Transcriptional Regulation

Mapping Dmef2-binding regulatory modules by using a ChIP-enriched in silico targets approach

Mapping the regulatory modules to which transcription factors bind in vivo is a key step toward understanding of global gene expression programs. A chromatin immunoprecipitation (ChIP)-chip strategy has been developed for identifying factor-specific regulatory regions acting in vivo. This method, called the ChIP-enriched in silico targets (ChEST) approach, combines immunoprecipitation of cross-linked protein-DNA complexes (X-ChIP) with in silico prediction of targets and generation of computed DNA microarrays. Use of ChEST in Drosophila is described to identify several previously unknown targets of myocyte enhancer factor 2 (MEF2), a key regulator of myogenic differentiation. The approach was validated by demonstrating that the identified sequences act as enhancers in vivo and are able to drive reporter gene expression specifically in MEF2-positive muscle cells. Presented here, the ChEST strategy was originally designed to identify regulatory modules in Drosophila, but it can be adapted for any sequenced and annotated genome (Junion, 2005).

To predict Dmef2-dependent CRMs, the Drosophila genome was scanned for modules containing one of the three previously described in vivo-acting Dmef2 binding sites. Because muscle differentiation events are controlled by the synergistic action of MADS-box (Mef2 family) and E-box (Twist and MyoD bHLH family) factors modules were sought containing Mef2- and E-box-binding sites. This scanning procedure led to the identification of ~1,243 potential Dmef2-binding CRMs, from which 99 modules were selected, amplified, and spotted to produce a computed Dmef2-CRM array. Three of four previously described Dmef2-CRMs, located in the vicinity of Paramyosin, Act57B, and Dmef2, were identified during genome scanning and selected for spotting as positive control. The fourth in vivo-acting Dmef2-CRM located close to b3-tubulin did not come out from the screen because of the high number of E-box sites used as the scanning criterion. The CRMs located in the vicinity of genes that are not expressed in the mesoderm or genes of unknown expression and function (some of CGs) were rejected. In parallel, X-ChIP was used to isolate DNA fragments to which Dmef2 binds in vivo. ChIP-DNA immunoprecipitated either with Dmef2 antibody or with nonimmune serum was then labeled and used to probe the Dmef2-CRM array. The three previously described in vivo-acting CRMs were found enriched in ChIP-DNA immunoprecipitated with Dmef2 antibody. Importantly, numerous other in silico-identified Dmef2-CRMs were enriched in ChIP-DNA, thus demonstrating the efficacy of the ChEST method (Junion, 2005).

Of the 99 in silico-predicted Dmef2-CRMs, 62 were enriched in the DNA immunoprecipitated with anti-Dmef2 antibody. The CRM-associated Dmef2 targets included genes expressed in all muscle cell types, in which Dmef2 has previously been reported to function. In addition to expected candidates encoding fusion (Lmd, Hibris) and structural (Ket, Pod1) muscle proteins, a large number of CRM-associated Dmef2 target genes coded for TFs and signal transduction proteins. For example, CRMs upstream of Fz2 and within the introns of Ci and Pan indicate a potential role of Dmef2 in transcriptional regulation of genes transducing to the mesodermal cells ectodermal Wg and Hh signals, whereas CRMs within the introns of If and Pka-C3 suggest that by regulating transcription of these genes Dmef2 is involved in the attachment of muscle fibers and in fiber contraction, respectively. In some cases (e.g., Kettin, NetB, N-cad), several Dmef2-dependent CRMs were mapped in the vicinity of the same gene, highlighting the complexity of transcriptional regulation in which Dmef2 is involved. Analysis of the position of CRMs in relation to adjacent genes revealed that the majority of ChIP-enriched modules are located upstream (42%) or within the introns (39%) of target genes. In these two categories, the most frequent positions of Dmef2-CRMs appear between 1 and 5 kb upstream of the gene and within the first intron (Junion, 2005).

To determine whether the ChEST-identified DNA fragments are able to act as regulatory modules in vivo, ten Dmef2-CRMs by reporter gene transgenesis were tested. Nine of 10 CRMs were found to drive reporter gene expression in Dmef2-positive muscle cells. In all transgenic Drosophila lines, lacZ reporter expression at least partially reproduced endogenous gene expression, indicating that the identified CRMs were bona fide enhancers of adjacent genes. For example, the Ket-1, Ket-2, and Ket-3 CRMs laying in the vicinity of the kettin gene, which encodes a giant muscle protein required for the formation and maintenance of normal sarcomere structure, were found to drive lacZ expression in distinct subsets of differentiating body wall muscles. These data indicate that the pan-muscular expression of kettin is regulated in a muscle-type-specific manner, and by multiple Dmef2-binding enhancers. Interestingly, four other analyzed CRMs located within the introns of N-cad and acon and upstream of fz2, sfl, and Meso18E also drive lacZ expression in discrete subsets of somatic muscle precursors. The muscle-type-restricted activity of these modules suggests that both CRM regulators (Dmef2, E-box factors) and their target genes are involved in different aspects of muscle precursors diversification, including muscle fiber shape and axial positioning. Alternatively, the observed muscle-type-specific expression of lacZ may result from the limited size (250-300 bp) of genomic sequences tested. In embryos carrying a Dmef2-dependent CRM found in the intron of If, lacZ expression is detected in a group of ventrolateral muscles. This lacZ pattern correlates with distribution of endogenous If, which accumulates at the extremities of ventrolateral muscle fibers and is required for their correct epidermal insertion. The reduced level of target gene expression in Dmef2 mutant embryos provides an additional support for Dmef2-dependent in vivo regulation of ChEST-identified CRMs (Junion, 2005).

Protein Interactions

In animal cells, GTPase signaling pathways are thought to generate cellular protrusions by modulating the activity of downstream actin-regulatory proteins. Although the molecular events linking activation of a GTPase to the formation of an actin-based process with a characteristic morphology are incompletely understood, Rac-GTP is thought to promote the activation of SCAR/WAVE, whereas Cdc42 is thought to initiate the formation of filopodia through WASP. SCAR and WASP then activate the Arp2/3 complex to nucleate the formation of new actin filaments, which through polymerization exert a protrusive force on the membrane. Using RNAi to screen for genes regulating cell form in an adherent Drosophila cell line, a set of genes, including Abi/E3B1 (an SH3 domain-containing Abl substrate), was identified that is absolutely required for the formation of dynamic protrusions. These genes delineate a pathway from Cdc42 and Rac to SCAR and the Arp2/3 complex. Efforts to place Abi in this signaling hierarchy reveal that Abi and two components of a recently identified SCAR complex, Sra1 (p140/PIR121/CYFIP) and Kette (Nap1/Hem), protect SCAR from proteasome-mediated degradation and are critical for SCAR localization and for the generation of Arp2/3-dependent protrusions. It is concluded that in Drosophila cells SCAR is regulated by Abi, Kette, and Sra1, components of a conserved regulatory SCAR complex. By controlling the stability, localization, and function of SCAR, these proteins may help to ensure that Arp2/3 activation and the generation of actin-based protrusions remain strictly dependant on local GTPase signaling (Kunda, 2003).

With the advent of RNAi it is possible to use Drosophila cells in culture as a model system to test the cell-biological function of genes identified by genomic sequencing; such genes include those involved in the generation of actin-based protrusions. S2R+ cells are particularly amenable to this type of loss-of-function analysis because a large number of distinct actin-related phenotypes can be readily distinguished in this cell type. Using such an approach, several genes were identified from a set of putative actin regulators that are absolutely required for the maintenance of S2R+ cell shape and for the formation of lamellipodia. In each case, the gene-specific dsRNA identified causes cells to assume a starfish-like morphology with multiple slender cell extensions. This change in form is accompanied by the loss of actin filaments from the cell periphery, resulting in a more diffuse, non-cortical F-actin distribution. Genes with this characteristic RNAi phenotype included a known activator of the Arp2/3 complex, the sole Drosophila SCAR/WAVE homolog, and Drosophila Abi, an SH3 domain-containing Abl substrate. In contrast, Drosophila WASP, another Arp2/3 complex activator, had no discernable RNAi phenotype in this assay and did not visibly accentuate the SCARRNAi phenotype. RNAi targeting of Arc-p34 and Arc-p20, two components of the Drosophila Arp2/3 complex, led to a similar change in S2R+ cell shape, implying that this spiky phenotype reflects the inability to nucleate new cortical actin filaments (Kunda, 2003).

