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Skeletal muscle metabolism has implications for swine feed efficiency (FE); however, it remains unclear if the metabolic profile of skeletal muscle changes during postnatal growth. To assess the metabolic changes, samples were collected from the longissimus dorsi (LD, glycolytic muscle), latissimus dorsi (LAT, mixed muscle), and masseter (MS, oxidative muscle) at 20, 53, 87, 120, and 180 days of age from barrows. Muscles were assessed to determine the abundance of several metabolic enzymes. Lactate dehydrogenase (LDHα) decreased in all muscles from 20 to 87 d (p < 0.01), which may be attributed to the muscles being more glycolytic at weaning from a milk-based diet. Pyruvate carboxylase (PC) increased in all muscles at 53 d compared to the other time points (p < 0.01), while pyruvate dehydrogenase α 1 (PDHα1) increased at 87 and 180 d in MS compared to LD (p < 0.05), indicating that potential changes occur in pyruvate entry into the tricarboxylic acid (TCA) cycle during growth. Isolated mitochondria from each muscle were incubated with 13C-labeled metabolites to assess isotopomer enrichment patterns of TCA intermediates. Citrate M + 2 and M + 4 derived from [13C3]-pyruvate increased at 87 d in LAT and MS mitochondria compared to LD mitochondria (p < 0.05). Regardless of the muscle, citrate M+3 increased at 87 d compared to 20, 53, and 120 d, while 180 d showed intermediate values (p < 0.01). These data support the notion that pyruvate metabolism is dynamic during growth. Our findings establish a metabolic fingerprint associated with postnatal muscle hypertrophy. [ABSTRACT FROM AUTHOR]

Repeated rounds of fusion between apposing myoblasts allow muscles to become multinucleated. New research finds that myoblasts undergoing fusion in the Drosophila embryo respond to hormone signaling from a nearby tissue, resulting in the activation of a myoblast-specific gene necessary for the fusion process. [ABSTRACT FROM AUTHOR]

Muscle attachment sites (MASs, apodemes) in insects and other arthropods involve specialized epithelial cells, called tendon cells or tenocytes, that adhere to apical extracellular matrices containing chitin. Here, we have uncovered a function for chitin deacetylases (CDAs) in arthropod locomotion and muscle attachment using a double-stranded RNA-mediated gene-silencing approach targeted toward specific CDA isoforms in the red flour beetle, Tribolium castaneum (Tc). Depletion of TcCDA1 or the alternatively spliced TcCDA2 isoform, TcCDA2a, resulted in internal tendon cuticle breakage at the femur-tibia joint, muscle detachment from both internal and external tendon cells, and defective locomotion. TcCDA deficiency did not affect early muscle development and myofiber growth toward the cuticular MASs but instead resulted in aborted microtubule development, loss of hemiadherens junctions, and abnormal morphology of tendon cells, all features consistent with a loss of tension within and between cells. Moreover, simultaneous depletion of TcCDA1 or TcCDA2a and the zona pellucida domain protein, TcDumpy, prevented the internal tendon cuticle break, further supporting a role for force-dependent interactions between muscle and tendon cells. We propose that in T. castaneum, the absence of N-acetylglucosamine deacetylation within chitin leads to a loss of microtubule organization and reduced membrane contacts at MASs in the femur, which adversely affect musculoskeletal connectivity, force transmission, and physical mobility. [ABSTRACT FROM AUTHOR]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2007.11.022 Byline: Erika R. Geisbrecht (a), Shruti Haralalka (a), Selene K. Swanson (b), Laurence Florens (b), Mike P. Washburn (b), Susan M. Abmayr (a) Keywords: Drosophila; ELMO; MBC; Rac; Myoblast fusion; Ommatidial organization Abstract: Members of the CDM (CED-5, Dock180, Myoblast city) superfamily of guanine nucleotide exchange factors function in diverse processes that include cell migration and myoblast fusion. Previous studies have shown that 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. Herein, we describe the isolation of Drosophila ELMO/CED-12, an [approximately equal to]82 kDa protein with a pleckstrin homology (PH) and proline-rich domain, by interaction with the MBC SH3 domain. 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. Author Affiliation: (a) Stowers Institute for Medical Research, 1000 E. 50th St., Kansas City, MO 64110, USA (b) Proteomics Core Facility, Stowers Institute for Medical Research, 1000 E. 50th St, Kansas City, MO 64110, USA Article History: Received 5 October 2007; Revised 7 November 2007; Accepted 17 November 2007

