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The cell cortex of syncytial Drosophila embryos is patterned into cap and intercap regions by centrosomes, specific sets of proteins that are restricted to their respective regions by unknown mechanisms. Here, we found that Kinesin-1 is required for the restriction of plus- and minus-ends of centrosomal and non-centrosomal microtubules to the cap region, marked by EB1 and Patronin/Shot, respectively. Kinesin-1 also directly or indirectly restricts proteins and Rho signaling to the intercap, including the RhoGEF Pebble, Dia, Myosin II, Capping protein-α, and the polarity protein Par-1. Furthermore, we found that Par-1 is required for cap restriction of Patronin/Shot, and vice versa Patronin, for Par-1 enrichment at the intercap. In summary, our data support a model that Kinesin-1 would mediate the restriction of centrosomal and non-centrosomal microtubules to a region close to the centrosomes and exclude Rho signaling and Par-1. In addition, mutual antagonistic interactions would refine and maintain the boundary between cap and intercap and thus generate a distinct cortical pattern.
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Proteínas de Drosophila , Drosophila , Glucógeno Sintasa Quinasa 3 , Cinesinas , Proteínas de la Membrana , Animales , Centrosoma , Proteínas del Citoesqueleto , Drosophila/embriología , Drosophila/genética , Proteínas de Drosophila/genética , Glucógeno Sintasa Quinasa 3/genética , Cinesinas/genética , Proteínas Asociadas a Microtúbulos/genética , Transducción de Señal , Proteínas de la Membrana/genéticaRESUMEN
Cell-cell adhesion is at the center of structure and dynamics of epithelial tissue. E-cadherin-catenin complexes mediate Ca2+-dependent trans-homodimerization and constitute the kernel of adherens junctions. Beyond the basic function of cell-cell adhesion, recent progress sheds light the dynamics and interwind interactions of individual E-cadherin-catenin complex with E-cadherin superclusters, contractile actomyosin and mechanics of the cortex and adhesion. The nanoscale architecture of E-cadherin complexes together with cis-interactions and interactions with cortical actomyosin adjust to junctional tension and mechano-transduction by reinforcement or weakening of specific features of the interactions. Although post-translational modifications such as phosphorylation and glycosylation have been implicated, their role for specific aspects of in E-cadherin function has remained unclear. Here, we provide an overview of the E-cadherin complex in epithelial cell and tissue morphogenesis focusing on nanoscale architectures by super-resolution approaches and post-translational modifications from recent, in particular in vivo, studies. Furthermore, we review the computational modelling in E-cadherin complexes and highlight how computational modelling has contributed to a deeper understanding of the E-cadherin complexes.
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The presence of farnesylated proteins at the inner nuclear membrane (INM), such as the Lamins or Kugelkern in Drosophila, leads to specific changes in the nuclear morphology and accelerated ageing on the organismal level reminiscent of the Hutchinson-Gilford progeria syndrome (HGPS). Farnesyl transferase inhibitors (FTIs) can suppress the phenotypes of the nuclear morphology in cultured fibroblasts from HGPS patients and cultured cells overexpressing farnesylated INM proteins. Similarly, FTIs have been reported to suppress the shortened lifespan in model organisms. Here, we report an experimental system combining cell culture and Drosophila flies for testing the activity of substances on the HGPS-like nuclear morphology and lifespan, with FTIs as an experimental example. Consistent with previous reports, we show that FTIs were able to ameliorate the nuclear phenotypes induced by the farnesylated nuclear proteins Progerin, Kugelkern, or truncated Lamin B in cultured cells. The subsequent validation in Drosophila lifespan assays demonstrated the applicability of the experimental system: treating adult Drosophila with the FTI ABT-100 reversed the nuclear phenotypes and extended the lifespan of experimentally induced short-lived flies. Since kugelkern-expressing flies have a significantly shorter average lifespan, half the time is needed for testing substances in the lifespan assay.