Although this analysis delineated a putative pathway (Cdc42>Rac>SCAR>Arp2/3 complex) that promotes the nucleation of actin filaments, it was not clear where to place Abi within this signaling hierarchy. A recent biochemical study, however, noted that Abi copurifies with mammalian homologs of Kette, Sra1, and HSPC300 (see Medline: 12181570 ) as part of a regulatory SCAR complex in extracts from mammalian brains. By using RNAi to test the functions of the equivalent Drosophila proteins, it was found that Abi, Sra1, and Kette are essential for the generation of protrusions and for the stability of SCAR protein. Similarly, in parallel studies in Drosophila and in Dictyostelium, reduced levels of SCAR protein were observed in cells lacking individual components of the complex. These data suggest that the presence of SCAR in a regulatory complex and its sensitivity to degradation have been highly conserved during evolution. Furthermore, the idea that these proteins form a physical complex in Drosophila is supported by the colocalization of Kette and SCAR at the tips of protrusions. The data concerning the function of the fifth component of the putative SCAR complex, HSPC300, are more equivocal. Although treatment of S2R+ cells with HSPC300 dsRNA compromised their ability to form lamellipodia and caused a reproducible, if partial, reduction in SCAR protein levels, it was not possible to measure the extent of RNAi-mediated HSPC300 silencing. Therefore, although the data support a role for HSPC300 in the regulation of SCAR, it has not yet been determined whether HSPC300 is absolutely required for the generation of SCAR-dependent protrusions, as are Abi, Kette, and Sra1 (Kunda, 2003).

Given the apparent sensitivity of SCAR to proteolysis, changes in local or global SCAR stability could modulate the rate of actin filament formation. Furthermore, if SCAR is released from the complex after the binding of Rac-GTP, as predicted, SCAR degradation could also act as a brake to limit SCAR-dependent actin filament nucleation. In either case, one would expect SCAR protein to exhibit a relatively short half-life in vivo. In actively ruffling S2R+ cells, however, SCAR appears to be relatively stable because proteasome inhibitors or inhibitors of transcription or translation have little effect on SCAR protein levels. These findings lead to the conclusion that most SCAR is present in stable complexes in wild-type cells. For this reason, the conserved instability of SCAR protein may simply provide cells with a mechanism to rapidly eliminate free, nascent, or mislocalized SCAR, protecting cells from the potentially adverse effects of this potent, constitutively active protein. Nevertheless, under special circumstances or in other cell types, proteasome-mediated degradation of SCAR may help to limit the extent of actin filament nucleation induced after a burst of Rac-GTP (see below) (Kunda, 2003).

Although Abi, Kette, and Sra1 are required for preventing SCAR degradation, the data clearly point to their having additional functions. Most importantly, the morphological changes observed in AbiRNAi cells precede the loss of SCAR protein. In addition, increasing the SCAR protein levels in AbiRNAi cells fails to rescue their morphological defects; it also fails to do so in KetteRNAi and Sra1RNAi cells. This might seem unexpected given that SCAR is able to activate the Arp2/3 complex on its own, both in vitro and in Drosophila cells. In the absence of complex components, however, SCAR fails to become properly localized at the cell cortex. So, by localizing SCAR at the cortex, the complex may play a critical role in harnessing its activity for the generation of protrusive force (Kunda, 2003).

Three recent genetic studies reported observations that conflict with data presented here. In particular, data presented in these studies show that Sra1/Kette and SCAR display an antagonistic relationship in the Drosophila nervous system. Although more work will have to be done to unravel such apparently contradictory data, some of these discrepancies may reflect differences in the relative levels of SCAR and components of the inhibitory complex in the model system under investigation. If the total cellular pool of Abi, Sra1, and Kette is bound up in stable, Rac-responsive SCAR complexes, a reduction in the level of any one component will lead to a reduction in Arp2/3-dependent actin nucleation (as observed in this study). In contrast, if Abi, Kette, and Sra1 are present in excess of SCAR, they will limit the ability of free, active SCAR to nucleate actin filaments (Kunda, 2003).

A speculative model is presented for the regulation of SCAR. This model attempts to reconcile the current findings with data from recent in vitro and genetic studies. It is proposed that nascent SCAR is rapidly incorporated into an inhibitory complex that contains Abi, Sra1, and Kette and protects the protein from proteolysis. The complex localizes at the cell cortex , where it is responsive to Rac signaling. The binding of Sra1 to Rac-GTP may induce a transient change in the makeup or conformation of the complex, which may free the SCAR VCA domain to interact with the Arp2/3 complex, whose activation triggers a burst of new actin filament formation. Finally, the extent of actin filament formation in response to a pulse of Rac-GTP may be limited by the presence of the inhibitory complex and to a lesser extent by proteasome-mediated degradation. In summary, it is proposed that cells regulate SCAR stability, localization, and activity to ensure that actin nucleation and the formation of cellular protrusions are precisely regulated in time and space (Kunda, 2003).

Sra-1 interacts with Kette and Wasp and is required for neuronal and bristle development in Drosophila

Regulation of growth cone and cell motility involves the coordinated control of F-actin dynamics. An important regulator of F-actin formation is the Arp2/3 complex, which in turn is activated by Wasp and Wave. A complex comprising Kette/Nap1, Sra-1/Pir121/CYFIP, Abi and HSPC300 modulates the activity of Wave and Wasp. This study presents the characterization of Drosophila Sra-1 (specifically Rac1-associated protein 1). sra-1 and kette are spatially and temporally co-expressed, and both encoded proteins interact in vivo. During late embryonic and larval development, the Sra-1 protein is found in the neuropile. Outgrowing photoreceptor neurons express high levels of Sra-1 also in growth cones. Expression of double stranded sra-1 RNA in photoreceptor neurons leads to a stalling of axonal growth. Following knockdown of sra-1 function in motoneurons, abnormal neuromuscular junctions were noted, similar to what was determined for hypomorphic kette mutations. Similar mutant phenotypes were induced after expression of membrane-bound Sra-1 that lacks the Kette-binding domain, suggesting that sra-1 function is mediated through kette. Furthermore, both proteins stabilize each other and directly control the regulation of the F-actin cytoskeleton in a Wasp-dependent manner (Bogdan, 2004).

Wasp proteins are auto-inhibited, whereas the Wave proteins are trans-inhibited. Both usually require small G proteins of the Rho family for activation. In the case of Wasp, activated, GTP-bound Cdc42 binds to the CRIB (Cdc42/Rac Interactive Binding) domain of Wasp, releasing the auto-inhibition and thereby leading to the activation of the Arp2/3 complex. However, a structure-function analysis of the Drosophila Wasp has demonstrated that the Cdc42-binding domain is not strictly necessary for function, suggesting that alternative pathways, such as phosphorylation can activate WASP. Indeed, some tyrosine kinases have been shown to activate Wasp by phosphorylation (Bogdan, 2004 and references therein).

In contrast to Wasp, Wave is not auto-inhibited. It is kept in an inactive state through association with a protein complex comprising Kette/Nap1, Sra1 (specifically Rac associated 1, Sra-1; also called CYFIP/p140Sra-1, from here on called Sra-1 according to FlyBase) and the Abelson-interactor protein (Abi) (Eden, 2002). Upon dissociation or conformational changes of this complex, Wave is assumed to be active. Thus, Kette or Sra-1 should antagonize Wave function. This is supported by genetic studies in Dictyostelium and Drosophila. Cell culture experiments show that Wave is degraded in a Ubiquitin-dependent manner following disruption of the Sra-1/Kette complex. These latter findings are likely to reflect the fast inactivation of Wave once activated. In vitro, activation of Wave can be mediated by Rac1 or SH3 domains, which presumably bind to Sra-1 (Bogdan, 2004 and references therein).

The Sra-1/Kette protein complex is not only required to negatively regulate the activity of Wave but is also able to activate Wasp function at the membrane. The interaction of Kette and Wasp is not direct but is likely to be mediated by the Abi, which can bind to both Kette and Wasp. Interestingly, the Nck adapter protein is also able to bind to Wasp via its third SH3 domain. Thus, Sra-1, which can bind to the first SH3 domain of Nck is a good candidate to locate the Sra-1/Kette complex to the membrane close to Wasp (Bogdan, 2004 and references therein).

To test whether the mutant phenotypes of sra-1 and kette are alike as predicted, and whether Sra-1 indeed acts through Kette to regulate actin dynamics, a functional characterization of Sra-1 during Drosophila development was conducted. Sra-1 and Kette are both required for axonal growth and perform common functions during formation and maturation of neuromuscular junctions (NMJ). Analysis of temporal and spatial distribution of the Sra-1 protein shows a prominent co-expression with Kette. Both proteins are maternally expressed and later in development become concentrated in the developing nervous system (CNS). Sra-1 is highly expressed in growth cones and neuromuscular synapses. Direct interaction of Sra-1 and Kette depends on a short C-terminal domain of the Sra-1 protein. Expression of a Sra-1 variant lacking the C-terminal domain leads to a dominant-negative phenotype that can be suppressed by expression of an activated Kette protein. In tissue culture cells as well as in vivo Sra-1 function is required for F-actin organization. Further genetic analyses demonstrate that Sra-1 function at the membrane depends on the presence of Wasp (Bogdan, 2004).

