RESUMEN
The number of adult myofibers in Drosophila is determined by the number of founder myoblasts selected from a myoblast pool, a process governed by fibroblast growth factor (FGF) signaling. Here, we show that loss of cabeza (caz) function results in a reduced number of adult founder myoblasts, leading to a reduced number and misorientation of adult dorsal abdominal muscles. Genetic experiments revealed that loss of caz function in both adult myoblasts and neurons contributes to caz mutant muscle phenotypes. Selective overexpression of the FGF receptor Htl or the FGF receptor-specific signaling molecule Stumps in adult myoblasts partially rescued caz mutant muscle phenotypes, and Stumps levels were reduced in caz mutant founder myoblasts, indicating FGF pathway deregulation. In both adult myoblasts and neurons, caz mutant muscle phenotypes were mediated by increased expression levels of Xrp1, a DNA-binding protein involved in gene expression regulation. Xrp1-induced phenotypes were dependent on the DNA-binding capacity of its AT-hook motif, and increased Xrp1 levels in founder myoblasts reduced Stumps expression. Thus, control of Xrp1 expression by Caz is required for regulation of Stumps expression in founder myoblasts, resulting in correct founder myoblast selection.
Asunto(s)
Proteínas de Drosophila/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Mioblastos/metabolismo , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Factor de Transcripción TFIID/metabolismo , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Drosophila , Proteínas de Drosophila/genética , Desarrollo de Músculos , Mioblastos/citología , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas de Unión al ARN/genética , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Factor de Transcripción TFIID/genéticaRESUMEN
Cells in developing organisms are subjected to particular mechanical forces that shape tissues and instruct cell fate decisions. How these forces are sensed and transmitted at the molecular level is therefore an important question, one that has mainly been investigated in cultured cells in vitro. Here, we elucidate how mechanical forces are transmitted in an intact organism. We studied Drosophila muscle attachment sites, which experience high mechanical forces during development and require integrin-mediated adhesion for stable attachment to tendons. Therefore, we quantified molecular forces across the essential integrin-binding protein Talin, which links integrin to the actin cytoskeleton. Generating flies expressing 3 Förster resonance energy transfer (FRET)-based Talin tension sensors reporting different force levels between 1 and 11 piconewton (pN) enabled us to quantify physiologically relevant molecular forces. By measuring primary Drosophila muscle cells, we demonstrate that Drosophila Talin experiences mechanical forces in cell culture that are similar to those previously reported for Talin in mammalian cell lines. However, in vivo force measurements at developing flight muscle attachment sites revealed that average forces across Talin are comparatively low and decrease even further while attachments mature and tissue-level tension remains high. Concomitantly, the Talin concentration at attachment sites increases 5-fold as quantified by fluorescence correlation spectroscopy (FCS), suggesting that only a small proportion of Talin molecules are mechanically engaged at any given time. Reducing Talin levels at late stages of muscle development results in muscle-tendon rupture in the adult fly, likely as a result of active muscle contractions. We therefore propose that a large pool of adhesion molecules is required to share high tissue forces. As a result, less than 15% of the molecules experience detectable forces at developing muscle attachment sites at the same time. Our findings define an important new concept of how cells can adapt to changes in tissue mechanics to prevent mechanical failure in vivo.