The type IIa family of receptor protein tyrosine phosphatases (RPTPs), including Lar, RPTPσ and RPTPδ, are well-studied in coordinating actin cytoskeletal rearrangements during axon guidance and synaptogenesis. To determine whether this regulation is conserved in other tissues, interdisciplinary approaches were utilized to study Lar-RPTPs in the Drosophila musculature. Here we find that the single fly ortholog, Drosophila Lar (Dlar), is localized to the muscle costamere and that a decrease in Dlar causes aberrant sarcomeric patterning, deficits in larval locomotion, and integrin mislocalization. Sequence analysis uncovered an evolutionarily conserved Lys-Gly-Asp (KGD) signature in the extracellular region of Dlar. Since this tripeptide sequence is similar to the integrin-binding Arg-Gly-Asp (RGD) motif, we tested the hypothesis that Dlar directly interacts with integrin proteins. However, structural analyses of the fibronectin type III domains of Dlar and two vertebrate orthologs that include this conserved motif indicate that this KGD tripeptide is not accessible and thus unlikely to mediate physical interactions with integrins. These results, together with the proteomics identification of basement membrane (BM) proteins as potential ligands for type IIa RPTPs, suggest a complex network of protein interactions in the extracellular space that may mediate Lar function and/or signaling in muscle tissue. [ABSTRACT FROM AUTHOR]

Woody breast (WB) is a myopathy observed in broiler Pectoralis major (PM) characterized by its tough and rubbery texture with greater level of calcium content. The objective of this study was to investigate the functionality/integrity of WB sarcoplasmic reticulum (SR), which may contribute to the elevated calcium content observed in WB and other factors that may influence WB texture. Fourteen Ross line broiler PM [7 severe WB and 7 normal (N)] were selected, packaged, and frozen at −20°C at 8 h postmortem from a commercial processing plant. Samples were used to measure pH, sarcomere length, proteolysis, calpain activity, collagenase activity, collagen content, collagen crosslinks density, and connective tissue peak transitional temperature. Exudate was also collected from each sample to evaluate free calcium concentration. The SR fraction of the samples was separated and utilized for proteomic and lipidomic analysis. The WB PM had a higher pH, shorter sarcomeres, lower % of intact troponin-T, more autolyzed μ/m calpain, more activated collagenase, greater collagen content, greater mature collagen crosslinks density, and higher connective tissue peak transitional temperature than the N PM (p ≤ 0.05). Exudate from WB PM had higher levels of free calcium than those from N PM (p < 0.05). Proteomics data revealed an upregulation of calcium transport proteins and a downregulation of proteins responsible for calcium release (p < 0.05) in WB SR. Interestingly, there was an upregulation of phospholipase A2 (PLA2), and cholinesterase exhibited a 7.6-fold increase in WB SR (p < 0.01). Lipidomics data revealed WB SR had less relative % of phosphatidylcholine (PC) and more lysophosphatidylcholine (LPC; p < 0.05). The results indicated that upregulation of calcium transport proteins and downregulation of calcium-release proteins in WB SR may be the muscle's attempt to regulate this proposed excessive signaling of calcium release due to multiple factors, such as upregulation of PLA2 resulting in PC hydrolysis and presence of cholinesterase inhibitors in the system prolonging action potential. In addition, the textural abnormality of WB may be the combined effects of shorter sarcomere length and more collagen with greater crosslink density being deposited in the broiler PM. [ABSTRACT FROM AUTHOR]

