ABSTRACT
The biological mechanisms linking sedentary lifestyles and metabolic derangements are incompletely understood. In this study, temporal muscle inactivation in Drosophila larvae carrying a temperature-sensitive mutation in the shibire (shi1) gene was induced to mimic sedentary behavior during early life and study its transcriptional outcome. Our findings indicated a significant change in the epigenetic profile, as well as the genomic profile, of RNA Pol II binding in the inactive muscles relative to control, within a relatively short time period. Whole-genome analysis of RNA-Pol II binding to DNA by muscle-specific targeted DamID (TaDa) protocol revealed that muscle inactivity altered Pol II binding in 121 out of 2010 genes (6%), with a three-fold enrichment of genes coding for lncRNAs. The suppressed protein-coding genes included genes associated with longevity, DNA repair, muscle function, and ubiquitin-dependent proteostasis. Moreover, inducing muscle inactivation exerted a multi-level impact upon chromatin modifications, triggering an altered epigenetic balance of active versus inactive marks. The downregulated genes in the inactive muscles included genes essential for muscle structure and function, carbohydrate metabolism, longevity, and others. Given the multiple analogous genes in Drosophila for many human genes, extrapolating our findings to humans may hold promise for establishing a molecular link between sedentary behavior and metabolic diseases.
Subject(s)
Drosophila , Transcriptome , Animals , Humans , Transcriptome/genetics , Epigenome , Larva/genetics , Sedentary Behavior , RNA Polymerase II , MusclesABSTRACT
We show evidence of the association of RNA polymerase II (RNAP) with chromatin in a core-shell organization, reminiscent of microphase separation where the cores comprise dense chromatin and the shell, RNAP and chromatin with low density. These observations motivate our physical model for the regulation of core-shell chromatin organization. Here, we model chromatin as a multiblock copolymer, comprising active and inactive regions (blocks) that are both in poor solvent and tend to be condensed in the absence of binding proteins. However, we show that the solvent quality for the active regions of chromatin can be regulated by the binding of protein complexes (e.g., RNAP and transcription factors). Using the theory of polymer brushes, we find that such binding leads to swelling of the active chromatin regions which in turn modifies the spatial organization of the inactive regions. In addition, we use simulations to study spherical chromatin micelles, whose cores comprise inactive regions and shells comprise active regions and bound protein complexes. In spherical micelles the swelling increases the number of inactive cores and controls their size. Thus, genetic modifications affecting the binding strength of chromatin-binding protein complexes may modulate the solvent quality experienced by chromatin and regulate the physical organization of the genome.
Subject(s)
Chromatin , Micelles , Chromosomes , Transcription Factors/genetics , RNA Polymerase II/genetics , SolventsABSTRACT
The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex transduces nuclear mechanical inputs suggested to control chromatin organization and gene expression; however, the underlying mechanism is currently unclear. We show here that the LINC complex is needed to minimize chromatin repression in muscle tissue, where the nuclei are exposed to significant mechanical inputs during muscle contraction. To this end, the genomic binding profiles of Polycomb, Heterochromatin Protein1 (HP1a) repressors, and of RNA-Pol II were studied in Drosophila larval muscles lacking functional LINC complex. A significant increase in the binding of Polycomb and parallel reduction of RNA-Pol-II binding to a set of muscle genes was observed. Consistently, enhanced tri-methylated H3K9 and H3K27 repressive modifications and reduced chromatin activation by H3K9 acetylation were found. Furthermore, larger tri-methylated H3K27me3 repressive clusters, and chromatin redistribution from the nuclear periphery towards nuclear center, were detected in live LINC mutant larval muscles. Computer simulation indicated that the observed dissociation of the chromatin from the nuclear envelope promotes growth of tri-methylated H3K27 repressive clusters. Thus, we suggest that by promoting chromatin-nuclear envelope binding, the LINC complex restricts the size of repressive H3K27 tri-methylated clusters, thereby limiting the binding of Polycomb transcription repressor, directing robust transcription in muscle fibers.
Subject(s)
Chromatin , Drosophila Proteins , Animals , Chromatin/metabolism , Computer Simulation , Cytoskeleton/metabolism , Transcription Factors/metabolism , Nuclear Matrix/metabolism , Polycomb-Group Proteins/genetics , Polycomb-Group Proteins/metabolism , Drosophila/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , RNA/metabolismABSTRACT
Chromatin organization in the nucleus represents an important aspect of transcription regulation. Most of the studies so far focused on the chromatin structure in cultured cells or in fixed tissue preparations. Here, we discuss the various approaches for deciphering chromatin 3D organization with an emphasis on the advantages of live imaging approaches.
