RESUMEN
The Rho family of GTPases plays a crucial role in cellular mechanics by regulating actomyosin contractility through the parallel induction of actin and myosin assembly and function. Using exocytosis of large vesicles in the Drosophila larval salivary gland as a model, we followed the spatiotemporal regulation of Rho1, which in turn creates distinct organization patterns of actin and myosin. After vesicle fusion, low levels of activated Rho1 reach the vesicle membrane and drive actin nucleation in an uneven, spread-out pattern. Subsequently, the Rho1 activator RhoGEF2 distributes as an irregular meshwork on the vesicle membrane, activating Rho1 in a corresponding punctate pattern and driving local myosin II recruitment, resulting in vesicle constriction. Vesicle membrane buckling and subsequent crumpling occur at local sites of high myosin II concentrations. These findings indicate that distinct thresholds for activated Rho1 create a biphasic mode of actomyosin assembly, inducing anisotropic membrane crumpling during exocrine secretion.
Asunto(s)
Proteínas de Drosophila , Exocitosis , Miosina Tipo II , Proteínas de Unión al GTP rho , Animales , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Miosina Tipo II/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Proteínas de Unión al GTP rho/genética , Exocitosis/fisiología , Drosophila melanogaster/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Larva/metabolismo , Glándulas Salivales/metabolismo , Glándulas Salivales/citología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Vesículas Secretoras/metabolismoRESUMEN
Exocrine cells utilize large secretory vesicles (LSVs) up to 10 µm in diameter. LSVs fuse with the apical surface, often recruiting actomyosin to extrude their content through dynamic fusion pores. The molecular mechanism regulating pore dynamics remains largely uncharacterized. We observe that the fusion pores of LSVs in the Drosophila larval salivary glands expand, stabilize, and constrict. Arp2/3 is essential for pore expansion and stabilization, while myosin II is essential for pore constriction. We identify several Bin-Amphiphysin-Rvs (BAR) homology domain proteins that regulate fusion pore expansion and stabilization. We show that the I-BAR protein Missing-in-Metastasis (MIM) localizes to the fusion site and is essential for pore expansion and stabilization. The MIM I-BAR domain is essential but not sufficient for localization and function. We conclude that MIM acts in concert with actin, myosin II, and additional BAR-domain proteins to control fusion pore dynamics, mediating a distinct mode of exocytosis, which facilitates actomyosin-dependent content release that maintains apical membrane homeostasis during secretion.
Asunto(s)
Actomiosina , Exocitosis , Vesículas Secretoras , Animales , Citoesqueleto de Actina , Membrana Celular , Proteínas del Citoesqueleto , Drosophila , Vesículas Secretoras/genéticaRESUMEN
Morphogen gradients are known to subdivide a naive cell field into distinct zones of gene expression. Here, we examine whether morphogens can also induce a graded response within such domains. To this end, we explore the role of the Dorsal protein nuclear gradient along the dorsoventral axis in defining the graded pattern of actomyosin constriction that initiates gastrulation in early Drosophila embryos. Two complementary mechanisms for graded accumulation of mRNAs of crucial zygotic Dorsal target genes were identified. First, activation of target-gene expression expands over time from the ventral-most region of high nuclear Dorsal to lateral regions, where the levels are lower, as a result of a Dorsal-dependent activation probability of transcription sites. Thus, sites that are activated earlier will exhibit more mRNA accumulation. Second, once the sites are activated, the rate of RNA Polymerase II loading is also dependent on Dorsal levels. Morphological restrictions require that translation of the graded mRNA be delayed until completion of embryonic cell formation. Such timing is achieved by large introns, which provide a delay in production of the mature mRNAs. Spatio-temporal regulation of key zygotic genes therefore shapes the pattern of gastrulation.
Asunto(s)
Proteínas de Drosophila/genética , Desarrollo Embrionario/genética , Morfogénesis/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , ARN Mensajero/genética , Factores de Transcripción/genética , Animales , Tipificación del Cuerpo/genética , Núcleo Celular/genética , Drosophila melanogaster/genética , Embrión no Mamífero , Gastrulación/genética , Regulación del Desarrollo de la Expresión Génica , Intrones/genética , ARN Polimerasa II/genéticaRESUMEN
Exocrine secretion commonly employs micron-scale vesicles that fuse to a limited apical surface, presenting an extreme challenge for maintaining membrane homeostasis. Using Drosophila melanogaster larval salivary glands, we show that the membranes of fused vesicles undergo actomyosin-mediated folding and retention, which prevents them from incorporating into the apical surface. In addition, the diffusion of proteins and lipids between the fused vesicle and the apical surface is limited. Actomyosin contraction and membrane crumpling are essential for recruiting clathrin-mediated endocytosis to clear the retained vesicular membrane. Finally, we also observe membrane crumpling in secretory vesicles of the mouse exocrine pancreas. We conclude that membrane sequestration by crumpling followed by targeted endocytosis of the vesicular membrane, represents a general mechanism of exocytosis that maintains membrane homeostasis in exocrine tissues that employ large secretory vesicles.
