/SH3 adaptor Dock/Nck transduces tyrosine phosphorylation signals to the actin cytoskeleton in regulating growth cone motility. The signaling cascade linking the action of Dock/Nck to the reorganization of cytoskeleton is poorly understood. Dock is shown to interact with the Ste20-like kinase Misshapen (Msn) in the Drosophila photoreceptor (R cell) growth cones. Loss of msn causes a failure of growth cones to stop at the target, a phenotype similar to loss of dock, whereas overexpression of msn induces pretarget growth cone termination. Physical and genetic interactions between Msn and Dock indicate a role for Msn in the Dock signaling pathway. It is proposed that Msn functions as a key controller of growth cone cytoskeleton in response to Dock-mediated signals (Ruan, 1999).

To investigate the potential role of msn in R cell growth cones, the effect of msn mutations on R cell projections was assessed. As strong loss-of-function alleles of msn are embryonic lethal, R cell projections were examined in third-instar larvae homozygous for a hypomorphic allele of msn [msn^l(3)03349]. While in msn mutants R cell growth cones are able to extend into the developing optic lobe, their innervation patterns within the lamina and the medulla are altered. The msn phenotype exhibits a certain similarity to that of dock loss-of-function mutants. In dock mutants, many R1-R6 growth cones pass over their normal target (i.e., lamina) and extend further into the medulla layer, generating gaps in the lamina R1-R6 termination site (a smooth continuous line of immunoreactivity in wild type). In addition, dock affects R cell fasciculation and growth cone morphology. Similarly, it has been found that loss of msn function causes defects in R cell targeting and fasciculation; gaps are observed in the R1-R6 termination site, coincident with projections of abnormal, large bundles into the medulla. R cell growth cone morphology is also altered in msn mutants. Unlike in dock, however, in msn, R cell growth cones are able to expand upon reaching the target. While all msn mutants examined exhibited defects in R cell innervation pattern, the severity of the phenotype varies from individual to individual (Ruan, 1999).

To determine whether msn is required in the developing eye for R cell projections, genetic mosaic analysis was carried out. Mutant eye clones homozygous for msn¹⁰², a strong loss of function allele, were generated in an otherwise wild-type fly by eye-specific mitotic recombination. R cell projections in mosaic larvae were visualized with mAb 24B10. Similar defects in R cell innervation pattern are observed. The percentage (~46%) of larvae showing obvious defects was close to the percentage (~50%) of individuals with relatively large mutant eye patches (identified as white eye tissue in adult). To specifically assess the role of Msn in R1-R6 growth cones, msn mutant eye patches were generated in msn heterozygous flies carrying the adult R1-R6-specific marker Rh1-lacZ. In wild-type adult flies, all R1-R6 axons terminate in the lamina, as assessed by lacZ staining. In contrast, in all mosaic adults examined, R1-R6 axons from msn mutant patches pass over the lamina and terminate abnormally in the medulla. These results indicate that msn, like dock, is genetically required in the eye for R1-R6 growth cone targeting. Similarly, no obvious defects are detected in the differentiation of the R1-R6 targeting region (i.e., lamina) in msn mutants, as assessed by anti-Dachshund staining. Moreover, eye-specific expression of a msn transgene rescues R cell projection defects in homozygous msn mutants (Ruan, 1999).

Dock protein is enriched in R cell axons and growth cones. If Msn has a functional relationship with Dock in R cell growth cones, it would be expected that Msn protein is expressed in R cells and is localized to growth cones. The expression pattern of Msn in third-instar larval eye-brain complexes was determined with a rabbit anti-Msn serum. Msn staining is seen in R cell axons along the path of projections (from the developing eye disc to the lamina) in wild-type whole-mount preparations. The lamina plexus is strongly stained as a continuous layer of immunoreactivity, a pattern that is indistinguishable from that stained with anti-Dock antibody. Since at this stage the vast majority of axonal processes in the lamina neuropil are expanded R1-R6 growth cones, the uniform staining of Msn and Dock in the lamina neuropil suggests strongly that Msn and Dock colocalize to R1-R6 growth cones.The localization of Msn in R1-R6 growth cones is consistent with a role for Msn in coordinating the response to target-derived signals (Ruan, 1999).

The fact that loss of msn caused the failure of R1-R6 growth cones to stop at their target lamina suggests a role for msn in the shutdown of growth cone motility when axons reach their target. Target-derived stop signals may activate Msn, which in turn coordinates cytoskeletal reorganization in decelerating growth cone motility. If this model is correct, one may predict that ectopic activation of Msn should induce abnormal termination of R cell growth cones. To test this, the endogenous msn gene was overexpressed in differentiating R cells using the eye-specific promoter GMR. Overexpression of Msn in R cell axons was confirmed by immunohistochemical staining. Compared with wild-type, overexpression of msn causes a large number of R1-R6 growth cones to stop before they reached their normal target lamina. Overexpression of msn also causes defects in the medulla terminal field. In contrast, neither the shape of R cells nor their localization on the developing eye disc is affected. Overexpression of msn from a transgene containing msn cDNA under control of the GMR promoter causes a similar early stop phenotype. The severity of the phenotype is dose dependent, because the increase in the copies of msn transgene enhanced the phenotype (Ruan, 1999).

