A C. elegans model for neurodegeneration in Cockayne syndrome.

Cockayne syndrome (CS) is a congenital syndrome characterized by growth and mental retardation, and premature ageing. The complexity of CS and mammalian models warrants simpler metazoan models that display CS-like phenotypes that could be studied in the context of a live organism. Here, we provide a characterization of neuronal and mitochondrial aberrations caused by a mutation in the csb-1 gene in Caenorhabditis elegans. We report a progressive neurodegeneration in adult animals that is enhanced upon UV-induced DNA damage. The csb-1 mutants show dysfunctional hyperfused mitochondria that degrade upon DNA damage, resulting in diminished respiratory activity. Our data support the role of endogenous DNA damage as a driving factor of CS-related neuropathology and underline the role of mitochondrial dysfunction in the disease

A Customized Light Sheet Microscope to Measure Spatio-Temporal Protein Dynamics in Small Model Organisms

We describe a customizable and cost-effective light sheet microscopy (LSM) platform for rapid three-dimensional imaging of protein dynamics in small model organisms. The system is designed for high acquisition speeds and enables extended time-lapse in vivo experiments when using fluorescently labeled specimens. We demonstrate the capability of the setup to monitor gene expression and protein localization during ageing and upon starvation stress in longitudinal studies in individual or small groups of adult Caenorhabditis elegans nematodes. The system is equipped to readily perform fluorescence recovery after photobleaching (FRAP), which allows monitoring protein recovery and distribution under low photobleaching conditions. Our imaging platform is designed to easily switch between light sheet microscopy and optical projection tomography (OPT) modalities. The setup permits monitoring of spatio-temporal expression and localization of ageing biomarkers of subcellular size and can be conveniently adapted to image a wide range of small model organisms and tissue samples.MR, GZ and AZ acknowledge funding from the Projects “Skin-DOCTor” Grant No. 1778 and “Neureka!” Grant No. LSF7-341 implemented under the "ARISTEIA" and "Supporting Postdoctoral Researchers" Actions respectively, of the "OPERATIONAL PROGRAMME EDUCATION AND LIFELONG LEARNING", (http://www.espa.gr/en/pages/staticOPEducationandLifelongLearning.aspx), which is co-funded by the European Social Fund and National Resources and from the EU Marie Curie Initial Training Network “OILTEBIA”, Grant No. PITNGA-2012-317526 (http://ec.europa.eu/research/mariecurieactions/, http://gdo.uc3m.es/oiltebia/index. php?option = com_content&view = frontpage). JR acknowledges support from EC FP7 CIG grant HIGHTHROUGHPUT TOMO PICIG12-GA-2012-333632, (http://cordis.europa.eu/projects/333632), and Spanish MINECO grant MESO-IMAGING FIS2013-41802-R, (https://sede.micinn.gob.es/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Improved Biocompatibility of Amino‐Functionalized Graphene Oxide in Caenorhabditis elegans

International audienc

Microscopic Optical Projection Tomography In Vivo

We describe a versatile optical projection tomography system for rapid three-dimensional imaging of microscopic specimens in vivo. Our tomographic setup eliminates the in xy and z strongly asymmetric resolution, resulting from optical sectioning in conventional confocal microscopy. It allows for robust, high resolution fluorescence as well as absorption imaging of live transparent invertebrate animals such as C. elegans. This system offers considerable advantages over currently available methods when imaging dynamic developmental processes and animal ageing; it permits monitoring of spatio-temporal gene expression and anatomical alterations with single-cell resolution, it utilizes both fluorescence and absorption as a source of contrast, and is easily adaptable for a range of small model organisms

Απεικόνιση του νηματώδους C. elegans με χρήση οπτικής προβολικής τομογραφίας.

Small sample spatial in vivo imaging techniques such as confocal microscopy, micro MRT (µMRT), Selective Plane Illumination Microscopy (SPIM) or contrast enhanced techniques such as DICM (Differential Interference Contrast Microscopy) are common tools for imaging fluorescent expression in the nematode Caenorhabditis elegans. However, these methods have limited capacity for high resolution, rapid, whole body 3D microscopic imaging and/or imaging of multiple contrast agents. The recently developed approach of Optical Projection Tomography (OPT) enables 3D visualization of whole specimens up to several millimetres in size as has already been shown in zebra fish, chick and mouse embryos. This is achieved by applying a filtered back projection algorithm on images taken from equidistant angles of a rotating specimen with magnification dependent resolution, down to 1-5 µm. Here I present a modified OPT setup for 3D imaging of GFP expressing neuronal cells in C. elegans. This study demonstrates that this novel technique allows rapid acquisition of whole-animal fluorescent expression patterns in the nematode with high accuracy. OPT visualization can easily be adapted to image multiple tissues and cell types, with a variety of chromophores, that allow multi-colour projections, in the nematod

Light Sheet Microscopy to Measure Protein Dynamics

Visualizing protein dynamics is the key to a quantitative understanding of molecular mechanisms in biological systems. Recent developments in fluorescent microscopy techniques allow for novel experimental approaches to study stochastic localization, distribution, and movement of fluorescently labeled molecules in high resolution as they occur over time in the context of living cells and whole organisms. Particularly suitable for such studies is the application of light sheet microscopy, as it enables rapid in vivo imaging of a wide range and size of specimen, from whole organisms and organs, down to the dynamics of subcellular structures and single molecules. This article summarizes the principles of light sheet microscopy and its advantages over other optical imaging techniques, such as light microscopy and confocal microscopy, and highlights recent innovations that significantly enhance spatio-temporal resolution. Also, this manuscript contains basic guidelines for the implementation of light sheet microscopy in the laboratory and presents thus far unpublished light sheet microscopy applications demonstrating the ability of this technology to measure protein dynamics in a whole-organism context. (C) 2016 Wiley Periodicals, Inc

Ο ρόλος της αποδόμησης του mRNA στη γήρανση του Caenorhabditis elegans.