Asunto(s)
Citoesqueleto de Actina/genética , Actomiosina/genética , Exocitosis/genética , Vesículas Secretoras/genética , Animales , Transporte Biológico/genética , Membrana Celular/genética , Clatrina/genética , Drosophila melanogaster/genética , Endocitosis/genética , Glándulas Exocrinas/metabolismo , Homeostasis/genética , Fusión de Membrana/genética , Ratones , Glándulas Salivales/metabolismo , Glándulas Salivales/fisiologíaRESUMEN
BACKGROUND: Muscle myofibrils and sarcomeres present exceptional examples of highly ordered cytoskeletal filament arrays, whose distinct spatial organization is an essential aspect of muscle cell functionality. We utilized ultra-structural analysis to investigate the assembly of myofibrils and sarcomeres within developing myotubes of the indirect flight musculature of Drosophila. RESULTS: A temporal sequence composed of three major processes was identified: subdivision of the unorganized cytoplasm of nascent, multi-nucleated myotubes into distinct organelle-rich and filament-rich domains; initial organization of the filament-rich domains into myofibrils harboring nascent sarcomeric units; and finally, maturation of the highly-ordered pattern of sarcomeric thick (myosin-based) and thin (microfilament-based) filament arrays in parallel to myofibril radial growth. Significantly, organized microtubule arrays were present throughout these stages and exhibited dynamic changes in their spatial patterns consistent with instructive roles. Genetic manipulations confirm these notions, and imply specific and critical guidance activities of the microtubule-based cytoskeleton, as well as structural interdependence between the myosin- and actin-based filament arrays. CONCLUSIONS: Our observations highlight a surprisingly significant, behind-the-scenes role for microtubules in establishment of myofibril and sarcomere spatial patterns and size, and provide a detailed account of the interplay between major cytoskeletal elements in generating these essential contractile myogenic units.
Asunto(s)
Citoesqueleto/metabolismo , Drosophila/crecimiento & desarrollo , Desarrollo de Músculos , Pupa/ultraestructura , Sarcómeros/metabolismo , Animales , Drosophila/ultraestructuraRESUMEN
Buffering variability in morphogen distribution is essential for reproducible patterning. A theoretically proposed class of mechanisms, termed "distal pinning," achieves robustness by combining local sensing of morphogen levels with global modulation of gradient spread. Here, we demonstrate a critical role for morphogen sensing by a gene enhancer, which ultimately determines the final global distribution of the morphogen and enables reproducible patterning. Specifically, we show that, while the pattern of Toll activation in the early Drosophila embryo is robust to gene dosage of its locally produced regulator, WntD, it is sensitive to a single-nucleotide change in the wntD enhancer. Thus, enhancer properties of locally produced WntD directly impinge on the global morphogen profile.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/embriología , Drosophila/genética , Drosophila/metabolismo , Elementos de Facilitación Genéticos/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Animales , Sitios de Unión , Tipificación del Cuerpo , Proteínas de Drosophila/genética , Desarrollo Embrionario/genética , Gástrula/fisiología , Dosificación de Gen , Regulación del Desarrollo de la Expresión Génica , Proteínas HMGB/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , Morfogénesis/genética , Morfogénesis/fisiología , Proteínas Represoras/metabolismo , Alineación de Secuencia , Transducción de Señal/genética , Transducción de Señal/fisiología , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMEN
Establishment of morphogen gradients in the early Drosophila embryo is challenged by a diffusible sextracellular milieu, and by rapid nuclear divisions that occur at the same time. To understand how a sharp gradient is formed within this dynamic environment, we followed the generation of graded nuclear Dorsal protein, the hallmark of pattern formation along the dorso-ventral axis, in live embryos. The dynamics indicate that a sharp extracellular gradient is formed through diffusion-based shuttling of the Spaetzle (Spz) morphogen that progresses through several nuclear divisions. Perturbed shuttling in wntD mutant embryos results in a flat activation peak and aberrant gastrulation. Re-entry of Dorsal into the nuclei at the final division cycle plays an instructive role, as the residence time of Dorsal in each nucleus is translated to the amount of zygotic transcript that will be produced, thereby guiding graded accumulation of specific zygotic transcripts that drive patterned gastrulation. We conclude that diffusion-based ligand shuttling, coupled with dynamic readout, establishes a refined pattern within the diffusible environment of early embryos.
