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.

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.

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.

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.

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.

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.

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.

TRIM32: A Multifunctional Protein Involved in Muscle Homeostasis, Glucose Metabolism, and Tumorigenesis.

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.

Costameric integrin and sarcoglycan protein levels are altered in a Drosophila model for Limb-girdle muscular dystrophy type 2H.

Mutations in two different domains of the ubiquitously expressed TRIM32 protein give rise to two clinically separate diseases, one of which is Limb-girdle muscular dystrophy type 2H (LGMD2H). Uncovering the muscle-specific role of TRIM32 in LGMD2H pathogenesis has proven difficult, as neurogenic phenotypes, independent of LGMD2H pathology, are present in TRIM32 KO mice. We previously established a platform to study LGMD2H pathogenesis using Drosophila melanogaster as a model. Here we show that LGMD2H disease-causing mutations in the NHL domain are molecularly and structurally conserved between fly and human TRIM32. Furthermore, transgenic expression of a subset of myopathic alleles (R394H, D487N, and 520fs) induce myofibril abnormalities, altered nuclear morphology, and reduced TRIM32 protein levels, mimicking phenotypes in patients afflicted with LGMD2H. Intriguingly, we also report for the first time that the protein levels of ßPS integrin and sarcoglycan δ, both core components of costameres, are elevated in TRIM32 disease-causing alleles. Similarly, murine myoblasts overexpressing a catalytically inactive TRIM32 mutant aberrantly accumulate α- and ß-dystroglycan and α-sarcoglycan. We speculate that the stoichiometric loss of costamere components disrupts costamere complexes to promote muscle degeneration.

Insect Cuticular Chitin Contributes to Form and Function.

Chitin contributes to the rigidity of the insect cuticle and serves as an attachment matrix for other cuticular proteins. Deficiency of chitin results in abnormal embryos, cuticular structural defects and growth arrest. When chitin is not turned over during molting, the developing insect is trapped inside the old cuticle. Partial deacetylation of cuticular chitin is also required for proper laminar organization of the cuticle and vertical pore canals, molting, and locomotion. Thus, chitin and its modifications strongly influence the structure of the exoskeleton as well as the physiological functions of the insect. Internal tendons and specialized epithelial cells called "tendon cells" that arise from the outer layer of epidermal cells provide attachment sites at both ends of adult limb muscles. Membrane processes emanating from both tendon and muscle cells interdigitate extensively to strengthen the attachment of muscles to the extracellular matrix (ECM). Protein ligands that bind to membrane-bound integrin complexes further enhance the adhesion between muscles and tendons. Tendon cells contain F-actin fiber arrays that contribute to their rigidity. In the cytoplasm of muscle cells, proteins such as talin and other proteins provide attachment sites for cytoskeletal actin, thereby increasing integrin binding and activation to mechanically couple the ECM with actin in muscle cells. Mutations in integrins and their ligands, as well as depletion of chitin deacetylases, result in defective locomotion and muscle detachment from the ECM. Thus, chitin in the cuticle and chitin deacetylases strongly influence the shape and functions of the exoskeleton as well as locomotion of insects.

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.

Drosophila TRIM32 cooperates with glycolytic enzymes to promote cell growth.

Cell growth and/or proliferation may require the reprogramming of metabolic pathways, whereby a switch from oxidative to glycolytic metabolism diverts glycolytic intermediates towards anabolic pathways. Herein, we identify a novel role for TRIM32 in the maintenance of glycolytic flux mediated by biochemical interactions with the glycolytic enzymes Aldolase and Phosphoglycerate mutase. Loss of Drosophila TRIM32, encoded by thin (tn), shows reduced levels of glycolytic intermediates and amino acids. This altered metabolic profile correlates with a reduction in the size of glycolytic larval muscle and brain tissue. Consistent with a role for metabolic intermediates in glycolysis-driven biomass production, dietary amino acid supplementation in tn mutants improves muscle mass. Remarkably, TRIM32 is also required for ectopic growth - loss of TRIM32 in a wing disc-associated tumor model reduces glycolytic metabolism and restricts growth. Overall, our results reveal a novel role for TRIM32 for controlling glycolysis in the context of both normal development and tumor growth.

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.

Thin is required for cell death in the Drosophila abdominal muscles by targeting DIAP1.

In holometabolous insects, developmentally controlled programmed cell death (PCD) is a conserved process that destroys a subset of larval tissues for the eventual creation of new adult structures. This process of histolysis is relatively well studied in salivary gland and midgut tissues, while knowledge concerning larval muscle destruction is limited. Here, we have examined the histolysis of a group of Drosophila larval abdominal muscles called the dorsal external oblique muscles (DEOMs). Previous studies have defined apoptosis as the primary mediator of DEOM breakdown, whose timing is controlled by ecdysone signaling. However, very little is known about other factors that contribute to DEOM destruction. In this paper, we examine the role of thin (tn), which encodes for the Drosophila homolog of mammalian TRIM32, in the regulation of DEOM histolysis. We find that loss of Tn blocks DEOM degradation independent of ecdysone signaling. Instead, tn genetically functions in a pathway with the death-associated inhibitor of apoptosis (DIAP1), Dronc, and death-associated APAF1-related killer (Dark) to regulate apoptosis. Importantly, blocking Tn results in the absence of active Caspase-3 immunostaining, upregulation of DIAP1 protein levels, and inhibition of Dronc activation. DIAP1 and Dronc mRNA levels are not altered in tn mutants, showing that Tn acts post-transcriptionally on DIAP1 to regulate apoptosis. Herein, we also find that the RING domain of Tn is required for DEOM histolysis as loss of this domain results in higher DIAP1 levels. Together, our results suggest that the direct control of DIAP1 levels, likely through the E3 ubiquitin ligase activity of Tn, provides a mechanism to regulate caspase activity and to facilitate muscle cell death.

Adult Muscle Formation Requires Drosophila Moleskin for Proliferation of Wing Disc-Associated Muscle Precursors.

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 ß-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.