RESUMO
During an immune response, T cells survey antigen presenting cells for antigenic peptides via the formation of an interface known as an immunological synapse. Among the complex and dynamic biophysical phenomena occurring at this interface is the trafficking of sub-synaptic vesicles carrying a variety of proximal signalling molecules. Here, we show that rather than being a homogeneous population, these vesicles display a diversity of membrane lipid order profiles, as measured using the environmentally sensitive dye di-4-ANEPPDHQ and multi-spectral TIRF microscopy. Using live-cell imaging, vesicle tracking and a variety of small molecule drugs to manipulate components of the actin and tubulin cytoskeleton, we show that the membrane lipid order of these vesicles correlate with their dynamics. Furthermore, we show that the key proximal signalling molecule Linker for Activation of T cells (LAT) is enriched in specific vesicle populations as defined by their higher membrane order. These results imply that vesicle lipid order may represent a novel regulatory mechanism for the sorting and trafficking of signalling molecules at the immunological synapse, and, potentially, other cellular structures.
Assuntos
Vesículas Citoplasmáticas/metabolismo , Sinapses Imunológicas/metabolismo , Lipídeos de Membrana/metabolismo , Linfócitos T/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Cultivadas , Humanos , Sinapses Imunológicas/química , Sinapses Imunológicas/ultraestrutura , Células Jurkat , Lipídeos de Membrana/química , Proteínas de Membrana/metabolismo , Linfócitos T/ultraestruturaRESUMO
During vertebrate embryogenesis, the germ layers are patterned by secreted Nodal signals. In the classical model, Nodals elicit signaling by binding to a complex comprising Type I/II Activin receptors (Acvr) and the co-receptor Tdgf1. However, it is currently unclear whether receptor binding can also affect the distribution of Nodals themselves through the embryo, and it is unknown which of the putative Acvr paralogs mediate Nodal signaling in zebrafish. Here, we characterize three Type I (Acvr1) and four Type II (Acvr2) homologs and show that - except for Acvr1c - all receptor-encoding transcripts are maternally deposited and present during zebrafish embryogenesis. We generated mutants and used them together with combinatorial morpholino knockdown and CRISPR F0 knockout (KO) approaches to assess compound loss-of-function phenotypes. We discovered that the Acvr2 homologs function partly redundantly and partially independently of Nodal to pattern the early zebrafish embryo, whereas the Type I receptors Acvr1b-a and Acvr1b-b redundantly act as major mediators of Nodal signaling. By combining quantitative analyses with expression manipulations, we found that feedback-regulated Type I receptors and co-receptors can directly influence the diffusion and distribution of Nodals, providing a mechanism for the spatial restriction of Nodal signaling during germ layer patterning.
Building a body is complicated. Cells must organise themselves head-to-tail, belly-to-back, and inside-to-outside. They do this by laying down a chemical map, which is made up of gradients of molecular signals, high in some places and lower in others. The amount of signal each cell receives helps to decide which part of the body it will become. One of the essential signals in developing vertebrates is Nodal. It helps cells to tell inside from outside and left from right. Cells detect Nodal using an activin receptor and co-receptor complex, which catch hold of passing Nodal proteins and transmit developmental signals into cells. An important model to study Nodal signals is the zebrafish embryo, but the identity of the activin receptors and their exact role in this organism has been unclear. To find out more, Preiß, Kögler, Mörsdorf et al. studied the activin receptors Acvr1 and Acvr2 in zebrafish embryos. The experiments revealed that two putative Acvr1 and four Acvr2 receptors were present during early development. To better understand their roles, Preiß et al. eliminated them one at a time, and in combination. Losing single activin receptors had no effect. But losing both Acvr1 receptors together stopped Nodal signalling and changed the distribution of the Nodal gradient. Loss of all Acvr2 receptors also caused developmental problems, but they were partly independent of Nodal. This suggests that Acvr1s seem to be able to transmit signals and to shape the Nodal gradient, and that Acvr2s might have another, so far unknown, role. Nodal signals guide the development of all vertebrates. Understanding how they work in a model species like zebrafish could shed light on their role in other species, including humans. A clearer picture could help to uncover what happens at a molecular level when development goes wrong.
Assuntos
Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Retroalimentação , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/metabolismo , Proteína Nodal/genética , Proteína Nodal/metabolismo , Padronização Corporal/genética , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex developmental processes. Zebrafish embryos are ideally suited for these manipulations since they are easily accessible, relatively large in size, and transparent. However, previously developed devices for cell removal and transplantation are cumbersome to use or expensive to purchase. In contrast, the transplantation device presented here is economical, easy to assemble, and simple to use. In this protocol, we first introduce the handling of the transplantation device as well as its assembly from commercially and widely available parts. We then present three applications for its use: generation of ectopic clones to study signal dispersal from localized sources, extirpation of cells to produce size-reduced embryos, and germline transplantation to generate maternal-zygotic mutants. Finally, we show that the tool can also be used for embryological manipulations in other species such as the Japanese rice fish medaka.