In addition to the CNS phenotypes, mutations in sra-1 and kette both lead to synaptic defects that are characterized by an overall reduction in size of the neuromuscular junction, as well as the induction of supernumerary buds in sra-1 and kette mutant synaptic boutons. It is known that new boutons often arise from existing ones by asymmetrical budding or symmetrical division, which in turn requires an intact regulation of the actin cytoskeleton. The increased number of branches as well as the bulged appearance of the synaptic boutons after depletion of kette or sra-1 function may reflect their function in regulating wasp. Indeed wasp mutants display synaptic phenotypes similar to those of sra-1 and kette. Recently, it has been found that the adaptor protein Nervous wreck (Nwk) binds Wasp and is also required for normal synapse morphology. Thus, Nwk might act as a scaffolding protein in the synapse assembling a Wasp activation complex comprising Sra-1, Kette and Abi (Bogdan, 2004).

As in Drosophila, mutations in several of the vertebrate orthologs of the above mentioned genes are associated with learning deficits, demonstrating the pivotal importance of F-actin dynamics for precise neuronal function (Bogdan, 2004).

Rapid remodeling of the F-actin cytoskeleton is mostly brought about by the Arp2/3 complex, which in turn is activated by members of the Wasp and Wave protein families. Wasp as well as Wave are potent F-actin nucleation factors. Obviously within the cell their activity must be tightly regulated. Whereas Wasp is auto-inhibited, Wave is trans-inhibited and requires the inhibiting Sra-1 Kette protein complex. Upon dissociation of this complex or conformational changes within the complex, Wave is active and presumably remains active until it is degraded via ubiquitination. This latter mechanism, which is frequently used in regulating the effective concentration of active proteins, ensures that Wave activity lasts for only a short time period (Bogdan, 2004).

Wave is not the only protein of the complex that is degraded upon disruption of the protein complex. Depletion of Kette not only leads to a loss of Wave but also of Sra-1. Vice versa, depletion of Sra-1 leads to a loss of Kette. Thus, ultimately the stability of all proteins of the inhibitory Sra-1 complex appears to be interdependent (Bogdan, 2004).

Kette can activate Wasp-mediated F-actin formation. Sra-1 function also depends on Wasp. In both cases, the membrane localization of Kette or Sra-1 is essential, indicating that in vivo regulation of membrane recruitment of Sra-1 and Kette is important for function. The data presented in this work also suggest that membrane-bound Sra-1 or Kette proteins are both able to activate Wasp independently of each other (Bogdan, 2004).

Vertebrate homologues of Sra-1 and Kette were first identified in a complex with the SH2 SH3 adapter Nck. The N-terminal SH3 domain of Nck is thought to bind to Sra-1, evoking a model where Nck recruits Sra-1 and the associated Kette protein to the membrane. kette and dock which encodes the Drosophila Nck homologue interact during axonal pathfinding. Cell signaling and cell adhesion leads to the activation of a number of receptor systems which in turn mediate anchorage-dependent recruitment of adapter proteins such as Nck. Nck in turn is able to connect cell-surface receptors via different signal cascades to the F-actin cytoskeleton (Bogdan, 2004 and references therein).

However, in vivo Nck cannot mediate all aspects of Sra-1/Kette function; the complete loss of maternal and zygotic Nck results in similar but not identical phenotypes when compared with kette or sra-1 loss-of-function phenotypes. In the developing synapse, the function of Nck in recruiting Sra-1 and Kette to the membrane may be fulfilled by Nwk, which as Nck also binds Wasp. Interestingly, both adaptor proteins are involved in Slit Robo signaling, where they may mediate different biological effects. This suggests that combinatorial and tissue specific factors are assembled in response to specific cues to activate Wasp in different cell types or compartments (Bogdan, 2004).

WAVE/SCAR, a multifunctional complex coordinating different aspects of neuronal connectivity

Although it is well established that the WAVE/SCAR complex transduces Rac1 signaling to trigger Arp2/3-dependent actin nucleation, regulatory mechanisms of this complex and its versatile function in the nervous system are poorly understood. The Drosophila proteins SCAR, CYFIP and Kette, orthologs of WAVE/SCAR complex components, all show strong accumulation in axons of the central nervous system and indeed form a complex in vivo. Neuronal defects of SCAR, CYFIP and Kette mutants are, despite the initially proposed function of CYFIP and Kette as SCAR silencers, indistinguishable and are as diverse as ectopic midline crossing and nerve branching as well as synapse undergrowth at the larval neuromuscular junction. The common phenotypes of the single mutants are readily explained by the finding that loss of any one of the three proteins leads to degradation of its partners. As a consequence, each mutant is unambiguously to be judged as defective in multiple components of the complex even though each component affects different signaling pathways. Indeed, SCAR-Arp2/3 signaling is known to control axonogenesis whereas CYFIP signaling to the Fragile X Mental Retardation Protein fly ortholog contributes to synapse morphology. Thus, these results identify the WAVE/SCAR complex as a multifunctional unit orchestrating different pathways and aspects of neuronal connectivity (Schenck, 2004).

Formal evidence is provided that SCAR, CYFIP and Kette proteins form a complex. Co-immunoprecipitation experiments were performed from cytoplasmic extracts of Drosophila Schneider (S2) cells using antibodies raised against CYFIP. Extract and co-immunoprecipitated material were subjected to Western blot analysis using antibodies against SCAR and Kette, the fly orthologs of WAVE and Hem-2/NAP125, respectively, which both associate with the human CYFIP2 protein. Anti-Kette antibody reveals a band of 112 kDa, whereas anti-SCAR reveals a doublet of about 66 and 70 kDa, one band of which may represent a post-translationally modified SCAR protein. The two Drosophila proteins are found to specifically co-immunoprecipitate with CYFIP. Using the same antibodies, it was found that, in wild-type embryos, Kette is present in longitudinal connectives as well as commissures during establishment of the axonal network, like SCAR and CYFIP. By late stages, all three proteins accumulate in longitudinal connectives, strongly suggesting that they act as a physical and functional unit during embryogenesis. In summary, the WAVE/SCAR complex is conserved in Drosophila and its members accumulate in axons of the nervous system (Schenck, 2004).

The mammalian WAVE/SCAR complex has been shown to be an integral part of Rac1 GTPase signaling pathways that coordinate actin cytoskeleton remodeling. Although mutations in single components call for a role of these proteins in construction of the nervous system, little is known about regulation and function of the WAVE/SCAR complex in this tissue. Evidence is provided that SCAR, CYFIP and Kette, which co-localise during embryogenesis, are submitted to interdependent, posttranscriptional, control. Moreover, the WAVE/SCAR complex acts as a functional unit coordinating different aspects of axonal and synapse development, revealing its role in core signaling pathways underlying neuronal connectivity (Schenck, 2004).

The analysis of CYFIP, SCAR and Kette mutant phenotypes and their genetic interaction with dFMR1 call for distinct pathways being triggered by the WAVE/SCAR unit. Better understanding of specific contribution requires a more complete knowledge on these signaling pathways. First conclusions, however, can be drawn. Guidance of embryonic central axons is, for example, affected in WAVE/SCAR complex but not in dFMR1 mutants and is hence controlled by dFMR1-independent pathways downstream of the WAVE/SCAR complex. In fact, central axons may be under control of the SCAR-Arp2/3 pathway, because mutations in different subunits of the Arp2/3 complex result in disruption of these axon tracts (Schenck, 2004).

In contrast, dFMR1 as well as WAVE/SCAR complex mutants affect NMJ morphology, suggesting a role of one or more complex components in this process. Indeed, overexpressed CYFIP rescues the dFMR1 gain of function phenotype, while overexpressed Kette and SCAR do not. This indicates that only CYFIP can signal to dFMR1 and suggests that the Kette and SCAR synaptic phenotypes are indirect consequences of CYFIP protein degradation. The fact that nevertheless, CYFIP, Kette or SCAR mutations compensate for the dFMR1 loss of function phenotype further supports the view that the WAVE/SCAR complex acts as an integral unit. While these studies do not exclude a direct role of WAVE/SCAR-mediated Arp2/3-dependent actin nucleation in synapse morphology, they clearly highlight the importance of CYFIP signaling to dFMR1. Interestingly, it has been recently shown that WASP, the second actin nucleation promoting factor, as well as its interacting protein Nervous wreck, control NMJ morphology. WASP is also directly linked to the WAVE/SCAR complex by its interaction with the Abi protein, indicating that proper synapse morphology requires integration of several related signaling pathways. Understanding the molecular basis of neuronal connectivity clearly implies evaluation of the specific contribution and integration of Arp2/3 and Fragile X Mental Retardation Protein mediated pathways at the synapse (Schenck, 2004).