The inability to remove protein aggregates in post-mitotic cells such as muscles or neurons is a cellular hallmark of aging cells and is a key factor in the initiation and progression of protein misfolding diseases. While protein aggregate disorders share common features, the molecular level events that culminate in abnormal protein accumulation cannot be explained by a single mechanism. Here we show that loss of the serine/threonine kinase NUAK causes cellular degeneration resulting from the incomplete clearance of protein aggregates in Drosophila larval muscles. In NUAK mutant muscles, regions that lack the myofibrillar proteins F-actin and Myosin heavy chain (MHC) instead contain damaged organelles and the accumulation of select proteins, including Filamin (Fil) and CryAB. NUAK biochemically and genetically interacts with Drosophila Starvin (Stv), the ortholog of mammalian Bcl-2-associated athanogene 3 (BAG3). Consistent with a known role for the co-chaperone BAG3 and the Heat shock cognate 71 kDa (HSC70)/HSPA8 ATPase in the autophagic clearance of proteins, RNA interference (RNAi) of Drosophila Stv, Hsc70-4, or autophagy-related 8a (Atg8a) all exhibit muscle degeneration and muscle contraction defects that phenocopy NUAK mutants. We further demonstrate that Fil is a target of NUAK kinase activity and abnormally accumulates upon loss of the BAG3-Hsc70-4 complex. In addition, Ubiquitin (Ub), ref(2)p/p62, and Atg8a are increased in regions of protein aggregation, consistent with a block in autophagy upon loss of NUAK. Collectively, our results establish a novel role for NUAK with the Stv-Hsc70-4 complex in the autophagic clearance of proteins that may eventually lead to treatment options for protein aggregate diseases. Author summary: Non-dividing muscle and nerve cells have limited options to clear harmful biological insults. One such insult is the progressive accumulation of damaged and misfolded proteins that ultimately destroy cellular function and may result in cell or organismal death. Understanding how and why normal protein turnover occurs in healthy tissue is essential for the eventual treatment of age-related and/or degenerative diseases that result from abnormal protein accumulation. Using the large, easily manipulated muscles in fruit fly larvae, we find that loss of the evolutionarily conserved NUAK protein results in cellular degeneration due to the abnormal accumulation of certain proteins, including Fil and CryAB. Moreover, we identify Stv/BAG3 and its binding partner Hsc70-4 to be crucial for NUAK function as overexpression of Stv rescues the NUAK degenerative muscle phenotype. This work is the first to uncover NUAK as a key regulator of protein degradation through autophagy and may provide a novel therapeutic target for the treatment of protein aggregate myopathies. [ABSTRACT FROM AUTHOR]

The migration defect is suppressed due to the overexpression of the Drosophila inhibitor of apoptosis 1 (DIAP1), which is encoded by the thread (th) gene. An apoptosis-independent role for DIAP1-mediated Dronc inhibition in Rac-mediated cell motility is suggested.

Establishment 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. We show that Drosophila importin-7 (Dim7) is an upstream regulator of the conserved Elmo-MbcRRac 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 those 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. Our results show that integrins function as upstream signals to mediate Dim7-Elmo enrichment to the MASs. [ABSTRACT FROM AUTHOR]

Background: Cell motility is essential for embryonic development and physiological processes such as the immune response, but also contributes to pathological conditions such as tumor progression and inflammation. However, our understanding of the mechanisms underlying migratory processes is incomplete. Drosophila border cells provide a powerful genetic model to identify the roles of genes that contribute to cell migration. Results: Members of the Hedgehog signaling pathway were uncovered in two independent screens for interactions with the small GTPase Rac and the polarity protein Par-1 in border cell migration. Consistent with a role in migration, multiple Hh signaling components were enriched in the migratory border cells. Interference with Hh signaling by several different methods resulted in incomplete cell migration. Moreover, the polarized distribution of E-Cadherin and a marker of tyrosine kinase activity were altered when Hh signaling was disrupted. Conservation of Hh-Rac and Hh-Par-1 signaling was illustrated in the wing, in which Hh-dependent phenotypes were enhanced by loss of Rac or par-1. Conclusions: We identified a pathway by which Hh signaling connects to Rac and Par-1 in cell migration. These results further highlight the importance of modifier screens in the identification of new genes that function in developmental pathways. Developmental Dynamics 242:414-431, 2013. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Abstract: It is the precise connectivity between skeletal muscles and their corresponding tendon cells to form a functional myotendinous junction (MTJ) that allows for the force generation required for muscle contraction and organismal movement. The Drosophila MTJ is composed of secreted extracellular matrix (ECM) proteins deposited between integrin-mediated hemi-adherens junctions on the surface of muscle and tendon cells. In this paper, we have identified a novel, cytoplasmic role for the canonical nuclear import protein Moleskin (Msk) in Drosophila embryonic somatic muscle attachment. Msk protein is enriched at muscle attachment sites in late embryogenesis and msk mutant embryos exhibit a failure in muscle–tendon cell attachment. Although the muscle–tendon attachment sites are reduced in size, components of the integrin complexes and ECM proteins are properly localized in msk mutant embryos. However, msk mutants fail to localize phosphorylated focal adhesion kinase (pFAK) to the sites of muscle–tendon cell junctions. In addition, the tendon cell specific proteins Stripe (Sr) and activated mitogen-activated protein kinase (MAPK) are reduced in msk mutant embryos. Our rescue experiments demonstrate that Msk is required in the muscle cell, but not in the tendon cells. Moreover, muscle attachment defects due to loss of Msk are rescued by an activated form of MAPK or the secreted epidermal growth factor receptor (Egfr) ligand Vein. Taken together, these findings provide strong evidence that Msk signals non-autonomously through the Vein-Egfr signaling pathway for late tendon cell late differentiation and/or maintenance. [Copyright &y& Elsevier]

