How to Download and Activate FULL Fusion Lifecycle 2012 with X-Force Keygen

Several recent studies have indicated that HIV depends on chemokine receptor signaling for efficient infection of target cells. First, Yoder and colleagues (2008) examined the ability of X4 HIV to infect resting CD4+ T cells and found that HIV-mediated, Gαi signaling through CXCR4 was required for entry. This pathway triggered the activation of a cellular actin-depolymerizing molecule, cofilin, altering cortical actin dynamics near the cell surface and facilitating viral fusion. Moreover, the study suggested a role for cofilin in movement of the viral preintegration complex (PIC) toward the nucleus. In contrast to the resting cell model used in this study, most activated and cycling CD4+ T cells disassemble cortical actin without the requirement of cofilin. Although this study was limited to CXCR4 viral entry, activation of cofilin may also be required for CCR5 viruses to enter resting, nondividing memory cells, such as the majority of CD4+ T cells present in the lamina propria of the gut.

FULL Fusion Lifecycle 2012 Activation

Download Zip

A second series of experiments from Harmon and colleagues (2010) showed that HIV also signals through the Gαq subunit, resulting in phospholipase C and Rac activation. Rac and the tyrosine kinase Abl then become linked to the Wave2 complex through the adapter proteins Tiam-1 and IRSp53, promoting Arp2/3-dependent actin nucleation and polymerization. Blocking activation of the Wave2 complex with small interfering RNAs (siRNAs) or Abl kinase inhibitors arrested HIV entry at the hemifusion stage. Together, these experiments suggest a critical role for envelope-coreceptor signaling-induced actin remodeling during HIV entry, particularly in the case of resting CD4+ T cells.

HN, before receptor engagement, prevents F activation. (A) Cell-to-cell fusion at 37C mediated by HPIV3 F coexpressed with uncleaved influenza virus HA and HN wt, uncleaved influenza virus HA and HN P111S/D216N, uncleaved influenza virus HA and HN-D216R, uncleaved influenza virus HA, or alone at 1, 4, 8, and 12 h in the presence of treatments as indicated on the x axis. Fusion, quantitated as described in Materials and Methods, is shown as a percentage of the fusion mediated by the HA, HN wt, and HPIV3 F in the no-treatment group ( standard deviation). (B) Fusion was visualized at 18 h by visible and fluorescence microscopy for the no-treatment group and the zanamivir-treated group. The data reflect experiments repeated at least three times.

Temperature dependence of fusion mediated by F in the presence of HA alone or with D216R HN, N551D HN, or P111S/D216N HN. For the F-activation assay, monolayers of 293T cells coexpressing wt F and uncleaved influenza virus HA, alone or in the presence of wt HN, D216R HN, N551D HN, or P111S/D216N HN were allowed to bind to receptor-bearing RBCs at 4C and then transferred to the indicated temperatures (x axis). After 60 min of incubation, fused RBCs were quantified, and results are expressed as a percentage of the total bound RBCs (y axis). The ordinate values are means of results ( standard deviations) of three experiments.

Respiratory Syncytial Virus (RSV) is a highly pathogenic paramyxovirus. We developed assays for RSV endocytosis, intracellular trafficking, membrane fusion, and infection. The results showed that RSV was rapidly and efficiently internalized, and that acid-independent membrane fusion occurred intracellularly after endocytosis. Cell biological studies demonstrated that endocytosis was macropinocytic, and that it was required for infection. The process involved activation of the EGF receptor and its downstream effectors including Cdc42, Pak1, and myosin II. RSV induced transient actin rearrangements accompanied by plasma membrane blebbing, elevated fluid uptake, and internalization of intact RSV particles into large macropinosomes. Expression of a dominant negative Rab5 mutant but not Rab7 decreased infection indicating that RSV penetration is intracellular, and takes place in Rab5 positive macropinosomes before fusion with endolysosomal compartments. The reason why RSV, unlike most paramyxoviruses, depended on endocytic entry was found to be the need for activation of the F protein by a second proteolytic cleavage. It occurred after endocytosis, and involved most likely a furin-like, vacuolar enzyme.

Perturbations that inhibited endocytic uptake caused a dramatic reduction in infection confirming a role for endocytosis in infection. EIPA inhibited both endocytosis and infection by 95%, and a similar level of inhibition was observed for agents that interfered with actin dynamics, a variety of kinases, and myosin II. The inhibitory effect of Rab5 D/N expression was also consistent with a role for endocytosis in infectious entry. Proteolytic activation of the F protein necessary for fusion and infection occurred in intracellular compartments. We concluded that RSV and VLPs were efficiently endocytosed, that penetration by membrane fusion occurred in endocytic vacuoles, and that at least 90% of infection was caused by endocytosed viruses.

A large number of cell wall proteins are encoded in the Neurospora crassa genome. Strains carrying gene deletions of 65 predicted cell wall proteins were characterized. Deletion mutations in two of these genes (wsc-1 and ham-7) have easily identified morphological and inhibitor-based defects. Their phenotypic characterization indicates that HAM-7 and WSC-1 function during cell-to-cell hyphal fusion and in cell wall integrity maintenance, respectively. wsc-1 encodes a transmembrane protein with extensive homology to the yeast Wsc family of sensor proteins. In N. crassa, WSC-1 (and its homolog WSC-2) activates the cell wall integrity MAK-1 MAP kinase pathway. The GPI-anchored cell wall protein HAM-7 is required for cell-to-cell fusion and the sexual stages of the N. crassa life cycle. Like WSC-1, HAM-7 is required for activating MAK-1. A Δwsc-1;Δham-7 double mutant fully phenocopies mutants lacking components of the MAK-1 MAP kinase cascade. The data identify WSC-1 and HAM-7 as the major cell wall sensors that regulate two distinct MAK-1-dependent cellular activities, cell wall integrity and hyphal anastomosis, respectively.

Despite extensive investigation, a clear, reproducible correlate of human immunity to M. tuberculosis infection has not yet been identified. There are several potential reasons for this. First, our knowledge of the full repertoire of T cell subsets and molecular mediators of protective immunity is still emerging, implying that one or more crucial determinants have not yet been examined. Second, it seems increasingly likely that no single parameter will mediate or correlate with protective immunity in tuberculosis, implying that increasing use of systems biology, bioinformatics and biostatistics will be needed to formulate optimal models and test them in expanded studies. Third, it is possible that using healthy subjects with latent TB infection as the 'gold standard' of protective immunity may lead to erroneous conclusions, as latent TB does not equate with sterile immunity, and latent TB progresses to reactivation TB in a substantial fraction of individuals. Therefore, there is clearly a great need for methods to reliably identify distinct states of infection and the corresponding immune responses after exposure to M. tuberculosis2.

The second well-characterized mechanism that is clearly associated with reactivation TB is the therapeutic neutralization of TNF65, especially by monoclonal antibodies87. Despite the strength of the association, the effects of TNF blockade that account for reactivation TB are not fully characterized, but they include: decreased macrophage-mediated anti-mycobacterial activity and the subsequent death of macrophages88; the induction of a higher frequency of regulatory T cells89; and the depletion of a subset of CD45RA+ effector memory CD8+ T cells that contain granulysin and have been shown to contribute to M. tuberculosis killing in vitro90.