Asunto(s)
Desarrollo de Músculos/fisiología , Sarcómeros/metabolismo , Talina/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Animales , Western Blotting , Células Cultivadas , Drosophila , Matriz Extracelular/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Adhesiones Focales/metabolismo , Adhesiones Focales/fisiología , Integrinas/genética , Integrinas/metabolismo , Masculino , Desarrollo de Músculos/genética , Fibras Musculares Esqueléticas/metabolismo , Unión Proteica , Talina/genética , Tendones/metabolismoRESUMEN
Sarcomeres are stereotyped force-producing mini-machines of striated muscles. Each sarcomere contains a pseudocrystalline order of bipolar actin and myosin filaments, which are linked by titin filaments. During muscle development, these three filament types need to assemble into long periodic chains of sarcomeres called myofibrils. Initially, myofibrils contain immature sarcomeres, which gradually mature into their pseudocrystalline order. Despite the general importance, our understanding of myofibril assembly and sarcomere maturation in vivo is limited, in large part because determining the molecular order of protein components during muscle development remains challenging. Here, we applied polarization-resolved microscopy to determine the molecular order of actin during myofibrillogenesis in vivo. This method revealed that, concomitantly with mechanical tension buildup in the myotube, molecular actin order increases, preceding the formation of immature sarcomeres. Mechanistically, both muscle and nonmuscle myosin contribute to this actin order gain during early stages of myofibril assembly. Actin order continues to increase while myofibrils and sarcomeres mature. Muscle myosin motor activity is required for the regular and coordinated assembly of long myofibrils but not for the high actin order buildup during sarcomere maturation. This suggests that, in muscle, other actin-binding proteins are sufficient to locally bundle or cross-link actin into highly regular arrays.
Asunto(s)
Citoesqueleto de Actina/ultraestructura , Actinas/metabolismo , Drosophila melanogaster/ultraestructura , Miofibrillas/ultraestructura , Pupa/ultraestructura , Sarcómeros/ultraestructura , Citoesqueleto de Actina/metabolismo , Actinas/ultraestructura , Animales , Fenómenos Biomecánicos , Conectina/metabolismo , Conectina/ultraestructura , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Vuelo Animal/fisiología , Microscopía de Polarización/métodos , Miofibrillas/metabolismo , Miosinas/metabolismo , Miosinas/ultraestructura , Pupa/crecimiento & desarrollo , Pupa/metabolismo , Sarcómeros/metabolismoRESUMEN
Muscle forces are produced by repeated stereotypical actomyosin units called sarcomeres. Sarcomeres are chained into linear myofibrils spanning the entire muscle fiber. In mammalian body muscles, myofibrils are aligned laterally, resulting in their typical cross-striated morphology. Despite this detailed textbook knowledge about the adult muscle structure, it is still unclear how cross-striated myofibrils are built in vivo Here, we investigate the morphogenesis of Drosophila abdominal muscles and establish them as an in vivo model for cross-striated muscle development. By performing live imaging, we find that long immature myofibrils lacking a periodic actomyosin pattern are built simultaneously in the entire muscle fiber and then align laterally to give mature cross-striated myofibrils. Interestingly, laser micro-lesion experiments demonstrate that mechanical tension precedes the formation of the immature myofibrils. Moreover, these immature myofibrils do generate spontaneous Ca2+-dependent contractions in vivo, which, when chemically blocked, result in cross-striation defects. Taken together, these results suggest a myofibrillogenesis model in which mechanical tension and spontaneous muscle twitching synchronize the simultaneous self-organization of different sarcomeric protein complexes to build highly regular cross-striated myofibrils spanning the length of large muscle fibers.
Asunto(s)
Drosophila melanogaster/fisiología , Músculo Esquelético/fisiología , Estrés Mecánico , Abdomen/fisiología , Animales , Rayos Láser , Modelos Biológicos , Morfogénesis , Contracción Muscular , Desarrollo de Músculos , Miofibrillas/metabolismo , Optogenética , Sarcómeros/metabolismoRESUMEN
Parkinson's disease (PD)-associated Pink1 and Parkin proteins are believed to function in a common pathway controlling mitochondrial clearance and trafficking. Glial cell line-derived neurotrophic factor (GDNF) and its signaling receptor Ret are neuroprotective in toxin-based animal models of PD. However, the mechanism by which GDNF/Ret protects cells from degenerating remains unclear. We investigated whether the Drosophila homolog of Ret can rescue Pink1 and park mutant phenotypes. We report that a signaling active version of Ret (Ret(MEN2B) rescues muscle degeneration, disintegration of mitochondria and ATP content of Pink1 mutants. Interestingly, corresponding phenotypes of park mutants were not rescued, suggesting that the phenotypes of Pink1 and park mutants have partially different origins. In human neuroblastoma cells, GDNF treatment rescues morphological defects of PINK1 knockdown, without inducing mitophagy or Parkin recruitment. GDNF also rescues bioenergetic deficits of PINK knockdown cells. Furthermore, overexpression of Ret(MEN2B) significantly improves electron transport chain complex I function in Pink1 mutant Drosophila. These results provide a novel mechanism underlying Ret-mediated cell protection in a situation relevant for human PD.