Cell morphogenesis, which requires rearrangement of the actin cytoskeleton, is essential to coordinate the development of tissues such as the musculature and nervous system during normal embryonic development. One class of signaling proteins that regulate actin cytoskeletal rearrangement is the evolutionarily conserved CDM (C. elegans Ced-5, human DOCK180, Drosophila Myoblast city, or Mbc) family of proteins, which function as unconventional guanine nucleotide exchange factors for the small GTPase Rac. This CDM-Rac protein complex is sufficient for Rac activation, but is enhanced upon the association of CDM proteins with the ELMO/Ced-12 family of proteins. We identified and characterized the role of Drosophila Sponge (Spg), the vertebrate DOCK3/DOCK4 counterpart as an ELMO-interacting protein. Our analysis shows Spg mRNA and protein is expressed in the visceral musculature and developing nervous system, suggesting a role for Spg in later embryogenesis. As maternal null mutants of spg die early in development, we utilized genetic interaction analysis to uncover the role of Spg in central nervous system (CNS) development. Consistent with its role in ELMO-dependent pathways, we found genetic interactions with spg and elmo mutants exhibited aberrant axonal defects. In addition, our data suggests Ncad may be responsible for recruiting Spg to the membrane, possibly in CNS development. Our findings not only characterize the role of a new DOCK family member, but help to further understand the role of signaling downstream of Ncadherin in neuronal development. [ABSTRACT FROM AUTHOR]

The evolutionarily conserved JAK/STAT signaling pathway is essential for the proliferation, survival and differentiation of many cells including cancer cells. Recent studies have implicated this transcriptional pathway in the process of cell migration in humans, mice, Drosophila and Dictyostelium. In the Drosophila ovary, JAK/STAT signaling is necessary and sufficient for the specification and migration of a group of cells called the border cells; however, it is not clear to what extent the requirement for cell fate is distinct from that for cell migration. We found that STAT protein is enriched in the migrating border cells throughout their migration and is an indicator of cells with highest JAK/STAT activity. In addition, stats mutants exhibited border cell migration defects after just 30 minutes at the non-permissive temperature, prior to any detectable change in the expression of cell fate markers. At later times, cell fate changes became evident, indicating that border cell fate is labile. JAK/STAT signaling was also required for organization of the border cell cluster. Finally, we show that both the accumulation of STAT protein and nuclear accumulation are positively regulated by JAK/STAT activity. The activity of the pathway is negatively regulated by overexpression of a SOCS protein and by blocking endocytosis. Together, our findings suggest that the requirement for STAT in border cells extends beyond the initial specification and delamination of cells from the epithelium. [ABSTRACT FROM AUTHOR]