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Drosophila blood cells called hemocytes form an efficient barrier against infections and tissue damage. During metamorphosis, hemocytes undergo tremendous changes in their shape and behavior, preparing them for tissue clearance. Yet, the diversity and functional plasticity of pupal blood cells have not been explored. Here, we combine single-cell transcriptomics and high-resolution microscopy to dissect the heterogeneity and plasticity of pupal hemocytes. We identified undifferentiated and specified hemocytes with different molecular signatures associated with distinct functions such as antimicrobial, antifungal immune defense, cell adhesion or secretion. Strikingly, we identified a highly migratory and immune-responsive pupal cell population expressing typical markers of the posterior signaling center (PSC), which is known to be an important niche in the larval lymph gland. PSC-like cells become restricted to the abdominal segments and are morphologically very distinct from typical Hemolectin (Hml)-positive plasmatocytes. G-TRACE lineage experiments further suggest that PSC-like cells can transdifferentiate to lamellocytes triggered by parasitoid wasp infestation. In summary, we present the first molecular description of pupal Drosophila blood cells, providing insights into blood cell functional diversification and plasticity during pupal metamorphosis.
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Proteínas de Drosophila , Drosophila , Animales , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Transcriptoma/genética , Diferenciación Celular , Células Sanguíneas/metabolismo , Proteínas de Drosophila/metabolismo , Hemocitos , Larva/metabolismoRESUMEN
Rods under mechanical stress are a classic example of dynamic instability. Axis elongation in Drosophila usually leads to a U-shaped axis, but folded or twisted axes are observed in certain mutants. Analysis of these mutants now reveals the source of the instability and the mechanism for maintaining left-right symmetry.
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Drosophila , Células Fotorreceptoras Retinianas Bastones , Animales , Drosophila/genética , Morfogénesis/genética , Estrés MecánicoRESUMEN
During embryonic development, dramatic cell shape changes and movements reshape the embryonic body plan. These require robust but dynamic linkage between the cell-cell adherens junctions and the force-generating actomyosin cytoskeleton. Our view of this linkage has evolved, and we now realize linkage is mediated by mechanosensitive multiprotein complexes assembled via multivalent connections. Here we combine genetic, cell biological, and modeling approaches to define the mechanism of action and functions of an important player, Drosophila polychaetoid, homologue of mammalian ZO-1. Our data reveal that Pyd reinforces cell junctions under elevated tension, and facilitates cell rearrangements. Pyd is important to maintain junctional contractility and in its absence cell rearrangements stall. We next use structured illumination microscopy to define the molecular architecture of cell-cell junctions during these events. The cadherin-catenin complex and Cno both localize to puncta along the junctional membrane, but are differentially enriched in different puncta. Pyd, in contrast, exhibits a distinct localization to strands that extend out from the region occupied by core junction proteins. We then discuss the implications for the protein network at the junction-cytoskeletal interface, suggesting different proteins localize and function in distinct ways, perhaps in distinct subcomplexes, but combine to produce robust connections.
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Uniones Adherentes , Proteínas de Drosophila , Animales , Citoesqueleto de Actina/metabolismo , Uniones Adherentes/metabolismo , Cadherinas/metabolismo , Citoesqueleto/metabolismo , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Mamíferos/metabolismo , Uniones Estrechas/metabolismoRESUMEN
During embryonic development dramatic cell shape changes and movements re-shape the embryonic body plan. These require robust but dynamic linkage between the cell-cell adherens junctions and the force-generating actomyosin cytoskeleton. Our view of this linkage has evolved, and we now realize linkage is mediated by a mechanosensitive multiprotein complex assembled via multivalent connections. Here we combine genetic, cell biological and modeling approaches to define the mechanism of action and functions of an important player, Drosophila Polychaetoid, homolog of mammalian ZO-1. Our data reveal that Pyd reinforces cell junctions under elevated tension, and facilitates cell rearrangements. Pyd is important to maintain junctional contractility and in its absence cell rearrangements stall. We next use structured illumination microscopy to define the molecular architecture of cell-cell junctions during these events. The cadherin-catenin complex and Cno both localize to puncta along the junctional membrane, but are differentially enriched in different puncta. Pyd, in contrast, exhibits a distinct localization to strands that extend out from the region occupied by core junction proteins. We then discuss the implications for the protein network at the junction-cytoskeletal interface, suggesting different proteins localize and function in distinct ways but combine to produce robust connections.
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Cell shape changes based on actomyosin contractility provide a driving force in tissue morphogenesis. The temporally and spatially coordinated constrictions of many cells result in changes in tissue morphology. Given the networks of complex and mutual cellular interactions, the mechanisms underlying the emergence in tissue behavior are challenging to pinpoint. Important in the analysis of such interactions are novel methods for noninvasive interference with single-cell resolution and sub-minute timescale temporal control. Here we characterize an optochemical approach of Ca2+ uncaging to control cell contractility in Drosophila embryos. We describe in detail the method of sample preparation, microinjection, Ca2+ uncaging, and data analysis.
