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The prefusion complex contains paired vesicles. Serial section electron micrographs through a prefusion complex in a wild-type stage 13 embryo. This complex contains about 45 pairs of vesicles distributed among three cells. Bar: (A) 200 nm.
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The events of myoblast fusion in Drosophila are dissected here by combining genetic analysis with light and electron microscopy. We describe a new and essential intermediate step in the process, the formation of a prefusion complex consisting of "paired vesicles." These pairs of vesicles from different cells align with each other across apposed pla...
Similar publications
Dynamic rearrangements of the actin cytoskeleton play a key role in numerous cellular processes. In Drosophila, fusion between a muscle founder cell and a fusion competent myoblast (FCM) is mediated by an invasive, F-actin-enriched podosome-like structure (PLS). Here, we show that the dynamics of the PLS is controlled by Blown fuse (Blow), a cytopl...
Citations
... Interesting candidates are I-BAR domain-containing proteins, well-documented for generating negative membrane curvature on PI(4,5)P 2rich membranes, linking direct membrane deformation to actin polymerization, and inhibiting the lateral diffusion of phosphoinositide molecules (51). Moreover, it was proposed that exocytosis of these vesicles may release fusogenic materials to trigger fusion (52). The analysis of the recent cryo-EM structure of the fusogen Myomaker (53) revealed clusters of positively charged amino acid residues (14 arginine and lysine) facing the cytosolic leaflet, compatible with its binding with phosphoinositides and that could be responsible for Myomaker clustering (Fig. 6F). ...
Myogenesis is a multistep process that requires a spatiotemporal regulation of cell events resulting finally in myoblast fusion into multinucleated myotubes. Most major insights into the mechanisms underlying fusion seem to be conserved from insects to mammals and include the formation of podosome-like protrusions (PLPs) that exert a driving force toward the founder cell. However, the machinery that governs this process remains poorly understood. In this study, we demonstrate that MTM1 is the main enzyme responsible for the production of phosphatidylinositol 5-phosphate, which in turn fuels PI5P 4-kinase α to produce a minor and functional pool of phosphatidylinositol 4,5-bisphosphate that concentrates in PLPs containing the scaffolding protein Tks5, Dynamin-2, and the fusogenic protein Myomaker. Collectively, our data reveal a functional crosstalk between a PI-phosphatase and a PI-kinase in the regulation of PLP formation.
... After myoblast recognition, the adhesion of both myoblast types is intensified ( Figure 2B). On subcellular levels, distinct ultrastructural features ( Figure 2C) have been described by using chemical fixation and freeze substitution methods [16][17][18][19][20][21][22]. However, the temporal appearance of some of these structures is still unknown (Figure 2(C1-3)). ...
... Following recognition and adhesion, electron-dense plaques and vesicles have been observed at the site of cell-cell contact (Figure 2(C1,C2)). About 50 of these vesicles can be found in adhering myoblasts where they line up with one another to form pairs across the apposing membranes [17,23]. The function of these vesicles remains unknown. ...
... In Drosophila, Wip is exclusively expressed in fusion-competent myoblasts where it localizes to the site of cell-cell contact [18,20,39]. Another fusion-relevant protein that can be found solely in fusion-competent myoblasts is the PH domain containing protein Blown fuse (Blow) [14,17]. After the recognition and adhesion of myoblasts, Blow is recruited to the site of cell-cell contact where it localizes with F-actin as a dense focus [14,36]. ...
Muscle fibers are multinucleated cells that arise during embryogenesis through the fusion of mononucleated myoblasts. Myoblast fusion is a lifelong process that is crucial for the growth and regeneration of muscles. Understanding the molecular mechanism of myoblast fusion may open the way for novel therapies in muscle wasting and weakness. Recent reports in Drosophila and mammals have provided new mechanistic insights into myoblast fusion. In Drosophila, muscle formation occurs twice: during embryogenesis and metamorphosis. A fundamental feature is the formation of a cell–cell communication structure that brings the apposing membranes into close proximity and recruits possible fusogenic proteins. However, genetic studies suggest that myoblast fusion in Drosophila is not a uniform process. The complexity of the players involved in myoblast fusion can be modulated depending on the type of muscle that is formed. In this review, we introduce the different types of multinucleated muscles that form during Drosophila development and provide an overview in advances that have been made to understand the mechanism of myoblast fusion. Finally, we will discuss conceptual frameworks in cell–cell fusion in Drosophila and mammals.