Myosin VI (MyoVI) is a pointed-end-directed, actin-based motor protein, and mutations in the gene result in disorganization of hair cell stereocilia and cause deafness in mice. MyoVI also localizes to the leading edges of growth-factor-stimulated fibroblast cells and has been suggested to be involved in cell motility. There has been no direct test of this hypothesis, however. Drosophila melanogaster MyoVI is expressed in a small group of migratory follicle cells, known as border cells. Here we show that depletion of MyoVI specifically from border cells severely inhibited their migration. Similar to MyoVI, E-cadherin is required for border cell migration. We found that E-cadherin and Armadillo (Arm, Drosophila β-catenin) protein levels were specifically reduced in cells lacking MyoVI, whereas other proteins were not. In addition, MyoVI protein levels were reduced in cells lacking DE-cadherin or Arm. MyoVI and Arm co-immunoprecipitated from ovarian protein extracts. These data suggest that MyoVI is required for border cell migration where it stabilizes E-cadherin and Arm. Mutations in MyoVIIA, another unconventional myosin protein, also lead to deafness, and MyoVIIA interacts with E-cadherin through a membrane protein called vezatin. Multiple biochemical mechanisms may exist, therefore, for cadherins to associate with diverse unconventional myosins that are required for normal stereocilium formation or maintenance. [ABSTRACT FROM AUTHOR]

Human tripartite motif family of proteins 32 (TRIM32) is a ubiquitous multifunctional protein that has demonstrated roles in differentiation, muscle physiology and regeneration, and tumor suppression. Mutations in TRIM32 result in two clinically diverse diseases. A mutation in the B-box domain gives rise to Bardet–Biedl syndrome (BBS), a disease whose clinical presentation shares no muscle pathology, while mutations in the NHL (NCL-1, HT2A, LIN-41) repeats of TRIM32 causes limb-girdle muscular dystrophy type 2H (LGMD2H). TRIM32 also functions as a tumor suppressor, but paradoxically is overexpressed in certain types of cancer. Recent evidence supports a role for TRIM32 in glycolytic-mediated cell growth, thus providing a possible mechanism for TRIM32 in the accumulation of cellular biomass during regeneration and tumorigenesis, including in vitro and in vivo approaches, to understand the broad spectrum of TRIM32 functions. A special emphasis is placed on the utility of the Drosophila model, a unique system to study glycolysis and anabolic pathways that contribute to the growth and homeostasis of both normal and tumor tissues. [ABSTRACT FROM AUTHOR]

Phosphorylation reactions performed by protein kinases are one of the most studied post-translational modifications within cells. Much is understood about conserved residues within protein kinase domains that perform catalysis of the phosphotransfer reaction, yet the identity of the target substrates and downstream biological effects vary widely among cells, tissues, and organisms. Here, we characterize key residues essential for NUAK kinase activity in Drosophila melanogaster myogenesis and homeostasis. Creation of a NUAK kinase-dead mutation using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 results in lethality at the embryo to larval transition, while loss of NUAK catalytic function later in development produces aggregation of the chaperone protein αB-crystallin/CryAB in muscle tissue. Yeast 2-hybrid assays demonstrate a physical interaction between NUAK and CryAB. We further show that a phosphomimetic version of NUAK promotes the phosphorylation of CryAB and this post-translational modification occurs at 2 previously unidentified phosphosites that are conserved in the primary sequence of human CryAB. Mutation of these serine residues in D. melanogaster NUAK abolishes CryAB phosphorylation, thus, proving their necessity at the biochemical level. These studies together highlight the importance of kinase activity regulation and provide a platform to further explore muscle tissue proteostasis. [ABSTRACT FROM AUTHOR]

myoblast city (mbc), a member of the CDM superfamily, is essential in the Drosophila melanogaster embryo for fusion of myoblasts into multinucleate fibers. Using germ line clones in which both maternal and zygotic contributions were eliminated and rescue of the zygotic loss-of-function phenotype, we established that mbc is required in the fusion-competent subset of myoblasts. Along with its close orthologs Dock180 and CED-5, MBC has an SH3 domain at its N terminus, conserved internal domains termed DHR1 and DHR2 (or "Docker"), and C-terminal proline-rich domains that associate with the adapter protein DCrk. The importance of these domains has been evaluated by the ability of MBC mutations and deletions to rescue the mbc loss-of-function muscle phenotype. We demonstrate that the SH3 and Docker domains are essential. Moreover, ethyl methanesulfonate-induced mutations that change amino acids within the MBC Docker domain to residues that are conserved in other CDM family members nevertheless eliminate MBC function in the embryo, which suggests that these sites may mediate interactions specific to Drosophila MBC. A functional requirement for the conserved DHR1 domain, which binds to phosphatidylinositol 3,4,5-triphosphate, implicates phosphoinositide signaling in myoblast fusion. Finally, the proline-rich C-terminal sites mediate strong interactions with DCrk, as expected. These sites are not required for MBC to rescue the muscle loss-of-function phenotype, however, which suggests that MBC's role in myoblast fusion can be carried out independently of direct DCrk binding. [ABSTRACT FROM AUTHOR]