Recent studies on fly and vertebrate cell cultures have shown that overexpressed SCAR or WAVE2 in cells that are knocked down for other components of the complex fail to be recruited to the cell periphery and do not rescue cytoskeletal defects. Loss and gain of function data show that WAVE/SCAR complex function relies on the integrity of all its components and that not only SCAR, but also its partners require proper control of protein stability and localisation. Surprisingly, the overexpressed SCAR protein can still accumulate, at least in part, at central axons, whereas excess CYFIP and Kette proteins cannot, suggesting the possibility that SCAR is directly connected to the translocation machinery responsible for axonal recruitment of the WAVE/SCAR complex (Schenck, 2004).

The observation that even upon simultaneous overexpression in pairwise or triple combinations SCAR is found in axons whereas excess CYFIP and Kette are not, is explained by the recent finding that CYFIP and Kette do not bind SCAR directly and must hence fail to travel piggybaggy with SCAR (Schenck, 2004).

Even properly localised excess of SCAR, however, is not capable of inducing an aberrant phenotype. Localisation of SCAR is hence a prerequisite but not sufficient to activate Arp2/3-dependent changes in the actin cytoskeleton, calling for an additional level of SCAR activity control. Whether this control occurs through phosphorylation, as in the case of the WAVE/SCAR related protein WASP and as suggested by the doublet revealed by anti-SCAR in immunoblotting, remains to be determined (Schenck, 2004).

Direct comparison of axonal and synaptic phenotypes displayed by CYFIP, Kette and SCAR mutant alleles has revealed that they are undistinguishable, a finding that suggested a common pathogenic mechanism. Indeed, in the developing nervous system, not only SCAR is subjected to protein turnover if either CYFIP or Kette are missing, as predictable from studies in cellular systems, but also CYFIP and Kette are lost if one of their partners is absent. These results demonstrate for the first time that not only SCAR levels are regulated by CYFIP and Kette dose, but also CYFIP and Kette levels depend on the dose of their protein partners. Thus, instead of being considered as single mutants, CYFIP, Kette and SCAR mutants have unambiguously to be judged as defective in multiple components of the WAVE/SCAR complex. This common biochemical basis (i.e., lack of all three proteins) clearly accounts for the identical observed phenotypes in the loss of function conditions, regardless of any effect these proteins may exert on each other in this tissue (Schenck, 2004).

An important question that has remained so far unanswered by studies on the WAVE/SCAR complex is why WAVE/SCAR requires four associated proteins to transduce Rac1 signaling to the Arp2/3 complex, whereas the WAVE/SCAR-related protein WASP is capable of doing this job on its own. It is speculated that the hetreopentameric WAVE/SCAR complex constitutes a checkpoint for a multitude of signaling pathways, which ensures their simultaneous activation. Several hints exist now in the literature indicating additional functions of Kette, Abi and CYFIP proteins. Whereas the functional significance of Kette interaction with signaling proteins like dynamin and Eps8 and Abi interaction with the Abl nonreceptor tyrosin kinase remain to be validated, this work has delineated a first pathway specific to one of the WAVE/SCAR-associated proteins, CYFIP signaling to dFMR1 (Schenck, 2004).

These data show that integrity of the WAVE/SCAR complex plays a pivotal function in nervous system development and that CYFIP and Kette do not simply function as SCAR silencers or proteins merely stabilising/localizing SCAR. This is of particular interest if one considers that a series of genes connected to the WAVE/SCAR complex and its associated signaling pathways are implicated in human mental retardation. First, several mutations directly affecting Rho/Rac regulatory or effector proteins cause X-linked mental retardation. Moreover, the most frequent cause of hereditary mental retardation is due to mutations in the Fragile X Mental Retardation gene, which is connected to Rac1 via CYFIP and thereby to the WAVE/SCAR complex. Finally, MEGAP (mental disorder-associated GAP protein), also known as WRP or srGAP3, encoded by one of the few so far identified autosomal mental retardation genes, is directly linked to the WAVE/SCAR complex. Indeed, MEGAP/WRP/srGAP3 is a negative regulator of the Rac1 GTPase and binds directly to WAVE1, suggesting that the protein terminates Rac1 signaling to the complex. The WAVE/SCAR complex is thus central to signaling pathways mutated in impaired conditions of neuronal functioning (Schenck, 2004).

In light of the data obtained in fly, one can speculate that also (some of) the different human genetic conditions mentioned above may have a common biochemical basis. If it can be formally proven that, analogous to flies, also the recently reported WAVE1 knockout mouse, notably characterised by cognitive deficits, is devoid of CYFIP and Kette proteins, this would provide the first direct evidence for the implication of this complex not only in neuronal connectivity but also in cognitive function (Schenck, 2004).

Blow acts with Kette to cause the stretching of the circular visceral muscles around the midgut

Circular visceral muscles of Drosophila are binuclear syncytia arising from fusion of two different kinds of myoblasts: a circular visceral founder cell and one visceral fusion-competent myoblast. In contrast to fusion leading to the somatic body-wall musculature, myoblast fusion for the circular visceral muscles does not result in massive syncytia but instead in syncytia interconnected with multiple cytoplasmic bridges, which differentiate into large web-shaped muscles. These syncytial circular visceral muscles build a gut-enclosing network with the interwoven longitudinal visceral muscles. At the ultrastructural level, during circular visceral myoblast fusion and the first step of somatic myoblast fusion prefusion complexes and electron-dense plaques were not detectable which was surprising as these structures are characteristic for the second step of somatic myoblast fusion. Moreover, Blown fuse (Blow), a cytoplasmic protein essential for the second step of somatic myoblast fusion, plays a different role in circular visceral myogenesis. Blow is known to be essential for progression beyond the prefusion complex in the somatic mesoderm; however, analysis of blow mutants established that it has a restricted role in stretching and outgrowth of the syncytia in the circular visceral muscles. Furthermore, in the visceral mesoderm, Blow is expressed in both the fusion-competent myoblasts and circular visceral founders, while expression in the somatic mesoderm is initially restricted to fusion-competent myoblasts. Different enhancer elements in the first intron of blow are responsible for this distinct expression pattern. Thus, a model for Blow is proposed in which this protein is involved in at least two clearly differing processes during Drosophila muscle formation, namely somatic myoblast fusion on the one hand and stretching and outgrowth of circular visceral muscles on the other (Schroter, 2006).

The ultrastructural analyses of the fusion mechanism of Drosophila circular visceral muscles indicates that fusion of the circular visceral muscles takes place without electron-dense vesicles or plaques and ends with incomplete membrane breakdown that is followed by the stretching of the syncytia around the gut. The start of this process resembles the observed contact structures between the somatic Fusion compentent myoblasts (FCMs) and founder cells during precursor formation which is different to the second fusion step during somatic myogenesis. However, there is also a clear difference in the ultrastructural features of fusing circular visceral muscles and the first somatic step of fusion: although cells first adhere in a very similar manner and fuse in both cases without a prefusion-complex or the formation of electron-dense plaques, the first somatic fusion proceeds to completion and results in a continuous syncytium, the precursor cell. Similarly, for vertebrate myogenesis, nascent myotubes containing about three nuclei are formed during the initial fusion steps. In contrast, the fusion of the visceral muscle founders (CVMs) is incomplete and leads to the formation of a web of interconnected strings, while no signs of string formation during the first somatic step of fusion were observed (Schroter, 2006).

Therefore, it is concluded that fusion of circular visceral muscles in Drosophila appears to be an unique process, sharing only similarities to somatic myogenesis in cell adhesion, but not in the presence of electron dense structures that precede fusion. This phenomenon is also reflected in the different use of the same proteins, e.g., Duf and Sns, in both mechanisms, namely somatic and visceral myoblast fusion (Schroter, 2006).

From an evolutionary view, however, both fusion mechanisms might have originated from an ancient general myoblast fusion mechanism, which is expected to be the case due to the molecular and ultrastructural similarities in cell adhesion, as well as the similarities in circular muscle stretching and somatic muscle attachment. The same ancient process might also be the evolutionary basis for vertebrate skeletal myogenesis but not for vertebrate smooth muscle formation. This is reflected in the expression of similar proteins, e.g., DOCK180, the vertebrate homolog to Mbc, and Hem2, a vertebrate Kette-homolog, although their involvement in vertebrate myoblast fusion remains to be confirmed (Schroter, 2006).