ABSTRACT
The three-dimensional organization of chromatin contributes to transcriptional control, but information about native chromatin distribution is limited. Imaging chromatin in live Drosophila larvae, with preserved nuclear volume, revealed that active and repressed chromatin separates from the nuclear interior and forms a peripheral layer underneath the nuclear lamina. This is in contrast to the current view that chromatin distributes throughout the nucleus. Furthermore, peripheral chromatin organization was observed in distinct Drosophila tissues, as well as in live human effector T lymphocytes and neutrophils. Lamin A/C up-regulation resulted in chromatin collapse toward the nuclear center and correlated with a significant reduction in the levels of active chromatin. Physical modeling suggests that binding of lamina-associated domains combined with chromatin self-attractive interactions recapitulate the experimental chromatin distribution profiles. Together, our findings reveal a novel mode of mesoscale organization of peripheral chromatin sensitive to lamina composition, which is evolutionary conserved.
Subject(s)
Cell Nucleus , Chromatin , Animals , Cell Nucleus/metabolism , Chromatin/metabolism , Chromosomes , Drosophila , Nuclear Lamina/metabolismABSTRACT
Intact-organism imaging of Drosophila larvae reveals and quantifies chromatin-aqueous phase separation. The chromatin can be organized near the lamina layer of the nuclear envelope, conventionally fill the nucleus, be organized centrally, or as a wetting droplet. These transitions are controlled by changes in nuclear volume and the interaction of chromatin with the lamina (part of the nuclear envelope) at the nuclear periphery. Using a simple polymeric model that includes the key features of chromatin self-attraction and its binding to the lamina, we demonstrate theoretically that it is the competition of these two effects that determines the mode of chromatin distribution. The qualitative trends as well as the composition profiles obtained in our simulations compare well with the observed intact-organism imaging and quantification. Since the simulations contain only a small number of physical variables we can identify the generic mechanisms underlying the changes in the observed phase separations.
Subject(s)
Cell Nucleus/physiology , Chromatin/physiology , Computer Simulation , Animals , Drosophila , LarvaABSTRACT
DNA endoreplication has been implicated as a cell strategy for cell growth and in tissue injury. Here, we demonstrate that barrier-to-autointegration factor (BAF) represses endoreplication in Drosophila myofibers. We show that BAF localization at the nuclear envelope is eliminated in flies with mutations of the linker of nucleoskeleton and cytoskeleton (LINC) complex in which the LEM-domain protein Otefin is excluded, or after disruption of the nucleus-sarcomere connections. Furthermore, BAF localization at the nuclear envelope requires the activity of the BAF kinase VRK1/Ball, and, consistently, non-phosphorylatable BAF-GFP is excluded from the nuclear envelope. Importantly, removal of BAF from the nuclear envelope correlates with increased DNA content in the myonuclei. E2F1, a key regulator of endoreplication, overlaps BAF localization at the myonuclear envelope, and BAF removal from the nuclear envelope results in increased E2F1 levels in the nucleoplasm and subsequent elevated DNA content. We suggest that LINC-dependent and phosphosensitive attachment of BAF to the nuclear envelope, through its binding to Otefin, tethers E2F1 to the nuclear envelope thus inhibiting its accumulation in the nucleoplasm.
Subject(s)
DNA-Binding Proteins/genetics , Drosophila Proteins/genetics , Endoreduplication/genetics , Membrane Proteins/genetics , Nuclear Proteins/genetics , Transcription Factors/genetics , Animals , Cytoskeleton/genetics , DNA Replication/genetics , Drosophila melanogaster/genetics , Gene Expression Regulation, Developmental/genetics , Mutation/genetics , Myofibrils/genetics , Nuclear Envelope/genetics , Nuclear Matrix/genetics , Protamine Kinase/geneticsABSTRACT
Muscle contractions produce reiterated cytoplasmic mechanical variations, which potentially influence nuclear mechanotransduction, however information regarding the dynamics of muscle nuclei (myonuclei) in the course of muscle contraction is still missing. Towards that end, a minimal constraint device was designed in which intact live Drosophila larva is imaged, while its muscles still contract. The device is placed under spinning disc confocal microscope enabling imaging of fluorescently labeled sarcomeres and nuclei during muscle contraction, without any external stimulation. As a proof of principle we studied myonuclei dynamics in wild-type, as well as in Nesprin/klar mutant larvae lacking proper nuclear-cytoskeletal connections. Myonuclei in control larvae exhibited comparable dynamics in the course of multiple contractile events, independent of their position along the muscle fiber. In contrast, myonuclei of mutant larvae displayed differential dynamics at distinct positions along individual myofibers. Moreover, we identified a linear link between myonuclear volume and its acceleration values during muscle contraction which, in Nesprin/klar mutants exhibited an opposite tendency relative to control. Estimation of the drag force applied on individual myonuclei revealed that force fluctuations in time, but not the average force, differed significantly between control and Nesprin/klar mutant, and were considerably higher in the mutant myonuclei. Taken together these results imply significant alterations in the mechanical dynamics of individual myonuclei in the Nesprin/klar myonuclei relative to control. Such differences provide novel mechanical insight into Nesprin function in contractile muscles, and might reveal the mechanical basis underlying Nesprin-related human diseases.