One possible explanation for the gain-of-function phenotype is that overexpression of Msn activates the Msn pathway prematurely: this sends a terminating signal to growth cone cytoskeleton to induce pretarget termination. Alternatively, the hyperactivation of the Msn pathway may cause some general defects in the reorganization of growth cone cytoskeleton, leading to the arrest of growth cones before they reach the target. The former interpretation, that Msn plays an instructive role in terminating R1-R6 growth cones, is favored for the following reasons: (1) in msn gain-of-function mutants, the early stop growth cones expand, similar to growth cones that terminate correctly in the lamina. In wild type, R cell growth cones expand only when they terminate in the target; (2) the fact that the early stop R cell growth cones are still able to expand in the lamina argues against a general defect in the reorganization of growth cone cytoskeleton (Ruan, 1999).

To examine whether Msn interacts with Dock physically, a glutathione S-transferase (GST) fusion protein was generated containing a fragment of Msn that encompasses multiple consensus PXXP motifs for SH3 domain-binding. The immobilized GST-Msn fusion protein precipitates Dock from adult fly lysates in a dose-dependent manner, indicating the direct association of Msn with Dock. To test whether Msn associates with Dock in intact flies, coimmunoprecipitation experiments were carried out. Fly lysates were prepared from third-instar larval eye-brain complexes or adult heads. Anti-Dock antibody was used to precipitate Dock and its interacting proteins from the lysates. Msn protein is detected in anti-Dock precipitates but not in control serum precipitates, indicating an in vivo association of Msn with Dock in flies at both developmental and adult stages (Ruan, 1999).

To define the domains of Dock and Msn that mediate the binding, the yeast two-hybrid system was used to analyze their interactions. Consistent with binding experiments using GST-Msn fusion protein, the PXXP fragment of Msn binds to Dock in yeast. The binding of Dock to Msn is mediated mainly by its SH3-1 and SH3-2 domains. Mutations in either SH3-1 or SH3-2 inhibit the association of Dock with Msn, indicating that a stable association requires the simultaneous binding of SH3-1 and SH3-2 to the PXXP sequence in the polypeptide of Msn, whereas SH3-3 is less necessary for the binding (Ruan, 1999).

To determine the biological relevance of the physical association of Msn with Dock, a test was performed to see whether dock and msn interact genetically. The dosage of dock gene was reduced in larvae homozygous for the hypomorphic msn allele [msn^l(3)03349]. The reduction by half of dock gene dosage dramatically enhances the msn phenotype. The R1-R6 termination site at the lamina becomes more disorganized. R cell growth cones are much less expanded and appear more similar to those of dock mutants. This enhanced phenotype is completely penetrant. It is estimated that in each hemisphere, ~70%-100% of growth cones are less expanded, as compared with those in controls. In dock and msn double mutants, R cell projections are indistinguishable from those in dock mutants. These results, together with the physical association of Msn with Dock, strongly suggest that Msn and Dock function in the same signaling pathway controlling R cell projections (Ruan, 1999).

That Dock/Nck is capable of binding activated receptor tyrosine kinases via its SH2 domain, together with the above phenotypic analysis of dock and msn mutants, suggests that Msn is activated by Dock-mediated stop signals in terminating R1-R6 growth cones in the lamina. This model makes the simple prediction that gain of function in msn should suppress the R1-R6 nonstop phenotype in dock mutants. To assess this possibility, the endogenous msn gene was overexpressed in homozygous dock mutants. In dock mutants, the medulla layer is hyperinnervated, as many R1-R6 axons fail to stop at the lamina termination site. Overexpression of Msn in dock mutants largely suppresses the R1-R6 nonstop phenotype ; R cell axons in the medulla are dramatically reduced in all larvae examined. The fact that gain of function in msn is capable of terminating R1-R6 growth cones in dock null mutants is consistent with the prediction that Dock functions upstream of Msn activation in decelerating R1-R6 growth cone motility. Surprisingly, overexpression of msn in the absence of dock also causes the premature termination of many R cell growth cones within the optic stalk, a phenotype that is not observed in wild-type flies overexpressing msn. This result raises the intriguing possibility that Dock is also able to negatively regulate the function of Msn at certain stages of axonal projections (Ruan, 1999).

To further investigate the relationship between msn and dock in the control of growth cone motility, the effect of overexpressing Dock on the msn gain-of-function phenotype was examined. Dock was overproduced in R cells under control of the GMR promoter. In wild type, overexpression of Dock has no effect on R cell projections, suggesting that Dock is not rate limiting in the termination of growth cones. Overexpression of Dock in msn gain-of-function mutants largely suppresses the pretarget termination phenotype, confirming that Dock also negatively regulates the function of msn. SH3 mutants incapable of binding Msn in yeast either completely fail to suppress the phenotype or only weakly suppress the phenotype. In contrast, the SH3-3 mutant, displaying Msn-binding activity, suppresses the phenotype as efficiently as wild-type Dock. These results argue that the physical association of Dock with Msn is essential for the regulation of Msn by Dock. Interestingly, although the R336Q mutation (eliminating phosphotyrosine-binding activity of the SH2 domain) does not affect the binding of Dock to Msn, it completely abolishes the ability of Dock to suppress the msn gain-of-function phenotype. These data suggest that the negative regulation of Msn function by Dock involves an SH2-dependent tyrosine phosphorylation signal (Ruan, 1999).