To further support the hypothesis that somatic and visceral myoblast fusion are two closely related but clearly different molecular processes, somatic myogenesis relevant mutants from Drosophila were analyzed. No effect on circular visceral myoblast fusion was observed in blow or kette mutants but rather a phenotype affecting the muscle shaping and stretching. This is a further clear difference to somatic myoblast fusion, where Kette and Blow are required for the second step of fusion. Clearly, loss-of-function mutants for blow and kette exhibit different phenotypes in the somatic and visceral mesoderm. Therefore, it is proposed that these proteins are involved in different mechanisms during the development of both mesodermic tissues (Schroter, 2006).

During somatic myoblast fusion, Blow might be involved in transducing the cell-cell-adhesion signal in FCMs from Sns together with Kette and Crk. This would lead to progression beyond the prefusion complex, the formation of electron-dense plaques, and finally the fusion of the FCM with the syncytial precursor cell (Schroter, 2006).

A possible function for Blow during development of circular visceral muscles is the organization and rearrangement of actin together with the actin-regulating factor Kette. If this is true, then it might also explain the observed stretching phenotype. In other words, if actin could not reorganize, then cells would lose their ability to change their shape and thus to stretch and move along the gut for gaining the web-shaped morphology of the mature circular visceral muscles (Schroter, 2006).

Neither Blow mRNA nor protein is detectable before stage 10 in wild-type embryos; thus, it is highly unlikely that Blow mRNA or protein is maternally contributed. In contrast, wild-type embryos contain maternally contributed Kette. Therefore, it cannot be excluded that the maternal contribution of Kette plays a part in the fusion of circular visceral muscles as well as in the first somatic fusion step. For Blow, only the stretching defect was obseerved. Blow is clearly not involved in fusion of the circular visceral muscles (Schroter, 2006).

This study shows that Blow is involved in different processes that are essential for the proper formation of the musculature. Firstly, Blow is essential in somatic myoblast fusion for progression after the prefusion complex. Secondly, Blow is necessary for the stretching and outgrowth of the circular visceral muscles. In addition, Blow could also possibly play a role in the attachment of the somatic muscles at their epidermal destination, Blow was detected in the somatic muscle tips. These different functions of Blow are reflected by the separate enhancer elements guiding expression at specific times and in specific places. Thus, it is proposed that circular visceral myoblast fusion is independent of Blow, similar to the first somatic step of fusion. For the second step of fusion in the somatic mesoderm Blow is required in the FCMs and acts together in a cascade with Mbc, Crk, and the Kette/Sra-1/Abi-complex, which seems to be specific for somatic myogenesis. While in precursor cells Rolling pebbles (Rols) mediates signal transduction after cell-;cell recognition from Duf to the molecular components essential for maintenance of cell-cell contact and myoblast fusion, in FCMs, the component responsible for the initial signal transduction from Sns to this cascade is still unknown. Although the exact role of Blow in this signaling cascade also remains unclear, Blow acts upstream of the Kette/Sra-1/Abi-complex (Schroter, 2006).

During visceral myogenesis, Blow is expressed in both cell types, the FCMs and the circular visceral muscle founders. Therefore, it is proposed that Blow acts together with Kette to cause the stretching of the circular visceral muscles around the midgut. A similar process might later on in the somatic mesoderm be essential for the outgrowth and attachment of the somatic muscles, which is reflected by the concentration of Blow at the muscle ends in stage 16 embryos. It is further proposed that the molecular cascade for this process is similar to one essential for axonal pathfinding and neuronal outgrowth, since attachment phenotypes were observed in kette mutants as well as in other components of this mechanism, e.g., dock-mutants and mutant members of the Wasp-family (Schroter, 2006).

Taken together it has been shown that during Drosophila embryogensis at least two different modes of myogenesis take place: (1) the two-stepped process of somatic myoblast fusion, requiring Blow and the actin-regulating factor Kette for fusion of the precursor cell with additional FCMs and (2) the incomplete fusion of circular visceral founder-cells with one FCM each, requiring Blow and Kette for developing cell morphology after fusion. While process (1) is a very suitable model for vertebrate myogenesis, further studies would be required to see whether process (2) is found in other systems apart from Drosophila. Therefore, it remains to be clarified in which way longitudinal visceral myoblasts of Drosophila fuse and whether this process is similar to either of the two proposed in this study or whether it represents a third way of myogenesis (Schroter, 2006).

The Hem protein mediates neuronal migration by inhibiting WAVE degradation and functions opposite of Abelson tyrosine kinase

In the nervous system, neurons form in different regions, then they migrate and occupy specific positions. RP2/sib, a well-studied neuronal pair in the Drosophila ventral nerve cord (VNC), has a complex migration route. This study shows that the Hem protein, via the WAVE complex, regulates migration of GMC-1 and its progeny RP2 neuron. In Hem or WAVE mutants, RP2 neuron either abnormally migrates, crossing the midline from one hemisegment to the contralateral hemisegment, or does not migrate at all and fails to send out its axon projection. Hem regulates neuronal migration through stabilizing WAVE. Since Hem and WAVE normally form a complex, the data argues that in the absence of Hem, WAVE, which is presumably no longer in a complex, becomes susceptible to degradation. It was also found that Abelson tyrosine kinase affects RP2 migration in a similar manner as Hem and WAVE, and appears to operate via WAVE. However, while Abl negatively regulates the levels of WAVE, it regulates migration via regulating the activity of WAVE. The results also show that during the degradation of WAVE, Hem function is opposite to that of and downstream of Abl (Zhu, 2011).

Several studies have suggested that Hem dynamically regulates polymerization of F-actin. Hem can play a crucial role in linking extracellular signals to the cytoskeleton. On the other hand, Hem is also part of the WAVE complex and it may regulate the activity of the WAVE complex to promote polymerization of F-actin. The result that the migration defect in Hem mutants can be completely rescued by expression of WAVE from a transgene indicates that Hem regulates neuronal migration via WAVE (Zhu, 2011).

How Hem regulates WAVE is controversial. It has been argued that Hem (together with PIP212) inhibits WAVE in the WAVE complex. Upon activation by Rac1 or Nck, the WAVE complex dissociates releasing an active WAVE-HSPC300 to mediate actin nucleation. This conclusion was also supported by the findings that loss-of-function for Hem leads to an excess of F-actin in the cytosol. Moreover, a reduction in the WAVE gene dosage suppressed axon guidance defects in Hem mutant embryos. But, in vitro studies using Drosophila tissue culture cells argue that Hem protects WAVE from proteasome-mediated degradation. The current in vivo results are consistent with these studies and show that WAVE is protected by Hem and the above alternate model may be incorrect (Zhu, 2011).

The WAVE protein was first identified in Dictyostelium discoideum as a suppressor of mutations in the cAMP receptor (SCAR) but it is present in flies to humans. All WAVEs contain a N-terminal WHD/SHD (WAVE/SCAR homologue domain), a central proline-rich region and a C-terminal VCA domain. WAVE protein regulates actin polymerization by mediating the signal of Rac to Arp2/3 in lamellipodia. It is involved in forming branched and cross-linked actin networks. Unlike WASp proteins, which are intrinsically inactive by autoinhibition and activated by directly binding to Cdc42, PIP2 etc., WAVE appears to be intrinsically active, at least in vitro.However, the majority of WAVE is in the 'WAVE complex' with four other proteins: Hem, Sra-1/PIR121/CYFIP, Abi and HSPC300/Brk1 (Zhu, 2011).

In the WAVE-complex, direct association between WAVE, Abi and HSPC300 represents the backbone of the complex. Hem binds to Sra-1 forming a sub-complex, which is able to bind to Rac through Sra-1. The interaction between Abi and Hem is what binds Hem and Sra-1 into the complex. Hem and Sra-1 are sequentially recruited to the WAVE complex. Free subunits and assembly intermediates of the WAVE-complex are usually not detected but supposedly degraded. Also, previous studies suggest that depletion of one component leads to degradation of others. Indeed, the current results, that in Hem mutants, the level of WAVE protein, but not the WAVE gene transcription, is drastically reduced supports this contention. Perhaps in the absence of Hem, WAVE complex is either not formed or partially formed, resulting in the degradation of WAVE and phenotypes such as mis-migration of neurons. When the levels of WAVE are supplemented using a WAVE transgene (UAS-WAVE), the migration defect in Hem mutants is promptly rescued (Zhu, 2011).

While a complete lack of WAVE (or Hem) function causes an arrest in the migration of RP2, a reduction in the levels of WAVE due to a reduction in the levels of Hem causes abnormal migration. For example, the lowest level of WAVE is seen in the Hem allele that has the strongest penetrance. Moreover, since this mis-migration defect is rescued by expressing WAVE from a transgene, it can be concluded that this mis-migration is also due to an effect on WAVE. It has been suggested that the WAVE-complex exists cytoplasmically and in membrane-bound forms. Through an interaction with Rac, WAVE gets recruited to the lamellipodia where actin polymerization required for membrane protrusion is initiated and regulated. The integrity of the complex is critical for its proper localization since removal of either WAVE or Abi prevents its translocation to the leading edge of the lamellipodia. It is possible that a reduction in the levels of WAVE in Hem mutant embryos causes non-translocation of the WAVE complex to the membrane, causing a non/mis-migration of RP2 (Zhu, 2011).