Mitochondria are dynamic organelles that change their architecture in normal physiological conditions. Mutations in genes that control mitochondrial fission or fusion, such as dynamin-related protein ( Drp1 ), Mitofusins 1 ( Mfn1 ) and 2 ( Mfn2 ), and Optic atrophy 1 ( Opa1 ), result in neuropathies or neurodegenerative diseases. It is increasingly clear that altered mitochondrial dynamics also underlie the pathology of other degenerative diseases, including Parkinson's disease (PD). Thus, understanding mitochondrial distribution, shape, and dynamics in all cell types is a prerequisite for developing and defining treatment regimens that may differentially affect tissues. The majority of Drosophila genes implicated in mitochondrial dynamics have been studied in the adult indirect flight muscle (IFM). Here, we discuss the utility of Drosophila third instar larvae (L3) as an alternative model to analyze and quantify mitochondrial behaviors. Advantages include large muscle cell size, a stereotyped arrangement of mitochondria that is conserved in mammalian muscles, and the ability to analyze muscle-specific gene function in mutants that are lethal prior to adult stages. In particular, we highlight methods for sample preparation and analysis of mitochondrial morphological features. [ABSTRACT FROM AUTHOR]

Cell growth and proliferation must be balanced during development to attain a final adult size with the appropriate proportions of internal organs to maximize fitness and reproduction. While multiple signaling pathways coordinate Drosophila development, it is unclear how multi-organ communication within and between tissues converge to regulate systemic growth. One such growth pathway, mediated by insulin-like peptides that bind to and activate the insulin receptor in multiple target tissues, is a primary mediator of organismal size. Here we uncover a signaling role for the NUAK serine/threonine kinase in muscle tissue that impinges upon insulin pathway activity to limit overall body size, including a reduction in the growth of individual organs. In skeletal muscle tissue, manipulation of NUAK or insulin pathway components influences sarcomere number concomitant with modulation of thin and thick filament lengths, possibly by modulating the localization of Lasp, a nebulin repeat protein known to set thin filament length. This mode of sarcomere remodeling does not occur in other mutants that also exhibit smaller muscles, suggesting that a sensing mechanism exists in muscle tissue to regulate sarcomere growth that is independent of tissue size control. [Display omitted] • NUAK overexpression in muscle tissue blunts insulin signaling and reduces organismal size. • Methuselah-like 8 (mthl8) is a new target of NUAK and FOXO-mediated transcription. • Reduced muscle growth can be uncoupled from alterations in sarcomere number and size. [ABSTRACT FROM AUTHOR]

The evolutionarily conserved Dock proteins function as unconventional guanine nucleotide exchange factors (GEFs). Upon binding to engulfment and cell motility (ELMO) proteins, Dock-ELMO complexes activate the Rho family of small GTPases to mediate a diverse array of biological processes, including cell motility, apoptotic cell clearance, and axon guidance. Overlapping expression patterns and functional redundancy among the 11 vertebrate Dock family members, which are subdivided into four families (Dock A, B, C, and D), complicate genetic analysis. In both vertebrate and invertebrate systems, the actin dynamics regulator, Rac, is the target GTPase of the Dock-A subfamily. However, it remains unclear whether Rac or Rap1 are the in vivo downstream GTPases of the Dock-B subfamily. Drosophila melanogaster is an excellent genetic model organism for understanding Dock protein function as its genome encodes one ortholog per subfamily: Myoblast city (Mbc; Dock A) and Sponge (Spg; Dock B). Here we show that the roles of Spg and Mbc are not redundant in the Drosophila somatic muscle or the dorsal vessel.Moreover, we confirm the in vivo role of Mbc upstream of Rac and provide evidence that Spg functions in concert with Rap1, possibly to regulate aspects of cell adhesion. Together these data show that Mbc and Spg can have different downstream GTPase targets. Our findings predict that the ability to regulate downstream GTPases is dependent on cellular context and allows for the fine-tuning of actin cytoskeletal or cell adhesion events in biological processes that undergo cell morphogenesis. [ABSTRACT FROM AUTHOR]