This Review proposes and presents evidence that immunity to M. tuberculosis develops and proceeds in a manner consistent with four distinct stages of a life cycle, in which the state of the bacteria and the nature of immune responses exhibit specific features. In some cases, the distinctions between the stages of the immunological life cycle are clear, as exemplified by the stage limited to innate immune responses and the stage of immunological equilibrium after adaptive immune responses develop and latency is established. However, much more knowledge is needed to fully understand the differences that occur in T cell phenotypes and functions, and in the targets of T cell responses, between the stage of immunological equilibrium and the reactivation and transmission stages. Although our understanding of the mechanisms and targets of immunity to M. tuberculosis has advanced considerably, a higher resolution understanding is needed, and some of this will require the development of new experimental models. The overall goal of this Review is to provide a conceptual framework for prioritizing, designing and interpreting the results of future studies, in order to derive the maximum benefit from efforts to decrease the global burden of tuberculosis.

myoblast city Regulation Asymmetric Mbc, active Rac1 and F-actin foci in the fusion-competent myoblasts during myoblast fusion in DrosophilaMyoblast fusion is an intricate process that is initiated by cell recognition and adhesion, and culminates in cell membrane breakdown and formation of multinucleate syncytia. In the Drosophila embryo, this process occurs asymmetrically between founder cells that pattern the musculature and fusion-competent myoblasts (FCMs) that account for the bulk of the myoblasts. The present studies clarify and amplify current models of myoblast fusion in several important ways. They demonstrate that the non-conventional guanine nucleotide exchange factor (GEF) Mbc plays a fundamental role in the FCMs, where it functions to activate Rac1, but is not required in the founder cells for fusion. Mbc, active Rac1 and F-actin foci are highly enriched in the FCMs, where they localize to the Sns:Kirre junction. Furthermore, Mbc is crucial for the integrity of the F-actin foci and the FCM cytoskeleton, presumably via its activation of Rac1 in these cells. Finally, the local asymmetric distribution of these proteins at adhesion sites is reminiscent of invasive podosomes and, consistent with this model, they are enriched at sites of membrane deformation, where the FCM protrudes into the founder cell/myotube. These data are consistent with models promoting actin polymerization as the driving force for myoblast fusion (Haralalka, 2011). Recent consideration of myoblast fusion have included a common model in which Mbc and Rac1 function downstream of Kirre in the founder cells to direct actin polymerization, and is based on studies showing that Mbc interacts with the Kirre-associated Rols/Ants protein. However, since founder cell-specific expression of Mbc is inadequate to rescue its loss of function fusion phenotype, this study reasoned that it must be required in the FCMs. It has also been suggested that F-actin foci are present symmetrically at points of contact between founder cells and FCMs. This symmetric F-actin-associated adhesive structure has been termed the FuRMAS (fusion-restricted myogenic-adhesive structure), and appears to be a ring of Sns and Kirre surrounding a central core of F-actin. The current results amplify these models in several important areas. First, the data reveal that Mbc is explicitly required in the FCMs and is not needed in the founder cells for fusion to occur. Thus, the essential function of Rols/Ants in fusion cannot be to direct recruitment of Mbc to Kirre in the founder cells. Second, high-resolution imaging has revealed that Mbc, active Rac1 and F-actin are concentrated in FCMs near Sns at the point of contact with founder cells, and are therefore localized asymmetrically in the fusion partners. Moreover, FCM-associated structures project into the founder cell and myotube at the Sns:Kirre adhesion site prior to fusion. Consistent with these observations, other approaches have recently reported the enrichment of actin foci in FCMs and the presence of invadopodia visible in EM that extend from the FCM into the founder cell/myotube. Finally, it was also found that Mbc is important for the integrity of the F-actin focus and for the overall integrity of the actin cytoskeleton in FCMs (Haralalka, 2011). Although very limited fusion occurs in embryos lacking Mbc, more than 80% of the founder cells do not undergo even a single fusion event. By contrast, FCM-directed expression of Mbc rescues an almost wild-type pattern of muscle fibers. Thus, Mbc expression in the FCMs is both necessary and sufficient for their fusion with founder cells. Robust fusion was also observed when activated Rac1 is expressed in the FCMs of mbcD11.2 embryos, indicating that the primary role of Mbc is to activate Rac1. The data do not support a mechanism in which Rols/Ants functions in the founder cells to recruit Mbc to the cytodomain of Kirre, a mechanism that is also inconsistent with the ability of Kirre to direct precursor formation in the absence of its cytodomain. Rather, these data support a mechanism in which Rols/Ants stabilizes Kirre, thereby ensuring that Kirre continues to be present on the myotube surface for fusion with FCMs. Unfortunately, it not possible to determine in the embryo whether Mbc or activated Rac1 is sufficient in the FCMs for later Mbc-dependent fusion between syncitia and FCMs, as the contents of the FCMs become incorporated into the syncitia following fusion. In primary cultures, however, wild-type founder cells as well as founder cells and binucleate precursors lacking Mbc all fuse with wild-type FCMs and at similar rates. Thus, the asymmetric distribution of Mbc in the FCMs is also sufficient for later fusion events, at least in cultured myoblasts (Haralalka, 2011). It was not possible to address whether Rac1 and Rac2 are specifically required in the FCMs as the founder cells of rac1J11, rac2? embryos are already syncytial owing to the perdurance of maternally provided gene product. Although localization of active Rac1 to points of cell contact in the FCMs may be an indication that, like Mbc, Rac1 is essential only in the FCMs, it is noted that Rac1 is required in both fusion partners in vertebrates. In either case, it can be concluded that any requirement for Rac1 in the founder cells must involve a GEF other than Mbc/Elmo (Haralalka, 2011). The data localize Mbc, active Rac1, and F-actin to FCMs at the Sns:Kirre adhesion site with either founder cells or developing myotubes. This asymmetric distribution is independent of whether the FCMs are contacting founder cells or myotubes and, in combination with features of fusion in primary myoblasts, suggests that the first fusion event does not differ from subsequent events with respect to these proteins. These data support a model in which both early and later stages of fusion are highly asymmetric, and that this asymmetry extends beyond recognition and adhesion at the cell surface to cytoplasmic events associated with polymerization of actin. Mbc, which is present but not localized in founder cells/myotubes, may serve a different purpose in these cells such as activating Rac1-dependent myotube guidance or attachment. In addition, though no large F-actin foci were observed on the founder cell/myotube side of the adhesion site, a strong layer of cortical actin was observed along the surface of the founder/developing myotube (Haralalka, 2011). The local accumulation of F-actin in FCMs is reminiscent of dynamic actin foci found at sites of fusion and associated with the WAVE/SCAR, Vrp/WASp and the Arp2/3 complex. F-actin is also present in the core of the muscle-specific fusion-restricted myogenic-adhesive structure (FuRMAS). Interestingly, similarities have been noted between the FuRMAS and the immunological synapse (IS), podosome and invadopodia. The data provides important new information in support of this analogy, as invasive podosomes, the IS and invadopodia are actually all associated with asymmetic F-actin. As noted earlier, strong support for this analogy has recently been demonstrated by the presence of invadopodia-like invasive structures at the level of EM, that extend multiple finger-like projections