Development of a chemical genetic approach for human aurora B kinase identifies novel substrates of the chromosomal passenger complex

To understand how the chromosomal passenger complex ensures chromosomal stability, it is crucial to identify its substrates and to find ways to specifically inhibit the enzymatic core of the complex, Aurora B. We therefore developed a chemical genetic approach to selectively inhibit human Aurora B. By mutating the gatekeeper residue Leu-154 in the kinase active site, the ATP-binding pocket was enlarged, but kinase function was severely disrupted. A unique second site suppressor mutation was identified that rescued kinase activity in the Leu-154 mutant and allowed the accommodation of bulky N(6)-substituted adenine analogs. Using this analog-sensitive Aurora B kinase, we found that retention of the chromosomal passenger complex at the centromere depends on Aurora B kinase activity. Furthermore, analog-sensitive Aurora B was able to use bulky ATPγS analogs and could thiophosphorylate multiple proteins in cell extracts. Utilizing an unbiased approach for kinase substrate mapping, we identified several novel substrates of Aurora B, including the nucleosomal-binding protein HMGN2. We confirmed that HMGN2 is a bona fide Aurora B substrate in vivo and show that its dynamic association to chromatin is controlled by Aurora B.     

Functions of the MAPK family in vertebrate-development

The mitogen activated protein kinase (MAPK) family, consisting of the extracellular signal regulated protein kinase, c-Jun amino terminal MAPK and p38 subfamilies, is conserved in evolution throughout the plant and animal kingdoms. These proteins have been implicated in diverse cellular processes including cell growth, migration, proliferation, differentiation, survival and development. Gene-targeting approaches in mice, chickens, frogs and zebrafish revealed crucial roles of MAPK in vertebrate development. Gene-disruption or -silencing often lead to lethal effects, therefore the zebrafish ex utero development provides an excellent in vivo model to study the function of MAPK in early embryogenesis. In this review, we summarize the current understanding of the MAPK family function in vertebrate-development and place this into the perspective of possibilities for future research.     

Circuit neuroscience in zebrafish

A central goal of modern neuroscience is to obtain a mechanistic understanding of higher brain functions under healthy and diseased conditions. Addressing this challenge requires rigorous experimental and theoretical analysis of neuronal circuits. Recent advances in optogenetics, high-resolution in vivo imaging, and reconstructions of synaptic wiring diagrams have created new opportunities to achieve this goal. To fully harness these methods, model organisms should allow for a combination of genetic and neurophysiological approaches in vivo. Moreover, the brain should be small in terms of neuron numbers and physical size. A promising vertebrate organism is the zebrafish because it is small, it is transparent at larval stages and it offers a wide range of genetic tools and advantages for neurophysiological approaches. Recent studies have highlighted the potential of zebrafish for exhaustive measurements of neuronal activity patterns, for manipulations of defined cell types in vivo and for studies of causal relationships between circuit function and behavior. In this article, we summarize background information on the zebrafish as a model in modern systems neuroscience and discuss recent results.     

A Purine Analog-Sensitive Protein Kinase Activity Associates with Trk Nerve Growth Factor Receptors

Abstract: Previous studies showed that purine analogs block with varying efficiency and specificity certain effects of nerve growth factor (NGF) on PC12 cells. These compounds also inhibit protein kinase activities. The analog 6-thioguanine has thus far been shown to inhibit only protein kinase N, an NGF-activated protein kinase, whereas 2-aminopurine also blocks other kinases. In the present study, immunoprecipitates of Trk NGF receptors from PC12 cells (NGF treatment) were assayed for protein kinase activity by using the substrates myelin basic protein and histone HF1 under phosphorylating conditions optimal for protein kinase N and in the presence or absence of purine analogs. Activity was detected and ∼50–80% was inhibited by these compounds. The purine analog-sensitive activity was maximally stimulated by NGF within 5 min, was partially decreased by 10 min, and still remained over basal levels after 15 h of NGF treatment. Analysis of myelin basic protein phosphorylated by anti-Trk immunoprecipitates revealed an NGF-stimulated increase in phosphothreonine and phosphotyrosine. Phosphorylation of threonine, but not of tyrosine residues, was inhibited by 6-thioguanine, which therefore inhibits a serine/threonine kinase associated with NGF receptor rather than the receptor kinase itself. Neither 2-aminopurine nor 6-thioguanine inhibited the NGF-dependent induction of Trk-associated kinase activity. Our findings thus indicate association of a purine analog-sensitive serine/threonine protein kinase activity with Trk NGF receptors.     