Abstract: Neurogenesis depends on exquisitely regulated interactions between macromolecules on the cell surface and in the extracellular matrix. In particular, interactions between proteoglycans and members of the type IIa subgroup of receptor protein tyrosine phosphatases underlie crucial developmental processes such as the formation of synapses at the neuromuscular junction and the migration of axons to their appropriate targets. We report the crystal structures of the first and second immunoglobulin-like domains of the Drosophila type IIa receptor Dlar and its mouse homolog LAR. These two domains adopt an unusual antiparallel arrangement that has not been reported in tandem repeats of immunoglobulin-like domains and that is presumably conserved in all type IIa receptor protein tyrosine phosphatases. [Copyright &y& Elsevier]

Adult muscle precursor (AMP) cells located in the notum of the larval wing disc undergo rapid amplification and eventual fusion to generate the Drosophila melanogaster indirect flight muscles (IFMs). Here we find that loss of Moleskin (Msk) function in these wing disc-associated myoblasts reduces the overall AMP pool size, resulting in the absence of IFM formation. This myoblast loss is due to a decrease in the AMP proliferative capacity and is independent of cell death. In contrast, disruption of Msk during pupal myoblast proliferation does not alter the AMP number, suggesting that Msk is specifically required for larval AMP proliferation. It has been previously shown that Wingless (Wg) signaling maintains expression of the Vestigial (Vg) transcription factor in proliferating myoblasts. However, other factors that influence Wg-mediated myoblast proliferation are largely unknown. Here we examine the interactions between Msk and the Wg pathway in regulation of the AMP pool size. We find that a myoblast-specific reduction of Msk results in the absence of Vg expression and a complete loss of the Wg pathway readout b-catenin/Armadillo (Arm). Moreover, msk RNA interference knockdown abolishes expression of the Wg target Ladybird (Lbe) in leg disc myoblasts. Collectively, our results provide strong evidence that Msk acts through the Wg signaling pathway to control myoblast pool size and muscle formation by regulating Arm stability or nuclear transport. [ABSTRACT FROM AUTHOR]

The organization and stability of higher order structures that form in the extracellular matrix (ECM) to mediate the attachment of muscles are poorly understood. We have made the surprising discovery that a subset of clotting factor proteins are also essential for muscle attachment in the model organism Drosophila melanogaster. One such coagulation protein, Fondue (Fon), was identified as a novel muscle mutant in a pupal lethal genetic screen. Fon accumulates at muscle attachment sites and removal of this protein results in decreased locomotor behavior and detached larval muscles. A sensitized genetic background assay reveals that fon functions with the known muscle attachment genes Thrombospondin (Tsp) and Tiggrin (Tig). Interestingly, Tig is also a component of the hemolymph clot. We further demonstrate that an additional clotting protein, Larval serum protein 1γ (Lsp1γ), is also required for muscle attachment stability and accumulates where muscles attach to tendons. While the local biomechanical and organizational properties of the ECM vary greatly depending on the tissue microenvironment, we propose that shared extracellular protein-protein interactions influence the strength and elasticity of ECM proteins in both coagulation and muscle attachment. [ABSTRACT FROM AUTHOR]

An efficient and low-cost method of examining larval movement in Drosophila melanogaster is needed to study how mutations and/or alterations in the muscular, neural, and olfactory systems affect locomotor behavior. Here, we describe the implementation of wrMTrck, a freely available ImageJ plugin originally developed for examining multiple behavioral parameters in the nematode C. elegans . Our optimized method is rapid, reproducible and does not require automated microscope setups or the purchase of proprietary software. To demonstrate the utility of this method, we analyzed the velocity and crawling paths of two Drosophila mutants that affect muscle structure and/or function. Additionally, we show that this approach is useful for tracking the behavior of adult insects, including Tribolium castaneum and Drosophila melanogaster . [ABSTRACT FROM AUTHOR]