into the founder cell/myotube. Interestingly, the IS, invadopodia and muscle-specific FuRMAS have common F-actin regulators that include WASp, HEM/Kette, SCAR/WAVE and Rho-family GTPases. Thus, the FCM appears to provide the primary F-actin associated force for myoblast fusion (Haralalka, 2011). Previous studies have reported that F-actin foci in mbc mutants are enlarged and increased in number. However, careful 3D reconstruction of F-actin in embryos and in primary cells suggests that the tight actin foci found in wild-type FCMs are less organized and more dispersed in mbc mutants. The current analysis also revealed that the cytoskeletal network at the periphery of the FCM has collapsed in the absence of mbc. Thus, it appears that Mbc positively regulates organization of the actin cytoskeleton and actin polymerization at the adhesion site (Haralalka, 2011). Although the mechanism(s) by which Mbc-activated Rac1 accomplishes this role have yet to be elucidated, Rac1 is known to interact with components of the WAVE/SCAR pathway in Drosophila and mammalian cells. In mammals, SCAR exists as part of a multiprotein complex composed of HEM/Kette, Abi, Sra1 and HSPC300. These subunits control SCAR stability and localization at the membrane. Moreover, the pentameric SCAR complex can be activated by GTP-bound Rac to promote actin polymerization by Arp2/3. HEM/Kette and SCAR play crucial roles in Drosophila myoblast fusion, and Rac1 has been shown to synergize with SCAR in the myoblast/myotube. Moreover, SCAR is absent from sites of fusion inrac1 mutant embryos. Notably, recent studies have shown that SCAR is required in both cell types, though it remains to be determined whether it plays similar roles in each. In summary, however, the current studies support a mechanism in which Mbc/Elmo mediates the cell-type specific activation of Rac1 and, in turn, activation of WAVE/SCAR to promote an invasive actin-associated structure in the FCMs (Haralalka, 2011). Protein InteractionsThe fusion of myoblasts leading to the formation of myotubes is an integral part of skeletal myogenesis in many organisms. In Drosophila, specialized founder myoblasts initiate fusion through expression of the receptor-like attractant Dumbfounded (Duf: Kin of irre/Kirre), which brings them into close contact with other myoblasts. Rolling pebbles (Rols), a gene expressed in founders, is an essential component for fusion during myotube formation. During fusion, Rols localizes in a Duf-dependent manner at membrane sites that contact other myoblasts. These sites are also enriched with D-Titin, which functions to maintain myotube structure and morphology. When Rols is absent or its localization is perturbed, the enrichment of D-Titin fails to occur. Rols encodes an ankyrin repeat-, TPR repeat-, and RING finger-containing protein. Rols, which is expressed specifically in founder cells, interacts with the cytoplasmic domain of Dumbfounded, a founder cell transmembrane receptor, and with Myoblast city, a cytoskeletal protein, both of which are also required for myoblast fusion. Thus Rols integrates the initial event of myoblast attraction with the downstream event of myotube structural organization by linking Duf to D-Titin (Chen, 2001).The aggregation of Rols in distinctive cytoplasmic locations in founder cells, and the presence of multiple protein-protein interaction motifs in the Rols protein prompted an examination of whether Rols plays a role during myoblast fusion by mediating interactions between molecules in the myoblast fusion pathway(s). To test whether Rols interacts with other fusion molecules, coimmunoprecipitation assays were performed in Drosophila S2 cells using MYC-tagged Rols and other fusion proteins, including Blow, Duf, Mbc, and Sns, tagged with the V5-epitope at their carboxyl termini. Rols interacts with the founder cell receptor Duf but not the fusion-competent cell receptor Sns, despite the high homology shared by Duf and Sns. This specific interaction between Rols and Duf is consistent with the founder cell-specific expression of Rols. A cleaved form of is generated when full-length Duf is expressed in S2 cells. This form migrates slightly slower than the Duf cytoplasmic domain alone, suggesting that it is likely to contain both the transmembrane and the cytoplasmic domains. Interestingly, this cleaved form also associates with Rols. However, when the Duf cytoplasmic domain alone was tested, no interaction was detected. These results suggest that the transmembrane domain of Duf is required for its interaction with Rols. In addition, protein-protein interaction was detected between an amino-terminal fragment of Mbc and Rols, while no interaction was detected between Blow and Rols. The interactions between full-length Mbc and Rols could not be tested, since the full-length Mbc was not expressed at a detectable level (Chen, 2001).Myoblast city and Rac are interaction partners of Rols7 in Malpighian tubule developmentDuring myoblast fusion, cell-cell recognition along with cellmigration and adhesion are essential biological processes. The factors involvedin these processes include members of the immunoglobulin superfamily like Sticksand stones (Sns), Dumbfounded (Duf) and Hibris (Hbs), SH3 domain-containingadaptor molecules like Myoblast city (Mbc) and multidomain proteins like Rollingpebbles (Rols). For rolling pebbles, two differentially expressedtranscripts have been defined (rols7 and rols6). However, to date,only a muscle fusion phenotype has been described and assigned to the lack ofthe mesoderm-specific expressed rols7 transcript. This study shows that aloss of the second rolling pebbles transcript, rols6, which isexpressed from the early bud to later embryonic stages during Malpighian tubule(MpT) development, leads to an abnormal MpT morphology that is not due todefects in cell determination or proliferation but to aberrant morphogenesis. Inaddition, when Myoblast city or Rac are knocked out, a similar phenotype isobserved. Myoblast city and Rac are essentially involved in the development ofthe somatic muscles and are proposed to be interaction partners of Rols7.Because of the predicted structural similarities of the Rols7 and Rols6proteins, it is argued that genetic interaction of rols6, mbc andrac might lead to proper MpT morphology. It is also proposed that theseinteractions result in stable cell connections due to rearrangement of thecytoskeleton (Putz, 2005).The Malpighian tubules (MpTs) of Drosophila arise as four buds fromthe hindgut anlage close to its boundary with the posterior midgut primordium.The cells of the four buds are characterised by the expression of thetranscription factor Cut (Ct) at stage 10 of embryogenesis.During germ band extension at stage 11, the cells of the four tubule primordiaundergo cell proliferation, and the tubules begin to bud out. By stage 13,proliferation is complete and short tubules have formed. From stage 13onwards, cells from the caudal mesoderm join the MpT primordia and later thestellate cells (SCs). From the end of germ band retraction, the tubules begin toelongate due to cell rearrangement. In stage 15 and 16 embryos, thecharacteristic stereotypic course of the four renal tubules through theembryonic body is clearly visible. The paired posterior tubules span theposterior abdominal and terminal segments of the embryo. The anterior tubulesextend forwards into abdominal segments 2/3 where the tubuleloops back on itself so that the tips of both anterior tubules lie moreposteriorly within the abdomen (Putz, 2005).Since Rols6 is expressed in the Malpighian tubules (MpTs) throughout theirdevelopment, the role of Rols6 in the generation of this tissue was investigated.For this purpose, a rols6-specific mutant was generated, in which themajority of the putative promoter region of rols6 was deleted, and thereby rols6 transcription was knocked out, while rols7 expression persisted as in the wild type. In this rols6-specific mutant, the early phase oforganogenesis is the same as in wild type, i.e. the MpTs consist of two celltypes, the principal cells (PCs) and the SCs. As the SCs originate from the mesoderm, one mightexpect that they would be affected in rols mutants. However, in thespecific rols6 mutation generated, the SCs are able to migrate andintegrate between the PCs as observed in the wild type. However, the PCs and SCsdo not arrange correctly, and therefore, the typical MpT arrangement as found inwild-type embryo is