It is proposed that Dock couples different signals to Msn at different stages of axonal projection. At an early stage, signals promoting growth cone extension may induce tyrosine phosphorylation on specific proteins (e.g., docking protein), which then recruit Msn through Dock (via the SH2 domain) to specific regions within the growth cone. Consequently, this may segregate Msn from its substrates, thus preventing the premature activation of the Msn pathway. In growth cones overexpressing Msn, however, excessive Msn that cannot be recruited by a limited amount of endogenous Dock may diffuse freely into certain regions to activate its (Msn's) substrates, which then induce pretarget growth cone termination. Similarly, the pretarget termination phenotype is enhanced by loss of dock and is suppressed by overexpression of dock. Once the growth cone reaches the target, upregulation of Msn may be accomplished in two steps through the combination of reducing the extension signal and increasing the stop signal. (1) The Dock-Msn complex needs to be released from those docking sites, which would be achieved by dephosphorylation through the activation of some protein tyrosine phosphatases. One such candidate is the receptor tyrosine phosphatase PTP69D, which has recently been shown to be required for the proper targeting of R1-R6 growth cones. (2) The stop signal activates the function of Msn through Dock by either positioning Msn close to its substrate or directly stimulating its activity, leading to the termination of the growth cone in the target. In the absence of Dock, endogenous Msn may not reach a threshold local concentration or activity required for growth cone termination. The observation that reduction of dock gene dosage enhances the hypomorphic msn loss-of-function phenotype is consistent with this view. While the above model fits with the results, understanding of the exact biochemical mechanism underlying the regulation of Msn by Dock awaits identification of upstream regulators of Dock in R cell growth cones (Ruan, 1999).

Recent studies suggest that Dock/Nck plays a highly conserved role in growth cone signaling. Nck can be recruited into signaling complexes in response to the activation of the vertebrate guidance receptors EphB1 and EphB2, two Eph receptor tyrosine kinase family members (see Drosophila Eph receptor tyrosine kinase). Moreover, Nck can functionally replace Dock in R cell growth cones. Furthermore, Dock, like Nck, is capable of binding ligand-activated EphB1. Given the extraordinary sequence conservation between Msn and NIK, it is highly likely that in vertebrate growth cones, NIK plays a similar role in response to Nck-mediated signals. Hence, the interaction between Dock/Nck and Msn/NIK may represent an evolutionarily conserved mechanism linking tyrosine phosphorylation to changes in growth cone behavior (Ruan, 1999 and references therein).

It has been proposed, based on mutational analyses of domain requirements for Dreadlocks in axon guidance, that Dock interacts with upstream guidance signals in a redundant fashion through both SH3 and SH2 domains. The Dock SH2 domain interacts directly with Dscam. Binding is disrupted by pretreatment with alkaline phosphatase. The SH3 domains of Dock also directly interact with Dscam. Interactions between different SH3 domains and Dscam were assessed in a yeast two-hybrid assay and in GST pulldown experiments. In yeast, full-length Dock interacts strongly with the cytoplasmic domain of Dscam. Each of the three SH3 domains tested individually in yeast show a comparatively reduced level of interaction. This suggests Dock interacts through multiple SH3 domains with Dscam (Schmucker, 2000).

The interaction sites between different SH3 domains and Dscam were mapped. Two putative SH3 binding sites (PXXP1 and PXXP2) separated by 40 amino acids are found in the N-terminal portion of the Dscam cytoplasmic domain; a C-terminal polyproline sequence (PEPPP) is also present. Site-directed mutagenesis of the PXXP sites revealed that the first SH3 domain (SH3-1) binds preferentially to PXXP1 and the third SH3 domain (SH3-3) binds to PXXP2. GST-SH3-1 and GST-SH3-3 interact with the N-terminal half of the cytoplasmic tail of Dscam containing the PXXP sites, but only weakly to the C-terminal half encompassing the polyproline sequence. Conversely, the second SH3 domain (SH3-2) binds preferentially to the C-terminal polyproline motif. That PXXP1 and PXXP2 sequences are the primary interaction sites between Dock and Dscam is strongly supported by the marked reduction in interaction between Dock and the cytoplasmic domain of Dscam carrying point mutations in both these sites. Residual binding may be due to interaction between SH3-2 and the C-terminal polyproline sequence. In summary, these data indicate that Dscam binds directly to Dock through both SH3 and SH2 domains, consistent with genetic studies arguing for redundancy between these domains (Schmucker, 2000).

How might binding of Dscam on a filopodial process to a ligand at P2 promote attraction? Two alternative models are proposed. (1) Dock and Dscam may form a complex in the absence of ligand. This interaction would be mediated by the SH3-1 and SH3-3 domains in Dock and two closely spaced PXXP sites in Dscam's cytoplasmic tail. Dock's SH3-2 domain may bind to the polyproline stretch in the C-terminal tail; this may prevent recruitment of Pak to the membrane in the absence of ligand. Upon encountering an attractive ligand at P2, however, it is envisioned that Dscam becomes tyrosine phosphorylated. This may promote rearrangement of the complex with Dock binding through its SH2 domain to phosphotyrosine. This conformational change cannot strictly depend upon the SH2 domain, as the SH3-1 and SH3-3 domains can functionally compensate for the SH2 domain. This conformational change would unmask the SH3-2 domain, thus facilitating recruitment of Pak to the complex. Pak, in turn, would promote actin reorganization and subsequent movement of the growth cone to P2. Alternatively, in the absence of ligand the cytoplasmic tail of Dscam may be in a conformation not accessible to Dock binding. In this model, ligand binding would promote both a conformational change in Dscam's cytoplasmic domain and tyrosine phosphorylation. Dock may then associate with Dscam and recruit Pak to the membrane. Candidates for additional components in this signaling pathway have been identified through biochemical and genetic studies and include a nonreceptor tyrosine kinase, an adaptor protein linked to actin, and a nonreceptor tyrosine phosphatase. A precise mechanistic understanding of the relationship between Dscam, Dock, and Pak and these additional components will require identification of the Dscam ligand and detailed biochemical studies (Schmucker, 2000).