Asunto(s)
Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/fisiología , Drosophila melanogaster/genética , Mitocondrias Musculares/ultraestructura , Atrofia Muscular/prevención & control , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Proto-Oncogénicas c-ret/fisiología , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Autofagia , Línea Celular Tumoral , Modelos Animales de Enfermedad , Dopamina/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/crecimiento & desarrollo , Complejo I de Transporte de Electrón/fisiología , Genes Letales , Factor Neurotrófico Derivado de la Línea Celular Glial/farmacología , Humanos , Neuroblastoma/patología , Neuronas/ultraestructura , Consumo de Oxígeno , Enfermedad de Parkinson , Fenotipo , Proteínas Quinasas/deficiencia , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas c-ret/genética , Pupa , Transducción de Señal/fisiología , Ubiquitina-Proteína Ligasas/deficiencia , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
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.
Asunto(s)
Proteínas de Drosophila/metabolismo , Músculos/citología , Músculos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Western Blotting , Movimiento Celular/genética , Movimiento Celular/fisiología , Drosophila , Proteínas de Drosophila/genética , Inmunoprecipitación , Modelos Teóricos , Proteínas del Tejido Nervioso/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Tendones/citología , Tendones/metabolismoRESUMEN
In Drosophila, fibrillar flight muscles (IFMs) enable flight, while tubular muscles mediate other body movements. Here, we use RNA-sequencing and isoform-specific reporters to show that spalt major (salm) determines fibrillar muscle physiology by regulating transcription and alternative splicing of a large set of sarcomeric proteins. We identify the RNA-binding protein Arrest (Aret, Bruno) as downstream of salm. Aret shuttles between the cytoplasm and nuclei and is essential for myofibril maturation and sarcomere growth of IFMs. Molecularly, Aret regulates IFM-specific splicing of various salm-dependent sarcomeric targets, including Stretchin and wupA (TnI), and thus maintains muscle fiber integrity. As Aret and its sarcomeric targets are evolutionarily conserved, similar principles may regulate mammalian muscle morphogenesis.
Asunto(s)
Empalme Alternativo/fisiología , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Músculo Esquelético/metabolismo , Miofibrillas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Empalme Alternativo/genética , Animales , Drosophila , Drosophila melanogasterRESUMEN
Flying insects oscillate their wings at high frequencies of up to 1,000 Hz and produce large mechanical forces of 80 W per kilogram of muscle. They utilize a pair of perpendicularly oriented indirect flight muscles that contain fibrillar, stretch-activated myofibres. In contrast, all other, more slowly contracting, insect body muscles have a tubular muscle morphology. Here we identify the transcription factor Spalt major (Salm) as a master regulator of fibrillar flight muscle fate in Drosophila. salm is necessary and sufficient to induce fibrillar muscle fate. salm switches the entire transcriptional program from tubular to fibrillar fate by regulating the expression and splicing of key sarcomeric components specific to each muscle type. Spalt function is conserved in insects evolutionarily separated by 280 million years. We propose that Spalt proteins switch myofibres from tubular to fibrillar fate during development, a function potentially conserved in the vertebrate heart--a stretch-activated muscle sharing features with insect flight muscle.