... These results coincide with our observations where the number of DLM fascicles was constant, but in DVMs in the groups 1 and 2, and despite the small number of samples analyzed, variations in myotube numbers were seen [56]. The regulation of muscle size and number during IFMs formation in D. melanogaster is controlled by a balance between fusion and proliferation during larval and pupal stages [56,[62][63][64]. ...
... Analyzing the DVM development in A. aegypti we observed cells with locations and "teardrop" and spindle morphologies, corresponding to D. melanogaster FCM which migrate from the wing imaginal disc to specific sites where primordia will be developed forming nascent fibers. Our observations of FCM-like cells in A. aegypti DVM primordia allow us to reasonably propose, that in this mosquito FCM cells go through a similar process with active division, migration and location out and inside the primordial myotubes [62]. In addition, in late L3 and early L4 the FCM associate to founder cells, which have distinguishable big and heterochromatic nuclei at the center of nascent myotubes, and, as result of cell fusion, syncytial myotubes are formed. ...
... During A. aegypti L3 instar, myoblasts migrate to form small clusters located at defined places, priming the construction of DVM. Inside the nascent fascicles, founder cells (FC) define the myotube formation recruiting fusion competent cells (FCM), which divide actively, and it is possible to recognize their presence by their characteristic morphologies, that are considered hallmarks of this kind of fusing cells [62]. Myoblast fusion process involve the formation of filopodia and podosome for the initial contact and the organization of prefusion vesicles that coalesce around points where the integration of myoblasts to the myotubes are happening [30,[32][33][34]74], generating syncytial structures. ...
Background
Flying is an essential function for mosquitoes, required for mating and, in the case of females, to get a blood meal and consequently function as a vector. Flight depends on the action of the indirect flight muscles (IFMs), which power the wings beat. No description of the development of IFMs in mosquitoes, including Aedes aegypti, is available.
Methods
A. aegypti thoraces of larvae 3 and larvae 4 (L3 and L4) instars were analyzed using histochemistry and bright field microscopy. IFM primordia from L3 and L4 and IFMs from pupal and adult stages were dissected and processed to detect F-actin labelling with phalloidin-rhodamine or TRITC, or to immunodetection of myosin and tubulin using specific antibodies, these samples were analyzed by confocal microscopy. Other samples were studied using transmission electron microscopy.
Results
At L3–L4, IFM primordia for dorsal-longitudinal muscles (DLM) and dorsal–ventral muscles (DVM) were identified in the expected locations in the thoracic region: three primordia per hemithorax corresponding to DLM with anterior to posterior orientation were present. Other three primordia per hemithorax, corresponding to DVM, had lateral position and dorsal to ventral orientation. During L3 to L4 myoblast fusion led to syncytial myotubes formation, followed by myotendon junctions (MTJ) creation, myofibrils assembly and sarcomere maturation. The formation of Z-discs and M-line during sarcomere maturation was observed in pupal stage and, the structure reached in teneral insects a classical myosin thick, and actin thin filaments arranged in a hexagonal lattice structure.
Conclusions
A general description of A. aegypti IFM development is presented, from the myoblast fusion at L3 to form myotubes, to sarcomere maturation at adult stage. Several differences during IFM development were observed between A. aegypti (Nematoceran) and Drosophila melanogaster (Brachyceran) and, similitudes with Chironomus sp. were observed as this insect is a Nematoceran, which is taxonomically closer to A. aegypti and share the same number of larval stages.
... After this, their actin filaments, which are anchored to the FAs, begin to form the myofibrils. During the next step, the differentiated myocytes fuse with each other to form multinucleated myotubes that contain strong myofibrils, with contractile capacity, essential for the muscle to function [4][5][6][7][8][9][10] . The main purpose of muscle fibers is to develop force by contracting relative to their surrounding ECM, such that the human body and other animals could support themselves and move. ...