Alterations of general or specific mRNA levels are a universal manifestation of the ageing process (Cookson, 2011). During their existence, mRNAs are constantly decorated by dynamically changing factors, which form messenger ribonucleoprotein (mRNP) complexes and determine the fate of an mRNA. Mechanisms that control mRNA turnover in the cytoplasm have been described in great detail but whether they might be involved in the regulation of ageing is unknown (Anderson and Kedersha, 2009; Decker and Parker, 2012). Bulk mRNA decay in eukaryotes is initiated by irreversible shortening of the poly(A)-tail, subsequent decapping and final 5’ to 3’ degradation (Houseley and Tollervey, 2009). We present compelling evidence that EDC-3, a highly conserved modulator of decapping, is a novel determinant of ageing in C. elegans. Decapping has been shown to regulate protein synthesis by competing with the mechanism of translation initiation. Congruently, we find that EDC-3 regulates protein synthesis and lifespan in interaction with the previously described translation initiation factor IFE-2, an isoform of the human eIF4E, which has a conserved role in the control of ageing. We demonstrate that EDC-3 and IFE-2 mediated regulation of C. elegans lifespan happens specifically in neuronal tissue and governs neural integrity. Further, we show that loss of EDC-3 protects from oxidative and heat induced stress and that lifespan extension depends on the activity of Nrf-like xenobiotic- response factor SKN-1 and heat shock response factor HSF-1. Also, longevity upon loss of EDC-3 triggers a ROS induced hormesis response that depends on SKN-1 activity. Most mRNPs accumulate in distinct cellular foci termed processing bodies (P-bodies) or stress granules, which store mRNAs stalled in modes of degradation or translation initiation (Sheth and Parker, 2003, reviewed in Decker and Parker, 2012; Franks and Lykke-Andersen, 2008). Decapping factors, including EDC-3, are part of P-bodies, while IFE-2 localizes to stress granules in C. elegans. We establish an increased formation of P-bodies and stress granules and their co-localization upon specific stress insults and during age in the nematode, thereby defining them as biomarkers of ageing. It is unknown, whether mRNP granule formation is cause or consequence of mRNA decay and stress response (Eulalio et al., 2007). We demonstrate, that loss of SKN-1 contributes to an increased formation of P-bodies upon oxidative stress. Curiously, down-regulation of HSF-1 prevents P-body assembly specifically upon heat stress and causes age-related granulation of IFE-2. These results implicate that mRNP aggregation is a transcriptionally controlled process that contributes to maintenance of cellular stress response and ageing. Unexpectedly, we find that HSF-1 suppresses transcription, stability and nuclear granulation of IFE-2 during ageing. These granules co-localize with components of P-bodies at the nuclear envelope, which also have been shown to be involved in transcription regulation in the nucleus (summarized in Reines, 2012). Excitingly, we observe decreased localization of IFE- 2 in the nucleus, upon depletion of EDC-3. Our findings suggest that HSF-1 modulates IFE-2 function and localization during ageing, and that IFE-2 also serves as nuclear mRNP export factor in C. elegans. Thus, IFE-2 likely mediates the effects of the heat stress response on both mRNA translation and degradation to influence ageing

P-body and Stress Granule Quantification in Caenorhabditis elegans

Eukaryotic cells contain various types of cytoplasmic, non-membrane bound ribonucleoprotein (RNP) granules that consist of non-translating mRNAs and a versatile set of associated proteins. One prominent type of RNP granules is Processing bodies (P bodies), which majorly harbors translationally inactive mRNAs and an array of proteins mediating mRNA degradation, translational repression and cellular mRNA transport (Sheth and Parker, 2003). Another type of RNP granules, the stress granules (SGs), majorly contain mRNAs associated with translation initiation factors and are formed upon stress-induced translational stalling (Kedersha et al., 2000 and 1999). Multiple evidence obtained from studies in unicellular organisms supports a model in which P bodies and SGs physically interact during cellular stress to direct mRNAs for transport, decay, temporal storage or reentry into translation (Anderson and Kedersha, 2008; Decker and Parker, 2012). The quantification, distribution and colocalization of P bodies and/or SGs are essential tools to study the composition of RNP granules and their contribution to fundamental cellular processes, such as stress response and translational regulation. In this protocol we describe a method to quantify P bodies and SGs in somatic tissues of the nematode Caenorhabditis elegans

Generation of Caenorhabditis elegans Transgenic Animals by DNA Microinjection

Microinjection is the most frequently used tool for genetic transformation of the nematode Caenorhabditis elegans, facilitating the transgenic expression of genes, genome editing by the clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 system, or transcription of dsRNA for RNA intereference (RNAi). Exogenous DNA is delivered into the developing oocytes in the germline of adult hermaphrodites, which then generate transgenic animals among their offspring. In this protocol, we describe the microinjection procedure and the subsequent selection of transgenic progeny