Chitin deacetylases are necessary for insect femur muscle attachment and mobility.

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.

Independent pathways control muscle tissue size and sarcomere remodeling.

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.

Drosophila NUAK functions with Starvin/BAG3 in autophagic protein turnover.

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.

Integration of proteomic and genetic approaches to assess developmental muscle atrophy.

Muscle atrophy, or a decline in muscle protein mass, is a significant problem in the aging population and in numerous disease states. Unraveling molecular signals that trigger and promote atrophy may lead to a better understanding of treatment options; however, there is no single cause of atrophy identified to date. To gain insight into this problem, we chose to investigate changes in protein profiles during muscle atrophy in Manduca sexta and Drosophila melanogaster. The use of insect models provides an interesting parallel to probe atrophic mechanisms as these organisms undergo a normal developmental atrophy process during the pupal transition stage. Leveraging the inherent advantages of each model organism, we first defined protein signature changes during M. sexta intersegmental muscle (ISM) atrophy and then used genetic approaches to confirm their functional importance in the D. melanogaster dorsal internal oblique muscles (DIOMs). Our data reveal an upregulation of proteasome and peptidase components and a general downregulation of proteins that regulate actin filament formation. Surprisingly, thick filament proteins that comprise the A-band are increased in abundance, providing support for the ordered destruction of myofibrillar components during developmental atrophy. We also uncovered the actin filament regulator ciboulot (Cib) as a novel regulator of muscle atrophy. These insights provide a framework towards a better understanding of global changes that occur during atrophy and may eventually lead to therapeutic targets.

A tissue communication network coordinating innate immune response during muscle stress.

Complex tissue communication networks function throughout an organism's lifespan to maintain tissue homeostasis. Using the genetic model Drosophila melanogaster, we have defined a network of immune responses that are activated following the induction of muscle stresses, including hypercontraction, detachment and oxidative stress. Of these stressors, loss of the genes that cause muscle detachment produced the strongest levels of JAK-STAT activation. In one of these mutants, fondue (fon), we also observe hemocyte recruitment and the accumulation of melanin at muscle attachment sites (MASs), indicating a broad involvement of innate immune responses upon muscle detachment. Loss of fon results in pathogen-independent Toll signaling in the fat body and increased expression of the Toll-dependent antimicrobial peptide Drosomycin. Interestingly, genetic interactions between fon and various Toll pathway components enhance muscle detachment. Finally, we show that JAK-STAT and Toll signaling are capable of reciprocal activation in larval tissues. We propose a model of tissue communication for the integration of immune responses at the local and systemic level in response to altered muscle physiology.

Drosophila clueless is involved in Parkin-dependent mitophagy by promoting VCP-mediated Marf degradation.

PINK1/Parkin-mediated mitochondrial quality control (MQC) requires valosin-containing protein (VCP)-dependent Mitofusin/Marf degradation to prevent damaged organelles from fusing with the healthy mitochondrial pool, facilitating mitochondrial clearance by autophagy. Drosophila clueless (clu) was found to interact genetically with PINK1 and parkin to regulate mitochondrial clustering in germ cells. However, whether Clu acts in MQC has not been investigated. Here, we show that overexpression of Drosophila Clu complements PINK1, but not parkin, mutant muscles. Loss of clu leads to the recruitment of Parkin, VCP/p97, p62/Ref(2)P and Atg8a to depolarized swollen mitochondria. However, clearance of damaged mitochondria is impeded. This paradox is resolved by the findings that excessive mitochondrial fission or inhibition of fusion alleviates mitochondrial defects and impaired mitophagy caused by clu depletion. Furthermore, Clu is upstream of and binds to VCP in vivo and promotes VCP-dependent Marf degradation in vitro Marf accumulates in whole muscle lysates of clu-deficient flies and is destabilized upon Clu overexpression. Thus, Clu is essential for mitochondrial homeostasis and functions in concert with Parkin and VCP for Marf degradation to promote damaged mitochondrial clearance.

Drosophila importin-7 functions upstream of the Elmo signaling module to mediate the formation and stability of muscle attachments.

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-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 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.

Thin, a Trim32 ortholog, is essential for myofibril stability and is required for the integrity of the costamere in Drosophila.

Myofibril stability is required for normal muscle function and maintenance. Mutations that disrupt myofibril stability result in individuals who develop progressive muscle wasting, or muscular dystrophy, and premature mortality. Here we present our investigations of the Drosophila l(2)thin [l(2)tn] mutant. The "thin" phenotype exhibits features of the human muscular disease phenotype in that tn mutant larvae show progressive muscular degeneration. Loss-of-function and rescue experiments determined that l(2)tn is allelic to the tn locus [previously annotated as both CG15105 and another b-box affiliate (abba)]. tn encodes a TRIM (tripartite motif) containing protein highly expressed in skeletal muscle and is orthologous to the human limb-girdle muscular dystrophy type 2H disease gene Trim32. Thin protein is localized at the Z-disk in muscle, but l(2)tn mutants showed no genetic interaction with mutants affecting the Z-line-associated protein muscle LIM protein 84B. l(2)tn, along with loss-of-function mutants generated for tn, showed no relative mislocalization of the Z-disk proteins α-Actinin and muscle LIM protein 84B. In contrast, tn mutants had significant disorganization of the costameric orthologs ß-integrin, Spectrin, Talin, and Vinculin, and we present the initial description for the costamere, a key muscle stability complex, in Drosophila. Our studies demonstrate that myofibrils progressively unbundle in flies that lack Thin function through progressive costamere breakdown. Due to the high conservation of these structures in animals, we demonstrate a previously unknown role for TRIM32 proteins in myofibril stability.