During differentiation, skeletal muscle develops mature multinucleated muscle fibers, which could contract to exert force on a substrate. Muscle dysfunction occurs progressively in patients with muscular dystrophy, leading to a loss of the ability to walk and eventually to death. The synthetic glucocorticoid dexamethasone (Dex) has been used therapeutically to treat muscular dystrophy by an inhibition of inflammation, followed by slowing muscle degeneration and stabilizing muscle strength. Here, in mice with muscle injury, we found that Dex significantly promotes muscle regeneration via promoting kinesin-1 motor activity. Nevertheless, how Dex promotes myogenesis through kinesin-1 motors remains unclear. We found that Dex directly increases kinesin-1 motor activity, which is required for the expression of a myogenic marker (muscle myosin heavy chain 1/2), and also for the process of myoblast fusion and the formation of polarized myotubes. Upon differentiation, kinesin-1 mediates the recruitment of integrin β1 onto microtubules allowing delivery of the protein into focal adhesions. Integrin β1-mediated focal adhesion signaling then guides myoblast fusion towards a polarized morphology. By imposing geometric constrains via micropatterns, we have proved that cell adhesion is able to rescue the defects caused by kinesin-1 inhibition during the process of myogenesis. These discoveries reveal a mechanism by which Dex is able to promote myogenesis, and lead us towards approaches that are more efficient in improving skeletal muscle regeneration.
... Review in Advance first posted on Blown fuse. blown fuse (blow) was first identified as an axon guidance mutant (125), but later studies found that the primary defect in the blow mutant embryos was in myoblast fusion (38). Blow is another FCM-specific protein colocalizing with the actin foci, like WASP and Sltr (73). ...
... Ultrastructural analyses of the fusogenic synapse. The first comprehensive EM study of embryonic myoblast fusion identified several characteristic structures along the muscle cell contact zone: paired electron-dense vesicles (termed prefusion complexes); relatively rare electron-dense plaques; and multiple membrane discontinuities (MMDs) with diameters between 50 and 250 nm (38). It was proposed that the vesicles release electron-dense materials to form the plaques, which induce the formation of multiple fusion pores along the muscle cell contact zone (38). ...
... The first comprehensive EM study of embryonic myoblast fusion identified several characteristic structures along the muscle cell contact zone: paired electron-dense vesicles (termed prefusion complexes); relatively rare electron-dense plaques; and multiple membrane discontinuities (MMDs) with diameters between 50 and 250 nm (38). It was proposed that the vesicles release electron-dense materials to form the plaques, which induce the formation of multiple fusion pores along the muscle cell contact zone (38). Subsequent studies over the next ten years reproduced these structures using the same conventional room temperature chemical fixation method, making this a prevailing model describing the distinct steps of myoblast fusion. ...
Cell–cell fusion is indispensable for creating life and building syncytial tissues and organs. Ever since the discovery of cell–cell fusion, how cells join together to form zygotes and multinucleated syncytia has remained a fundamental question in cell and developmental biology. In the past two decades, Drosophila myoblast fusion has been used as a powerful genetic model to unravel mechanisms underlying cell–cell fusion in vivo. Many evolutionarily conserved fusion-promoting factors have been identified and so has a surprising and conserved cellular mechanism. In this review, we revisit key findings in Drosophila myoblast fusion and highlight the critical roles of cellular invasion and resistance in driving cell membrane fusion.
Expected final online publication date for the Annual Review of Genetics Volume 53 is November 25, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... Finally, a critical step of membrane fusion is pore formation. Although in the late 90's electron microscopy studies suggested the presence of multiple membrane pores between fusing cells (Doberstein et al., 1997), a more recent work has proposed that a single pore forms at the tip of one invasive protrusion and expands to engulf the fusing FCM . However, fusion pore formation remains to be fully elucidated. ...
Muscle regeneration relies on a pool of muscle-resident stem cells called satellite cells (MuSCs). MuSCs are quiescent and can activate following muscle injury to give rise to transient amplifying progenitors (myoblasts) that will differentiate and finally fuse together to form new myofibers. During this process, a complex network of signalling pathways is involved, among which, Transforming Growth Factor beta (TGFβ) signalling cascade plays a fundamental role. Previous reports proposed several functions for TGFβ signalling in muscle cells including quiescence, activation and differentiation. However, the impact of TGFβ on myoblast fusion has never been investigated. In this study, we show that TGFβ signalling reduces muscle cell fusion independently of the differentiation step. In contrast, inhibition of TGFβ signalling enhances cell fusion and promotes branching between myotubes. Pharmacological modulation of the pathway in vivo perturbs muscle regeneration after injury. Exogenous addition of TGFβ protein results in a loss of muscle function while inhibition of the TGFβ pathway induces the formation of giant myofibres. Transcriptome analyses and functional assays revealed that TGFβ acts on actin dynamics to reduce cell spreading through modulation of actin-based protrusions. Together our results reveal a signalling pathway that limits mammalian myoblast fusion and add a new level of understanding to the molecular regulation of myogenesis.