Subject(s)
Drosophila Proteins , Drosophila , Animals , Cell Nucleus/metabolism , Humans , Larva/metabolism , Mechanotransduction, Cellular , Membrane Transport Proteins , Muscle Contraction , MusclesABSTRACT
Differentiation of germline stem cells (GSCs) in the Drosophila ovary is induced by somatic escort cells (ECs), which extend membrane protrusions encapsulating the germline cells (GCs). Germline encapsulation requires activated epidermal growth factor receptor (Egfr) signaling within the ECs, following secretion of its ligands from the GCs. We show that the conserved family of irre cell recognition module (IRM) proteins is essential for GC encapsulation by ECs, with a requirement for roughest (rst) and kin of irre (kirre) in the germline and for sticks and stones (sns) and hibris (hbs) in ECs. In the absence of IRM components in their respective cell types, EC extensions are reduced concomitantly with a decrease in Egfr signaling in these cells. Reintroducing either activated Egfr in the ECs, or overexpressing its ligand Spitz (Spi) from the germline, rescued the requirement for IRM proteins in both cell types. These experiments introduce novel essential components, the IRM proteins, into the process of inductive interactions between GCs and ECs, and imply that IRM-mediated activity is required upstream of the Egfr signaling.
ABSTRACT
The cytoplasm of striated myofibers contains a large number of membrane organelles, including sarcoplasmic reticulum (SR), T-tubules and the nuclear membrane. These organelles maintain a characteristic juxtaposition that appears to be essential for efficient inter-membranous exchange of RNA, proteins and ions. We found that the membrane-associated Muscle-specific α2/δ (Ma2/d) subunit of the Ca2+ channel complex localizes to the SR and T-tubules, and accumulates at the myonuclear surfaces. Furthermore, Ma2/d mutant larval muscles exhibit nuclear positioning defects, disruption of the nuclear-SR juxtapositioning, as well as impaired larval locomotion. Ma2/d localization at the nuclear membrane depends on the proper function of the nesprin ortholog Msp300 and the BAR domain protein Amphiphysin (Amph). Importantly, live imaging of muscle contraction in intact Drosophila larvae indicated altered distribution of Sarco/Endoplamic Reticulum Ca2+-ATPase (SERCA) around the myonuclei of Ma2/d mutant larvae. Co-immunoprecipitation analysis supports association between Ma2/d and Amph, and indirectly with Msp300. We therefore suggest that Ma2/d, in association with Msp300 and Amph, mediates interactions between the SR and the nuclear membrane.
Subject(s)
Biological Transport/physiology , Calcium Channels/metabolism , Drosophila Proteins/metabolism , Microfilament Proteins/metabolism , Muscle Proteins/metabolism , Myofibrils/metabolism , Nerve Tissue Proteins/metabolism , Nuclear Envelope/metabolism , Sarcoplasmic Reticulum/metabolism , Animals , Animals, Genetically Modified , Calcium/metabolism , Drosophila , Muscle Contraction/physiology , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolismABSTRACT
Nuclear mechanotransduction has been implicated in the control of chromatin organization; however, its impact on functional contractile myofibers is unclear. We found that deleting components of the linker of nucleoskeleton and cytoskeleton (LINC) complex in Drosophila melanogaster larval muscles abolishes the controlled and synchronized DNA endoreplication, typical of nuclei across myofibers, resulting in increased and variable DNA content in myonuclei of individual myofibers. Moreover, perturbation of LINC-independent mechanical input after knockdown of ß-Integrin in larval muscles similarly led to increased DNA content in myonuclei. Genome-wide RNA-polymerase II occupancy analysis in myofibers of the LINC mutant klar indicated an altered binding profile, including a significant decrease in the chromatin regulator barrier-to-autointegration factor (BAF) and the contractile regulator Troponin C. Importantly, muscle-specific knockdown of BAF led to increased DNA content in myonuclei, phenocopying the LINC mutant phenotype. We propose that mechanical stimuli transmitted via the LINC complex act via BAF to regulate synchronized cell-cycle progression of myonuclei across single myofibers.