Dock, an adaptor protein that functions in Drosophila axonal guidance, consists of three tandem Src homology 3 (SH3) domains preceding an SH2 domain. To develop a better understanding of axonal guidance at the molecular level, the SH2 domain of Dock was used to purify a protein complex from fly S2 cells. Five proteins were obtained in pure form from this protein complex. The largest protein in the complex was identified as Dscam (Down syndrome cell adhesion molecule), which has been shown to play a key role in directing neurons of the fly embryo to correct positions within the nervous system. The smallest protein in this complex p63) has now been identified. p63 has been named SH3PX1 because it appears to be the Drosophila ortholog of the human protein known as SH3PX1. DSH3PX1 is comprised of an NH(2)-terminal SH3 domain, an internal PHOX homology (PX) domain, and a carboxyl-terminal coiled-coil region. Because of its PX domain, DSH3PX1 is considered to be a member of a growing family of proteins known collectively as sorting nexins, some of which have been shown to be involved in vesicular trafficking. DSH3PX1 immunoprecipitates with Dock and Dscam from S2 cell extracts. The domains responsible for the in vitro interaction between DSH3PX1 and Dock were also identified. DSH3PX1 interacts with the Drosophila ortholog of Wasp, a protein component of actin polymerization machinery, and DSH3PX1 co-immunoprecipitates with AP-50, the clathrin-coat adapter protein. This evidence places DSH3PX1 in a complex linking cell surface receptors like Dscam to proteins involved in cytoskeletal rearrangements and/or receptor trafficking (Worby, 2001).

Drosophila Ack targets its substrate, the sorting nexin DSH3PX1, to a protein complex involved in axonal guidance

Dock, the Drosophila ortholog of Nck, is an adaptor protein that is known to function in axonal guidance paradigms in the fly including proper development of neuronal connections in photoreceptor cells and axonal tracking in Bolwig's organ. To develop a better understanding of axonal guidance at the molecular level, proteins in a complex with the SH2 domain of Dock were purified from fly Schneider 2 cells. A protein designated p145 was identified and shown to be a tyrosine kinase with sequence similarity to mammalian Cdc-42-associated tyrosine kinases. It was demonstrated that Drosophila Ack (DAck) can be co-immunoprecipitated with Dock and the sorting nexin DSH3PX1 from fly cell extracts. The domains responsible for the in vitro interaction between Drosophila Ack and Dock were identified, and direct protein-protein interactions between complex members were established. It is concluded that DSH3PX1 is a substrate for DAck in vivo and in vitro, and one of the major in vitro sites of DSH3PX1 phosphorylation was found to be Tyr-56. Tyr-56 is located within the SH3 domain of DSH3PX1, placing it in an important position for regulating the binding of proline-rich targets. It was demonstrated that Tyr-56 phosphorylation by DAck diminishes the DSH3PX1 SH3 domain interaction with the Wiskott-Aldrich Syndrome protein while enabling DSH3PX1 to associate with Dock. Furthermore, when Tyr-56 is mutated to aspartate or glutamate, the binding to Wiskott-Aldrich Syndrome protein is abrogated. These results suggest that the phosphorylation of DSH3PX1 by DAck targets this sorting nexin to a protein complex that includes Dock, an adaptor protein important for axonal guidance (Worby, 2001).

This study has identified DAck as a member of a complex of proteins involved in axonal guidance via its association with Dock. It is important to note that experiments in fly embryos using dsRNAs directed against Dock and DAck result in similar axonal pathfinding defects as assayed by Bolwig's organ development. Given the ability of DAck to interact with DSH3PX1, a potential sorting nexin that associates with the clathrin-coated adaptor protein 50, and the ability of mammalian ACK1 to interact with clathrin, it is tempting to speculate that DAck is involved in regulating the extracellular presentation of Dscam and/or other Dock-associated receptors by endocytosis via clathrin-coated pits. This speculation is further supported by the role of C. elegans ACK-related tyrosine kinase-1 in down-regulating Let-23, the C. elegans epidermal growth factor receptor orthologue. Furthermore, DSH3PX1 was identified as a substrate for DAck, and it was demonstrated that phosphorylation of DSH3PX1 probably increases its interaction with Dock while decreasing its interaction with WASP. In this scenario, DAck acts as a molecular switch to control DSH3PX1 protein-protein interactions. Nonphosphorylated DSH3PX1 interacts strongly with WASP, a known modulator of the actin cytoskeleton. When phosphorylated, DSH3PX1 interacts preferentially with Dock. In addition, the PX domain of DSH3PX1 interacts with phospholipids, thereby targeting DSH3PX1 to specific cellular membranes. It would be interesting to understand how the Dock SH2 domain chooses among its binding partners, i.e. Dscam versus DAck versus DSH3PX1, and how the resulting protein complexes ultimately influence neurite outgrowth. For now, the complexity of the protein-protein interactions involving Dock preclude directly linking a specific Dock protein complex to specific changes in the actin cytoskeleton. Nevertheless, Dock and the proteins recruited by Dock are clearly instrumental in signaling changes in the actin cytoskeleton that are required for directed axonal growth (Worby, 2001).

