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1.
EMBO J ; 43(9): 1822-1842, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38565947

RESUMO

A key question in plant biology is how oriented cell divisions are integrated with patterning mechanisms to generate organs with adequate cell type allocation. In the root vasculature, a gradient of miRNA165/6 controls the abundance of HD-ZIP III transcription factors, which in turn control cell fate and spatially restrict vascular cell proliferation to specific cells. Here, we show that vascular development requires the presence of ARGONAUTE10, which is thought to sequester miRNA165/6 and protect HD-ZIP III transcripts from degradation. Our results suggest that the miR165/6-AGO10-HDZIP III module acts by buffering cytokinin responses and restricting xylem differentiation. Mutants of AGO10 show faster growth rates and strongly enhanced survival under severe drought conditions. However, this superior performance is offset by markedly increased variation and phenotypic plasticity in sub-optimal carbon supply conditions. Thus, AGO10 is required for the control of formative cell division and coordination of robust cell fate specification of the vasculature, while altering its expression provides a means to adjust phenotypic plasticity.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas Argonautas , Divisão Celular , Regulação da Expressão Gênica de Plantas , MicroRNAs , Raízes de Plantas , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/citologia , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Divisão Celular/genética , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Diferenciação Celular , Xilema/citologia , Xilema/metabolismo , Xilema/crescimento & desenvolvimento , Xilema/genética
2.
Plant Physiol ; 178(1): 40-53, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30026289

RESUMO

Understanding the context-specific role of gene function is a key objective of modern biology. To this end, we generated a resource for inducible cell type-specific transactivation in Arabidopsis (Arabidopsis thaliana) based on the well-established combination of the chimeric GR-LhG4 transcription factor and the synthetic pOp promoter. Harnessing the flexibility of the GreenGate cloning system, we produced a comprehensive set of transgenic lines termed GR-LhG4 driver lines targeting most tissues in the Arabidopsis shoot and root with a strong focus on the indeterminate meristems. When we combined these transgenic lines with effectors under the control of the pOp promoter, we observed tight temporal and spatial control of gene expression. In particular, inducible expression in F1 plants obtained from crosses of driver and effector lines allows for rapid assessment of the cell type-specific impact of an effector with high temporal resolution. Thus, our comprehensive and flexible method is suitable for overcoming the limitations of ubiquitous genetic approaches, the outputs of which often are difficult to interpret due to the widespread existence of compensatory mechanisms and the integration of diverging effects in different cell types.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Arabidopsis/citologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Clonagem Molecular/métodos , Meristema/citologia , Meristema/genética , Meristema/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Brotos de Planta/citologia , Brotos de Planta/genética , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genética , Ativação Transcricional
3.
Dev Biol ; 386(2): 371-84, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24368071

RESUMO

The Drosophila embryo undergoes a developmental transition in the blastoderm stage switching from syncytial to cellular development. The cleavage furrow, which encloses nuclei into cells, is a prominent morphological feature of this transition. It is not clear how the pattern of the furrow array is defined and how zygotic genes trigger the formation and invagination of interphase furrows. A key to these questions is provided by the gene slam, which has been previously implicated in controlling furrow invagination. Here we investigate the null phenotype of slam, the dynamics of Slam protein, and its control by the recycling endosome. We find that slam is essential for furrow invagination during cellularisation and together with nullo, for specification of the furrow. During cellularisation, Slam marks first the furrow, which is derived from the metaphase furrow of the previous mitosis. Slightly later, Slam accumulates at new furrows between daughter cells early in interphase. Slam is stably associated with the furrow canal except for the onset of cellularisation as revealed by FRAP experiments. Restriction of Slam to the furrow canal and Slam mobility during cellularisation is controlled by the recycling endosome and centrosomes. We propose a three step model. The retracting metaphase furrow leaves an initial mark. This mark and the border between corresponding daughter nuclei are refined by vesicular transport away from pericentrosomal recycling endosome towards the margins of the somatic buds. Following the onset of zygotic gene expression, Slam and Nullo together stabilise this mark and Slam triggers invagination of the cleavage furrow.