These results show that WAVE protein exists as three different molecular weight forms. Treatment of the extract with phosphatase collapses these three forms into a single band, indicating that WAVE protein is phosphorylated, with varying degrees of phosphorylation to yield different molecular weight species. Whether there are any changes in the three different forms with respect to their relative contributions in Hem and Abl mutants was examined. However, no changes were found in their relative contributions and the levels of all the forms were reduced in Hem mutants. Therefore, it may be that the reduction in all the forms, or that the reduction in one or two of the forms is responsible for the migration defect. In Abl mutants, the level of WAVE is modestly increased, which is the opposite to that of the effect of Hem on WAVE. Thus, it seems more likely that the activity of WAVE is affected in Abl mutants. Being a protein kinase, it was possible that Abl phosphorylates WAVE, thus affecting either its activity or level. However, no significant changes in were seen in the relative levels of the different phosphorylated forms of WAVE in Abl mutants. It has been shown in vitro that Abl is recruited to WAVE by Abi following cell stimulation, triggering the translocation of Abl together with the WAVE complex to the leading edge of the membrane. Thus, Abl might affect WAVE activity, either directly or indirectly, via the translocation of the WAVE complex to the membrane of an actively migrating RP2. It is also possible that Abl affects migration in a pathway that does not involve WAVE (Zhu, 2011).

In contrast, the effect of loss-of-function for Abl on WAVE levels is more pronounced in older embryos. These results indicate that Abl directly or indirectly regulates the levels of WAVE. Furthermore, though modest, ectopic expression of Abl does down-regulate WAVE. Interestingly, the results also show that Hem regulation of WAVE levels is downstream of the Abl regulation of WAVE since the Hem; Abl double mutants had the same levels of WAVE as Hem single mutants. It seems likely that in the absence of Hem, WAVE protein gets degraded, resulting in the loss of migration or abnormal migration. Whereas in Abl mutants, the most likely scenario is that the activity of WAVE is affected, resulting in the same migration defect (Zhu, 2011).



Drosophila Hem was analysed in detail for its spatial expression pattern during development and for its mutant phenotype. Hem is expressed maternally in the oocyte and shows uniform expression during the first half of embryogenesis, but becomes restricted to the brain and the nervous system during late embryogenesis, consistent with the expression of its vertebrate ortholog in the brain. One P-element insertion, located 39 base-pairs downstream from the Hem transcription start site, causes female sterility, due to the fact that developmental processes in the oocyte are disturbed. Of the vertebrate HEM family members, the mammalian Hem-1 gene is expressed only in cells of hematopoietic origin, while Hem-2 is preferentially expressed in brain, heart, liver and testis (Baumgartner, 1995).

In Northern blots, Hem transcript is detected throughout development, with higher levels in the early embryo, in early first larval instar, and in adults. The expression detected in adults is likely due to ovarian accumulation. Hem transcript is observed in oogenesis and embryogenesis using in situ hybridization. In stage 10 oocytes, the follicle cell and the oocyte express Hem. Hem is ubiquitous in the early embryo. At embryonic stages 7-10, the presumptive procephalic neuroblasts express Hem. Expression is once again ubiquitous in stage 13 embryos. At stage 14, expression is strongest in the pharynx, supraoesophageal ganglion, and the ventral nerve cord. In stage 17 embryos, transcript is detected only in the brain and ventral cord. This pattern persists in the early larval stages (Baumgartner, 1995).

In contrast to the restricted ß-Gal expression pattern of the kette enhancer trap, the endogenous kette gene shows a much broader expression pattern. As determined by use of a digoxygenin-labeled kette cDNA, high levels of kette RNA are provided maternally. Ubiquitous kette RNA distribution is found until stage 13, when kette expression becomes restricted to the CNS. The expression of kette in distinct groups of midline cells can be detected from stage 13 onward on the basis of their position, which appears to correspond to that of the midline neurons. The discrepancy of the RNA distribution and the specific ß-Gal expression pattern might indicate a complex regulation of the kette gene (Hummel, 2000).

Effects of Mutation or Deletion

Five different kette mutations were recovered on the basis of a characteristic CNS axon pattern phenotype consisting of fused commissures and reduced longitudinal connectives. In an independent screen, a large number of P-element-induced lethal mutations were screened for defects in axonal pattern formation. The P-element insertion l(3)03335 leads to strong fused commissure phenotype and failed to complement all EMS-induced kette alleles. The P element mapped at the cytological position 79E, which corresponds well to the meiotic position 3-46.8 determined for the EMS-induced kette mutations (Hummel, 1999a). Both the kette phenotype and the lethality associated with the P-element insertion could be reverted by mobilizing the P element. From 160 independent excision lines, 53 remained associated with a lethal mutation or showed reduced viability. In 36 lines, the mutations led to embryonic lethality; the original l(3)03335 P-element insertion and 13 excision mutations led to larval or pupal lethality. In four lines <10% of the expected numbers of homozygous adult flies eclosed. The largest deficiency isolated, ketteDelta14-2, removes the entire kette gene and shows the same axonal phenotype in homozygous condition and in transheterozygosity to ketteC3-20 or ketteJ4-48, indicating that ketteC3-20 and ketteJ4-48 are amorphic mutations (Hummel, 2000).

Strong kette alleles show a fused commissure phenotype, which is generally due to a defect in the migration of midline glial cells. To analyze the migration of these cells, the midline glia marker AA142 was used. In late stage 12 embryos, the midline glial cells are found anterior to where commissural axons cross the midline. In wild-type embryos, these cells initiate a posterior-ward migration. In stage 16 embryos, two midline glial cells have migrated between anterior and posterior commissure and separated the two axon bundles. In kette mutant embryos, the midline glial cells are specified normally, however, their migration appears abnormal. Contrary to the wild type, the midline glial cells migrate laterally away from the midline along the forming commissural axon bundles. This is reminiscent of the phenotype of embryos lacking pointed, which encodes a glial differentiation factor. Because kette appears to act in the VUM midline neurons (Hummel, 1999b), the cause of this phenotype is different from the one in pointed embryos (Hummel, 2000).

Glial migration defects could be due to alterations in the axonal projection pattern of the VUM neurons. The axonal processes of the VUM neurons are in direct contact with the migrating glial cells and presumably provide the substrate for their migration. Differentiation of the VUM neurons can be analyzed by use of the mAb 22C10. The VUM neurons reside ventrally in the CNS midline and project through the posterior commissure to the anterior commissure, where they turn and grow toward the periphery. In kette mutant embryos, the projection of the VUM neurons is frequently affected. Pathfinding defects are found at positions in which wild-type VUM axons change their growth directions. Possibly due to maternal kette contribution, this early kette phenotype is variable. In ~20% of the embryos, the VUM axons project normally to the posterior commissure but do not follow the correct path once they have reached the anterior commissure. In ~30% of the mutant embryos, severe VUM projection defects are found from early stages onward. In the remaining embryos, the projection defects are such that VUM axons cannot be discriminated from other neuronal processes. However, in all cases, the number of the midline neurons is normal in mutant kette embryos, but their position is slightly altered (Hummel, 2000).

Beside the fusion of commissures, a pronounced reduction of longitudinal connectives is observed in strong kette mutants. Using an antibody directed against the Fasciclin II protein, it was found that longitudinal connectives do not properly form, and axons occasionally are observed crossing the midline. Defects can already be detected during early stages of axonal outgrowth. No gross alterations in the number as well as fate of cortical neurons were detected using different antibodies (anti-Eve, anti-Elav, and anti-Eagle). The longitudinal glial cells that normally are found on the entire longitudinal connective are present in their normal number but are displaced and concentrated at the commissures (Hummel, 2000).

Additional kette phenotypes were observed in the embryonic PNS. A slight reduction is found in the number of all sensory neurons as well as changes in their cellular morphology. In accordance with the reduction in cell number, the size of the segmental nerves is reduced, the axonal projections toward the ventral nerve cord, however, appear normal (Hummel, 2000).

A strong disorganization of motoaxons is found that correlates with defects in the somatic musculature. Using a MHC-lacZ reporter construct, a regular pattern of 30 individual polyploid muscles per abdominal hemisegment can be recognized in wild-type embryos. In kette mutant embryos, a severe reduction in the number of muscle fibers can be observed. The remaining muscle cells appear much smaller as in wild type. In addition, kette also affects midgut formation (Hummel, 2000).