Gastrulation dynamics: cells move into focus

During vertebrate gastrulation, a relatively limited number of blastodermal cells undergoes a stereotypical set of cellular movements that leads to formation of the three germ layers: ectoderm, mesoderm and endoderm. Gastrulation, therefore, provides a unique developmental system in which to study cell movements in vivo in a fairly simple cellular context. Recent advances have been made in elucidating the cellular and molecular mechanisms that underlie cell movements during zebrafish gastrulation. These findings can be compared with observations made in other model systems to identify potential general mechanisms of cell migration during development.     

Real‐time imaging and genetic dissection of host–microbe interactions in zebrafish

Many aspects of host interactions with microbes can only be studied in the context of a whole organism. The zebrafish as a model organism has shown to be highly successful for studies of infection biology and the interactions of commensal microbiota with their hosts. Zebrafish are transparent during embryo and larval development and these early life stages are optimally suited for high‐resolution imaging of host–microbe interactions in a vertebrate organism. This is facilitated by the development of a variety of fluorescent reporter lines that mark different immune cell types or subcellular compartments where pathogens reside. The zebrafish is an excellent vertebrate model for forward genetic screening and efficient tools for gene knock‐down and targeted mutagenesis add further to the strength of this model organism. The use of zebrafish larvae for studying microbial infections has recently led to important new insights in host defence mechanisms, which are highlighted in this review focused on bacterial pathogens. Considering the highly conserved nature of the processes involved, including innate immune recognition, immunometabolism and autophagy, it is to be expected that these recent findings in zebrafish will have great translational value for biomedical applications.     

An ATP analog-sensitive version of the tomato cell death suppressor protein kinase Adi3 for use in substrate identification

Adi3 is a protein kinase from tomato that functions as a cell death suppressor and its substrates are not well defined. As a step toward identifying Adi3 substrates we developed an ATP analog-sensitive version of Adi3 in which the ATP-binding pocket is mutated to allow use of bulky ATP analogs. Met385 was identified as the “gatekeeper” residue and the M385G mutation allows for the use of two bulky ATP analogs. Adi3^M385G can also specifically utilize N⁶-benzyl-ATP to phosphorylate a known substrate and provides a tool for identifying Adi3 substrates.     

The zebrafish myotome contains tonic muscle fibers: Morphological characterization and time course of formation

It is long known that the skeletal muscle of teleost fish contains muscle fibers which are in all probability of a tonic type according to morphological criteria. However, the evidence for the existence of teleost tonic fibers is still confined to a very small number of species, and knowledge concerning their ontogeny and possible functions is even more restricted. A remarkable deficit in this context is that it is not even exactly known whether the zebrafish, which is widely used to study vertebrate developmental biology, has such fibers, or how they arise. The present study demonstrates the existence of tonic fibers in the zebrafish myotome. They are identical with a fiber population previously termed “red muscle rim” fibers. A combined histochemical, immunocytochemical, and ultrastructural approach is used to characterize the morphology and development of these fibers. This study provides a basis for using the zebrafish model system in the future research on the developmental regulation and the functions of tonic fibers. J. Morphol., 2013. © 2012 Wiley Periodicals, Inc.     

High-throughput imaging of adult fluorescent zebrafish with an LED fluorescence macroscope

Zebrafish are a useful vertebrate model for the study of development, behavior, disease and cancer. A major advantage of zebrafish is that large numbers of animals can be economically used for experimentation; however, high-throughput methods for imaging live adult zebrafish had not been developed. Here, we describe protocols for building a light-emitting diode (LED) fluorescence macroscope and for using it to simultaneously image up to 30 adult animals that transgenically express a fluorescent protein, are transplanted with fluorescently labeled tumor cells or are tagged with fluorescent elastomers. These protocols show that the LED fluorescence macroscope is capable of distinguishing five fluorescent proteins and can image unanesthetized swimming adult zebrafish in multiple fluorescent channels simultaneously. The macroscope can be built and used for imaging within 1 day, whereas creating fluorescently labeled adult zebrafish requires 1 hour to several months, depending on the method chosen. The LED fluorescence macroscope provides a low-cost, high-throughput method to rapidly screen adult fluorescent zebrafish and it will be useful for imaging transgenic animals, screening for tumor engraftment, and tagging individual fish for long-term analysis.     