Asunto(s)
Evolución Biológica , Secuencia Conservada , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/crecimiento & desarrollo , Proteínas de Homeodominio/metabolismo , Músculos/anatomía & histología , Músculos/fisiología , Factores de Transcripción/metabolismo , Empalme Alternativo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Systematic genetic approaches have provided deep insight into the molecular and cellular mechanisms that operate in simple unicellular organisms. For multicellular organisms, however, the pleiotropy of gene function has largely restricted such approaches to the study of early embryogenesis. With the availability of genome-wide transgenic RNA interference (RNAi) libraries in Drosophila, it is now possible to perform a systematic genetic dissection of any cell or tissue type at any stage of the lifespan. Here we apply these methods to define the genetic basis for formation and function of the Drosophila muscle. We identify a role in muscle for 2,785 genes, many of which we assign to specific functions in the organization of muscles, myofibrils or sarcomeres. Many of these genes are phylogenetically conserved, including genes implicated in mammalian sarcomere organization and human muscle diseases.
Asunto(s)
Drosophila melanogaster/embriología , Genes de Insecto/genética , Animales , Biología Computacional , Estudio de Asociación del Genoma Completo , Biblioteca Genómica , Larva , Masculino , Músculos/embriología , Interferencia de ARNRESUMEN
The development and molecular composition of muscle tissue is evolutionarily conserved. Drosophila is a powerful in vivo model system to investigate muscle morphogenesis and function. Here, we provide a short and comprehensive overview of the important developmental steps to build Drosophila body muscle in embryos, larvae and pupae. We describe key methods, including muscle histology, live imaging and genetics, to study these steps at various developmental stages and include simple behavioural assays to assess muscle function in larvae and adults. We list valuable antibodies and fly strains that can be used for these different methods. This overview should guide the reader to choose the best marker or the appropriate method to obtain high quality muscle morphogenesis data in Drosophila.
Asunto(s)
Biología Evolutiva/métodos , Drosophila/crecimiento & desarrollo , Desarrollo de Músculos/genética , Animales , Drosophila/genética , Embrión no Mamífero , Larva/genética , Larva/crecimiento & desarrollo , Pupa/genética , Pupa/crecimiento & desarrolloRESUMEN
Drosophila is a well-established genetic model organism: thousands of point mutations, deficiencies or transposon insertions are available from stock centres. However, to date, it is still difficult to modify a specific gene locus in a defined manner. A potential solution is the application of transcription activator-like effector nucleases (TALENs), which have been used successfully to mutate genes in various model organisms. TALENs are constructed by fusion of TALE proteins to the endonuclease FokI, resulting in artificial, sequence-specific endonucleases. They induce double strand breaks, which are either repaired by error-prone non-homologous end joining (NHEJ) or homology directed repair (HDR). We developed a simple TALEN-based protocol to mutate any gene of interest in Drosophila within approximately 2 months. We inject mRNA coding for two TALEN pairs targeting the same gene into embryos, employ T7 endonuclease I screening of pooled F1 flies to identify mutations and generate a stable mutant stock in the F3 generation. We illustrate the efficacy of our strategy by mutating CG11617, a previously uncharacterized putative transcription factor with an unknown function in Drosophila. This demonstrates that TALENs are a reliable and efficient strategy to mutate any gene of interest in Drosophila.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila/genética , Mutagénesis Sitio-Dirigida/métodos , Animales , Desoxirribonucleasas/química , ARN Mensajero/química , Genética InversaRESUMEN
Muscles coordinate body movements throughout the animal kingdom. Each skeletal muscle is built of large, multi-nucleated cells, called myofibers, which are classified into several functionally distinct types. The typical fiber-type composition of each muscle arises during development, and in mammals is extensively adjusted in response to postnatal exercise. Understanding how functionally distinct muscle fiber-types arise is important for unraveling the molecular basis of diseases from cardiomyopathies to muscular dystrophies. In this review, we focus on recent advances in Drosophila and mammals in understanding how muscle fiber-type specification is controlled by the regulation of transcription and alternative splicing. We illustrate the cooperation of general myogenic transcription factors with muscle fiber-type specific transcriptional regulators as a basic principle for fiber-type specification, which is conserved from flies to mammals. We also examine how regulated alternative splicing of sarcomeric proteins in both flies and mammals can directly instruct the physiological and biophysical differences between fiber-types. Thus, research in Drosophila can provide important mechanistic insight into muscle fiber specification, which is relevant to homologous processes in mammals and to the pathology of muscle diseases.