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Proteínas de Drosophila , Drosophila , Actomiosina , Animales , Forma de la Célula , MorfogénesisRESUMEN
Dorsal closure is a prominent morphogenetic process during Drosophila embryogenesis, which involves two epithelial tissues, that is, the squamous amnioserosa and the columnar lateral epidermis. Non-muscle myosin II-driven constriction in the amnioserosa leads to a decrease in the apical surface area and pulls on the adjacent lateral epidermis, which subsequently moves dorsally. The pull by the amnioserosa becomes obvious in an elongation of the epidermal cells, especially of those in the first row. The contribution of the epidermal cell elongation has remained unclear to dorsal closure. Cell elongation may be a mere passive consequence or an active response to the pulling by the amnioserosa. Here, we found that the lateral epidermis actively responds. We analyzed tensions within tissues and cell junctions by laser ablation before and during dorsal closure, the elliptical and dorsal closure stages, respectively. Furthermore, we genetically and optochemically induced chronic and acute cell contraction, respectively. In this way, we found that tension in the epidermis increased during dorsal closure. A correspondingly increased tension was not observed at individual junctions, however. Junctional tension even decreased during dorsal closure in the epidermis. We strikingly observed a strong increase of the microtubule amount in the epidermis, while non-muscle myosin II increased in both tissues. Our data suggest that the epidermis actively antagonizes the pull from the amnioserosa during dorsal closure and the increased microtubules might help the epidermis bear part of the mechanical force.
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Changes in cell morphology require the dynamic remodeling of the actin cytoskeleton. Calcium fluxes have been suggested as an important signal to rapidly relay information to the actin cytoskeleton, but the underlying mechanisms remain poorly understood. Here, we identify the EF-hand domain containing protein EFhD2/Swip-1 as a conserved lamellipodial protein strongly upregulated in Drosophila macrophages at the onset of metamorphosis when macrophage behavior shifts from quiescent to migratory state. Loss- and gain-of-function analysis confirm a critical function of EFhD2/Swip-1 in lamellipodial cell migration in fly and mouse melanoma cells. Contrary to previous assumptions, TIRF-analyses unambiguously demonstrate that EFhD2/Swip-1 proteins efficiently cross-link actin filaments in a calcium-dependent manner. Using a single-cell wounding model, we show that EFhD2/Swip-1 promotes wound closure in a calcium-dependent manner. Mechanistically, our data suggest that transient calcium bursts reduce EFhD2/Swip-1 cross-linking activity and thereby promote rapid reorganization of existing actin networks to drive epithelial wound closure.
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Actinas , Proteínas de Unión al Calcio , Calcio , Cicatrización de Heridas , Actinas/metabolismo , Animales , Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Movimiento Celular , Drosophila , Proteínas de Drosophila , Ratones , Transducción de SeñalRESUMEN
Cortical domains are characterized by spatially restricted polarity proteins. The pattern of cortical domains is dynamic and changes during cell differentiation and development. Although there is a good understanding for how the cortical pattern is maintained, e. g. by mutual antagonism, less is known about how the initial pattern is established, and its dynamics coordinated with developmental progression. Here we investigate the initial restriction of subapical marker proteins during the syncytial-cellular transition in Drosophila embryos. The subapical markers Canoe/Afadin, the complex ELMO-Sponge, Baz and Arm become initially restricted between apical and lateral domains during cellularization. We define the role of zygotic genome activation as a timer for subapical domain formation. Subapical markers remained widely spread in embryos treated with α-amanitin and became precociously restricted in mutant embryos with premature zygotic transcription. In contrast, remodeling of the nuclear division cycle without cytokinesis to a full cell cycle is not a prerequisite for subapical domain formation, since we observed timely subapical restriction in embryos undergoing an extra nuclear cycle. We provide evidence that earliest subapical markers ELMO-Sponge and Canoe are required for subapical accumulation of Baz. Supporting an important role of cortical F-actin in subapical restriction, we found that the formin Dia was required for Baz restriction, and its distribution depended on the onset of zygotic gene expression. In summary, we define zygotic transcription as a timer, to which subapical markers respond in a dia-dependent mechanism.