Assuntos
Fase de Clivagem do Zigoto/fisiologia , Proteínas do Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Centrossomo/metabolismo , Clonagem Molecular , Drosophila/metabolismo , Embrião não Mamífero/metabolismo , Embrião não Mamífero/fisiologia , Recuperação de Fluorescência Após Fotodegradação , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Membrana , Microinjeções , Modelos Biológicos , Imagem com Lapso de Tempo
4.
J Cell Sci ; 126(Pt 8): 1796-805, 2013 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-23424199

RESUMO

During Drosophila embryogenesis, the first epithelium with defined cortical compartments is established during cellularization. Actin polymerization is required for the separation of lateral and basal domains as well as suppression of tubular extensions in the basal domain. The actin nucleator mediating this function is unknown. We found that the formin Diaphanous (Dia) is required for establishing and maintaining distinct lateral and basal domains during cellularization. In dia mutant embryos lateral marker proteins, such as Discs-large and Armadillo/ß-Catenin spread into the basal compartment. Furthermore, high-resolution and live-imaging analysis of dia mutant embryos revealed an increased number of membrane extensions and endocytic activity at the basal domain, indicating a suppressing function of dia on membrane invaginations. Dia function might be based on an antagonistic interaction with the F-BAR protein Cip4/Toca-1, a known activator of the WASP/WAVE-Arp2/3 pathway. Dia and Cip4 physically and functionally interact and overexpression of Cip4 phenocopies dia loss-of-function. In vitro, Cip4 inhibits mainly actin nucleation by Dia. Thus, our data support a model in which linear actin filaments induced by Dia stabilize cortical compartmentalization by antagonizing membrane turnover induced by WASP/WAVE-Arp2/3.


Assuntos
Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Animais , Proteínas de Transporte/genética , Drosophila , Proteínas de Drosophila/genética , Forminas , Ligação Proteica , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo
5.
Cells Dev ; 175: 203850, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37182581

RESUMO

Plant growth is driven by apical meristems at the shoot and root growth points, which comprise continuously active stem cell populations. While many of the key factors involved in homeostasis of the shoot apical meristem (SAM) have been extensively studied under artificial constant growth conditions, only little is known how variations in the environment affect the underlying regulatory network. To shed light on the responses of the SAM to ambient temperature, we combined 3D live imaging of fluorescent reporter lines that allowed us to monitor the activity of two key regulators of stem cell homeostasis in the SAM namely CLAVATA3 (CLV3) and WUSCHEL (WUS), with computational image analysis to derive morphological and cellular parameters of the SAM. Whereas CLV3 expression marks the stem cell population, WUS promoter activity is confined to the organizing center (OC), the niche cells adjacent to the stem cells, hence allowing us to record on the two central cell populations of the SAM. Applying an integrated computational analysis of our data we found that variations in ambient temperature not only led to specific changes in spatial expression patterns of key regulators of SAM homeostasis, but also correlated with modifications in overall cellular organization and shoot meristem morphology.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Brotos de Planta/metabolismo , Imageamento Tridimensional , Temperatura , Proteínas de Homeodomínio/metabolismo , Células-Tronco
6.
Nat Commun ; 14(1): 8001, 2023 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-38049411

RESUMO

Despite the importance of Nitric Oxide (NO) as signaling molecule in both plant and animal development, the regulatory mechanisms downstream of NO remain largely unclear. Here, we show that NO is involved in Arabidopsis shoot stem cell control via modifying expression and activity of ARGONAUTE 4 (AGO4), a core component of the RNA-directed DNA Methylation (RdDM) pathway. Mutations in components of the RdDM pathway cause meristematic defects, and reduce responses of the stem cell system to NO signaling. Importantly, we find that the stem cell inducing WUSCHEL transcription factor directly interacts with AGO4 in a NO dependent manner, explaining how these two signaling systems may converge to modify DNA methylation patterns. Taken together, our results reveal that NO signaling plays an important role in controlling plant stem cell homeostasis via the regulation of de novo DNA methylation.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Metilação de DNA/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Óxido Nítrico/metabolismo , Meristema/genética , Meristema/metabolismo , Arabidopsis/metabolismo , RNA/metabolismo , Regulação da Expressão Gênica de Plantas
7.
Nat Commun ; 14(1): 2128, 2023 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-37059727