Asunto(s)
Empalme Alternativo , Diferenciación Celular , Regulación del Desarrollo de la Expresión Génica , Desarrollo de Músculos/genética , Fibras Musculares Esqueléticas/citología , Proteínas Musculares/metabolismo , Transcripción Genética , Animales , Linaje de la Célula/genética , Drosophila , Mamíferos , Fibras Musculares Esqueléticas/metabolismoRESUMEN
Regular spatial patterns are ubiquitous forms of organization in nature. In animals, regular patterns can be found from the cellular scale to the tissue scale, and from early stages of development to adulthood. To understand the formation of these patterns, how they assemble and mature, and how they are affected by perturbations, a precise quantitative description of the patterns is essential. However, accessible tools that offer in-depth analysis without the need for computational skills are lacking for biologists. Here, we present PatternJ, a novel toolset to analyze regular one-dimensional patterns precisely and automatically. This toolset, to be used with the popular imaging processing program ImageJ/Fiji, facilitates the extraction of key geometric features within and between pattern repeats in static images and time-lapse series. We validate PatternJ with simulated data and test it on images of sarcomeres from insect muscles and contracting cardiomyocytes, actin rings in neurons, and somites from zebrafish embryos obtained using confocal fluorescence microscopy, STORM, electron microscopy, and brightfield imaging. We show that the toolset delivers subpixel feature extraction reliably even with images of low signal-to-noise ratio. PatternJ's straightforward use and functionalities make it valuable for various scientific fields requiring quantitative one-dimensional pattern analysis, including the sarcomere biology of muscles or the patterning of mammalian axons, speeding up discoveries with the bonus of high reproducibility.
Asunto(s)
Axones , Procesamiento de Imagen Asistido por Computador , Sarcómeros , Somitos , Pez Cebra , Animales , Axones/fisiología , Procesamiento de Imagen Asistido por Computador/métodos , Sarcómeros/ultraestructura , Somitos/embriología , Programas Informáticos , AlgoritmosRESUMEN
Forward genetic screens in model organisms have provided important insights into numerous aspects of development, physiology and pathology. With the availability of complete genome sequences and the introduction of RNA-mediated gene interference (RNAi), systematic reverse genetic screens are now also possible. Until now, such genome-wide RNAi screens have mostly been restricted to cultured cells and ubiquitous gene inactivation in Caenorhabditis elegans. This powerful approach has not yet been applied in a tissue-specific manner. Here we report the generation and validation of a genome-wide library of Drosophila melanogaster RNAi transgenes, enabling the conditional inactivation of gene function in specific tissues of the intact organism. Our RNAi transgenes consist of short gene fragments cloned as inverted repeats and expressed using the binary GAL4/UAS system. We generated 22,270 transgenic lines, covering 88% of the predicted protein-coding genes in the Drosophila genome. Molecular and phenotypic assays indicate that the majority of these transgenes are functional. Our transgenic RNAi library thus opens up the prospect of systematically analysing gene functions in any tissue and at any stage of the Drosophila lifespan.
