Genetically encoded thiol redox-sensors in the zebrafish model: lessons for embryonic development and regeneration.

Important roles for reactive oxygen species (ROS) and redox signaling in embryonic development and regenerative processes are increasingly recognized. However, it is difficult to obtain information on spatiotemporal dynamics of ROS production and signaling in vivo. The zebrafish is an excellent model for in vivo bioimaging and possesses a remarkable regenerative capacity upon tissue injury. Here, we review data obtained in this model system with genetically encoded redox-sensors targeting H2O2 and glutathione redox potential. We describe how such observations have prompted insight into regulation and downstream effects of redox alterations during tissue differentiation, morphogenesis and regeneration. We also discuss the properties of the different sensors and their consequences for the interpretation of in vivo imaging results. Finally, we highlight open questions and additional research fields that may benefit from further application of such sensor systems in zebrafish models of development, regeneration and disease.

High-level recombinant protein expression in transgenic plants by using a double-inducible viral vector.

We describe here a unique ethanol-inducible process for expression of recombinant proteins in transgenic plants. The process is based on inducible release of viral RNA replicons from stably integrated DNA proreplicons. A simple treatment with ethanol releases the replicon leading to RNA amplification and high-level protein production. To achieve tight control of replicon activation and spread in the uninduced state, the viral vector has been deconstructed, and its two components, the replicon and the cell-to-cell movement protein, have each been placed separately under the control of an inducible promoter. Transgenic Nicotiana benthamiana plants incorporating this double-inducible system demonstrate negligible background expression, high (over 0.5 × 10(4)-fold) induction multiples, and high absolute levels of protein expression upon induction (up to 4.3 mg/g fresh biomass). The process can be easily scaled up, supports expression of practically important recombinant proteins, and thus can be directly used for industrial manufacturing.

CoA Synthase is phosphorylated on tyrosines in mammalian cells, interacts with and is dephosphorylated by Shp2PTP.

CoA Synthase (CoASy, 4'-phosphopantetheine adenylyltransferase/dephospho-CoA kinase) mediates two final stages of de novo coenzyme A (CoA) biosynthesis in higher eukaryotes. Unfortunately very little is known about regulation of this important metabolic pathway. In this study, we demonstrate that CoASy interacts in vitro with Src homology-2 (SH2) domains of a number of signaling proteins, including Src homology-2 domains containing protein tyrosine phosphatase (Shp2PTP). Complexes between CoASy and Shp2PTP exist in vivo in mammalian cells and this interaction is regulated in a growth-factor-dependent manner. We have also demonstrated that endogenous CoASy is phosphorylated on tyrosine residues in vivo, and that cytoplasmic protein tyrosine kinases can mediate this phosphorylation in vitro and in vivo. Importantly, Shp2PTP-mediated CoASy in vitro dephosphorylation leads to an increase in CoASy enzymatic phosphopantetheine adenylyltransferase (PPAT) activity. We therefore argue that CoASy is a novel potential substrate of Shp2PTP and phosphorylation of CoASy at tyrosine residue(s) could represent unrecognized before mechanism of modulation intracellular CoA level in response to hormonal and (or) other extracellular stimuli.

CoA synthase is in complex with p85alphaPI3K and affects PI3K signaling pathway.

The complex interplay between cellular signaling and metabolism in eukaryotic cells just start to emerge. Coenzyme A (CoA) and its derivatives play a key role in cell metabolism and also participate in regulatory processes. CoA synthase (CoASy) is a mitochondria-associated enzyme which mediates two final stages of de novo CoA biosynthesis. Here, we report that CoASy is involved in signaling events in the cell and forms a functional complex with p85alphaPI3K in vivo. Importantly, observed interaction of endogenous CoASy and p85alphaPI3K is regulated in a growth factor dependent manner. Surprisingly, both catalytic p110alpha and regulatory p85alpha subunits of PI3K were detected in mitochondrial fraction where mitochondria-localized p85alphaPI3K was found in complex with CoASy. Unexpectedly, significant changes of PI3K signaling pathway activity were observed in experiments with siRNA-mediated CoASy knockdown pointing on the role of CoA biosynthetic pathway in signal transduction.

Distinct roles for BAI1 and TIM-4 in the engulfment of dying neurons by microglia.