Son of sevenless directly links the Robo receptor to rac activation to control axon repulsion at the midline

Son of sevenless (Sos) is a dual specificity guanine nucleotide exchange factor (GEF) that regulates both Ras and Rho family GTPases and thus is uniquely poised to integrate signals that affect both gene expression and cytoskeletal reorganization. Sos is recruited to the plasma membrane, where it forms a ternary complex with the Roundabout receptor and the SH3-SH2 adaptor protein Dreadlocks (Dock) to regulate Rac-dependent cytoskeletal rearrangement in response to the Slit ligand. Intriguingly, the Ras and Rac-GEF activities of Sos can be uncoupled during Robo-mediated axon repulsion; Sos axon guidance function depends on its Rac-GEF activity, but not its Ras-GEF activity. These results provide in vivo evidence that the Ras and RhoGEF domains of Sos are separable signaling modules and support a model in which Robo recruits Sos to the membrane via Dock to activate Rac during midline repulsion (Yang, 2006).

Sos was identified in Drosophila as a GEF for Ras in the sevenless signaling pathway during the development of the Drosophila compound eye, where it activates the Ras signaling cascade to determine R7 photoreceptor specification. Studies in mammalian cell culture demonstrated that Sos functions as a GEF for both Ras and Rac in the growth factor-induced receptor tyrosine kinase (RTK) signaling cascade. Upon RTK activation, the SH3/SH2 adaptor protein Grb2/Drk recruits Sos to autophosphorylated receptors at the plasma membrane, where Sos activates membrane-bound Ras. In a later event downstream of RTK activation, Sos is thought to be targeted to submembrane actin filaments by interaction with another SH3 adaptor, E3b1(Abi-1), where Sos activates Rac . Whether the activation of Rac by Sos is strictly dependent on prior activation of Ras remains controversial, nor is it clear how Sos coordinates the activity of its two GEF domains in vivo (Yang, 2006 and references therein).

Evidence is provided that Sos functions as a Rac-specific GEF during Drosophila midline guidance. Sos is enriched in developing axons, and sos exhibits dosage-sensitive genetic interactions with slit and robo. Strikingly, genetic rescue experiments show that the Dbl homology (DH) RhoGEF domain of Sos, but not its RasGEF domain, is required for its midline guidance function. Biochemical experiments show that Sos physically associates with the Robo receptor through Dock in both mammalian cells and Drosophila embryos. Furthermore, Slit stimulation of cultured cells results in the rapid recruitment of Sos to membrane Robo receptors. These results provide a molecular link between the Robo receptor and Rac activation, reveal an independent in vivo axon guidance function of the DH RhoGEF domain of Sos, and support the model that Slit stimulation recruits Sos to the membrane Robo receptor via Dock to activate Rac-dependent cytoskeletal changes within the growth cone during axon repulsion (Yang, 2006).

These data support the idea that Sos provides a direct molecular link between the Robo receptor and the activation of Rac during Drosophila midline guidance. Genetic interactions between sos, robo, dock, crGAP/vilse, and the Rho family of small GTPases strongly suggest that Sos functions in vivo to regulate Rac activity during Robo signaling. Genetic rescue experiments indicate that sos is required specifically in neurons to mediate its axon guidance function. Furthermore, genetic data establish that, in the context of midline axon guidance, the Ras-GEF and Rac-GEF activities of Sos can be functionally uncoupled. Biochemical experiments in cultured cells and Drosophila embryos show that Sos is recruited into a multiprotein complex consisting of the Robo receptor, the SH3-SH2 adaptor protein Dock, and Sos, in which Dock bridges the physical association between Robo and Sos. Finally, experiments in cultured cells support the idea that Slit activation of Robo can recruit Sos to the submembrane actin cytoskeleton to regulate cell morphology. Together, these results suggest a model in which Slit stimulation recruits Sos to the Robo receptor via Dock to regulate Rac-dependent cytoskeletal changes within the growth cone during axon repulsion (Yang, 2006).

Based on previous work implicating rac in Robo repulsion, as well as in vitro studies demonstrating that Sos exhibits GEF activity for Rac, but not Rho or Cdc42, Rac seemed the most likely Sos substrate. However, rho has also been implicated in mediating Robo repulsion, and genetic interactions between sos and dominant-negative Rho have been interpreted to suggest that Sos could act as a GEF for Rho. This question was investigated further, and two types of genetic evidence have been presented that suggest that indeed Rac is the favored substrate of Sos. First, ectopic expression experiments in the eye reveal interactions exclusively between sos and rac. Second, genetic interaction experiments using loss of function mutations in rac and rho (rather than the more problematic dominant-negative forms of the GTPases) reveal strong dose-dependent interactions between sos and rac, but not sos and rho during midline axon guidance. Together, these observations argue in favor of Rac as the primary in vivo Sos substrate. Nevertheless, the possibilities that Sos also contributes to Rho activation and that the combined activation of Rac and Rho is instrumental in mediating the Robo response cannot be excluded (Yang, 2006).