... Of the three SH2-SH3 adaptor proteins in Drosophila (Crk, Dock and Drk), both Crk and Dock have been shown to interact with CAMs and actin cytoskeletal regulators, suggesting that they may link cell adhesion with the actin cytoskeleton ( Kim et al., 2007;Kaipa et al., 2013). The stability of the WASP-WIP complex is regulated by a PH domain-containing protein, Blown fuse (Blow) ( Doberstein et al., 1997;Jin et al., 2011). Blow colocalizes with the F-actin focus at the fusogenic synapse and competes with WASP for WIP binding (Jin et al., 2011). ...
Cell-cell fusion is a fundamental process underlying fertilization, development, regeneration and physiology of metazoans. It is a multi-step process involving cell recognition and adhesion, actin cytoskeletal rearrangements, fusogen engagement, lipid mixing and fusion pore formation, ultimately resulting in the integration of two fusion partners. Here, we focus on the asymmetric actin cytoskeletal rearrangements at the site of fusion, known as the fusogenic synapse, which was first discovered during myoblast fusion in Drosophila embryos and later also found in mammalian muscle and non-muscle cells. At the asymmetric fusogenic synapse, actin-propelled invasive membrane protrusions from an attacking fusion partner trigger actomyosin-based mechanosensory responses in the receiving cell. The interplay between the invasive and resisting forces generated by the two fusion partners puts the fusogenic synapse under high mechanical tension and brings the two cell membranes into close proximity, promoting the engagement of fusogens to initiate fusion pore formation. In this Cell Science at a Glance article and the accompanying poster, we highlight the molecular, cellular and biophysical events at the asymmetric fusogenic synapse using Drosophila myoblast fusion as a model.
... The data plotted for 0.2 < ΔA/A 0 < 1.0 are only for theoretical discussion. Experimental observations demonstrate that many vesicles diffuse to the fusion sites and provide additional lipid molecules for the area expansion during cell-cell fusion of Drosophila myoblasts [62], which greatly reduces the area strain of the plasma membrane near the fusion site such that membrane rupture is avoided. ...
Myosin II and spectrin β display mechanosensitive accumulations in invasive protrusions during cell-cell fusion of Drosophila myoblasts. The biochemical inhibition and deactivation of these proteins results in significant fusion defects. Yet, a quantitative understanding of how the protrusion geometry and fusion process are linked to these proteins is still lacking. Here we present a quantitative model to interpret the dependence of the protrusion size and the protrusive force on the mechanical properties and microstructures of the actin cytoskeleton and plasma membrane based on a mean-field theory. We build a quantitative linkage between mechanosensitive accumulation of myosin II and fusion pore formation at the tip of the invasive protrusion through local area dilation. The mechanical feedback loop between myosin II and local deformation suggests that myosin II accumulation possibly reduces the energy barrier and the critical radius of fusion pores. We also analyse the effect of spectrin β on maintaining the proper geometry of the protrusions required for the success of cell-cell fusion.
... Формирование синцития мышечных волокон у эмбриона дрозофилы происходит в результате слияния коммитированных к этому процессу миобластов (fusion-competent myoblasts -FCM) с главным миобластом, так называемой клеткойосновательницей (founder cell -FC) или с растущей миотубой (рис. 1) [1,2]. Процесс слияния миобластов начинается с миграции и адгезии гетеротипичных клеток и заканчивается формированием "синапса слияния" ("fusogenic synapse") [3][4][5][6]. В это время у FCM появляются F-актиновые структуры, называемые "фокусами схождения пучков актина". Они обогащаются молекулами клеточной адгезии и формируют вырост размером ~2 мкм с динамичными пальцевидными отростками, которые внедряются в FC или в рас-Сокращения: FC -клетка-основательница (Founder Cell); FCM -коммитированные к слиянию миобласты (Fusion Competent Myoblasts); PLS -подосома-подобные структуры (Рodosome Like Structures); NPFфактор нуклеации (Nucleation Promoting Factor); CAM -молекулы клеточной адгезии (Сell Adhesion Molecules); DAH -гипотеза дифференциальной адгезии (Differential Adhesion Hypothesis). ...