Genomic kette sequences were cloned by inverse PCR using the P-element insertion l(3)03335. Sequence analysis shows that the transcription unit flanking the P-element insertion site had been described before as the Dhem-2 gene (Baumgartner, 1995). The P-element insertion occurs at position 39 within the transcribed region. Dhem2 encodes a putative transmembrane protein homologous to the vertebrate Hem-2/NAP1 protein and a deduced Caenorhabditis elegans hem-2 protein (Baumgartner, 1995; Hummel, 2000).

The P-element insertion l(3)03335 into the kette gene shows restricted ß-galactosidase (ß-Gal) expression in the VUM midline neurons at stage 12. The midline expression persists until stage 14, and thus is present throughout the growth period of the VUM axons, which is in agreement with the predicted function of kette in the midline neurons (Hummel, 1999b).

To further analyze the requirement of kette in commissure formation, the GAL4 system was used to express kette in midline cells. The above-described results indicate that kette is required in the midline neurons. In agreement with this notion, no phenotypic rescue of the kette-fused commissure phenotype was observed following expression of kette in the midline glial cells using the slit-GAL4 driver line. However, when kette was expressed in all midline cells using the single minded-GAL4 driver line, a partial phenotypic rescue of the ketteJ4-048 phenotype was obtained. Following CNS midline expression of kette, commissures were separated and the connectives were found at a further distance from the midline. In particular, the VUM neurons show a normal projection pattern. A complete rescue of the kette mutant CNS phenotype was never obtained. The remaining axonal defects could either originate from the function of kette in lateral neurons or could be due to an inappropriate expression of kette in the midline cells. The importance of the exact expression level is also supported by the observation that expression of high levels of kette in all CNS midline cells in a wild-type background results in an abnormal axonal projection phenotype of the VUM neurons, similar to the mutant kette phenotype. In older embryos, the appearance of the overall axon pattern resembles a phenotype of hypomorphic kette or amorphic dock mutations (Hummel, 2000).

The vertebrate homolog of KETTE has been shown to interact with the first SH3 domain of the Nck adapter protein (Kitamura, 1996). The Drosophila homolog of Nck is encoded by dreadlocks (Garrity, 1996). dock was identified in a screen for mutations affecting axonal pathfinding and targeting of the adult photoreceptor neurons. In wild-type third instar larvae, the different photoreceptor cells stop their axonal growth in two distinct layers of the optic lobe, the lamina and the medulla. In contrast, dock mutant photoreceptor cells fail to establish this specific targeting, leading to a disruption of the lamina neuropile organization. In ~70% of the third instar larvae homozygous for the hypomorphic ketteDelta2-6 allele (n = 25), a weak disorganization was found of the lamina plexus and an abnormal bundling of R-cell axons in the medulla. The remaining larvae showed a stronger disorganization of the R-cell axons (20%) or were indistinguishable from wild type (10%). Further reduction of the kette gene function results in an enhancement of this axonal phenotype in 50% of the analyzed transheterozygous mutant kette larva. If one copy of dock is removed in the background of a hypomorphic kette mutation, a considerable enhancement of the larval projection phenotype is observed in 60% of the individuals. In addition, a significant enhancement of the homozygous dock phenotype is observed when removing one copy of kette in a dock mutant background (Hummel, 2000).

A reduction in the size of the longitudinal connectives in the embryonic CNS is observed in dock mutants. This phenotype resembles a hypomorphic kette connective phenotype. In mutant dockP2 embryos, commissure separation is also affected, comparable with the hypomorphic phenotype seen in ketteJ1-70 embryos. In correlation with the commissural phenotype, the VUM axons do not project properly in mutant dock embryos (Hummel, 2000).

In summary, these data show that both kette and dock mutants genetically interact and share a number of phenotypic traits. This suggests that these genes might be acting in the same genetic pathway during axonal pathfinding (Hummel, 2000).

The Rho family of small GTPases constitutes important regulatory factors also interacting with Nck. To analyze the functional interaction of KETTE with members of the Rho family, both the activated as well as the dominant-negative versions of Cdc42 and Rac1 were expressed in the midline cells of wild-type and kette mutant embryos. Expression of both of these mutant proteins in all midline cells using the simGAL4 driver line results in similar axonal defects. The projection of the VUM neurons resembles the phenotype observed in kette mutant embryos. In addition, the cell bodies of the VUM neurons appear sometimes displaced. In stage 16 embryos, the segmental commissures appear fused, which again indicates the importance of the midline neurons for the migration of the midline glia. Only a weak commissural disorganization is observed when the different Cdc42 or Rac1 proteins are expressed in the midline glial cells only. In all experiments, the expression of Rac1 appears to have more pronounced effects on the axonal morphology (Hummel, 2000).

To further test the interaction of kette and Rac1, activated Rac1 was expressed in all midline cells of mutant ketteJ4-48 embryos. The commissures appeared separated, indicating that the midline glial cells are able to migrate between anterior and posterior commissures. Concomitantly, the connectives are further distant from the midline), indicating that expression of activated Rac1 can partially rescue the kette phenotype (Hummel, 2000).

Among others, the Nck adapter protein transduces signals via CDC42 and Rac1 to the Actin cytoskeleton. A GFP-moesin transgene was used to analyze the Actin cytoskeleton of mutant kette embryos. This protein binds to the F-actin fibers and thus allows a determination of their subcellular distribution using confocal microscopy. In wild-type embryos, F actin is found in axonal processes that are arranged in the typical ladder-like pattern. Prominent expression is also detected in the epidermis and the somatic musculature. In similar focal planes, kette embryos appear very different. Within the CNS, the typical fused commissure phenotype of mutant kette embryos is evident. Frequent intense granular staining is observed in the CNS and in the lateral body wall. Furthermore, the regular appearance of the cytoskeleton is disrupted in both mesoderm and ectoderm. In a tangential section of the dorsal epidermis, individual cells can be seen in wild-type embryos. Some cells form hairs, characterized by thin F-actin bundles. In mutant ketteC3-20 embryos, pronounced F-actin bundles are found, which often have a wavy appearance. In addition, the cortical actin cytoskeleton appears to stain weaker compared with wild-type embryos (Hummel, 2000).

Thus, mutations in kette affect the organization of the cytoskeleton. kette is expressed in neurons and is needed for correct axonal pathfinding. The KETTE protein seems to interact with the SH2-SH3 adapter Dock and at least part of the kette function might be mediated via small GTPases such as Rac1 (Hummel, 2000).

In addition to Kette function in axonal pathfinding, defects are observed in the morphology of trichomes and bristles in flies homozygous for the weak hypomorphic ketteDelta2-6 allele. Around 10% of the bristles appear wavy or do bend sharply and wing trichomes are enlarged and sometimes split. These phenotypes resemble those observed for mutations affecting the organization of the F-actin bundles or following expression of mutated Cdc42 or Rac1. Similarly, elevated levels of GTPase function in the developing eye cause late developmental defects as observed in hypomorphic kette mutations. cdc42 mutations have been isolated, but, presumably due to maternal contribution, loss of cdc42 function does not lead to an embryonic CNS phenotype. Both Cdc42 and Rac1 are important regulators of the actin cytoskeleton. The transduction of extracellular signaling to small GTPases is believed to involve Nck-type adapter proteins. Several phenotypic traits of kette are shared by mutations in the Drosophila gene dock, which encodes a Nck homolog. Furthermore, dock and kette genetically interact. The genetic data in combination with the kette loss-of-function and kette overexpression phenotypes led to the proposal of a model relating Dhem2/NAP1 function to cytoskeleton organization (Hummel, 2000).

The Drosophila Hem protein is predicted to be an integral transmembrane protein with six transmembrane domains (Baumgartner, 1995). The amorphic mutations ketteJ4-48 and ketteC3-20 delete the carboxy-terminal half of the protein. Thus, an important function must reside in this part. In the CNS, the membrane protein Kette could be participating directly in the neuron-glial interaction at the midline, where it could act as a signal to direct glial migration. Alternatively, Kette could serve as a receptor of possibly glial-derived signals during VUM growth cone guidance. The experimental data suggest that Kette transduces information to the neuronal cytoskeleton, which is in agreement with a receptor function (Hummel, 2000).

Binding on a hypothesized ligand would lead to a concentration of Kette at the interaction zone, where it could initiate further signaling to induce cytoskeletal reorganization. Interestingly, the loss-of-function phenotype of kette resembles the gain-of-function phenotype, which could indicate that the correct amount of Kette is important (Hummel, 2000).