Previous studies have demonstrated that Slit stimulation of the Robo receptor leads to a rapid increase in Rac activity in cultured cells. However, the mechanism by which Rac is activated downstream of Robo was not clear. This study provides direct genetic and biochemical evidence that Sos is coupled to the Robo receptor through the Dock/Nck SH3-SH2 adaptor, where it can regulate local Rac activation. Studies in cultured mammalian cells have highlighted the importance of distinct Sos/adaptor protein complexes in controlling the subcellular localization and substrate specificity of Sos. In the context of Rac activation, the E3b1 (Abi-1) adaptor has been shown to play a critical and rate-limiting role in Sos-dependent Rac activation and subsequent formation of membrane ruffles (Innocenti, 2002). Could Sos regulation of Rac activity during Robo repulsion be similarly limited by the availability of specific adaptor proteins? It is interesting to note in this context that overexpression of dock does not lead to ectopic axon repulsion, suggesting that Dock may not be limiting for Robo signaling. However, although dock mutants do have phenotypes indicative of reduced Robo repulsion, their phenotype is considerably milder than that seen in robo mutants, raising the possibility that there may be additional links between Robo and Sos (Yang, 2006).

A number of studies in cultured mammalian cells have suggested that Rac activation induced by activated growth factor receptors requires the prior activation of Ras. For example, PDGF-induced membrane ruffling can be promoted or inhibited by expression of constitutively active or dominant-negative Ras, respectively. However, other studies have suggested that in Swiss 3T3 cell lines RTK activation of Rac is Ras independent. In addition, the observation that Ras activation and Rac activation display very different kinetics, with Rac activation persisting long after Ras activity has returned to basal levels, has been used to argue against an obligate role for Ras in Rac activation. In this study, using a genetic rescue approach, whether the ability of Sos to activate Rac during axon guidance in an intact organism requires its Ras-GEF function was directly tested. Genetic data indicate that the RasGEF domain of Sos is dispensable for axon guidance, while the DH RhoGEF domain is strictly required. This observation argues strongly in favor of the model that in vivo Sos activation of Rac does not strictly require Sos activation of Ras (Yang, 2006).

It is clear that subcellular localization plays a major role in regulating Sos activity and that different protein complexes containing Sos exist in different locations in the cell. This study has shown that activation of the Robo receptor by Slit triggers the recruitment of Sos to Robo receptors at the plasma membrane. Biochemical data argue that the adaptor Dock/Nck is instrumental in bridging this interaction, and given the diverse interactions between Dock/Nck and guidance receptors, it seems likely that Dock/Nck could fulfill this role in many guidance receptor contexts. This bridging function of Dock/Nck and guidance receptors is analogous to the role of Grb2 for growth factor receptors only insomuch as it brings signaling molecules to the receptor—the mechanism of interaction is distinct, since it is mediated through SH3 domain contacts rather than SH2/phosphotyrosine interactions. These observations suggest that there may be an additional pool of Sos that can function in a distinct adaptor protein/guidance receptor complex to regulate cell morphology in response to extracellular guidance cues (Yang, 2006).

Is regulating subcellular localization the only mechanism by which Sos activity is controlled? This seems unlikely. Indeed, a recent study has implicated tyrosine phosphorylation of Sos by Abl as an additional mechanism to activate the Rac-specific GEF activity of Sos in vertebrate cell culture models. This raises the intriguing possibility that Abl may fulfill a similar role for Robo signaling. This is a particularly appealing idea given the well-documented genetic and physical interactions between Robo and Abl. Indeed, sos and abl exhibit dose-dependent genetic interactions during midline axon guidance. A clear genetic test of whether Abl activates the Rac-GEF activity of Sos downstream of Robo may be complicated by the fact that Abl appears to play a dual role in Robo repulsion: both increasing and decreasing abl function lead to disruptions in Robo function. Nevertheless, it should be possible in the future to generate mutant versions of Sos that are refractory to Abl activation and to test whether these alterations disrupt the Sos guidance function. It will also be of great interest to determine whether the redistribution of Sos can also be observed in response to guidance receptor signaling in navigating growth cones, and if so, then what changes in actin dynamics and growth cone behavior are elicited (Yang, 2006).

Dissecting muscle and neuronal disorders in a Drosophila model of muscular dystrophy

Perturbation in the Dystroglycan (Dg)-Dystrophin (Dys) complex results in muscular dystrophies and brain abnormalities in human. Drosophila is an excellent genetically tractable model to study muscular dystrophies and neuronal abnormalities caused by defects in this complex. Using a fluorescence polarization assay, a high conservation in Dg-Dys interaction between human and Drosophila is demonstrated. Genetic and RNAi-induced perturbations of Dg and Dys in Drosophila cause cell polarity and muscular dystrophy phenotypes: decreased mobility, age-dependent muscle degeneration and defective photoreceptor path-finding. Dg and Dys are required in targeting glial cells and neurons for correct neuronal migration. Importantly, Dg interacts with insulin receptor and Nck/Dock SH2/SH3-adaptor molecule in photoreceptor path-finding. This is the first demonstration of a genetic interaction between Dg and InR (Shcherbata, 2007).