... Затем в течение 5-30 мин актиновые фокусы исчезают, поры расширяются и сливаются, процесс полного слияния клеток завершается [5,6]. Последовательность этих событий описана во многом благодаря генетическому скринингу партнеровучастников этого процесса [1,2], получению прижизненных и фиксированных образцов, позволяющих найти отличия в организации и динамике накопления актина в FC по сравнению с FCM [5][6][7]11], и ультраструктурному анализу сливающихся клеток [3,6,12]. Чтобы показать возможности дрозофилы в такого рода исследованиях, мы остановимся на механизмах, лежащих в основе формирования фокусов актина в готовых к слиянию миобластах. ...
... Прояснить роль WASP в центрах накопления актина помогло изучение мутантов по гену blown fuse (blow) [11]. Мутанты blow были обнаружены в результате скрининга генов, нарушающих слияние миобластов [3], однако функция Blow, белка с доменом, гомологичным плекстрину (PH -pleckstrin homology), долгое время оставалась неизвестной, пока не было выявлено непрямое взаимодействие Blow со специфической молекулой клеточной адгезии Sticks and Stones (Sns) [11], и показан прямой контакт Blow с dWIP. Сам dWIP связывается с WASP и регулирует его стабильность и направленность на структуры клетки [21]. ...
This review analyzes three studies carried out on Drosophila, which resulted in discoveries that would be impossible while using other subjects. Thanks to these discoveries, events accompanying the myoblast fusion process, the oocyte polarization, and the functioning of supracellular linear actomyosin cable-like structures coordinating the polarization of the cytoskeleton of the cell can be described in detail.
... FCMs (Doberstein et al., 1997 ;Richardson et al., 2008). Après reconnaissance et adhésion des FCs/FCMs via les complexes Sns-Hbs/Duf-Rhs une synapse fusogénique (SF) se forme à l'interface entre les deux cellules. ...
... Cette tension va alors induire le recrutement des protéines fusogéniques et de protéines impliquées dans la formation des pores membranaires indispensables à la fusion (Shilagardi et al., 2013). Cette dernière étape de la fusion est moins détaillée et demande encore de nouvelles investigations, même si on connait déjà le rôle des modulateurs du cytosquelette d'actine dans la formation et l'extension des pores ainsi que celui de l'exocytose (Doberstein et al., 1997 ;Kim et al., 2007). ...
Les précurseurs de muscles adultes (ou AMPs) représentent une population transitoire de cellules souches musculaires chez la Drosophile. Ces cellules dérivent du mésoderme embryonnaire et sont caractérisées par l’activation de la voie Notch ainsi que par le maintien d’une forte expression du facteur de transcription Twist (de type basic helix loop helix). La répartition des AMPs chez la larve est établie au cours de l’embryogénèse de manière stéréotypée : on distingue 6 AMPs par hémisegment abdominal en position ventrale, latérale, dorso-latérale et dorsale. Après spécification et jusqu’au début du deuxième stade larvaire, les AMPs sont maintenues dans un état de quiescence, au cours duquel, les AMPs sont en contact étroit avec les fibres musculaires et les axones moteurs. L’objectif principal de mon projet de thèse a été de caractériser le rôle et le comportement de ces cellules AMPs au cours de l’embryogenèse. Dans un premier temps, j’ai pu montrer que les AMPs sont capables d’attirer les axones moteurs responsables de l’innervation des muscles. Cette attraction passe par l’établissement de contacts entre ces deux tissus via une forte dynamique filopodiale. De plus, une sous-population d’AMPs dans la région latérale est responsable de la formation d’une des branches nerveuses qui innervera le muscle de bordure de segment. La deuxième partie de ce projet s’est concentrée sur l’étude du comportement de ces cellules, ainsi que sur l’analyse du rôle des gènes exprimés dans les AMPs. J’ai ainsi pu mettre en évidence de nouvelles interactions entre les AMPs latérales et leur environnement musculaire. De plus, j’ai identifié de nouveaux marqueurs des AMPs tels que le récepteur Unc-5, la métalloprotéase MMP1 et la protéine de guidance Sidestep. L’une des contributions majeures de ce projet a été de pouvoir établir pour la première fois le rôle des cellules souches musculaires AMPs dans la mise en place du système nerveux moteur. L’ajout de ce nouvel acteur va permettre une meilleure compréhension des mécanismes de guidance des axones au cours de l’innervation musculaire.
