RESUMO
Developmental biology research would benefit greatly from tools that enable protein function to be regulated, both systematically and in a precise spatial and temporal manner, in vivo In recent years, functionalized protein binders have emerged as versatile tools that can be used to target and manipulate proteins. Such protein binders can be based on various scaffolds, such as nanobodies, designed ankyrin repeat proteins (DARPins) and monobodies, and can be used to block or perturb protein function in living cells. In this Primer, we provide an overview of the protein binders that are currently available and highlight recent progress made in applying protein binder-based tools in developmental and synthetic biology.
Assuntos
Ligação Proteica , Proteínas/metabolismo , Animais , Repetição de Anquirina , Biologia do Desenvolvimento , Humanos , Modelos Moleculares , Biblioteca de Peptídeos , Modificação Traducional de Proteínas , Transporte Proteico , Proteínas/química , Proteínas/genética , Proteólise , Anticorpos de Domínio Único/metabolismo , Biologia SintéticaRESUMO
Drosophila Decapentaplegic (Dpp) has served as a paradigm to study morphogen-dependent growth control. However, the role of a Dpp gradient in tissue growth remains highly controversial. Two fundamentally different models have been proposed: the 'temporal rule' model suggests that all cells of the wing imaginal disc divide upon a 50% increase in Dpp signalling, whereas the 'growth equalization model' suggests that Dpp is only essential for proliferation control of the central cells. Here, to discriminate between these two models, we generated and used morphotrap, a membrane-tethered anti-green fluorescent protein (GFP) nanobody, which enables immobilization of enhanced (e)GFP::Dpp on the cell surface, thereby abolishing Dpp gradient formation. We find that in the absence of Dpp spreading, wing disc patterning is lost; however, lateral cells still divide at normal rates. These data are consistent with the growth equalization model, but do not fit a global temporal rule model in the wing imaginal disc.
Assuntos
Padronização Corporal/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/metabolismo , Animais , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Drosophila melanogaster/citologia , Masculino , Proteínas Repressoras/metabolismo , Transdução de Sinais , Anticorpos de Cadeia Única , Fatores de Transcrição/metabolismo , Asas de Animais/citologiaRESUMO
The Drosophila tracheal system consists of an interconnected network of monolayered epithelial tubes that ensures oxygen transport in the larval and adult body. During tracheal dorsal branch (DB) development, individual DBs elongate as a cluster of cells, led by tip cells at the front and trailing cells in the rear. Branch elongation is accompanied by extensive cell intercalation and cell lengthening of the trailing stalk cells. Although cell intercalation is governed by Myosin II (MyoII)-dependent forces during tissue elongation in the Drosophila embryo that lead to germ-band extension, it remained unclear whether MyoII plays a similar active role during tracheal branch elongation and intercalation. Here, we have used a nanobody-based approach to selectively knock down MyoII in tracheal cells. Our data show that, despite the depletion of MyoII function, tip cell migration and stalk cell intercalation (SCI) proceed at a normal rate. This confirms a model in which DB elongation and SCI in the trachea occur as a consequence of tip cell migration, which produces the necessary forces for the branching process.
Assuntos
Proteínas de Drosophila/metabolismo , Miosina Tipo II/metabolismo , Traqueia/embriologia , Traqueia/metabolismo , Animais , Proliferação de Células/genética , Proliferação de Células/fisiologia , Drosophila , Proteínas de Drosophila/genética , Embrião não Mamífero/metabolismo , Feminino , Masculino , Morfogênese/genética , Morfogênese/fisiologia , Miosina Tipo II/genéticaRESUMO
Final organ size and shape result from volume expansion by growth and shape changes by contractility. Complex morphologies can also arise from differences in growth rate between tissues. We address here how differential growth guides the morphogenesis of the growing Drosophila wing imaginal disc. We report that 3D morphology results from elastic deformation due to differential growth anisotropy between the epithelial cell layer and its enveloping extracellular matrix (ECM). While the tissue layer grows in plane, growth of the bottom ECM occurs in 3D and is reduced in magnitude, thereby causing geometric frustration and tissue bending. The elasticity, growth anisotropy and morphogenesis of the organ are fully captured by a mechanical bilayer model. Moreover, differential expression of the Matrix metalloproteinase MMP2 controls growth anisotropy of the ECM envelope. This study shows that the ECM is a controllable mechanical constraint whose intrinsic growth anisotropy directs tissue morphogenesis in a developing organ.