Subject(s)
DNA Replication , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Mechanotransduction, Cellular , Multiprotein Complexes/metabolism , Muscle Cells/metabolism , Nuclear Matrix/metabolism , Animals , Cell Cycle , DNA/metabolism , DNA-Binding Proteins/metabolism , Down-Regulation , Drosophila Proteins/genetics , Drosophila melanogaster/cytology , Endoreduplication , Larva/metabolism , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Microtubules/metabolism , Muscles/metabolism , Mutation/genetics , Nuclear Proteins/metabolism , RNA Polymerase II/metabolism , Time Factors , Transcription Factors/metabolismABSTRACT
The musculoskeletal and proprioceptive sensory systems exhibit intricate crosstalk between force generation, force sensation and force transmission, all of which are critical for coordinated animal locomotion. Recent developmental studies of the musculoskeletal and proprioceptive units of the invertebrate Drosophila embryo, have revealed several common molecular and structural principles mediating the formation of each of these systems. These common principles, as well as the differences between the developmental programs of the two systems, are discussed. Interestingly, a molecular pathway triggered by the Neuregulin/Vein ligand-dependent activation of the epidermal growth factor receptor (EGFR) pathway, which upregulates the early growth response (EGR)-like transcription factor Stripe, is utilized not only by the Drosophila muscle-tendon and proprioceptive organ-ectoderm attachment, but also by their vertebrate counterparts. An additional theme that has been observed during the development of the musculoskeletal system in both invertebrates and vertebrates is the functional importance of the extracellular matrix and its adhesion receptors. The contribution of mechanical forces to proper junction formation between muscles and tendons and between the sensory cap/ligament cells and their epidermal attachment cells is discussed. The structural and genetic similarities between the musculoskeletal and the proprioceptive systems offer new perspectives as to their common developmental nature.
Subject(s)
Drosophila melanogaster/embryology , Gene Expression Regulation, Developmental , Movement , Animals , Cell Differentiation , DNA-Binding Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/genetics , Ectoderm/metabolism , Embryo, Nonmammalian/metabolism , ErbB Receptors/metabolism , Extracellular Matrix/metabolism , Fibroblast Growth Factors/metabolism , Ligands , Muscles/embryology , Muscles/metabolism , Neuregulin-1/metabolism , Sensation , Signal Transduction , Tendons/embryology , Tendons/metabolism , Transcription Factors/metabolism , Up-RegulationABSTRACT
Slit cleavage into N-terminal and C-terminal polypeptides is essential for restricting the range of Slit activity. Although the Slit cleavage site has been characterized previously and is evolutionally conserved, the identity of the protease that cleaves Slit remains elusive. Our previous analysis indicated that Slit cleavage is essential to immobilize the active Slit-N at the tendon cell surfaces, mediating the arrest of muscle elongation. In an attempt to identify the protease required for Slit cleavage we performed an RNAi-based assay in the ectoderm and followed the process of elongation of the lateral transverse muscles toward tendon cells. The screen led to the identification of the Drosophila homolog of pheromone convertase 2 (PC2), Amontillado (Amon), as an essential protease for Slit cleavage. Further analysis indicated that Slit mobility on SDS polyacrylamide gel electrophoresis (SDS-PAGE) is slightly up-shifted in amon mutants, and its conventional cleavage into the Slit-N and Slit-C polypeptides is attenuated. Consistent with the requirement for amon to promote Slit cleavage and membrane immobilization of Slit-N, the muscle phenotype of amon mutant embryos was rescued by co-expressing a membrane-bound form of full-length Slit lacking the cleavage site and knocked into the slit locus. The identification of a novel protease component essential for Slit processing may represent an additional regulatory step in the Slit signaling pathway.