Asunto(s)
Proteínas de Drosophila/fisiología , Drosophila/embriología , Gástrula/metabolismo , Gastrulación , Regulación del Desarrollo de la Expresión Génica , Morfogénesis , Animales , Tipificación del Cuerpo , Núcleo Celular/fisiología , Proteínas de Drosophila/genética , Embrión no Mamífero/fisiología , Femenino , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Mutación , Proteínas Nucleares/fisiología , Fosfoproteínas/fisiología , Transducción de Señal , Factores de Transcripción/fisiologíaRESUMEN
Secretion of adhesive glycoproteins to the lumen of Drosophila melanogaster larval salivary glands is performed by contraction of an actomyosin network assembled around large secretory vesicles, after their fusion to the apical membranes. We have identified a cycle of actin coat nucleation and disassembly that is independent of myosin. Recruitment of active Rho1 to the fused vesicle triggers activation of the formin Diaphanous and actin nucleation. This leads to actin-dependent localization of a RhoGAP protein that locally shuts off Rho1, promoting disassembly of the actin coat. When contraction of vesicles is blocked, the strict temporal order of the recruited elements generates repeated oscillations of actin coat formation and disassembly. Interestingly, different blocks to actin coat disassembly arrested vesicle contraction, indicating that actin turnover is an integral part of the actomyosin contraction cycle. The capacity of F-actin to trigger a negative feedback on its own production may be widely used to coordinate a succession of morphogenetic events or maintain homeostasis.
Asunto(s)
Actinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Retroalimentación Fisiológica , Vesículas Secretoras/metabolismo , Proteínas de Unión al GTP rho/metabolismo , Actomiosina/metabolismo , Amidas/farmacología , Animales , Depsipéptidos/farmacología , Drosophila melanogaster/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Modelos Biológicos , Profilinas/metabolismo , Piridinas/farmacología , Vesículas Secretoras/efectos de los fármacosRESUMEN
Morphogen gradients determine tissue pattern by triggering differential cell responses to distinct morphogen concentrations. The strict quantitative dependence of the emerging patterns on morphogen distribution raises the challenge of buffering variability in morphogen profile to ensure a reproducible outcome. We describe the underlying principles of two modules for buffering morphogen distribution: buffering morphogen amplitude by storing excess morphogen in a limited spatial region, and buffering morphogen spread by pinning morphogen levels at a distal position through global feedback that adjusts morphogen diffusion or degradation across the tissue. We also present concrete examples of patterning systems that implement these modules.
Asunto(s)
Tipificación del Cuerpo , Morfogénesis , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Difusión , Modelos Biológicos , Transducción de SeñalRESUMEN
Activation of extracellular signal regulated kinase (ERK) is used by many signaling pathways to control tissue patterning in a broad range of multicellular organisms. In this issue of Developmental Cell, Johnson et al. (2017) provide an optogenetic approach to manipulate this pathway with high precision and explore its signaling code.
Asunto(s)
Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Luz , Optogenética , Activación Enzimática , Sistema de Señalización de MAP Quinasas , Fosforilación , Transducción de SeñalRESUMEN
Actin-based thin filament arrays constitute a fundamental core component of muscle sarcomeres. We have used formation of the Drosophila indirect flight musculature for studying the assembly and maturation of thin-filament arrays in a skeletal muscle model system. Employing GFP-tagged actin monomer incorporation, we identify several distinct phases in the dynamic construction of thin-filament arrays. This sequence includes assembly of nascent arrays after an initial period of intensive microfilament synthesis, followed by array elongation, primarily from filament pointed-ends, radial growth of the arrays via recruitment of peripheral filaments and continuous barbed-end turnover. Using genetic approaches we have identified Fhos, the single Drosophila homolog of the FHOD sub-family of formins, as a primary and versatile mediator of IFM thin-filament organization. Localization of Fhos to the barbed-ends of the arrays, achieved via a novel N-terminal domain, appears to be a critical aspect of its sarcomeric roles.