not observed for stage 15 embryos onwards. The anteriortubules often show abnormal curves and lasso-like structures and fail to extendthrough the abdominal cavity. These navigation defects might well result fromincorrect cell rearrangements, indicated by thickened regions of the tubules,whereas other parts seem to have a typical wild-type organisation (Putz, 2005).Evidence is presented that correct cell rearrangement is dependenton Rols6 and proteins such as Mbc and Rac. These factors have been proposed toact with Rols7 in a common signalling cascade during myoblast fusion. Anadditional defect is the disorientation of MpTs in the body cavity, which againis characteristic for rols6, mbc and rac mutants (Putz, 2005).Homozygous rols6 mutants are viable, indicating that the physiological functions of principal cells and stellate cells are largely unaffected. Loss of rols6 expression only moderately affects embryonic viability. Furthermore, homozygous EP(3)3330*5a flies do not die prematurely in contrast to those lacking another gene essential for MpT formation, hibris. The strongest hibris allelic combination die early as adults. Also, in contrast to rols6 mutants, in hibris mutants, the number of SCs is strongly reduced. This might cause defects in excretory function of the tubules, and thus leads to the observed lethality (Putz, 2005).rols6-specific mutants show no distortion in rols7transcription and in muscle development indicating that rols6 is specificfor MpT development, while rols7 is essential for myogenesis. This isconsistent with the observatio that Rols6 is not able to rescue the myogenic defect in rols mutants (Putz, 2005).myoblast city mutants, rolling pebbles mutants and rac1/rac2 double mutants show late defects in Malpighian tubule differentiation. mbc mutants exhibit a MpT phenotype and it is proposed that this might be due to a failure to complete cell rearrangement; a phenomenon which is more apparent in mbc mutants than for rolling pebbles ones. Mbc, the homologue of vertebrate DOCK180 in Drosophila, associates with the adapter protein Crk. This interaction regulates cell migration and cytoskeleton organisation in a Rac-dependent manner. This agrees with the finding that rac1/rac2 double mutants exhibit the characteristic MpT defects as rols6 and mbc mutants do. Rols7 and Duf have been shown to interact in myogenesis. The strong similarity between the Rols proteins and their proposed functions leads to the hypothesis that Rols6 interacts with a so far unknown partner in the PCs. It is proposed that Rols6 initiates a signalling cascade via Mbc and Rac that leads to the correct rearrangement of cells, presumedly by rearranging the cytoskeleton, as has been proposed for Rols7 in the myogenic precursor cells. In the development of thesomatic musculature, rearrangement of cytoskeleton is mediated by Blown fuse(Blow) and Kette in the second fusion wave (Putz, 2005).Individual factors and protein complexes involved in cell migration and cytoskeleton arrangement have been described from many model organisms as well as from cell culture experiments. DOCK180/CED-5, the homologues of Drosophila Myoblast city (Mbc) in vertebrates and in C. elegans, form a complex withELMO1/CED-12 that functions as a guanine nucleotide exchange factor (GEF). Thisfunctional GEF promotes Rac activation, and thus facilitates cell migration andrearrangement of the cytoskeleton. In vertebrates, additional proteincomplexes are built via DOCK180/p130Cas/Crk interaction and regulatecell migration and cytoskeletal organisation in a Rac-dependent manner. From kidneycells of human and mouse, the signalling molecule NEPHRIN is known to be ofmajor importance in the podocyte for slit-diaphragm formation. Mutations in thenephrin gene are the major cause of congenital nephrotic syndrome inhumans. In Drosophila, the homologue of vertebrate Nephrin, Hibris (hbs), is expressed duringMpT development specifically in SCs. Therefore, it is likely that during MpTdifferentiation, Hibris mediates cell adhesion and arrangement between the PCsand the SCs, a mechanism comparable to myogenesis. In vertebrates, CMS/CD2AP hasbeen identified as an interaction partner for Nephrin. The CMS/CD2AP homologuein Drosophila can be detected in silico as CG11316. CD2AP knock-out mice die due to kidney failure. Moreover, the Nephrin/CD2AP complex is able to bind to actin and to p130Cas (corresponding to CG1212). In Drosophila,homologues have been identified for all the above-mentioned factors involved inthese protein complexes. However, little is known about their role in thedevelopmental processes taking place during MpT development (Putz, 2005).In Drosophila, a group of immunoglobulin-like proteins act incell-cell recognition and attraction during myogenesis. These processesare also of importance in MpT development. Rolling pebbles is a multidomain and adapter-like protein. It is proposed that Rols6 interacts in Malpighiantubule development with proteins also involved in myogenesis such as Mbc andRac. It is assumed that Rolling pebbles interacts with Mbc, and thus activates Rac.This hypothesis is supported by the observations that mbc and racmutants exhibit defects in MpT development which might be linked to cellorganisation in this tissue (Putz, 2005).The mechanisms underlying the stereotypic courseof the MpTs through the body cavity are still unclear. However, studies ofphenotypes of early determination mutants like numb show that the tipcell and its sibling might both play a critical role in controlling the spatialarrangement of the growing tubules. This is indicated by the MpT phenotypes ofnumb mutants and UAS-numb embryos, where numb isoverexpressed. These embryos lack either the tip cell or the sibling cell butform elongated MpTs with normally rearranged PCs. Although the PCs rearrange normally in these numb alleles, the MpTs are misrouted through the body cavity, as has been observed for rols and rac mutants. This raises the question whether determination of the tip cells is affected in rolsmutants (Putz, 2005).Essential transcription factors for Tip cell determination and PCcell proliferation are the A-SC, Krüppel and Seven up. These factors could be required for rols6 expression in the MpTs. However, since rols6 is expressed in the rudimentary primordia of MpTs in Krüppel mutants and in seven up mutants, this is unlikely. It is assumed, therefore, that Rolling pebbles is not a signalling molecule involved incell specification through direct regulation of early genes, but rather that itplays a role as an adapter molecule in a protein complex connecting the cells inthe tissue as Rols7 does in myogenesis. Since rols6, mbc and racmutant embryos exhibit the described MpT phenotype, it is likely that theybelong to a group of genes that can be helpful in discovering the mechanisms inMpT development that lead to the typical thin tubule morphology through cellrearrangement (Putz, 2005).Drosophila ELMO/CED-12 interacts with Myoblast city to direct myoblast fusion and ommatidial organizationMembers of the CDM (CED-5, Dock180, Myoblast city) superfamily of guanine nucleotide exchange factors function as RAC activators in diverse processes that include cell migration and myoblast fusion. The SH3, DHR1 and DHR2 domains of Myoblast city (MBC) are essential for it to direct myoblast fusion in the Drosophila embryo, while the conserved DCrk-binding proline rich region is expendable. This study describes the isolation of Drosophila ELMO/CED-12, an 82 kDa protein with a pleckstrin homology (PH) and proline-rich domain, by interaction with the MBC SH3 domain. ELMO has been shown to modulate the Rac activation by Dock180 by means of at least three distinct mechanisms: helping Dock180 stabilize Rac in its nucleotide-free transition state; relieving a self-inhibition of Dock180; and targeting Dock180 to the plasma membrane to gain access to Rac. Thus, Dock180 and ELMO function together as a bipartite GEF to optimally activate Rac, upon upstream stimulation, to mediate the engulfment of apoptotic cells and cell migration (Geisbrecht, 2008; Lu, 2006).Mass spectrometry confirms the presence of an MBC/ELMO complex within the embryonic musculature at the time of myoblast fusion and embryos maternally and/or zygotically mutant for elmo exhibit defects in myoblast fusion. Overexpression of MBC and ELMO in the embryonic mesoderm causes defects in myoblast fusion reminiscent of those seen with constitutively-activated