Asunto(s)
Drosophila melanogaster/genética , Biblioteca Genómica , Interferencia de ARN , Animales , Animales Modificados Genéticamente , Drosophila melanogaster/metabolismo , Exones , Femenino , Masculino , Músculos/metabolismo , Neuronas/metabolismo , Especificidad de Órganos , ARN Mensajero , Ribonucleasa III/metabolismoRESUMEN
Measuring the positions and dynamics of proteins in intact tissues or whole animals is key to understanding protein function. However, to date, this is challenging, as the accessibility of large antibodies to dense tissues is often limited, and fluorescent proteins inserted close to a domain of interest may affect protein function. These complications apply in particular to muscle sarcomeres, arguably one of the most protein-dense assemblies in nature, which complicates studying sarcomere morphogenesis at molecular resolution. Here, we introduce a toolbox of nanobodies recognising various domains of the two Drosophila titin homologs, Sallimus and Projectin, as well as the key sarcomeric proteins Obscurin, α-Actinin, and Zasp52. We verified the superior labelling qualities of our nanobodies in muscle tissue as compared to antibodies. By applying our toolbox to larval muscles, we found a gigantic Sallimus isoform stretching more than 2 µm to bridge the sarcomeric I-band, while Projectin covers almost the entire myosin filaments in a polar orientation. Transgenic expression of tagged nanobodies confirmed their high affinity-binding without affecting target protein function. Finally, adding a degradation signal to anti-Sallimus nanobodies suggested that it is difficult to fully degrade Sallimus in mature sarcomeres; however, expression of these nanobodies caused developmental lethality. These results may inspire the generation of similar toolboxes for other large protein complexes in Drosophila or mammals.
Our muscles are not just for lifting weights. They also keep us alive. For example, our heartbeat is powered by the muscles in the heart wall. Just like other organs in the body, muscles are made up of cells called muscle fibres. Each muscle fibre is divided into many smaller units, or 'sarcomeres', which contain specialised proteins that pull on each other to produce muscle contractions. Although the structure of mature muscles is rather well understood, we know much less about how muscles develop or how they are maintained throughout adult life. Understanding this is especially important in the case of the heart, because its muscle cells are not replaced throughout our lives. Instead, the heart muscle cells we are born with are maintained as we age while working continuously. This means that the proteins within the heart muscle sarcomeres are continuously under mechanical stress and may need to be repaired. How this repair might happen is not well understood. Nanobodies are very small versions of antibodies that recognise and bind to specific protein targets. In biological research, they are used as a tool to observe proteins of interest within cells. This is done by labelling nanobodies, for example, with chemical fluorophores or fluorescent proteins; once labelled, the nanobody binds to its target protein, and scientists can monitor its location and behaviour within the cell. Cells, and even flies, can also be genetically manipulated to produce labelled nanobodies themselves, which has the advantage of visualising the dynamic behaviour of the target protein in the living cell or organism. To better study the proteins in muscle cells, scientists from two different research groups developed a nanobody 'toolbox' that specifically targets sarcomere proteins. First, Loreau et al. made a 'library' of labelled nanobodies targeting different sarcomere proteins in Drosophila melanogaster fruit flies. Second, they used this library of nanobodies to locate several sarcomere proteins in the mature sarcomeres of different fly muscles. Third, using flies that had been genetically altered to produce the labelled nanobodies in their muscle cells, Loreau et al. were able to observe the behaviour of the target proteins in the living muscle. Together, these experiments showed that one protein in Drosophila that is similar to the human sarcomere protein titin has a similar size to the human version, whereas a second Drosophila titin-like protein is shorter and located at a different place in the sarcomere. Both of these proteins work together to stabilise muscle fibres, which is also the role of human titin. The nanobodies generated here are a significant contribution to the tools available to study muscle development and maintenance. Loreau et al. hope that they will help reveal how sarcomere proteins like titin are maintained, especially in the heart, and ultimately how the heart muscle manages to continue working throughout our lives.
Asunto(s)
Sarcómeros , Anticuerpos de Dominio Único , Animales , Conectina/genética , Conectina/metabolismo , Sarcómeros/metabolismo , Drosophila , Anticuerpos de Dominio Único/metabolismo , Animales Modificados Genéticamente , MamíferosRESUMEN
Sarcomeres are the force-producing units of all striated muscles. Their nanoarchitecture critically depends on the large titin protein, which in vertebrates spans from the sarcomeric Z-disc to the M-band and hence links actin and myosin filaments stably together. This ensures sarcomeric integrity and determines the length of vertebrate sarcomeres. However, the instructive role of titins for sarcomeric architecture outside of vertebrates is not as well understood. Here, we used a series of nanobodies, the Drosophila titin nanobody toolbox, recognising specific domains of the two Drosophila titin homologs Sallimus and Projectin to determine their precise location in intact flight muscles. By combining nanobodies with DNA-PAINT super-resolution microscopy, we found that, similar to vertebrate titin, Sallimus bridges across the flight muscle I-band, whereas Projectin is located at the beginning of the A-band. Interestingly, the ends of both proteins overlap at the I-band/A-band border, revealing a staggered organisation of the two Drosophila titin homologs. This architecture may help to stably anchor Sallimus at the myosin filament and hence ensure efficient force transduction during flight.