RESUMO

Spatial specificity of cell fate decisions is central for organismal development. The phloem tissue mediates long-distance transport of energy metabolites along plant bodies and is characterized by an exceptional degree of cellular specialization. How a phloem-specific developmental program is implemented is, however, unknown. Here we reveal that the ubiquitously expressed PHD-finger protein OBE3 forms a central module with the phloem-specific SMXL5 protein for establishing the phloem developmental program in Arabidopsis thaliana. By protein interaction studies and phloem-specific ATAC-seq analyses, we show that OBE3 and SMXL5 proteins form a complex in nuclei of phloem stem cells where they promote a phloem-specific chromatin profile. This profile allows expression of OPS, BRX, BAM3, and CVP2 genes acting as mediators of phloem differentiation. Our findings demonstrate that OBE3/SMXL5 protein complexes establish nuclear features essential for determining phloem cell fate and highlight how a combination of ubiquitous and local regulators generate specificity of developmental decisions in plants.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Floema/metabolismo , Arabidopsis/metabolismo , Proteínas de Membrana/metabolismo , Diferenciação Celular , Regulação da Expressão Gênica de Plantas
8.
Elife ; 92020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32379043

RESUMO

Positional information is essential for coordinating the development of multicellular organisms. In plants, positional information provided by the hormone auxin regulates rhythmic organ production at the shoot apex, but the spatio-temporal dynamics of auxin gradients is unknown. We used quantitative imaging to demonstrate that auxin carries high-definition graded information not only in space but also in time. We show that, during organogenesis, temporal patterns of auxin arise from rhythmic centrifugal waves of high auxin travelling through the tissue faster than growth. We further demonstrate that temporal integration of auxin concentration is required to trigger the auxin-dependent transcription associated with organogenesis. This provides a mechanism to temporally differentiate sites of organ initiation and exemplifies how spatio-temporal positional information can be used to create rhythmicity.


Plants, like animals and many other multicellular organisms, control their body architecture by creating organized patterns of cells. These patterns are generally defined by signal molecules whose levels differ across the tissue and change over time. This tells the cells where they are located in the tissue and therefore helps them know what tasks to perform. A plant hormone called auxin is one such signal molecule and it controls when and where plants produce new leaves and flowers. Over time, this process gives rise to the dashing arrangements of spiraling organs exhibited by many plant species. The leaves and flowers form from a relatively small group of cells at the tip of a growing stem known as the shoot apical meristem. Auxin accumulates at precise locations within the shoot apical meristem before cells activate the genes required to make a new leaf or flower. However, the precise role of auxin in forming these new organs remained unclear because the tools to observe the process in enough detail were lacking. Galvan-Ampudia, Cerutti et al. have now developed new microscopy and computational approaches to observe auxin in a small plant known as Arabidopsis thaliana. This showed that dozens of shoot apical meristems exhibited very similar patterns of auxin. Images taken over a period of several hours showed that the locations where auxin accumulated were not fixed on a group of cells but instead shifted away from the center of the shoot apical meristems faster than the tissue grew. This suggested the cells experience rapidly changing levels of auxin. Further experiments revealed that the cells needed to be exposed to a high level of auxin over time to activate genes required to form an organ. This mechanism sheds a new light on how auxin regulates when and where plants make new leaves and flowers. The tools developed by Galvan-Ampudia, Cerutti et al. could be used to study the role of auxin in other plant tissues, and to investigate how plants regulate the response to other plant hormones.


Assuntos
Arabidopsis/metabolismo , Ácidos Indolacéticos/metabolismo , Organogênese Vegetal , Reguladores de Crescimento de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Técnicas Biossensoriais , Regulação da Expressão Gênica de Plantas , Genes Reporter , Microscopia Confocal , Organogênese Vegetal/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Fatores de Tempo , Transcrição Gênica
9.
Elife ; 92020 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-32723478

RESUMO

Quantitative analysis of plant and animal morphogenesis requires accurate segmentation of individual cells in volumetric images of growing organs. In the last years, deep learning has provided robust automated algorithms that approach human performance, with applications to bio-image analysis now starting to emerge. Here, we present PlantSeg, a pipeline for volumetric segmentation of plant tissues into cells. PlantSeg employs a convolutional neural network to predict cell boundaries and graph partitioning to segment cells based on the neural network predictions. PlantSeg was trained on fixed and live plant organs imaged with confocal and light sheet microscopes. PlantSeg delivers accurate results and generalizes well across different tissues, scales, acquisition settings even on non plant samples. We present results of PlantSeg applications in diverse developmental contexts. PlantSeg is free and open-source, with both a command line and a user-friendly graphical interface.