Kette could be a more general factor that helps to recruit Nck-like adapter proteins to the membrane, where it interacts with different receptor tyrosine kinases via its SH2 domain. In this scenario, the specific interaction between the midline glia and the midline neurons would be detected by a still-unknown receptor also interacting with the Nck adapter homolog. The FGF receptor is capable of signaling via Nck. In Drosophila, the FGF receptor homolog Breathless is expressed in neuronal midline cells and mutations in this gene result in a mild migration defect of the midline glial cells. Whether this phenotype (which is reminiscent of hypomorphic kette or amorphic dock mutations) results from an interference with Nck/DOCK signaling remains to be shown (Hummel, 2000).

The vertebrate KETTE homolog is HEM-2/NAP1 with 86% amino acid identity over the entire ORF, indicating that presumed protein-protein interactions are also conserved. To date, no hem-2 mutation has been described in vertebrates. The first SH3 domain of Nck was used to isolate Nck-associated proteins (NAP) and led to the identification of HEM-2/NAP1. Binding of HEM-2/NAP1 to Nck appears to be mediated by a 140-kD protein (Kitamura, 1996). Interestingly, in a screen for proteins interacting with activated Rac1, a complex consisting of HEM-2/NAP1 and a 140-kD protein was isolated (Kitamura, 1997). Thus, the 140-kD protein might be a novel adapter linking HEM-2/NAP1 signaling along two routes to the small GTPases. It will be of interest to identify Drosophila genes interacting with kette (Hummel, 2000).

The Drosophila Nck homolog is encoded by dock. dock function appears highly specialized for growth-cone guidance since no mutant phenotypes have been reported in the mesoderm or the ectoderm. Because kette shows more pleiotropic defects, other adapter proteins may interact with the Kette protein (Hummel, 2000).

During axonal pathfinding, coordinated cytoskeletal remodeling occurs at the tip of the extending neurites, the growth cone. The Rho family of GTPases mediates the regulation of the reorganization of the actin cytoskeleton induced by extracellular signals: Cdc42, Rac1, and RhoA. In fibroblast cells, the different GTPases induce different cellular responses. Similarly, different functions appear to be associated with the different Drosophila GTPases. Rho as well as Cdc42 function is needed for cell shape changes during gastrulation, dorsal closure, bristle, and hair formation. Bristle and hair formation are similarly affected by kette (Hummel, 2000).

These data suggest that Kette provides a novel mechanism linking extracellular signals to the neuronal cytoskeleton. Central relay proteins are SH2-SH3 adapter proteins that control the organization of the actin cytoskeleton via a number of downstream proteins. Biochemical data suggest that additional proteins (p140 kD) may bypass the function of SH2-SH3 adapter proteins (Kitamura, 1996, 1997), but a detailed analysis awaits its isolation. The Kette protein might interact with extracellular signals, which, in the CNS, might possibly be presented by glial cells. To gain further insight in the neuron-glial interaction at the midline, future work will be directed toward the identification of these components (Hummel, 2000).

kette and blown fuse interact genetically during the second fusion step of myogenesis in Drosophila

Drosophila myoblast fusion proceeds in two steps. The first step gives rise to small syncytia, the muscle precursor cells, which then recruit further fusion competent myoblasts to reach the final muscle size. Kette has been identified as an essential component for myoblast fusion. In kette mutants, founder cells and fusion-competent myoblasts are determined correctly and overcome the very first fusion. But then, at the precursor cell stage, fusion is interrupted. At the ultrastructural level, fusion is characterized by cell-cell recognition, alignment, formation of prefusion complexes, electron dense plaques and membrane breakdown. In kette mutants, electron dense plaques of aberrant length accumulate and fusion is interrupted owing to a complete failure of membrane breakdown. Furthermore, kette interacts genetically with blown fuse (blow) which encodes a novel cytoplasmic protein and is to be required to proceed from prefusion complexes to the formation of the electron dense plaques. Interestingly, a surplus of Kette can replace Blow function during myogenesis. A model is proposed in which Dumbfounded/Sticks and stones-dependent cell adhesion is mediated over Rolling Pebbles, Myoblast city, Crk, Blown fuse and Kette, and thus induces membrane fusion (Schröter, 2004).

kette mutants exhibit a strong myoblast fusion phenotype. Null alleles of kette show numerous unfused myoblasts, while in hypomorphic alleles the fusion phenotype is less severe but defects in muscle attachment become obvious. These fusion defects are due to the intrinsic function of Kette in the myogenic mesoderm (Schröter, 2004).

Furthermore, it has been shown that founder cells and fusion-competent myoblasts are correctly determined in kette mutants and muscle precursor cells are properly formed during the first myoblast fusion step. Electron microscopic analysis of kette mutants revealed that the second myoblast fusion step is interrupted during formation of the electron-dense plaques and thus kette mutants stop development shortly after blow but before sns15 mutants (Schröter, 2004).

Myoblast fusion requires intensive membrane rearrangements and thus an active modulation of the F-actin cytoskeleton, e.g., as seen during endocytosis. In support of the idea that muscle development depends on a F-actin dynamics, myofibre atrophy is observed in the Wave1 knockout mice. The Wave regulator Kette has been shown to be required for myoblast fusion in the fly embryo. Previous biochemical analyses showed that Kette fulfils a dual role in the regulation of the actin cytoskeleton. In one case, Kette promotes F-actin formation at the cell-membranes via Wasp; in the other, Kette inhibits Scar/Wave function in the cytosol (Schröter, 2004).

In addition to a role of Kette during myoblast fusion, high expression of Kette is found at the growing tips of mature myotubes. These structures are rich in F-actin and, like growth cones, migrate towards the muscle-attachment sites (Schröter, 2004).

Within the two-step model of myoblast fusion, kette can be placed relative to other components of the fusion process. The initial recognition between founder cells and fusion-competent myoblasts is mediated by the Ig-domain proteins Duf/Kirre and Rst in the founder cell. The extracellular domain of Duf/Kirre interacts with Sns, another member of the immunoglobulin superfamily, which is expressed in fusion competent myoblasts. This interaction may signal into both cell types and thus initiate the first fusion step that leads to the formation of precursor cells (Schröter, 2004).

It is possible that Duf/Kirre and Rst, as well as Sns, are also active in the second series of fusion events leading from the precursor cells to the mature myotubes. In the precursor cells, the Rols/Ants protein concentrates at the membrane (Chen, 2003; Menon, 2001) and it is proposed that Rols/Ants is needed to start the second series of fusion (Rau, 2001). Chen (2001) has shown that in vitro Rols/Ants binds to the intracellular domain of Duf/Kirre, and it is suggested that this might be the signal in the precursor cell that recruits further FCMs for fusion. In the precursor cell, this interaction might initiate the formation of the prefusion complex and subsequently the formation of the electron-dense plaques and finally to membrane breakdown (Schröter, 2004).

It is assumed that Blow and Kette mediate the Duf/Kirre-Rols/Ants interaction signal in the precursor cell. Blow and Kette are also present in the fusion-competent cells, where it is proposed that Rols/Ants function is taken over by an, as yet, unidentified protein that interacts with Sns. In the precursor cell, Rols/Ants is proposed to mediate rearrangement of the cytoskeleton via Mbc/Dock180. The electron-dense plaques and their connection to microfilaments are symmetrical structures at opposing plasma membranes between precursor and fusion-competent myoblasts. It is proposed that the rearrangement of the actin filaments and their connection to electron-dense plaques is dependent on Kette and its interaction with Blow (Schröter, 2004).

The Blow protein is characterised by a pleckstrin homology (PH) domain that is often involved in mediation of membrane binding and in regulation of the cytoskeleton. Because membrane association is important for Kette function, the observed genetic interaction may reflect a contribution of Blow in activating Kette. Interestingly, it has been recently reported that Blow binds to Crk, which in turn is able to associate with the Dock180 homolog Mbc. This adapter protein is proposed to link to the Duf/Kirre protein via Rols/Ants in precursor cells. It is proposed that a similar link between Sns and Mbc in the fusion-competent cells is mediated by a yet unidentified protein. This scenario would link the activation of membrane-bound receptors to the regulation of F-actin dynamics (Schröter, 2004).

The SH2-SH3 adaptor protein Crk has not yet been studied at the functional level in Drosophila but it is known from vertebrates that its orthologue CrkII and Dock180 form a complex after external stimulation and the complex is able to promote Rac1 activation. Rac1, in turn, is acting on the activation of Wave (Schröter, 2004).

The interaction between Blow and Crk is supported by the finding of several potential binding motives in Blow that are described as potential recognition sites by both Crk-SH3-domains. Therefore, it is postulated that the function of Blow in myoblast fusion is dependent on its binding to Crk for which no mutants exist. It is proposed that this interaction leads to the activation of kette (Schröter, 2004).


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HEM-protein/Kette: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 15 December 2011

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