From ants to humans, the muscles that set an organism in motion are formed of bundles of fiber-like cells which can shorten and lengthen at will. At the microscopic level, changes in muscle cell lengths are underpinned by contractile filaments formed of multiple repeats of a basic unit, known as the sarcomere. Each unit is bookended by intricate 'Z-discs' and features an 'M-band' in its center. Three protein types give a sarcomere its ability to shorten and expand at will: two types of filaments (myosin and actin), which can slide on one another; and a spring-like molecule known as titin, which ensures that the unit does not fall apart by mechanically connecting myosin and actin. More specifically, actin filaments are anchored to the Z-discs and extend towards the M-band, while myosin filaments are centered around the M-band and extend towards the Z-discs. As myosin and actin slide alongside each other, the overlap between the two types of filaments increases or decreases and the whole unit changes its length. In vertebrates, one gigantic molecule of titin spans from the Z-disc to the M-band, linking together actin and myosin filaments and determining the length of the sarcomere. In insects and other invertebrates, however, this single molecule is replaced by two titin proteins known as Projectin and Sallimus. Understanding how these titins work together remains unclear and difficult to study. Traditional approaches are unable to precisely label titin in an environment teaming with other molecules, and they cannot offer the nanometer resolution required to dissect sarcomere organization. As a response, Schueder, Mangeol et al. combined super-resolution microscopy and a new toolbox of labelling molecules known as nanobodies to track the position of Sallimus and Projectin in the flight muscles of fruit flies. These experiments revealed that the two proteins are arranged in tandem along the length of the sarcomere, forming a structure that measures about 350 nm. Sallimus is anchored in the Z-disc and it runs alongside actin until it reaches the end of a myosin filament; there, it overlaps with Projectin for about 10 nm. Projectin then stretches for 250 nm along the length of the beginning myosin filament. These findings confirm the importance of titin in dictating the length of a sarcomere; they suggest that, in invertebrates, this role is split between two proteins, each possibly ruling over a section of the sarcomere. In addition, the work by Schueder, Mangeol et al. demonstrate the value of combining nanobodies and super-resolution microscopy to study complex structures in tissues.