Asunto(s)
Actinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/fisiología , Sustancias Macromoleculares/metabolismo , Proteínas de Microfilamentos/metabolismo , Multimerización de Proteína , Sarcómeros/metabolismo , Actinas/genética , Animales , Forminas , Genes Reporteros , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Indirect flight muscles (IFMs) in Drosophila are generated during pupariation by fusion of hundreds of myoblasts with larval muscle templates (myotubes). Live observation of these muscles during the fusion process revealed multiple long actin-based protrusions that emanate from the myotube surface and require Enabled and IRSp53 for their generation and maintenance. Fusion is blocked when formation of these filopodia is compromised. While filopodia are not required for the signaling process underlying critical myoblast cell-fate changes prior to fusion, myotube-myoblast adhesion appears to be filopodia dependent. Without filopodia, close apposition between the cell membranes is not achieved, the cell-adhesion molecule Duf is not recruited to the myotube surface, and adhesion-dependent actin foci do not form. We therefore propose that the filopodia are necessary to prime the heterotypic adhesion process between the two cell types, possibly by recruiting the cell-adhesion molecule Sns to discrete patches on the myoblast cell surface.
Asunto(s)
Adhesión Celular/fisiología , Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Fibras Musculares Esqueléticas/citología , Mioblastos/citología , Seudópodos/fisiología , Actinas/metabolismo , Animales , Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular , Fusión Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Femenino , Masculino , Fibras Musculares Esqueléticas/fisiología , Mioblastos/fisiologíaRESUMEN
Rhomboid proteins have emerged as one of the most tantalizing and diverse families of proteases. Gene duplication events and structural alterations have sculpted the varied roles of this protein family, maintaining a conserved structural core throughout the bacterial, plant and animal kingdoms. Unresolved questions pop up at many junctions. This review will focus on a distinct class of Rhomboid proteins that plays an essential role in development. It will outline the diverse mechanisms by which these proteins are regulated, and the implications on the biological processes they control. While most of the review will deal with Rhomboids in Drosophila, a system that has been studied in the greatest detail, it will also explore parallels and differences in the function of Rhomboids in the flour beetle T. casteneum and the worm C. elegans.
Asunto(s)
Desarrollo Embrionario , Proteínas de la Membrana/metabolismo , Péptido Hidrolasas/metabolismo , Animales , Receptores ErbB/metabolismo , Humanos , Ligandos , Especificidad por SustratoRESUMEN
The discovery of a handful of conserved signaling pathways that dictate most aspects of embryonic and post-embryonic development of multicellular organisms has generated a universal view of animal development (Perrimon, N., Pitsouli, C., and Shilo, B. Z. (2012)Cold Spring Harb. Perspect. Biol.4, a005975). Although we have at hand most of the "hardware" elements that mediate cell communication events that dictate cell fate choices, we are still far from a comprehensive mechanistic understanding of these processes. One of the next challenges entails an analysis of developmental signaling pathways from the cell biology perspective. Where in the cell does signaling take place, and how do general cellular machineries and structures contribute to the regulation of developmental signaling? Another challenge is to examine these signaling pathways from a quantitative perspective, rather than as crude on/off switches. This requires more precise measurements, and incorporation of the time element to generate a dynamic sequence instead of frozen snapshots of the process. The quantitative outlook also brings up the issue of precision, and the unknown mechanisms that buffer variability in signaling between embryos, to produce a robust and reproducible output. Although these issues are universal to all multicellular organisms, they can be effectively tackled in theDrosophilamodel, by a combination of genetic manipulations, biochemical analyses, and a variety of imaging techniques. This review will present some of the recent advances that were accomplished by utilizing the versatility of theDrosophilasystem.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila/crecimiento & desarrollo , Transducción de Señal , Animales , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Receptores de Péptidos de Invertebrados/genética , Receptores de Péptidos de Invertebrados/metabolismo , Vía de Señalización WntRESUMEN
Patterning by morphogen gradients relies on the capacity to generate reproducible distribution profiles. Morphogen spread depends on kinetic parameters, including diffusion and degradation rates, which vary between embryos, raising the question of how variability is controlled. We examined this in the context of Toll-dependent dorsoventral (DV) patterning of the Drosophila embryo. We find that low embryo-to-embryo variability in DV patterning relies on wntD, a Toll-target gene expressed initially at the posterior pole. WntD protein is secreted and disperses in the extracellular milieu, associates with its receptor Frizzled4, and inhibits the Toll pathway by blocking the Toll extracellular domain. Mathematical modeling predicts that WntD accumulates until the Toll gradient narrows to its desired spread, and we support this feedback experimentally. This circuit exemplifies a broadly applicable induction-contraction mechanism, which reduces patterning variability through a restricted morphogen-dependent expression of a secreted diffusible inhibitor.