The Dg-Dys binding interface is highly conserved in humans and Drosophila. Both proteins are required for oocyte cellular polarity and interact in this process. Futhermore, mutants of both Dg and Dys genes show symptoms observed in muscular dystrophy. Reduction of Dg and Dys proteins results in age-dependent mobility defects. Eliminating Dg and Dys specifically in mesoderm derived tissues reveals that these proteins are required for muscle maintenance in adult flies: age-dependent muscle degeneration was observed in mutant tissues. Dg-Dys complex is also required for neuron path-finding and has both cell autonomous and non-cell autonomous functions for this process. Further, in neuronal path-finding process Dg interacts with InR and an SH2/SH3-domain adapter molecule Nck/Dock (Shcherbata, 2007).

Animal models have been used efficiently in muscular dystrophy studies. Some of the models are naturally occurring mutations (mdx-mouse, muscular dystrophy dog, cat and hamster), others have been generated by gene targeting. However, the regulation and the control of Dg-Dys complex are not understood, and no successful therapeutics exist yet for muscular dystrophies. Recently developed models for muscular dystrophy exist in C. elegans and zebrafish. In C. elegans Dys mutant, the transporter snf-6 that normally participates in eliminating acetylcholine from the cholinergic synapses, is not properly localized, resulting in an increased acetylcholine concentration at the neuromuscular junction and muscle wasting (Kim, 2004). The function of Dys in neuromuscular junctions has been addressed in Drosophila. These results bring up the possibility that muscular dystrophies in humans might also at least partly be attributed to the altered kinetics of acetylcholine transmission through neuromuscular junctions (Shcherbata, 2007).

Drosophila acts as a remarkably good model for age-dependent progression of muscular dystrophy. Dg and Dys reduction in Drosophila show age-dependent muscle degeneration and lack of climbing ability. It is tempting to speculate that the common denominator between different defects observed in Dg-Dys mutants in Drosophila and C. elegans is defective cellular polarity. The defects observed in C. elegans could be due to a defect in polarization of a cell, which will generate a neuromuscular junction that leads to miss-targeted snf-6. Similarly, Drosophila Dg-Dys complex is required for cellular polarity in the oocyte. In addition, neural defects observed are plausibly due to polarity defects in the growing axon (Shcherbata, 2007).

Similar to neuronal defects observed in human muscular dystrophy patients, neuronal defects were also found in Drosophila Dg and Dys mutant brains. In vertebrate brains, Dg affects neuronal migration (Montanaro, 2003; Qu, 2004) possibly through interaction of neurons with their glial guides. The neuronal migration and process outgrowth have been shown to require supportive input from glial cells and involve the formation of adhesion junctions along the length of the soma. Also, the outgrowth of the leading process involves rapid extension and contraction over the length of the glial fiber. Disruption of the cytoskeletal organization within the neuron, either of actin filaments, has been shown to inhibit glial-mediated neuronal migration. The glial function in this process is less well studied (Shcherbata, 2007).

Drosophila photoreceptor path-finding provides an excellent system for genetic dissection of neuronal outgrowth and target recognition. During the formation of the nervous system, newly born neurons send out axons to find their targets. Each axon is led by a growth cone that responds to extracellular axon guidance cues and chooses between different extracellular substrates upon which to migrate. Recent work has also identified a variety of intracellular signaling pathways by which these cues induce cytoskeletal rearrangements, but the proteins connecting signals from cell surface receptors to actin cytoskeleton have not been clearly determined. Dg is a good candidate for linking receptor signaling to the remodeling of the actin cytoskeleton and thereby polarizing the growth cone. Perturbation of Dg-Dys complex causes phenotypes that resemble Nck/Dock-Pak-Trio axon path-finding phenotypes, suggesting that Dg may be one of the key players in Nck/Dock signaling pathway for axon guidance and target recognition in Drosophila (Shcherbata, 2007).

Interestingly, Insulin receptor-tyrosine kinase (InR) mutants also show similar phenotypes to those of Nck/Dock signaling in photoreceptor axon path-finding and these two proteins show genetic and biochemical interactions. These data have led to speculations of mammalian InR acting in conjunction with Nck/Dock pathway in learning, memory and eating behavior. The current data now add Dg-Dys complex to this pathway; similar to what is seen in the case of Dg and Dys photoreceptor mutants, InR mutants show no obvious defects in patterning of the photoreceptors. However, the guidance of photoreceptor cell axons from the retina to the brain is aberrant. Furthermore, genetic and biochemical evidence suggests that InR function in axon guidance involves the Dock-Pak pathway rather than the PI3K-Akt/PKB pathway. Independently, biochemical interaction between Nck/Dock and Dg has been reported supporting the hypothesis that InR, Dg and Nck/Dock interaction regulates Dg-Dys complex. Furthermore, Dg interacts genetically with InR and Dock in photoreceptor axon path-finding. Since Dys interacts with Dg but not with InR and Dock, it is tempting to speculate that Dg can selectively interact with either Dys or InR and Dock. One possibility is that the tyrosine kinase activity of InR could regulate the Dg-Dys interaction by tyrosine phosphorylation in the Dg-Dys binding interphase. This tyrosine phosphorylation could prohibit the Dg-Dys interaction and thereby result in rearrangements in the actin cytoskeleton. Alternatively, other components observed in Dg-Dys complex might be involved in this regulation. However, it is also possible that potential polarity defects in the Dg mutant axons result in defective InR membrane localization. Interestingly, in another cell type, the Drosophila oocyte, InR, Dg and Dys also show similar phenotypes. In addition, insulin-like growth factors (IGF) and InR are important in maintaining muscle mass in vertebrates. Further connection of InR to Dg-Dys complex comes from experiments showing that muscle specific expression of IGF counters muscle decline in mdx-mice. The work presented in this study is the first demonstration of genetic interaction between Dg and InR. Future biochemical studies should unravel the molecular mechanism of this interaction (Shcherbata, 2007).