Rac1, supporting the previous finding that both the absence of and an excess of Rac activity are deleterious to myoblast fusion. Overexpression of MBC and ELMO/CED-12 in the eye causes perturbations in ommatidial organization that are suppressed by mutations in Rac1 and Rac2, demonstrating genetically that MBC and ELMO/CED-12 cooperate to activate these small GTPases in Drosophila (Geisbrecht, 2008).Drosophila myoblast city (MBC), Caenorhabditis elegans CED-5, and vertebrate DOCK180, are closely related members of the evolutionarily conserved CDM family of proteins. They serve as key players in a signaling complex that includes the SH2- SH3 domain-containing adaptor protein CrkII/CED-2 and the PH domain containing protein ELMO/CED-12 (reviewed in Meller, 2005). This complex then acts at the membrane to relay signals to the small GTPase Rac1/CED-10. MBC/DOCK180/CED-5 function as non-conventional Guanine nucleotide Exchange Factors (GEFs) for Rac. Conventional GEFs bind to nucleotide-free Rac via a Dbl-homology (DH) domain, thereby facilitating exchange of GDP for GTP. DOCK180/CED-5, which lack DH domains, associate with nucleotide-free Rac through a conserved Dock-Homology Region (DHR2) (Brugnera, 2002; Cote, 2002). Deletion of this domain results in loss of Rac binding and the inability to direct formation of GTP-bound Rac (Brugnera, 2002; Geisbrecht, 2008 and references therein).In addition to DHR2, CDM proteins have in common an N-terminal SH3 domain, a second Dock Homology Region (DHR1), and a C-terminal proline rich region. The C-terminal region directs interaction with the SH3 domain of CrkII/CED-2. The CrkII SH2 domain can then direct interaction with upstream proteins that are phosphorylated on tyrosine, such as transmembrane receptors and components of focal adhesions. In addition to membrane recruitment through Crk-related interactions, the C-terminal PH domain of ELMO/CED-12 can also mediate DOCK180 membrane localization (deBakker, 2004; Grimsley, 2004). The DHR1 region of DOCK180, which binds to phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], is also required for its membrane localization (Cote, 2005; Kobayashi, 2001). The N-terminal SH3 domains of DOCK180 and CED-5 mediate interaction with the C-terminal proline-rich region of ELMO and CED-12, respectively (Lu, 2004; Lu, 2005; Wu, 2001). In vitro studies demonstrate that DOCK180 binding to Rac can be sufficient for its activation (Cote, 2005), but that this activation can be significantly enhanced by DOCK180 bound to ELMO (Brugnera, 2002; Katoh, 2003; Lu, 2004). Thus, the DOCK180/ELMO complex is a key component in CDM signaling to Rac (Geisbrecht, 2008).In addition to extensive homology and conserved biochemical interactions between the DOCK180/CED-5 and ELMO/CED-12 protein families, complexes of these proteins perform similar biological functions. For example, genetic studies have shown that C. elegans CED-10 acts with CED-2/Crk, CED-5/DOCK180 and CED-12/ELMO to promote cell migration of the distal tip cells during development of the somatic gonad and engulfment of cell corpses following apoptosis. Membrane targeted DOCK180 increases cell spreading, and overexpression of wild-type DOCK180 in mammalian cells enhances cell migration and phagocytosis of apoptotic cells. Lastly, a reduction in wild-type DOCK180 or overexpression of mutant forms of DOCK180 decrease activated Rac and cause defects in cell spreading and cell migration (Geisbrecht, 2008).As in C. elegans and vertebrates, Drosophila MBC interacts genetically with other molecules required for CDM pathway function. For example, mutations in mbc delay border cell migration and influence PVR mediated F-actin accumulation in the follicle cells during ovary development, reflecting its proposed role in the PVR-Rac pathway (Duchek, 2001). Studies utilizing RNAi constructs have demonstrated a genetic interaction between MBC, Drosophila Crk (DCrk) and ELMO in adult thorax closure (Ishimaru, 2004). In the Drosophila eye, in which Rac is required for proper actin organization, the effects of Rac1 overexpression are suppressed by loss of one copy of mbc. Rac1 was initially implicated in myoblast fusion in the embryo by the demonstration that overexpression of either dominant-negative or constitutively-active Rac1 interferes with myoblast fusion. More recently, loss-of-function studies have established that Rac1 and Rac2 play a redundant but essential role in this process. MBC is absolutely essential for myoblast fusion, where it colocalizes in founder myoblasts with the immunoglobulin superfamily member Duf/Kirre. MBC is also expressed in, and apparently required in, the fusion competent myoblasts. Structure/function analysis of MBC identified the protein domains that are essential for myoblast fusion. This analysis revealed that, like Dock180, the DHR1 domain binds to PtdIns(3,4,5)P3 and is essential for myoblast fusion. The N-terminal SH3 domain and the putative Rac binding DHR2 domain are essential for MBC transgenes to rescue the myoblast fusion defects in mbc mutant embryos. Surprisingly, however, while the C-terminal proline-rich region directs a strong interaction with DCrk (Balagopalan, 2006; Ishimaru, 2004), this region is not required for MBC function in the embryonic musculature (Balagopalan, 2006). Thus, the canonical Crk-associated CDM pathway that has been described for other organisms and used in other Drosophila tissues does not appear to be used in Drosophila myoblast fusion. This finding raised the broader question of whether other components of the canonical pathway functioned along with MBC in the embryonic muscle, or whether MBC functions in this tissue through different protein interactions (Geisbrecht, 2008).To address the involvement of Drosophila ELMO in signaling from MBC, its role in myoblast fusion, border cell migration and ommatidial organization has been isolated and characterized. ELMO is expressed in the ovary, where it interacts genetically with MBC and plays a role in border cell migration. Multiple loss-of-function alleles were generate and analyzed to demonstrate the importance of maternal and zygotic ELMO in myoblast fusion. It was confirmed, through a targeted mass spectrometry approach, that these proteins form a stable complex in the embryonic musculature. ELMO and MBC act cooperatively in the mesoderm, such that an excess of both causes serious defects in myoblast fusion reminiscent of those seen with excess Rac activity. Finally, it was established genetically that ELMO and MBC can cooperate to activate Rac GTPases in the adult eye (Geisbrecht, 2008).Thus, the conserved PH-domain that is normally present within conventional GEFs is actually provided for CDM proteins by a separate protein family represented by ELMO/CED-12 (Brugnera, 2002; Lu, 2004). One member of each of these two protein families can combine to form an unconventional bipartite GEF. While the small adaptor protein Crk often forms a critical component of this complex, recent studies have suggested that both DOCK180 and MBC can function in its absence (Balagopalan, 2006; Tosello-Trampont, 2007]). Moreover, Crk has been shown to function in pathways that are totally independent of this bipartite GEF. Reminiscent of this diversity of interactions, recent studies have suggested that CDM and ELMO/CED-12 family proteins may also function through independent interactions. For example, DOCK180 is capable of activating Rac on its own and has positive effects on cell migration and phagocytosis, albeit enhanced by binding of ELMO (Brugnera; Katoh, 2003]; Lu, 2004), and ELMO interacts with radixin independent of its interaction with DOCK180 (Grimsley, 2005). Drosophila MBC functions in a wide variety of processes that include border cell migration in the ovary and myoblast fusion in the embryo. This study has demonstrated, through its biochemical and genetic analysis, that Drosophila elmo functions in concert with MBC in these processes. Like MBC, decreased levels of ELMO impair border cell migration and myoblast fusion (Bianco, 2007). Moreover, MBC interacts stoichiometrically in the mesoderm with ELMO. Coincident over-expression of this complex impairs myoblast fusion, reinforcing the model from constitutively-active Rac1 that excess active Rac1 also interferes with myoblast fusion. Co-expression of MBC and ELMO also impacts development of the adult eye, resulting in a rough eye phenotype that is suppressed by decreasing endogenous