Assuntos
Oryzias , Peixe-Zebra , Animais , Células Germinativas , ZigotoRESUMO
Opposing sources of bone morphogenetic protein (BMP) and Nodal signaling molecules are sufficient to induce the formation of a full axis in zebrafish embryos. To address how these signals orchestrate patterning, we transplant sources of fluorescently tagged Nodal and BMP into zebrafish embryos, robustly inducing the formation of secondary axes. Nodal and BMP signal non-cell-autonomously and form similar protein gradients in this context, but the signaling range of Nodal (pSmad2) is shorter than the BMP range (pSmad5). This yields a localized region of pSmad2 activity around the Nodal source, overlapping with a broad domain of pSmad5 activity across the embryo. Cell fates induced in various regions stereotypically correlate with pSmad2-to-pSmad5 ratios and can even be induced BMP- and Nodal-independently with different ratios of constitutively active Smad2 and Smad5. Strikingly, we find that Smad2 and Smad5 antagonize each other for specific cell fates, providing a mechanism for how cells integrate and discriminate between overlapping signals during development.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteína Nodal/metabolismo , Peixe-Zebra/embriologia , Animais , Padronização Corporal/genética , Proteínas Morfogenéticas Ósseas/fisiologia , Linhagem da Célula/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteína Nodal/fisiologia , Transdução de Sinais/fisiologia , Proteína Smad2/metabolismo , Proteína Smad5/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Morphogens are signaling molecules that provide positional information to cells during development. They must move through embryonic tissues in order to coordinate patterning. The rate of a morphogen's movement through a tissue-its effective diffusivity-affects the morphogen's distribution and therefore influences patterning. Fluorescence recovery after photobleaching (FRAP) is a powerful method to measure the effective diffusion of molecules through cells and tissues, and has been successfully employed to examine morphogen mobility and gain important insights into embryogenesis. Here, we provide detailed protocols for FRAP assays in vitro and in living zebrafish embryos, and we explain how to analyze FRAP data using the open-source software PyFRAP to determine effective diffusion coefficients.
Assuntos
Embrião não Mamífero/citologia , Embrião não Mamífero/fisiologia , Recuperação de Fluorescência Após Fotodegradação/métodos , Processamento de Imagem Assistida por Computador/métodos , Morfogênese , Peixe-Zebra/embriologia , Peixe-Zebra/fisiologia , Animais , Transporte Biológico , Difusão , Modelos Biológicos , Transdução de Sinais , SoftwareRESUMO
Fluorescence Recovery After Photobleaching (FRAP) and inverse FRAP (iFRAP) assays can be used to assess the mobility of fluorescent molecules. These assays measure diffusion by monitoring the return of fluorescence in bleached regions (FRAP), or the dissipation of fluorescence from photoconverted regions (iFRAP). However, current FRAP/iFRAP analysis methods suffer from simplified assumptions about sample geometry, bleaching/photoconversion inhomogeneities, and the underlying reaction-diffusion kinetics. To address these shortcomings, we developed the software PyFRAP, which fits numerical simulations of three-dimensional models to FRAP/iFRAP data and accounts for bleaching/photoconversion inhomogeneities. Using PyFRAP we determined the diffusivities of fluorescent molecules spanning two orders of magnitude in molecular weight. We measured the tortuous effects that cell-like obstacles exert on effective diffusivity and show that reaction kinetics can be accounted for by model selection. These applications demonstrate the utility of PyFRAP, which can be widely adapted as a new extensible standard for FRAP analysis.
RESUMO
Individuals can vary substantially in size, but the proportions of their body plans are often maintained. We generated smaller zebrafish by removing 30% of their cells at the blastula stages and found that these embryos developed into normally patterned individuals. Strikingly, the proportions of all germ layers adjusted to the new embryo size within 2 hours after cell removal. As Nodal-Lefty signalling controls germ-layer patterning, we performed a computational screen for scale-invariant models of this activator-inhibitor system. This analysis predicted that the concentration of the highly diffusive inhibitor Lefty increases in smaller embryos, leading to a decreased Nodal activity range and contracted germ-layer dimensions. In vivo studies confirmed that Lefty concentration increased in smaller embryos, and embryos with reduced Lefty levels or with diffusion-hindered Lefty failed to scale their tissue proportions. These results reveal that size-dependent inhibition of Nodal signalling allows scale-invariant patterning.
Assuntos
Blástula/metabolismo , Padronização Corporal , Fatores de Determinação Direita-Esquerda/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Padronização Corporal/genética , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Determinação Direita-Esquerda/genética , Proteínas de Membrana/genética , Transdução de Sinais , Fatores de Tempo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
During vertebrate embryogenesis, dorsal-ventral patterning is controlled by the BMP/Chordin activator/inhibitor system. BMP induces ventral fates, whereas Chordin inhibits BMP signaling on the dorsal side. Several theories can explain how the distributions of BMP and Chordin are regulated to achieve patterning, but the assumptions regarding activator/inhibitor diffusion and stability differ between models. Notably, 'shuttling' models in which the BMP distribution is modulated by a Chordin-mediated increase in BMP diffusivity have gained recent prominence. Here, we directly test five major models by measuring the biophysical properties of fluorescently tagged BMP2b and Chordin in zebrafish embryos. We found that BMP2b and Chordin diffuse and rapidly form extracellular protein gradients, Chordin does not modulate the diffusivity or distribution of BMP2b, and Chordin is not required to establish peak levels of BMP signaling. Our findings challenge current self-regulating reaction-diffusion and shuttling models and provide support for a graded source-sink mechanism underlying zebrafish dorsal-ventral patterning.