Assuntos
Drosophila , Matriz Extracelular , Animais , Anisotropia , Tamanho do Órgão , ElasticidadeRESUMO
The size and proportions of animals are tightly controlled during development. How this is achieved remains poorly understood. The control of organ size entails coupling of cellular growth and cell division on one hand, and the measure of organ size on the other. In this review we focus on three layers of growth control consisting of genetic patterning, notably chemical gradients, mechanics and energetics which are complemented by a systemic control unit that modulates growth in response to the nutritional conditions and coordinates growth between different organs so as to maintain proportions. Growth factors, often present as concentration dependent chemical gradients, are positive inducers of cellular growth that may be considered as deterministic cues, hence acting as organ-intrinsic controllers of growth. However, the exponential growth dynamics in many developing tissues necessitate more stringent growth control in the form of negative feedbacks. Feedbacks endow biological systems with the capacity to quickly respond to perturbations and to correct the growth trajectory to avoid overgrowth. We propose to integrate chemical, mechanical and energetic control over cellular growth in a framework that emphasizes the self-organizing properties of organ-autonomous growth control in conjunction with systemic organ non-autonomous feedback on growth.
Assuntos
Fenômenos Biológicos , Animais , Ciclo Celular , Divisão Celular , Retroalimentação , Tamanho do ÓrgãoRESUMO
Interfaces between cells with distinct genetic identities elicit signals to organize local cell behaviors driving tissue morphogenesis. The Drosophila embryonic axis extension requires planar polarized enrichment of myosin-II powering oriented cell intercalations. Myosin-II levels are quantitatively controlled by GPCR signaling, whereas myosin-II polarity requires patterned expression of several Toll receptors. How Toll receptors polarize myosin-II and how this involves GPCRs remain unknown. Here, we report that differential expression of a single Toll receptor, Toll-8, polarizes myosin-II through binding to the adhesion GPCR Cirl/latrophilin. Asymmetric expression of Cirl is sufficient to enrich myosin-II, and Cirl localization is asymmetric at Toll-8 expression boundaries. Exploring the process dynamically, we reveal that Toll-8 and Cirl exhibit mutually dependent planar polarity in response to quantitative differences in Toll-8 expression between neighboring cells. Collectively, we propose that the cell surface protein complex Toll-8/Cirl self-organizes to generate local asymmetric interfaces essential for planar polarization of contractility.
Assuntos
Proteínas de Drosophila/genética , Desenvolvimento Embrionário/genética , Morfogênese/genética , Miosina Tipo II/genética , Receptores Acoplados a Proteínas G/genética , Receptores de Peptídeos/genética , Receptor 8 Toll-Like/genética , Animais , Polaridade Celular/genética , Proteínas do Citoesqueleto/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Membrana/genética , Complexos Multiproteicos/genética , Contração Muscular/genéticaRESUMO
The role of protein localization along the apical-basal axis of polarized cells is difficult to investigate in vivo, partially due to lack of suitable tools. Here, we present the GrabFP system, a collection of four nanobody-based GFP-traps that localize to defined positions along the apical-basal axis. We show that the localization preference of the GrabFP traps can impose a novel localization on GFP-tagged target proteins and results in their controlled mislocalization. These new tools were used to mislocalize transmembrane and cytoplasmic GFP fusion proteins in the Drosophila wing disc epithelium and to investigate the effect of protein mislocalization. Furthermore, we used the GrabFP system as a tool to study the extracellular dispersal of the Decapentaplegic (Dpp) protein and show that the Dpp gradient forming in the lateral plane of the Drosophila wing disc epithelium is essential for patterning of the wing imaginal disc.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Entomologia/métodos , Biologia Molecular/métodos , Transporte Proteico , Anticorpos de Domínio Único/metabolismo , Animais , Drosophila/genética , Drosophila/fisiologia , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genéticaRESUMO
Bone morphogenetic proteins (BMPs) act as morphogens to control patterning and growth in a variety of developing tissues in different species. How BMP morphogen gradients are established and interpreted in the target tissues has been extensively studied in Drosophila melanogaster. In Drosophila, Decapentaplegic (Dpp), a homologue of vertebrate BMP2/4, acts as a morphogen to control dorsal-ventral patterning of the early embryo and anterior-posterior patterning and growth of the wing imaginal disc. Despite intensive efforts over the last twenty years, how the Dpp morphogen gradient in the wing imaginal disc forms remains controversial, while gradient formation in the early embryo is well understood. In this review, we first focus on the current models of Dpp morphogen gradient formation in these two tissues, and then discuss new strategies using genome engineering and nanobodies to tackle open questions.