levels of Rac. These data indicate that MBC and ELMO function together in a complex, and as a RacGEF (Geisbrecht, 2008).CDM family proteins are required for a diverse array of biological processes. If ELMO functions with MBC in these processes, one would expect it to be expressed in a temporal and spatial pattern coincident with that of MBC. Consistent with this expectation, RT-PCR throughout the fly life cycle, embryonic in situ hybridizations and antibody stainings at multiple stages of Drosophila development, reveal that elmo is broadly expressed. Interestingly, however, the MBC/ELMO complex may serve distinct roles in each of the tissues in which it is expressed. MBC and ELMO are required for migration of the border cells in the ovary (Bianco, 2007; Duchek, 2001); however, myoblast migration appears to occur normally in mbc mutant embryos, as the fusion competent cells in mbc mutants can be found clustered and aligned with the founder cells (Geisbrecht, 2008). The CDM/ELMO(Ced-12) complexes in both vertebrate and C. elegans function upstream of Rac as unconventional bipartite GEFs to promote exchange of GDP for GTP in activation of monomeric GTPases (Meller, 2005). These studies support a similar role for Drosophila MBC/ELMO. First, during ommatidial development, the rough eye phenotype resulting from co-expression of MBC and ELMO can be suppressed by removing half the gene dosage contributed by Rac1 and Rac2. Also, overexpression of the MBC/ELMO complex is sufficient to provide enough GEF activity to overcome the effect of its sequestration by RacN17 in the eye. In both the musculature and eye, expression of neither MBC nor ELMO alone has a phenotypic consequence, yet co-expression of MBC and ELMO phenocopies activated Rac (Geisbrecht, 2008).Notably, embryos that are completely lacking both maternal and zygotic elmo die before muscle development occurs, possibly reflecting an earlier role for the protein. Interestingly, however, the development of mutant embryos that lack both maternally and zygotically provided mbc continues until myogenesis (Balagopalan, 2006). These data suggest that, like its vertebrate counterparts, Drosophila ELMO has multiple binding partners. In addition to DOCK180, vertebrate ELMOs bind to three of the five additional CDM family members, suggesting that ELMO binding is a general feature of these proteins (Grimsley, 2004; Sanui, 2003). Based upon primary sequence homology, the fly genome contains at least four potential CDM superfamily members in addition to MBC. The predicted transcripts of two of these are most closely related to vertebrate DOCK9/11 and DOCK 7/8. The Drosophila protein most closely related to vertebrate DOCK4 has been reported as the protein product of the sponge locus, but has not been studied extensively. It remains to be seen whether Drosophila ELMO is capable of binding to these other CDM-like molecules, and functions in concert with them in other tissues. One such place to examine in this regard is the embryonic CNS, where ELMO expression is quite strong but MBC is strikingly low. Thus, alternative CDM/ELMO-like complexes may be present and required in different tissues throughout Drosophila development, or in the same tissues to regulate different GTPases (Geisbrecht, 2008).The above studies, combined with recent reports of ELMO binding to non-CDM family members, may reflect a role for ELMO proteins in integrating signals from different pathways. Interaction of the N-terminal region of ELMO with RhoG is capable of translocating the ELMO/DOCK180 complex to the membrane to regulate neurite outgrowth and cell migration (Katoh, 2003). Simultaneously, the N-terminal region of ELMO can bind to both the inactive and active forms of the ERM protein radixin (Grimsley, 2005). Interestingly, this ELMO/radixin interaction does not affect the ability of the ELMO/DOCK180 complex to promote Rac activation. Hence, ELMO may be functioning at the membrane to regulate the actin cytoskeleton via Rac, while recruiting radixin and ERM family members to perform their recognized roles in cross-linking the actin cytoskeleton to the plasma membrane (Geisbrecht, 2008).In addition to Rac activation via the CDM/ELMO proteins, the ARF (ADP-ribosylation factor) family of GTPases has been shown to signal through Rac. Both DOCK180 and ELMO colocalize with ARNO (an ARF-GEF) and overexpression of mutant forms of DOCK180 and ELMO mutants block ARNO-induced Rac activation (Santy, 2005). However, RhoG signaling does not seem to be required for the ARNO-ELMO activation of Rac. This suggests Rac activation is differentially regulated in cell or tissue-specific manners or that within a cell there are localized mechanisms defined by crosstalk between signaling pathways that are responsible for Rac membrane localization. Intriguingly, in the Drosophila musculature, expression of a dominant-negative ARF6 results in myoblast fusion defects while the corresponding ARF-GEF Loner/Schizo is required for membrane localization of Rac. More work is required to see if ARF6, Loner/Schizo, and the ELMO/MBC proteins function in a signaling pathway in Drosophila to activate Rac (Geisbrecht, 2008).Numerous studies have established a crucial role for Rac activation in myoblast fusion. More than a decade ago, it was demonstrated that a dominant-negative form of Rac1 interferes with myoblast fusion. A genetic analysis of the small Rac GTPases proved that the fusion defects observed with dominant-negative Rac1 reflect the Rac1, Rac2 loss-of-function phenotype. The need for activated Rac in myoblast fusion was further supported by the phenotype of embryos mutant for mbc and elmo. Rac, in turn, likely functions to direct rearrangement of the actin cytoskeleton through regulation of the Arp2/3 complex via Kette and WAVE. Surprisingly, however, constitutively-active forms of Rac negatively impact this process in a manner that, on the surface, have the same phenotypic consequence as the absence of active Rac. The specific relevance of excess activated Rac1 to normal myoblast fusion remains unclear, and it cannot be ruled out that excess Rac interferes with other signaling pathways that do not normally impact myoblast fusion. However, perturbation of this phenotype also has the potential to uncover key components and regulators that contribute to the normal process (Geisbrecht, 2008).These data establish, for example, that monomeric Rac GTPases are in excess, and are therefore available to be activated when the level of the MBC/ELMO GEF complex is increased. Under normal circumstances, then, the ability of the cell to activate this endogenous Rac remains low. Potential mechanisms for this include the direct regulation of MBC and/or ELMO levels through synthesis or turnover. Though little is known about the synthesis of either MBC or ELMO or their levels in the cell, it is intriguing that DOCK180 is ubiquitinated and this modification ensures its rapid turnover (Makino, 2006). Alternatively, GAPs may be present to ensure that Rac does not remain active. Finally, the MBC/ELMO pathway may be integrating with the loner-ARF6 associated pathway, in such a way that perturbation of MBC and ELMO is impacting Rac1 activity through components of the ARF6 pathway. Either way, the fact that this myoblast fusion phenotype is occurring in response to perturbation of the endogenous pathway by wild-type proteins in a stoichiometric manner suggests that it may be possible to modulate it in ways that provide insights into the myoblast fusion process (Geisbrecht, 2008).Drosophila Importin-7 functions upstream of the Elmo signaling module to mediate the formation and stability of muscle attachmentsEstablishment and maintenance of stable muscle attachments is essential for coordinated body movement. Studies in Drosophila have pioneered a molecular understanding of the morphological events in the conserved process of muscle attachment formation, including myofiber migration, muscle-tendon signaling, and stable junctional adhesion between muscle cells and their corresponding target insertion sites. In both Drosophila and vertebrate models, integrin complexes play a key role in the biogenesis and stability of muscle attachments through the interactions of integrins with extracellular matrix (ECM) ligands. This study shows that Drosophila Importin7 (Dim7) is an upstream regulator of the conserved Elmo-Mbc-->Rac signaling pathway in the formation of embryonic muscle attachment