Asunto(s)
Tipificación del Cuerpo/fisiología , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Transducción de Señal/fisiología , Receptores Toll-Like/metabolismo , Animales , Tipificación del Cuerpo/genética , Embrión no Mamífero/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Generation of periodic patterns is fundamental to the differentiation of multiple tissues during development. How such patterns form robustly is still unclear. The Drosophila eye comprises â¼750 units, whose crystalline order is set during differentiation of the eye imaginal disc: an activation wave sweeping across the disc is coupled to lateral inhibition, sequentially selecting pro-neural cells. Using mathematical modelling, here we show that this template-based lateral inhibition is highly sensitive to spatial variations in biochemical parameters and cell sizes. We reveal the basis of this sensitivity, and suggest that it can be overcome by assuming a short-range diffusible activator. Clonal experiments identify Scabrous, a previously implicated inhibitor, as the predicted activator. Our results reveal the mechanism by which periodic patterning in the fly eye is stabilized against spatial variations, highlighting how the need to maintain robustness shapes the design of patterning circuits.
Asunto(s)
Diferenciación Celular/genética , Ojo Compuesto de los Artrópodos/embriología , Proteínas de Drosophila/genética , Drosophila/embriología , Glicoproteínas/genética , Células Fotorreceptoras de Invertebrados/metabolismo , Animales , Animales Modificados Genéticamente , Ojo Compuesto de los Artrópodos/metabolismo , Inmunohistoquímica , Modelos Teóricos , PeriodicidadRESUMEN
Releasing content from large vesicles measuring several micrometres in diameter poses exceptional challenges to the secretory system. An actomyosin network commonly coats these vesicles, and is thought to provide the necessary force mediating efficient cargo release. Here we describe the spatial and temporal dynamics of the formation of this actomyosin coat around large vesicles and the resulting vesicle collapse, in live Drosophila melanogaster salivary glands. We identify the Formin family protein Diaphanous (Dia) as the main actin nucleator involved in generating this structure, and uncover Rho as an integrator of actin assembly and contractile machinery activation comprising this actomyosin network. High-resolution imaging reveals a unique cage-like organization of myosin II on the actin coat. This myosin arrangement requires branched-actin polymerization, and is critical for exerting a non-isotropic force, mediating efficient vesicle contraction.
Asunto(s)
Actomiosina/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Exocitosis , Proteínas del Pegamento Salivar de Drosophila/metabolismo , Proteínas de la Membrana/metabolismo , Glándulas Salivales/metabolismo , Vesículas Secretoras/metabolismo , Actinas/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas Portadoras/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/ultraestructura , Forminas , Cinética , Proteínas de la Membrana/genética , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Microscopía por Video , Miosina Tipo II/metabolismo , Tamaño de los Orgánulos , Glándulas Salivales/ultraestructura , Vesículas Secretoras/ultraestructura , Imagen de Lapso de Tiempo , Quinasas Asociadas a rho/metabolismoRESUMEN
Fusion of individual myoblasts to form multinucleated myofibers constitutes a widely conserved program for growth of the somatic musculature. We have used electron microscopy methods to study this key form of cell-cell fusion during development of the indirect flight muscles (IFMs) of Drosophila melanogaster. We find that IFM myoblast-myotube fusion proceeds in a stepwise fashion and is governed by apparent cross talk between transmembrane and cytoskeletal elements. Our analysis suggests that cell adhesion is necessary for bringing myoblasts to within a minimal distance from the myotubes. The branched actin polymerization machinery acts subsequently to promote tight apposition between the surfaces of the two cell types and formation of multiple sites of cell-cell contact, giving rise to nascent fusion pores whose expansion establishes full cytoplasmic continuity. Given the conserved features of IFM myogenesis, this sequence of cell interactions and membrane events and the mechanistic significance of cell adhesion elements and the actin-based cytoskeleton are likely to represent general principles of the myoblast fusion process.