Assuntos
Arabidopsis/anatomia & histologia , Imageamento Tridimensional/métodos , Células Vegetais , Software , Arabidopsis/citologia , Redes Neurais de Computação
10.
Curr Biol ; 16(6): 543-52, 2006 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-16458513

RESUMO

BACKGROUND: The shape of a nucleus depends on the nuclear lamina, which is tightly associated with the inner nuclear membrane and on the interaction with the cytoskeleton. However, the mechanism connecting the differentiation state of a cell to the shape changes of its nucleus are not well understood. We investigated this question in early Drosophila embryos, where the nuclear shape changes from spherical to ellipsoidal together with a 2.5-fold increase in nuclear length during cellularization. RESULTS: We identified two genes, kugelkern and kurzkern, required for nuclear elongation. In kugelkern- and kurzkern-depleted embryos, the nuclei reach only half the length of the wild-type nuclei at the end of cellularization. The reduced nuclear size affects chromocenter formation as marked by Heterochromatin protein 1 and expression of a specific set of genes, including early zygotic genes. kugelkern contains a putative coiled-coil domain in the N-terminal half of the protein, a nuclear localization signal (NLS), and a C-terminal CxxM-motif. The carboxyterminal CxxM motif is required for the targeting of Kugelkern to the inner nuclear membrane, where it colocalizes with lamins. Depending on the farnesylation motif, expression of kugelkern in Drosophila embryos or Xenopus cells induces overproliferation of nuclear membrane. CONCLUSIONS: Kugelkern is so far the first nuclear protein, except for lamins, that contains a farnesylation site. Our findings suggest that Kugelkern is a rate-determining factor for nuclear size increase. We propose that association of farnesylated Kugelkern with the inner nuclear membrane induces expansion of nuclear surface area, allowing nuclear growth.


Assuntos
Núcleo Celular/ultraestrutura , Proteínas de Drosophila/fisiologia , Drosophila/embriologia , Drosophila/ultraestrutura , Proteínas Nucleares/fisiologia , Motivos de Aminoácidos , Animais , Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Regulação da Expressão Gênica , Cinética , Larva/metabolismo , Larva/ultraestrutura , Membrana Nuclear/metabolismo , Membrana Nuclear/fisiologia , Membrana Nuclear/ultraestrutura , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fenótipo , RNA/metabolismo , Xenopus
11.
J Vis Exp ; (146)2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-31058906

RESUMO

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Clonagem Molecular/métodos , Transativadores/metabolismo , Fatores de Transcrição/genética , Animais , Arabidopsis/metabolismo , Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Ratos , Receptores de Glucocorticoides/metabolismo
12.
Nat Commun ; 10(1): 5093, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31704928

RESUMO

To maintain the balance between long-term stem cell self-renewal and differentiation, dynamic signals need to be translated into spatially precise and temporally stable gene expression states. In the apical plant stem cell system, local accumulation of the small, highly mobile phytohormone auxin triggers differentiation while at the same time, pluripotent stem cells are maintained throughout the entire life-cycle. We find that stem cells are resistant to auxin mediated differentiation, but require low levels of signaling for their maintenance. We demonstrate that the WUSCHEL transcription factor confers this behavior by rheostatically controlling the auxin signaling and response pathway. Finally, we show that WUSCHEL acts via regulation of histone acetylation at target loci, including those with functions in the auxin pathway. Our results reveal an important mechanism that allows cells to differentially translate a potent and highly dynamic developmental signal into stable cell behavior with high spatial precision and temporal robustness.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Diferenciação Celular , Autorrenovação Celular , Proteínas de Homeodomínio/metabolismo , Ácidos Indolacéticos/metabolismo , Meristema/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proliferação de Células , Meristema/citologia , Brotos de Planta , Plantas Geneticamente Modificadas , Células-Tronco Pluripotentes/citologia , Transdução de Sinais
13.
Curr Opin Plant Biol ; 35: 117-123, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27918940