Asunto(s)
Anticuerpos de Dominio Único , Animales , Conectina/genética , Conectina/metabolismo , Drosophila/fisiología , Músculo Esquelético/metabolismo , Miosinas/metabolismo , Sarcómeros/metabolismo , Anticuerpos de Dominio Único/metabolismo , ADN/químicaRESUMEN
Oxidative metabolism is the predominant energy source for aerobic muscle contraction in adult animals. How the cellular and molecular components that support aerobic muscle physiology are put in place during development through their transcriptional regulation is not well understood. Using the Drosophila flight muscle model, we show that the formation of mitochondria cristae harbouring the respiratory chain is concomitant with a large-scale transcriptional upregulation of genes linked with oxidative phosphorylation (OXPHOS) during specific stages of flight muscle development. We further demonstrate using high-resolution imaging, transcriptomic and biochemical analyses that Motif-1-binding protein (M1BP) transcriptionally regulates the expression of genes encoding critical components for OXPHOS complex assembly and integrity. In the absence of M1BP function, the quantity of assembled mitochondrial respiratory complexes is reduced and OXPHOS proteins aggregate in the mitochondrial matrix, triggering a strong protein quality control response. This results in isolation of the aggregate from the rest of the matrix by multiple layers of the inner mitochondrial membrane, representing a previously undocumented mitochondrial stress response mechanism. Together, this study provides mechanistic insight into the transcriptional regulation of oxidative metabolism during Drosophila development and identifies M1BP as a critical player in this process.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Drosophila/genética , Drosophila/metabolismo , Proteínas Portadoras/metabolismo , Factores de Transcripción/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Fosforilación Oxidativa , Estrés Oxidativo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismoRESUMEN
Directed cell migration and target recognition are critical for the development of both the nervous and muscular systems. Molecular mechanisms that control these processes in the nervous system have been intensively studied, whereas those that act during muscle development are still largely uncharacterized. Here we identify a transmembrane protein, Kon-tiki (Kon), that mediates myotube target recognition in the Drosophila embryo. Kon is expressed in a specific subset of myotubes and is required autonomously for these myotubes to recognize their tendon cell targets and to establish a stable connection. Kon is enriched at myotube tips during targeting and signals through the intracellular adaptor Dgrip in a conserved molecular pathway. Forced overexpression of Kon stimulates muscle motility. We propose that Kon promotes directed myotube migration and transduces a target-derived signal that initiates the formation of a stable connection.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Proteínas de la Membrana/metabolismo , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Proteínas Portadoras/química , Movimiento Celular , Secuencia Conservada , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/embriología , Embrión no Mamífero/citología , Regulación del Desarrollo de la Expresión Génica , Masculino , Datos de Secuencia Molecular , Mutación/genética , Mioblastos Esqueléticos/citología , Proteínas del Tejido Nervioso/química , Fenotipo , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Seudópodos/metabolismo , Transducción de SeñalRESUMEN
Proteins are typically not expressed homogeneously in all cells of a complex organism. Within cells, proteins can dynamically change locations, be transported to their destinations, or be degraded upon external signals. Thus, revealing the cellular and subcellular localizations as well as the temporal dynamics of a protein provides important insights into the possible function of the studied protein. Tagging a protein of interest with a genetically encoded fluorophore enables us to follow its expression dynamics in the living organism. Here, we summarize the genetic resources available for tagged Drosophila proteins that assist in studying protein expression and dynamics. We also review the various techniques used in the past and at present to tag a protein of interest with a genetically encoded fluorophore. Comparing the pros and cons of the various techniques guides the reader to judge the suitable applications possible with these tagged proteins in Drosophila.
Asunto(s)
Proteínas de Drosophila , Animales , Sistemas CRISPR-Cas , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Colorantes FluorescentesRESUMEN
Human muscle is a hierarchically organised tissue with its contractile cells called myofibers packed into large myofiber bundles. Each myofiber contains periodic myofibrils built by hundreds of contractile sarcomeres that generate large mechanical forces. To better understand the mechanisms that coordinate human muscle morphogenesis from tissue to molecular scales, we adopted a simple in vitro system using induced pluripotent stem cell-derived human myogenic precursors. When grown on an unrestricted two-dimensional substrate, developing myofibers spontaneously align and self-organise into higher-order myofiber bundles, which grow and consolidate to stable sizes. Following a transcriptional boost of sarcomeric components, myofibrils assemble into chains of periodic sarcomeres that emerge across the entire myofiber. More efficient myofiber bundling accelerates the speed of sarcomerogenesis suggesting that tension generated by bundling promotes sarcomerogenesis. We tested this hypothesis by directly probing tension and found that tension build-up precedes sarcomere assembly and increases within each assembling myofibril. Furthermore, we found that myofiber ends stably attach to other myofibers using integrin-based attachments and thus myofiber bundling coincides with stable myofiber bundle attachment in vitro. A failure in stable myofiber attachment results in a collapse of the myofibrils. Overall, our results strongly suggest that mechanical tension across sarcomeric components as well as between differentiating myofibers is key to coordinate the multi-scale self-organisation of muscle morphogenesis.