Approaches to measuring calcium in zebrafish: focus on neuronal development

Calcium ions are known to act as important cellular signals during nervous system development. In vitro studies have provided significant information on the role of calcium signals during neuronal development; however, the function of this messenger in nervous system maturation in vivo remains to be established. The zebrafish has emerged as a valuable model for the study of vertebrate embryogenesis. Fertilisation is external and the rapid growth of the transparent embryo, including development of internal organs, can be observed easily making it well suited for imaging studies. The developing nervous system is relatively simple and has been well characterised, allowing individual neurons to be identified. Using the zebrafish model, both intracellular and intercellular calcium signals throughout embryonic development have been characterised. This review summarises technical approaches to measure calcium signals in developing embryonic and larval zebrafish, and includes recent developments that will facilitate the study of calcium signalling in vivo. The application of calcium imaging techniques to investigate the action of this messenger during embryogenesis in intact zebrafish is illustrated by discussion of their contribution to our understanding of neuronal development in vivo.     

Zebrafish xenografts as a tool for in vivo studies on human cancer

The zebrafish has become a powerful vertebrate model for genetic studies of embryonic development and organogenesis and increasingly for studies in cancer biology. Zebrafish facilitate the performance of reverse and forward genetic approaches, including mutagenesis and small molecule screens. Moreover, several studies report the feasibility of xenotransplanting human cells into zebrafish embryos and adult fish. This model provides a unique opportunity to monitor tumor-induced angiogenesis, invasiveness, and response to a range of treatments in vivo and in real time. Despite the high conservation of gene function between fish and humans, concern remains that potential differences in zebrafish tissue niches and/or missing microenvironmental cues could limit the relevance and translational utility of data obtained from zebrafish human cancer cell xenograft models. Here, we summarize current data on xenotransplantation of human cells into zebrafish, highlighting the advantages and limitations of this model in comparison to classical murine models of xenotransplantation.     

Chemical discovery and global gene expression analysis in zebrafish

The zebrafish (Danio rerio) provides an excellent model for studying vertebrate development and human disease because of its ex utero, optically transparent embryogenesis and amenability to in vivo manipulation. The rapid embryonic developmental cycle, large clutch sizes and ease of maintenance at large numbers also add to the appeal of this species. Considerable genomic data has recently become publicly available that is aiding the construction of zebrafish microarrays, thus permitting global gene expression analysis. The zebrafish is also suitable for chemical genomics, in part as a result of the permeability of its embryos to small molecules and consequent avoidance of external confounding maternal effects. Finally, there is increasing characterization and analysis of zebrafish models of human disease. Thus, the zebrafish offers a high-quality, high-throughput bioassay tool for determining the biological effect of small molecules as well as for dissecting biological pathways.     

A model 450 million years in the making: zebrafish and vertebrate immunity

Since its first splash 30 years ago, the use of the zebrafish model has been extended from a tool for genetic dissection of early vertebrate development to the functional interrogation of organogenesis and disease processes such as infection and cancer. In particular, there is recent and growing attention in the scientific community directed at the immune systems of zebrafish. This development is based on the ability to image cell movements and organogenesis in an entire vertebrate organism, complemented by increasing recognition that zebrafish and vertebrate immunity have many aspects in common. Here, we review zebrafish immunity with a particular focus on recent studies that exploit the unique genetic and in vivo imaging advantages available for this organism. These unique advantages are driving forward our study of vertebrate immunity in general, with important consequences for the understanding of mammalian immune function and its role in disease pathogenesis.     