RESUMO

Developmental plasticity is a defining feature of plants allowing them to colonize a wide range of different ecosystems by promoting environmental adaptation. Their postembryonic development requires life-long maintenance of stem cells, which are embedded into specialized tissues, called meristems. The shoot apical meristem gives rise to all above ground tissues and is a complex and dynamic three-dimensional structure harboring cells of different clonal origins and fates. Functionally divergent subdomains are stably maintained despite permanent cell division, however their relative sizes are modified in response to developmental and environmental signals. In this review, we briefly describe the core regulatory program of the shoot apical meristem and discuss progress in the fields of mechanical and environmental control of its activity.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Meristema/crescimento & desenvolvimento , Células-Tronco/citologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Divisão Celular/genética , Meio Ambiente , Meristema/genética , Meristema/metabolismo , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Células-Tronco/metabolismo
14.
Elife ; 62017 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-29058667

RESUMO

Plant meristems carry pools of continuously active stem cells, whose activity is controlled by developmental and environmental signals. After stem cell division, daughter cells that exit the stem cell domain acquire transit amplifying cell identity before they are incorporated into organs and differentiate. In this study, we used an integrated approach to elucidate the role of HECATE (HEC) genes in regulating developmental trajectories of shoot stem cells in Arabidopsis thaliana. Our work reveals that HEC function stabilizes cell fate in distinct zones of the shoot meristem thereby controlling the spatio-temporal dynamics of stem cell differentiation. Importantly, this activity is concomitant with the local modulation of cellular responses to cytokinin and auxin, two key phytohormones regulating cell behaviour. Mechanistically, we show that HEC factors transcriptionally control and physically interact with MONOPTEROS (MP), a key regulator of auxin signalling, and modulate the autocatalytic stabilization of auxin signalling output.


Assuntos
Arabidopsis/fisiologia , Diferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Células Vegetais/fisiologia , Reguladores de Crescimento de Plantas/metabolismo , Células-Tronco/fisiologia , Genes de Plantas , Células Vegetais/efeitos dos fármacos , Brotos de Planta/fisiologia , Células-Tronco/efeitos dos fármacos , Transcrição Gênica
15.
PLoS One ; 8(12): e83043, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24376629

RESUMO

Building expression constructs for transgenesis is one of the fundamental day-to-day tasks in modern biology. Traditionally it is based on a multitude of type II restriction endonucleases and T4 DNA ligase. Especially in case of long inserts and applications requiring high-throughput, this approach is limited by the number of available unique restriction sites and the need for designing individual cloning strategies for each project. Several alternative cloning systems have been developed in recent years to overcome these issues, including the type IIS enzyme based Golden Gate technique. Here we introduce our GreenGate system for rapidly assembling plant transformation constructs, which is based on the Golden Gate method. GreenGate cloning is simple and efficient since it uses only one type IIS restriction endonuclease, depends on only six types of insert modules (plant promoter, N-terminal tag, coding sequence, C-terminal tag, plant terminator and plant resistance cassette), but at the same time allows assembling several expression cassettes in one binary destination vector from a collection of pre-cloned building blocks. The system is cheap and reliable and when combined with a library of modules considerably speeds up cloning and transgene stacking for plant transformation.


Assuntos
Arabidopsis/genética , Clonagem Molecular/métodos , Vetores Genéticos , Nicotiana/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Sequência de Bases , Desoxirribonucleases de Sítio Específico do Tipo II/química , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Proteínas de Plantas/química , Regiões Promotoras Genéticas , Regiões Terminadoras Genéticas , Transformação Genética , Transgenes
16.
Mech Dev ; 127(7-8): 371-84, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20060902