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Proteínas de Drosophila , Cigoto , Animales , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Forminas , Morfogénesis , Cigoto/metabolismoRESUMEN
Rho signaling with its major targets the formin Dia, Rho kinase (Rok) and non-muscle myosin II (MyoII, encoded by zip in flies) control turnover, amount and contractility of actomyosin. Much less investigated has been a potential function for the distribution of F-actin plus and minus ends. In syncytial Drosophila embryos, Rho1 signaling is high between actin caps, i.e. the cortical intercap region. Capping protein binds to free plus ends of F-actin to prevent elongation of the filament. Capping protein has served as a marker to visualize the distribution of F-actin plus ends in cells and in vitro. In the present study, we probed the distribution of plus ends with capping protein in syncytial Drosophila embryos. We found that capping proteins are specifically enriched in the intercap region similar to Dia and MyoII but distinct from overall F-actin. The intercap enrichment of Capping protein was impaired in dia mutants and embryos, in which Rok and MyoII activation was inhibited. Our observations reveal that Dia and Rok-MyoII control Capping protein enrichment and support a model that Dia and Rok-MyoII control the organization of cortical actin cytoskeleton downstream of Rho1 signaling. This article has an associated First Person interview with the first authors of the paper.
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Proteínas de Drosophila , Forminas , Quinasas Asociadas a rho , Citoesqueleto de Actina/genética , Actinas/genética , Animales , Drosophila/genética , Proteínas de Drosophila/genética , Forminas/genética , Proteínas de la Membrana , Cadenas Pesadas de Miosina , Quinasas Asociadas a rho/genéticaRESUMEN
Mechanosensitive ion channels mediate the neuronal sensation of mechanical signals such as sound, touch, and pain. Recent studies point to a function of these channel proteins in cell types and tissues in addition to the nervous system, such as epithelia, where they have been little studied, and their role has remained elusive. Dynamic epithelia are intrinsically exposed to mechanical forces. A response to pull and push is assumed to constitute an essential part of morphogenetic movements of epithelial tissues, for example. Mechano-gated channels may participate in sensing and responding to such forces. In this review, focusing on Drosophila, we highlight recent results that will guide further investigations concerned with the mechanistic role of these ion channels in epithelial cells.
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Células Epiteliales/citología , Células Epiteliales/fisiología , Activación del Canal Iónico , Canales Iónicos/fisiología , Mecanotransducción Celular , Morfogénesis , Animales , HumanosRESUMEN
Cell proliferation in early embryos by rapid cell cycles and its abrupt pause after a stereotypic number of divisions present an attractive system to study the timing mechanism in general and its coordination with developmental progression. In animals with large eggs, such as Xenopus, zebrafish, or Drosophila, 11-13 very fast and synchronous cycles are followed by a pause or slowdown of the cell cycle. The stage when the cell cycle is remodeled falls together with changes in cell behavior and activation of the zygotic genome and is often referred to as mid-blastula transition. The number of fast embryonic cell cycles represents a clear and binary readout of timing. Several factors controlling the cell cycle undergo dynamics and gradual changes in activity or concentration and thus may serve as temporal gradients. Recent studies have revealed that the gradual loss of Cdc25 protein, gradual depletion of free deoxyribonucleotide metabolites, or gradual depletion of free histone proteins impinge on Cdk1 activity in a threshold-like manner. In this review, we will highlight with a focus on Drosophila studies our current understanding and recent findings on the generation and readout of these temporal gradients, as well as their position within the regulatory network of the embryonic cell cycle.
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Cell and tissue functions rely on the genetic programmes and cascades of biochemical signals. It has become evident during the past decade that the physical properties of soft material that govern the mechanics of cells and tissues play an important role in cellular function and morphology. The biophysical properties of cells and tissues are determined by the cytoskeleton, consisting of dynamic networks of F-actin and microtubules, molecular motors, crosslinkers and other associated proteins, among other factors such as cell-cell interactions. The Drosophila syncytial embryo represents a simple pseudo-tissue, with its nuclei orderly embedded in a structured cytoskeletal matrix at the embryonic cortex with no physical separation by cellular membranes. Here, we review the stereotypic dynamics and regulation of the cytoskeleton in Drosophila syncytial embryos and how cytoskeletal dynamics underlies biophysical properties and the emergence of collective features. We highlight the specific features and processes of syncytial embryos and discuss the applicability of biophysical approaches.