A chemical-genetic approach for functional analysis of plant protein kinases

Plant genomes encode hundreds of protein kinases, yet only for a small fraction of them precise functions and phosphorylation targets have been identified. Recently, we applied a chemical-genetic approach to sensitize the tomato serine/threonine kinase Pto to analogs of PP1, an ATP-competitive and cell-permeable small-molecule inhibitor. The Pto kinase confers resistance to Pst bacteria by activating immune responses upon specific recognition of bacterial effectors. By using PP1 analogs in combination with the analog-sensitive Pto, we shed new light on the role of Pto kinase activity in effector recognition and signal transduction. Here we broaden the use of this chemical-genetic approach to another defense-related plant protein kinase, the MAP kinase LeMPK3. In addition, we show that analog-sensitive but not wild-type kinases are able to use unnatural N⁶-modified ATP analogs as phosphodonors that can be exploited for tagging direct phosphorylation targets of the kinase of interest. Thus, sensitization of kinases to analogs of the small-molecule inhibitor PP1 and ATP can be an effective tool for the discovery of cellular functions and phosphorylation substrates of plant protein kinases.Key words: chemical genetics, gatekeeper, LeMPK3, protein kinase, Pto, small-molecule inhibitor     

In vivo biofluid dynamic imaging in the developing zebrafish

Flow-structure interactions are ubiquitous in nature, and are important factors in the proper development of form and function in living organisms. In order to uncover the mechanisms by which flow-structure interactions affect vertebrate development, we first need to establish the techniques necessary to quantitatively describe the fluid flow environment within the embryo. To do this, we must bring dynamic, in vivo imaging methods to bear on living systems. Traditional avian and mammalian model systems can be problematic in this regard. The zebrafish (Danio rerio) is widely accepted as an excellent model organism for the study of vertebrate biology, as it shows substantial anatomical and genetic conservation with higher vertebrates, including humans. Their small size, optical transparency, and external development make zebrafish the ideal model system for dynamic imaging. This article reviews the current state of research in imaging biofluid flow within and around developing zebrafish embryos, with an emphasis on dynamic imaging modalities.     

斑马鱼胸苷酸合成酶与自身mRNA的相互作用

斑马鱼(zebrafish,Danio rerio)是生物学领域中公认的研究脊椎类生物的模式生物.胸苷酸合成酶(thymidylate synthase,TS)是DNA从头合成的限速酶,多年来一直作为肿瘤化疗的重要靶酶.前期的研究表明,人和大肠杆菌中TS能与自身的mRNA结合,在翻译水平上具有反馈抑制自调控现象.斑马鱼作为药物模型的研究已成为热点研究领域,为了探讨斑马鱼的胸苷酸合成酶的调控规律,以及与人TS的相关性,利用原核表达,纯化获得高均一性斑马鱼TS蛋白,采用凝胶迁移研究了TS和其mRNA的体外结合,采用免疫共沉淀:RT-PCR技术研究了它们在体内的相互作用,实验结果表明,斑马鱼的TS在体内外均与自身的mRNA存在特异性的相互作用.研究说明,斑马鱼和人的TS具有高度生物学过程相关性,为构建斑马鱼抗肿瘤药理模型提供了理论基础.     

Strategies for the identification of kinase substrates using analog-sensitive kinases

Phosphorylation of proteins is a prevalent post-translational modification, which affects intracellular signaling in many ways. About 2% of all eukaryotic genes code for protein kinases catalyzing phosphorylation events. Despite technological advances that have made it possible to identify thousands of phosphorylation sites simultaneously, identification of the substrates of a given kinase remains an exceptionally challenging task. Here, we summarize approaches for substrate identification that make use of genetically engineered ‘analog-sensitive’ kinases.     

Behavioral screening for neuroactive drugs in zebrafish

The larval zebrafish has emerged asa vertebrate model system amenable to small molecule screens for probing diverse biological pathways. Two large-scale small molecule screens examined the effects of thousands of drugs on larval zebrafish sleep/wake and photomotor response behaviors. Both screens identified hundreds of molecules that altered zebrafish behavior in distinct ways. The behavioral profiles induced by these small molecules enabled the clustering of compounds according to shared phenotypes. This approach identified regulators of sleep/wake behavior and revealed the biological targets for poorly characterized compounds. Behavioral screening for neuroactive small molecules in zebrafish is an attractive complement to in vitro screening efforts, because the complex interactions in the vertebrate brain can only be revealed in vivo.