RESUMO

Essential for proper function of small GTPases of the Rho family, which control many aspects of cytoskeletal and membrane dynamics, is their temporal and spatial control by activating GDP exchange factors (GEFs) and deactivating GTPase-activating-proteins (GAPs). The regulatory mechanisms controlling these factors are not well understood, especially during development, when the organization and behaviour of cells change in a stage dependent manner. During Drosophila cellularization Rho signalling and RhoGEF2 are involved in furrow canal formation and the organization of actin and myosin. Here we analyze, how RhoGEF2 is localized at the sites of membrane invagination. We show that the PDZ domain is necessary for localization and function of RhoGEF2 and identify Slam as a factor that is necessary for RhoGEF2 localization. We also demonstrate that Slam can recruit RhoGEF2 to ectopic sites. Furthermore we find that the PDZ domain of RhoGEF2 can form a complex with Slam invivo and that Slam transcripts and protein colocalize at the furrow canal and in basal particles. Based on these findings, we propose that accumulation of slam mRNA and protein at the presumptive invagination site provides a spatial and temporal trigger for RhoGEF2-Rho1 signalling.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intracelular/genética , Dados de Sequência Molecular , Fenótipo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Relação Estrutura-Atividade , Proteínas rho de Ligação ao GTP/química
17.
Genes Dev ; 22(7): 878-83, 2008 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-18381892

RESUMO

Xenopus Paraxial Protocadherin (xPAPC) has signaling functions that are essential for convergent extension (CE) movements and tissue separation during gastrulation. PAPC modulates components of the planar cell polarity (PCP) pathway, but it is not clear how PAPC is connected to beta-catenin-independent Wnt-signaling. By yeast two-hybrid screen, we found that the intracellular domain of PAPC interacts with Sprouty (Spry), an inhibitor of CE movements. Upon binding to PAPC, Spry function is inhibited and PCP signaling is enhanced. Our data indicate that PAPC promotes gastrulation movements by sequestration of Spry and reveal a novel mechanism by which protocadherins modulate beta-catenin-independent Wnt-signaling.


Assuntos
Caderinas/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Proteínas Adaptadoras de Transdução de Sinal , Animais , Caderinas/genética , Linhagem Celular , Imunoprecipitação da Cromatina , Drosophila/genética , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Larva/genética , Proteínas de Membrana/genética , Microscopia de Fluorescência , Morfogênese , Fosfoproteínas/genética , Ligação Proteica , Protocaderinas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Leveduras/genética
18.
Development ; 132(5): 1009-20, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15689371

RESUMO

The physical interaction of the plasma membrane with the associated cortical cytoskeleton is important in many morphogenetic processes during development. At the end of the syncytial blastoderm of Drosophila the plasma membrane begins to fold in and forms the furrow canals in a regular hexagonal pattern. Every furrow canal leads the invagination of membrane between adjacent nuclei. Concomitantly with furrow canal formation, actin filaments are assembled at the furrow canal. It is not known how the regular pattern of membrane invagination and the morphology of the furrow canal is determined and whether actin filaments are important for furrow canal formation. We show that both the guanyl-nucleotide exchange factor RhoGEF2 and the formin Diaphanous (Dia) are required for furrow canal formation. In embryos from RhoGEF2 or dia germline clones, furrow canals do not form at all or are considerably enlarged and contain cytoplasmic blebs. Both Dia and RhoGEF2 proteins are localised at the invagination site prior to formation of the furrow canal. Whereas they localise independently of F-actin, Dia localisation requires RhoGEF2. The amount of F-actin at the furrow canal is reduced in dia and RhoGEF2 mutants, suggesting that RhoGEF2 and Dia are necessary for the correct assembly of actin filaments at the forming furrow canal. Biochemical analysis shows that Rho1 interacts with both RhoGEF2 and Dia, and that Dia nucleates actin filaments. Our results support a model in which RhoGEF2 and dia control position, shape and stability of the forming furrow canal by spatially restricted assembly of actin filaments required for the proper infolding of the plasma membrane.


Assuntos
Actinas/metabolismo , Proteínas de Transporte/fisiologia , Proteínas de Drosophila/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas rho de Ligação ao GTP/fisiologia , Junções Aderentes , Animais , Proteínas de Ciclo Celular , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Citoesqueleto/metabolismo , Drosophila melanogaster , Forminas , Glutationa/metabolismo , Glutationa Transferase/metabolismo , Microscopia de Fluorescência , Modelos Biológicos , Mutação , Fenótipo , Interferência de RNA , Fatores de Tempo
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