The removal of dying neurons by microglia has a key role during both development and in several diseases. To date, little is known about the cellular and molecular processes underlying neuronal engulfment in the brain. Here we took a live imaging approach to quantify neuronal cell death progression in embryonic zebrafish brains and studied the response of microglia. We show that microglia engulf dying neurons by extending cellular branches that form phagosomes at their tips. At the molecular level we found that microglia lacking the phosphatidylserine receptors BAI1 and TIM-4, are able to recognize the apoptotic targets but display distinct clearance defects. Indeed, BAI1 controls the formation of phagosomes around dying neurons and cargo transport, whereas TIM-4 is required for phagosome stabilization. Using this single-cell resolution approach we established that it is the combined activity of BAI1 and TIM-4 that allows microglia to remove dying neurons.

Generation and characterization of monoclonal antibodies to mTOR kinase.

Abstract Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that plays a critical role in the regulation of basic cellular functions, including cellular growth and proliferation. In this study we describe the generation and characterization of novel monoclonal antibodies directed against mTOR protein kinase. A GST-tagged fragment of mTOR expressed in bacteria was used as an antigen. Antibody-producing hybridoma cells were obtained by fusing SP2/0 myeloma cells with splenocytes from immunized mice. Anti-mTOR antibody-producing hybridoma cell lines were first identified by enzyme-linked immunosorbent assay and then subcloned by limiting dilution. Antibodies produced by selected clones were further tested for their reactivity towards the GST/mTOR 1334-1504 recombinant protein. Furthermore, antibody produced by F11 clone was shown to recognize specifically mTOR in different tissues and cell lines in Western blotting, immunoprecipitation, and immunohistochemistry. In addition, mTOR F11 antibody was suitable for immunoprecipitating and testing mTOR activity in in vitro kinase assay. In summary, generated antibodies will be useful for investigating mTOR signaling complexes in normal and pathological states.

Generation and characterization of monoclonal antibodies to TDRD7 protein.

TDRD7 is a scaffold protein whose specific function is unknown. It has been identified in complexes with proteins that regulate cytoskeleton dynamics and centrosomal movements, mRNA transport, and protein translation apparatus. Here we report the generation and characterization of monoclonal antibodies against TDRD7 protein. Bacterially expressed His-tagged fragments of TDRD7 were used as antigens. Spleen cells from immunized mice were collected and fused with SP2/0 myeloma cells using PEG 2000. High titer anti-TDRD7 antibody-producing hybridoma cell lines were identified by enzyme-linked immunosorbent assay (ELISA) and then subcloned by limiting dilution. Antibodies produced by E6 clone were further tested for their reactivity with the TDRD7 recombinant proteins. The results obtained clearly indicate that E6 anti-TDRD7 antibodies recognize specifically recombinant 6His-tagged TDRD7 proteins and endogenous TDRD7 in Western blotting, immunoprecipitation, and immunocytochemistry. In summary, these antibodies will be useful for researchers investigating TDRD7 and molecular complexes involving this protein.

Identification of a novel CoA synthase isoform, which is primarily expressed in the brain.

CoA and its derivatives Acetyl-CoA and Acyl-CoA are important players in cellular metabolism and signal transduction. CoA synthase is a bifunctional enzyme which mediates the final stages of CoA biosynthesis. In previous studies, we have reported molecular cloning, biochemical characterization, and subcellular localization of CoA synthase (CoASy). Here, we describe the existence of a novel CoA synthase isoform, which is the product of alternative splicing and possesses a 29aa extension at the N-terminus. We termed it CoASy beta and originally identified CoA synthase, CoASy alpha. The transcript specific for CoASy beta was identified by electronic screening and by RT-PCR analysis of various rat tissues. The existence of this novel isoform was further confirmed by immunoblot analysis with antibodies directed to the N-terminal peptide of CoASy beta. In contrast to CoASy alpha, which shows ubiquitous expression, CoASy beta is primarily expressed in the brain. Using confocal microscopy, we demonstrated that both isoforms are localized on mitochondria. The N-terminal extension does not affect the activity of CoA synthase, but possesses a proline-rich sequence which can bring the enzyme into complexes with signalling proteins containing SH3 or WW domains. The role of this novel isoform in CoA biosynthesis, especially in the brain, requires further elucidation.