Dg-Dys complex is required both in neural and in targeting glial cells for correct neuronal axon path-finding in Drosophila brain. These data reveal that Dg-Dys complex also has a non-cell autonomous effect on axon path-finding and suggest that Dg-Dys-controlled ECM both from neuron and glial cells regulate neuronal axon path-finding. Further experiments are required to reveal whether long-range Laminin fibers are involved in this process, as has been shown in epithelial planar polarity, or whether glial processes are observed in close proximity to the neural growth cone. Interestingly, similar phenotypes are observed with Integrin mutants, suggesting that, as in planar polarity, Integrin and Dg-Dys complex might act in concert to regulate the process of ECM organization that will regulate the cytoskeleton of the cells involved (Shcherbata, 2007).

Taken together, the phenotypes caused by Drosophila Dg and Dys mutations are remarkably similar to phenotypes observed in human muscular dystrophy patients, and therefore suggest that functional dissection of Dg-Dys complex in Drosophila should provide new insights into the origin and potential treatment of these fatal neuromuscular diseases. As a proof of principle, using Drosophila as a model InR has now been determined as a signaling pathway that genetically interacts with Dg. Future studies are directed to unravel the molecular mechanism of Dg and InR-Dock interactions in invertebrates as well as vertebrates (Shcherbata, 2007).

Src64B phosphorylates Dumbfounded and regulates slit diaphragm dynamics: Drosophila as a model to study nephropathies

Drosophila nephrocytes are functionally homologous to vertebrate kidney podocytes. Both share the presence of slit diaphragms that function as molecular filters during the process of blood and haemolymph ultrafiltration. The protein components of the slit diaphragm are likewise conserved between flies and humans, but the mechanisms that regulate slit diaphragm dynamics in response to injury or nutritional changes are still poorly characterised. This study shows that Dumbfounded/Neph1, a key diaphragm constituent, is a target of the Src kinase Src64B. Loss of Src64B activity leads to a reduction in the number of diaphragms, and this effect is in part mediated by loss of Dumbfounded/Neph1 tyrosine phosphorylation. The phosphorylation of Duf by Src64B, in turn, regulates Duf association with the actin regulator Dock. Diaphragm damage induced by administration of the drug puromycin aminonucleoside (PAN model) directly associates with Src64B hyperactivation, suggesting that diaphragm stability is controlled by Src-dependent phosphorylation of diaphragm components. These findings indicate that the balance between diaphragm damage and repair is controlled by Src-dependent phosphorylation of diaphragm components, and point to Src family kinases as novel targets for the development of pharmacological therapies for the treatment of kidney diseases that affect the function of the glomerular filtration barrier (Tutor, 2013).

Many of the vertebrate slit diaphragm proteins are tyrosine phosphorylated in normal glomeruli. Furthermore, tyrosine phosphorylation of nephrin and Neph1 by the SFK Fyn is crucial for the stability of this protein complex and therefore for glomerular filtration function. Thus, upon phosphorylation, the cytoplasmic regions of nephrin and Neph1 recruit the intracellular adaptors Nck and Grb2, among others, that in turn regulate actin cytoskeleton reorganisation. This study found that the post-translational regulation by phosphorylation of Duf, the orthologue of Neph1, is conserved and that Src64B, a member of the non-receptor Src family tyrosine kinases, is responsible for Duf tyrosine phosphorylation in nephrocytes. Furthermore, Src64B function is necessary for the structural integrity of the filtration diaphragm and for normal nephrocyte morphology. In Src64B loss-of-function or knockdown conditions, there is a reduction in the density of filtration diaphragms at the nephrocyte cell membrane. Presumably, this is due in part to their internalisation, as suggested by the accumulation of both Duf and Pyd at protrusions extending inwards from the membrane and the presence in these locations of structures dense to electrons reminiscent of filtration diaphragms. In addition, delocalisation was observed of Duf/Pyd complexes to cell contact membranes that, at the ultrastructural level, are rich in adherens junctions. The presence of adherens junctions in apposed membranes is never found in wild-type mature nephrocytes but is characteristic of embryonic nephrocytes prior to the formation of filtration diaphragms. All these alterations are concomitant with changes in nephrocyte architecture, revealed by their smoother surface and their agglutination. As the loss of filtration diaphragms result in regression of labyrinthine channels, and this is always associated with nephrocyte agglutination, these morphological changes are interpreted as being due to reallocation of Duf/Pyd complexes from SD to adherens junctions. It is suggested that these morphological alterations are the nephrocyte equivalent of podocyte foot process effacement, a feature common to all proteinuric diseases and believed to be initiated by changes in the actin cytoskeleton (Tutor, 2013).

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