ABSTRACT
Skeletal muscle is highly sensitive to mutations in genes that participate in membrane stability and cellular attachment, which often leads to muscular dystrophy. Here we show that Thrombospondin-4 (Thbs4) regulates skeletal muscle integrity and its susceptibility to muscular dystrophy through organization of membrane attachment complexes. Loss of the Thbs4 gene causes spontaneous dystrophic changes with aging and accelerates disease in 2 mouse models of muscular dystrophy, while overexpression of mouse Thbs4 is protective and mitigates dystrophic disease. In the myofiber, Thbs4 selectively enhances vesicular trafficking of dystrophin-glycoprotein and integrin attachment complexes to stabilize the sarcolemma. In agreement, muscle-specific overexpression of Drosophila Tsp or mouse Thbs4 rescues a Drosophila model of muscular dystrophy with augmented membrane residence of ßPS integrin. This functional conservation emphasizes the fundamental importance of Thbs' as regulators of cellular attachment and membrane stability and identifies Thbs4 as a potential therapeutic target for muscular dystrophy.
Subject(s)
Gene Expression , Membranes/metabolism , Muscle, Skeletal/metabolism , Myofibrils/metabolism , Thrombospondins/metabolism , Animals , Disease Models, Animal , Drosophila , Mice , Muscular Dystrophies/physiopathology , Muscular Dystrophies/prevention & controlABSTRACT
Contractile muscle fibers produce enormous intrinsic forces during contraction/relaxation waves. These forces are directly applied to their cytoplasmic organelles including mitochondria, sarcoplasmic reticulum, and multiple nuclei. Data from our analysis of Drosophila larval somatic muscle fibers suggest that an intricate network of organized microtubules (MT) intermingled with Spectrin-Repeat-Containing Proteins (SRCPs) are major structural elements that protect muscle organelles and maintain their structure and position during muscle contraction. Whereas the perinuclear MT network provides structural rigidity to the myonucleus, the SRCPs Nesprin and Spectraplakin form semiflexible filamentous biopolymer networks, providing nuclei with the elasticity required to resist the contractile cytoplasmic forces produced by the muscle. Spectrin repeats are domains found in numerous structural proteins, which are able to unfold under tension and are subject to mechanical stresses in the cell. This unique composite scaffold combines rigidity and resilience in order to neutralize the oscillating cellular forces occurring during muscle contraction/relaxation waves and thereby protect myonuclei. We suggest that the elastic properties of SRCPs are critical for nuclear protection and proper function in muscle fibers.
Subject(s)
Biopolymers , Cell Nucleus , Microfilament Proteins , Microtubules , Muscles , Animals , Drosophila , Microfilament Proteins/metabolism , Tissue EngineeringABSTRACT
The formation of functional musculoskeletal system relies on proper connectivity between muscles and their corresponding tendon cells. In Drosophila, larval muscles are born during early embryonic stages, and elongate toward tendons that are embedded within the ectoderm in later. The Slit/Robo signaling pathway had been implicated in the process of muscle elongation toward tendons. Here we discuss our recent findings regarding the critical contribution of Slit cleavage for immobilization and stabilization of the Slit signal on the tendon cells. Slit cleavage produces 2 polypeptides, the N-terminal Slit-N, which is extremely stable, undergoes oligomerization, and associates with the tendon cell surfaces, and the C-terminal Slit-C, which rapidly degrades. Slit cleavage leads to immobilization of Slit signaling on tendons, leading to a short-range repulsion, which eventually arrest further muscle elongation. Robo2, which is co-expressed with Slit by the tendon cells facilitates Slit cleavage. This activity does not require the cytoplasmic signaling domain of Robo2. We suggest that Robo2-dependent Slit cleavage, and the formation of Slit-N oligomers on the tendon cell surfaces direct muscle elongation, and provide a stop signal for the approaching muscle, through binding to Robo and Robo3 receptors expressed by the muscles.
Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/embryology , Gene Expression Regulation, Developmental/physiology , Muscles/embryology , Nerve Tissue Proteins/metabolism , Animals , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Gene Knock-In Techniques , Muscles/metabolism , Nerve Tissue Proteins/genetics , Protein Binding , Receptors, Cell Surface/metabolismABSTRACT
Coordinated locomotion of an organism relies on the development of proper musculoskeletal connections. In Drosophila, the Slit-Robo signaling pathway guides muscles to tendons. Here, we show that the Slit receptor Roundabout 2 (Robo2) plays a non-cell-autonomous role in directing muscles to their corresponding tendons. Robo2 is expressed by tendons, and its non-signaling activity in these cells promotes Slit cleavage, producing a cleaved Slit N-terminal guidance signal that provides short-range signaling into muscles. Consistently, robo2 mutant embryos exhibited a muscle phenotype similar to that of slit, which could not be rescued by muscle-specific Robo2 expression but rather by ectodermally derived Robo2. Alternatively, this muscle phenotype could be induced by tendon-specific robo2 RNAi. We further show that membrane immobilization of Slit or its N-terminal cleaved form (Slit-N) on tendons bypasses the functional requirement for Robo2 in tendons, verifying that the major role of Robo2 is to promote the association of Slit with the tendon cell membrane. Slit-N tends to oligomerize whereas full-length uncleavable Slit does not. It is therefore proposed that Slit-N oligomers, produced at the tendon membrane by Robo2, signal to the approaching muscle by combined Robo1 and Robo3 activity. These findings establish a Robo2-mediated mechanism, independent of signaling, that is essential to limiting Slit distribution and which might be relevant to the regulation of Slit-mediated short-range signaling in additional systems.
Subject(s)
Drosophila Proteins/physiology , Gene Expression Regulation, Developmental , Muscles/embryology , Mutation , Nerve Tissue Proteins/physiology , Receptors, Immunologic/physiology , Tendons/embryology , Animals , Body Patterning , Drosophila Proteins/genetics , Drosophila melanogaster/embryology , Gene Knock-In Techniques , Heterozygote , Homozygote , Nerve Tissue Proteins/genetics , Phenotype , Protein Structure, Tertiary , RNA Interference , Receptors, Immunologic/genetics , Signal Transduction , Roundabout ProteinsABSTRACT
Muscle nuclei are exposed to variable cytoplasmic strain produced by muscle contraction and relaxation, but their morphology remains stable. Still, the mechanism responsible for maintaining myonuclear architecture, and its importance, is currently elusive. Herein, we uncovered a unique myonuclear scaffold in Drosophila melanogaster larval muscles, exhibiting both elastic features contributed by the stretching capacity of MSP300 (nesprin) and rigidity provided by a perinuclear network of microtubules stabilized by Shot (spectraplakin) and EB1. Together, they form a flexible perinuclear shield that protects myonuclei from intrinsic or extrinsic forces. The loss of this scaffold resulted in significantly aberrant nuclear morphology and subsequently reduced levels of essential nuclear factors such as lamin A/C, lamin B, and HP1. Overall, we propose a novel mechanism for maintaining myonuclear morphology and reveal its critical link to correct levels of nuclear factors in differentiated muscle fibers. These findings may shed light on the underlying mechanism of various muscular dystrophies.
Subject(s)
Cell Nucleus/physiology , Drosophila Proteins/metabolism , Drosophila Proteins/physiology , Drosophila melanogaster/metabolism , Microfilament Proteins/metabolism , Microfilament Proteins/physiology , Microtubule-Associated Proteins/metabolism , Muscle Proteins/physiology , Actins/metabolism , Animals , Drosophila melanogaster/ultrastructure , Elasticity , Lamins/metabolism , Larva/metabolism , Larva/ultrastructure , Mitochondria, Muscle/metabolism , Mitochondria, Muscle/ultrastructure , Muscle, Striated/metabolism , Muscle, Striated/ultrastructure , Protein TransportABSTRACT
During organogenesis, secreted signaling proteins direct cell migration towards their target tissue. In Drosophila embryos, developing muscles are guided by signals produced by tendons to promote the proper attachment of muscles to tendons, essential for proper locomotion. Previously, the repulsive protein Slit, secreted by tendon cells, has been proposed to be an attractant for muscle migration. However, our findings demonstrate that through tight control of its distribution, Slit repulsion is used for both directing and arresting muscle migration. We show that Slit cleavage restricts its distribution to tendon cells, allowing it to function as a short-range repellent that directs muscle migration and patterning, and promotes their halt upon reaching the target site. Mechanistically, we show that Slit processing produces a rapidly degraded C-terminal fragment and an active, stable N-terminal polypeptide that is tethered to the tendon cell membrane, which further protects it from degradation. Consistently, the requirement for Slit processing can be bypassed by providing an uncleavable, membrane-bound form of Slit that is stable and is retained on expressing tendon cells. Moreover, muscle elongation appears to be extremely sensitive to Slit levels, as replacing the entire full-length Slit with the stable Slit-N-polypeptide results in excessive repulsion, which leads to a defective muscle pattern. These findings reveal a novel cleavage-dependent regulatory mechanism controlling Slit spatial distribution, which may operate in other Slit-dependent processes.