sites (MASs). Dim7 is encoded by the moleskin (msk) locus and was identified as an Elmo-interacting protein. Both Dim7 and Elmo localize to the ends of myofibers coincident with the timing of muscle-tendon attachment in late myogenesis. Phenotypic analysis of elmo mutants reveal muscle attachment defects similar to that previously described for integrin mutants. Furthermore, Elmo and Dim7 interact both biochemically and genetically in the developing musculature. The muscle detachment phenotype resulting from mutations in the msk locus can be rescued by components in the Elmo-signaling pathway, including the Elmo-Mbc complex, an activated Elmo variant, or a constitutively active form of Rac. In larval muscles, the localization of Dim7 and activated Elmo to the sites of muscle attachment is attenuated upon RNAi knockdown of integrin heterodimer complex components. These results show that integrins function as upstream signals to mediate Dim7-Elmo enrichment to the MASs (Liu, 2013).Previous studies have shown that Dim7 localizes to developing muscle-tendon insertion sitesand removal of Dim7 has severe consequences in muscle attachment maintenance (Liu, 2011). The current studies extend these observations to elucidate the functional contribution of Dim7-Elmo in regulating Drosophila muscle attachment. The results show that Dim7 is an upstream adaptor protein that recruits Elmo in response to integrin adhesion and/or signaling. Thus, it is proposed that the spatial and temporal regulation Elmo-Mbc activity results in regulation of the Rac-mediated actin cytoskeleton changes at the MASs (Liu, 2013).The 'myospheroid' phenotype in elmo or msk mutants resemble attachment defects first characterized in mutated genes that encode for integrins, ILK and Talin, and is not due to earlier developmental defects in myogenesis. A similar number of cells expressing the muscle differentiation factor DMef2 was present in elmo or msk mutants, indicating that muscle specification was not affected (Geisbrecht, 2008; Liu, 2011). Genes essential for muscle migration and targeting also lead to detached muscles. For example, in kon/perd or grip mutants, the early arrest of migrating myotubes resulting from defective migration eventually leads to a linkage failure between the muscle and tendon cells. In mutant embryos with reduced levels of Elmo or Dim7, the muscle detachment phenotype did not appear to result from muscle migration defects. First, the spatial-temporal accumulation of Elmo and Dim7 is developmentally regulated. Both proteins are not detected at the leading edges of migrating muscles, but begin to accumulate at MASs after stage 15. Second, failure of muscle ends contactingtheir corresponding attachment sites was not observed in elmo or msk mutants at late stage 15, when muscle migration was almost complete (Liu and Geisbrecht, 2011) (Liu, 2013).Both membrane localization and Rac-dependent cell spreading of the uninhibited, activeversion of Elmo is enhanced compared to native WT Elmo in cultured mammalian cells (Patel, 2010). These in vitro results are in agreement with the current in vivo analysis, where ElmoEDE (a mutation that prevents the autoinhibitory interaction of Elmo) is enriched at larval muscle ends compared to the poor accumulation of full-length Elmo-YFP. This may reflect a potential regulatory mechanism controlling the subcellular localization of Elmo from the cytoplasm to the muscle ends upon the release of Elmo autoinhibition. Within different cells or tissues, various proteins may regulate Elmo localization to the cell periphery, or other sites where active Elmo is needed. In cultured mammalian epithelial cells, membrane recruitment of the Elmo-Dock180 complex is dependent on active RhoG for cell spreading. Consistent with a functional role for membrane-targeted Elmo, active Elmo promotes cell elongation in Hela cells, when co-expressed with RhoG (Patel, 2010; Liu, 2013 and references therein).The data argues that adaptor proteins may be required in muscle cells for activated Elmo membrane recruitment. Decreased levels of ElmoEDE are observed at the polarized ends of muscle insertion sites when Dim7 levels are decreased. It is still not clear if Dim7 binding is required for the conformational change that results in Elmo activation or if an activated Dim7-Elmo complex already exists within the cell and gets recruited as a complex upon integrin activation. Furthermore, complete loss of Elmo-EDE protein levels is not observed, suggesting that either Dim7 protein levels are not depleted enough or other proteins in addition to Dim7 play a role in Elmo membrane recruitment. Alternatively, post-translational modification(s), such as phosphorylation,could be an additional mechanism for the relief of Elmo autoinhibition. Thus, it is concluded that in muscle, Dim 7 is an essential adaptor protein for the polarized membrane localization of active Elmo or the active Elmo-Mbc complex downstream of integrin signaling pathway (Liu, 2013).What is the relationship between the integrin adhesome and the Dim7-Elmo complex? Two explanations are proposed that are not mutually exclusive. One possibility is that the Dim7-Elmo-Mbc complex assembles at MASs via integrin-mediated 'outside-in' signaling. Upon ligand binding to ECM molecules, integrin activation results in Dim7-Elmo-Mbc complex localization for the spatial-temporal regulation of Rac activity to maintain dynamic actin filament adhesion at the MASs. It is predicted that localization of activated Elmo to the MASs is a prerequisite regulatory mechanism for actin cytoskeleton remodeling via Rac to maintain stable attachments. This hypothesis is supported by three lines of evidence: (1) muscle attachment defects upon loss of Dim7 or Elmo are only observed after the establishment of the integrin adhesion complex and onset of musclecontraction; (2) muscle detachment in msk mutants can be rescued by expressing lowlevels of activated Rac; and (3) the enrichment of Dim7 and Elmo-EDE proteins at the ends of muscle fibers is greatly reduced in integrin-deficient larvae (Liu, 2013).It is also possible that accumulation of the Dim7-Elmo complex to the ends of muscles regulates 'inside-out' signaling to dynamically regulate integrin affinity for strong ligand binding and stable muscle attachments. Previously, it was reported that Dim7 acts upstream of the Vein-Egfr signaling pathway in muscle to tendon cell signaling (Liu, 2011). Combined with previous results that muscle-specific Vein secretion is dependent on the adhesive role of βPS integrin, the Dim7-Elmo complex may be internally required for integrins to regulate Vein secretion. A decrease in Vein-Egfr signaling and loss of tendon cell terminal fate results in a reduction in ECM secretion and weakened integrin-ECM attachment. This is consistent with the observation that msk or elmo mutants phenocopy embryos with reduced or excessive amounts of the αPSβPS integrin complex, wherepointed muscle ends result in smaller muscle attachments. Future studies analyzing Dim7-Elmo-Mbc complex localization and function in the background of integrin deletion constructs which separate the 'inside-out' and 'outside-in' signaling pathways will be essential to uncover more detailed molecular mechanisms (Liu, 2013).What is the relationship between the Dim7-Elmo-Mbc-->Rac signaling pathway and the integrin mediated adhesome complex assembly (including the Talin, IPP [integrin linked kinase (ILK)-PINCH-Parvin-α) complex]? It is proposed that actin filaments within the muscle cell are anchored to the muscle cell membrane via the IPP complex, while regulation of MAS-actin remodeling iscontrolled by the Dim7-Elmo-Mbc-->Rac pathway. The data suggests that these two complexes assemble independently at the muscle ends. In msk mutant embryos, both ILK and Talin properly accumulate at the MASs, suggesting that Dim7 is not responsible for their localization (Liu, 2011). Similarly, both MAS-enriched Dim7 and active Elmo can be detected at two ends of the muscles in ILK-deficient larva, even in fully detached muscles. In a vertebrate cell culture model, Elmo2 was found to physically interact with ILK for the establishment for cell polarity (Ho and Dagnino, 2012; Ho, 2009). Thus, it is possible that the current approaches have not fully knocked down Ilk levels or that the Dim7-Elmo recruitment by Ilk is redundant with another attachment site protein. Alternatively, an upstream scaffold protein may