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Proteínas de Drosophila , Drosophila , Citoesqueleto de Actina , Actinas , Animales , Citoesqueleto , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Embrión no Mamífero , MicrotúbulosRESUMEN
Many aspects in tissue morphogenesis are attributed to a collective behavior of the participating cells. Yet, the mechanism for emergence of dynamic tissue behavior is not well understood. Here, we report that the "yo-yo"-like nuclear movement in the Drosophila syncytial embryo displays emergent features indicative of collective behavior. Following mitosis, the array of nuclei moves away from the wave front by several nuclear diameters only to return to its starting position about 5 min later. Based on experimental manipulations and numerical simulations, we find that the ensemble of elongating and isotropically oriented spindles, rather than individual spindles, is the main driving force for anisotropic nuclear movement. ELMO-dependent F-actin restricts the time for the forward movement and ELMO- and dia-dependent F-actin is essential for the return movement. Our study provides insights into how the interactions among the cytoskeleton as individual elements lead to collective movement of the nuclear array on a macroscopic scale.
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Núcleo Celular/fisiología , Citoesqueleto/fisiología , Drosophila melanogaster/fisiología , Embrión no Mamífero/fisiología , Mitosis/fisiología , Morfogénesis , Animales , Drosophila melanogaster/embriologíaRESUMEN
The protein phosphatase Cdc25 is a key regulator of the cell cycle by activating Cdk-cyclin complexes. Cdc25 is regulated by its expression levels and post-translational mechanisms. In early Drosophila embryogenesis, Cdc25/Twine drives the fast and synchronous nuclear cycles. A pause in the cell cycle and the remodeling to a more generic cell cycle mode with a gap phase are determined by Twine inactivation and destruction in early interphase 14, in response to zygotic genome activation. Although the pseudokinase Tribbles contributes to the timely degradation of Twine, Twine levels are controlled by additional yet unknown post-translational mechanisms. Here, we apply a non-invasive method based on fluorescence fluctuation analysis (FFA) to record the absolute concentration profiles of Twine with minute-scale resolution in single living embryos. Employing this assay, we found that Protein phosphatase V (PpV), the homologue of the catalytic subunit of human PP6, ensures appropriately low Twine protein levels at the onset of interphase 14. PpV controls directly or indirectly the phosphorylation of Twine at multiple serine and threonine residues as revealed by phosphosite mapping. Mutational analysis confirmed that these sites are involved in control of Twine protein dynamics, and cell cycle remodeling is delayed in a fraction of the phosphosite mutant embryos. Our data reveal a novel mechanism for control of Twine protein levels and their significance for embryonic cell cycle remodeling.
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Proteínas de Drosophila/genética , Embrión no Mamífero/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Animales , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Drosophila , Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Microscopía Fluorescente/métodos , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , ProteolisisRESUMEN
Imaging of complex (biological) samples in the near-infrared (NIR) is beneficial due to reduced light scattering, absorption, phototoxicity, and autofluorescence. However, there are few NIR fluorescent materials known and suitable for biomedical applications. Here we exfoliate the layered pigment CaCuSi4O10 (Egyptian Blue, EB) via ball milling and facile tip sonication into NIR fluorescent nanosheets (EB-NS). The size of EB-NS can be tailored to diameters <20 nm and heights down to 1 nm. EB-NS fluoresce at 910 nm and the fluorescence intensity correlates with the number of Cu2+ ions. Furthermore, EB-NS display no bleaching and high brightness compared with other NIR fluorophores. The versatility of EB-NS is demonstrated by in-vivo single-particle tracking and microrheology measurements in Drosophila melanogaster embryos. EB-NS can be uptaken by plants and remotely detected in a low-cost stand-off detection setup. In summary, EB-NS have the potential for a wide range of bioimaging applications.
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Colorantes Fluorescentes/efectos de la radiación , Rayos Infrarrojos , Imagen Óptica/métodos , Óptica y Fotónica/métodos , Silicatos/efectos de la radiación , Animales , Cobre , Drosophila melanogaster/embriología , Fluorescencia , Iones , Modelos Teóricos , NanopartículasRESUMEN
Planar cell polarity and anisotropic cell behavior play critical roles in large-scale epithelial morphogenesis, homeostasis, wound repair, and regeneration. Cell-Cell communication and mechano-transduction in the second to minute scale mediated by E-cadherin complexes play a central role in the coordination and self-organization of cellular activities, such as junction dynamics, cell shape changes, and cell rearrangement. Here we review the current understanding in the interplay of cell polarity and cell dynamics during body axis elongation and dorsal closure in Drosophila embryos with a focus on E-cadherin dynamics in linking cell and tissue polarization and tissue-scale shape changes.