Genetic interaction screens identify a role for hedgehog signaling in Drosophila border cell migration.

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.

Cell fusion: Inter-organ tissue communication promotes a union between myoblasts.

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.

Protocol for affinity purification-mass spectrometry interactome profiling in larvae of Drosophila melanogaster.

Many techniques exist for the identification of protein interaction networks. We present a protocol that relies on an affinity purification-mass spectrometry (AP-MS) approach to detect proteins that co-purify with a tagged bait of interest from Drosophila melanogaster larval muscles using the GAL4/upstream activating sequence (UAS) expression system. We also describe steps for the isolation and identification of protein complexes, followed by streamlined bioinformatics analysis for rapid and reproducible results. This protocol can be extended to investigate protein interactions in other tissues. For complete details on the use and execution of this protocol, please refer to Guo et al.1.

Skeletal Muscle Metabolism Is Dynamic during Porcine Postnatal Growth.

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.

Orchestration of autophagosome fusion by STRIPAK complex components in muscle tissue.

Autophagy is a central process responsible for the disposal of normal as well as damaged cellular proteins and organelles. Proper regulation of multiple steps - including initiation and the fusion between autophagosomes and lysosomes - is essential for the completion of cargo disposal. While the function of many proteins that mediate canonical autophagy has been characterized, the identification of new autophagy regulators may shed light on differences between tissues and/or responses to cellular stresses. In this punctum, we discuss our recent findings about how the Striatin-Interacting Phosphatase and Kinase (STRIPAK)-NUAK-Starvin (Stv) complex coordinately regulates autophagy in the muscle tissue of Drosophila melanogaster.

Identification of CryAB as a target of NUAK kinase activity in Drosophila muscle tissue.

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 phospho-mimetic 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.

A conserved STRIPAK complex is required for autophagy in muscle tissue.

Autophagy is important for cellular homeostasis and to prevent the abnormal accumulation of proteins. While many proteins that comprise the canonical autophagy pathway have been characterized, the identification of new regulators may help understand tissue and/or stress-specific responses. Using an in-silico approach, we identified Striatin interacting protein (Strip), MOB kinase activator 4, and fibroblast growth factor receptor 1 oncogene partner 2 as conserved mediators of muscle tissue maintenance. We performed affinity purification-mass spectrometry (AP-MS) experiments with Drosophila melanogaster Strip as a bait protein and copurified additional Striatin-interacting phosphatase and kinase (STRIPAK) complex members from larval muscle tissue. NUAK family kinase 1 (NUAK) and Starvin (Stv) also emerged as Strip-binding proteins and these physical interactions were verified in vivo using proximity ligation assays. To understand the functional significance of the STRIPAK-NUAK-Stv complex, we employed a sensitized genetic assay combined with RNA interference (RNAi) to demonstrate that both NUAK and stv function in the same biological process with genes that encode for STRIPAK complex proteins. RNAi-directed knockdown of Strip in muscle tissue led to the accumulation of ubiquitinated cargo, p62, and Autophagy-related 8a, consistent with a block in autophagy. Indeed, autophagic flux was decreased in Strip RNAi muscles, while lysosome biogenesis and activity were unaffected. Our results support a model whereby the STRIPAK-NUAK-Stv complex coordinately regulates autophagy in muscle tissue.

Moleskin is essential for the formation of the myotendinous junction in Drosophila.

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.

Gene expression profiling of NUAK kinase overexpression in Drosophila larval muscle development.

Signal transduction pathways mediated by kinases control diverse biological outputs at the level of cells and tissues to regulate a diverse array of biological and developmental events. To gain insight into how muscle expression of the evolutionarily conserved NUAK kinase regulates the transcriptional landscape during Drosophila melanogaster development, we performed high-throughput sequencing of RNA from either whole larvae or dissected muscle fillets at the end of larval development. Raw data was generated using the Illumina HiSeq 4000 platform. After trimming and mapping to the Drosophila reference genome, differential gene expression and GO enrichment analysis were completed. Raw data are deposited in the NCBI Gene Expression Ominbus (GEO) repository under GEO accession GSE204894.

Complex protein interactions mediate Drosophila Lar function in muscle tissue.

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.

An Investigation of the Altered Textural Property in Woody Breast Myopathy Using an Integrative Omics Approach.

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.