function to recruit both the IPP andDim7-Elmo complex to the MASs. It is likely that these two complexes are temporally regulated in embryogenesis, where the actin remodeling complex is not needed until initial muscle-tendon initiation has been established (Liu, 2013). DEVELOPMENTAL BIOLOGY Embryonic Northern analysis reveals that mbc is expressed early in development, in embryos 0-4 h after egg laying. mbc transcript levels remain relatively high during embryogenesis, with the possible exception of a decline from 8-12 h that may be, in part, an artifact of slightly degraded mRNA. Expression is not evident during larval stages, but the transcript does reappear during pupation, suggesting a possible role in adult development. A form of mbc with slightly altered mobility appears late in metamorphosis. This transcript may reflect alternative splicing and is under further investigation. PCR amplification of two different regions from the mRNA of unfertilized embryos reveals a small but detectable signal, and suggests that the transcript is maternally provided. Finally, the transcript was expressed in adult males and females, as evidenced by PCR analysis of cDNA (Erickson, 1997).The earliest expression of the mbc transcript is in the pole cells. It is later found in lateral portions of the embryo during cellularization but is not evident at the termini. Surprisingly, the ventral furrow, which will invaginate during gastrulation to form the mesoderm, shows no expression at this time. At germband elongation, expression is still quite strong in the ectoderm. By late stage 12, the mRNA appears to be decreasing in the ectoderm, leaving a pattern of stripes. mbc is expressed in both the mesoderm and endoderm during stage 12. Expression decreases in both the epidermal layer and the somatic mesoderm during stage 14 but remains strong in the visceral musculature. Examination of a stage 16 embryo reveals mRNA in both the cardial and pericardial cells of the dorsal vessel. Of note, the mbc transcript is not observed in mature muscle fibers (Erickson, 1997).The expression pattern of MBC was analyzed by fluorescent immunohistochemistry and confocal microscopy using an antiserum directed against the COOH-terminal portion of the protein. While slight temporal differences were evident between maximal levels of mRNA and maximal levels of protein in the pole cells, the expression of the protein essentially correlates with that of the mRNA. Mbc appears to be localized in the cytoplasm, consistent with its human counterpart DOCK180. Mbc is also present in the visceral musculature and the dorsal vessel. Cross reactivity of the Mbc antiserum is observed in the filtzkorper but does not correlate with the presence of transcript. Although mRNA was not evident in mature muscles, the protein can be detected in mature muscle at a low level (Erickson, 1997). Fluorescent immunohistochemistry and confocal microscopy were used to confirm that Mbc is present in myoblasts. For this analysis, the embryos were hybridized with antibodies to both Mbc and Mef2. The mef2 gene encodes a transcription factor that appears to be expressed throughout the mesoderm, including somatic muscle precursors and all muscle fibers. As anticipated from the expression pattern of mRNA, Mbc is present in ectodermal and endodermal germ layers. Of note, expression in the ectoderm is concentrated in the epidermal layer and appears to be absent from the underlying neuroectoderm. Mbc is also clearly present in presumptive myoblasts, coincident with the Mef2-expressing nuclei (Erickson, 1997). OogenesisSimilar to the Drosophila Egfr and to the mammalian PDGFR family, stimulation of PDGF- and VEGF-receptor related (Pvr) activates the MAP-kinase pathway in Schneider cells as well as in border cells. However, it has been shown, by loss-of-function and gain-of-function experiments, that MAP-kinase signaling does not affect border cell migration. In addition, no effect of phospholipase C-gamma (PLC-gamma) or phosphatidylinositol 3' kinase (PI3K) has been demonstrated on this migration, using loss-of-function mutants (PLC-gamma) or border cell expression of dominant negative and dominant activated forms (PI3K). This was somewhat unexpected, since PLC-gamma and PI3K have been implicated in motility and guidance effects of RTKs (in particular PDGFR) in tissue culture cells. To address how Pvr signaling might be affecting cell migration in vivo, the effect of Pvr signaling on cell morphology and cytoskeleton was tested. In border cells as well as in other follicle cells, expression of lambda-Pvr has a dramatic effect on the actin cytoskeleton. Massive F-actin accumulation, actin-rich extensions, and changes in cell shape were produced in lambda-Pvr expressing follicle cells. The normal cells have modest cortical F-actin accumulation. This result was likely to be relevant to the guidance function of Pvr, because direct control of F-actin accumulation would allow receptor activation to control cell migration (Duchek, 2001).The actin cytoskeleton has been shown to be affected by small GTPases of the Rho superfamily in many systems, with the exact effects depending on the cellular context. In the border cell migration system, Rac is an attractive candidate for mediating the effect of activated Pvr, since dominant negative Rac (RacN17) has been shown to inhibit border cell migration. Epistasis experiments could not be done by quantifying border cell migration because activated Pvr and dominant negative Rac have the same effect. Instead, whether Rac is required for the effect of Pvr on the actin cytoskeleton in follicle cells was tested. Coexpression of dominant negative Rac suppresses the effect of activated Pvr on the actin cytoskeleton. In addition, follicle cells expressing activated Rac (RacV12) have dramatic accumulation of F-actin, resembling that caused by activated Pvr. Finally, if Rac were directly downstream of Pvr, one would expect activated Rac to inhibit border cell migration, as observed for the activated receptor. Although a previous study reported that activated Rac does not affect border cell migration (Murphy, 1996), this was reexamined using the slboGal4 driver and it was found that activated Rac completely blocks border cell migration. These results are consistent with a role of Rac in the guidance pathway downstream of Pvr (Duchek, 2001).In mammalian tissue culture cells, PDGF stimulation can cause Rac-dependent F-actin accumulation, suggesting that the effect observed in follicle cells may reflect a conserved pathway. PI3K has been implicated as a mediator of the effect of PDGFR on Rac in Swiss 3T3 cells. However, PI3K does not appear to play a key role in guidance of border cell migration as discussed above. To investigate how Pvr might lead to activation of Rac, two groups of Drosophila mutants were tested for their effect on border cell migration: mutants in genes shown to be downstream of receptor tyrosine kinases in other contexts, and mutants linked to Rac activation. Most mutations were homozygous lethal, so their effect in border cells was tested by generating mutant clones in a heterozygous animal (mosaic analysis). Of the 8 genes tested, only myoblast city (mbc) has a detectable effect on border cell migration. Mbc is homologous to mammalian DOCK180 and C. elegans CED-5. Mbc/DOCK180/CED-5 acts as an activator of Rac (Duchek, 2001 and references therein).mbc has been independently identified in a screen for gain-of-function suppressors of the slbo mutant phenotype. slbo mutant border cells migrate poorly. Increased expression of mbc in slbo mutant border cells improves their migration, suggesting that mbc has a positive role in promoting border cell migration. Mbc protein is detected in follicle cells, including border cells, and is overexpressed upon induction of the EP element EPg36390 located upstream of mbc. Removing mbc function from border cells by generating mutant clones causes severe delays in their migration. At stage 10, when 100% of control (GFP) clones have reached the oocyte, only 10% of mbc mutant border cell clusters have done so, and these are the oldest egg chambers. Thus, mbc is not absolutely required for border cell migration, but, contrary to the other genes implicated in RTK and Rac signaling, loss of mbc function severely impairs this cell migration (Duchek, 2001). myoblast city: Biological Overview Evolutionary Homologs Regulation Effects of Mutation References 2ff7e9595c