This article is available online at http://www.jlr.orgWith short lifespan, rapid reproduction cycle, amenable genetics, and a remarkable conservation of human disease genes and pathways, Caenorhabditis elegans has become an ideal model to study the mechanisms of disease pathogenesis ( 1) . Being optically transparent, C. elegans has been extensively employed to visualize lipid storage with fl uorescent imaging of lipids stained with Nile Red ( 2, 3) or recently with label-free single-frequency coherent anti-Stokes Raman scattering (CARS) imaging ( 4) . However, either fl uorescent or single-frequency CARS imaging lacks spectral information critical for lipid composition analysis. Although spontaneous Raman microscopy ( 5) and multiplex CARS microscopy ( 6) could analyze the composition of single lipid droplets, their image acquisition speeds are too slow for live-cell or animal study. Alternatively, third harmonic generation microscopy can visualize lipid droplets using optical heterogeneity of biological samples for contrast mechanism ( 7) . However, third harmonic generation lacks chemical selectivity to analyze lipid composition. Consequently, a microscopy tool to study critical aspects of lipid metabolism, including lipid storage and composition, is lacking. By employing a multifunctional microscope that permits high-speed CARS imaging, two-photon excited fl uorescence (TPEF) imaging, and confocal Raman spectral analysis with a picosecond laser source ( 8, 9) , we demonstrate the capability to study the dynamic interactions between lipid storage, peroxidation, and desaturation in wild-type and mutant C. elegans .Abstract The ubiquity of lipids in biological structures and functions suggests that lipid metabolisms are highly regulated. However, current invasive techniques for lipid studies prevent characterization of the dynamic interactions between various lipid metabolism pathways. Here, we describe a noninvasive approach to study lipid metabolisms using a multifunctional coherent anti-Stokes Raman scattering (CARS) microscope. Using living Caenorhabditis elegans as a model organism, we report label-free visualization of coexisting neutral and autofl uorescent lipid species. We fi nd that the relative expression level of neutral and autofl uorescent lipid species can be used to assay the genotypephenotype relationship of mutant C. elegans with deletions in the genes encoding lipid synthesis transcription factors, LDL receptors, transforming growth factor  receptors, lipid desaturation enzymes, and antioxidant enzymes . T. L. and H.M.D. designed and performed experiments. T.T.L. and M.N.S. analyzed data. J-X.C. and C-D.H. contributed analytical tools, reagents, and research guidance. T.T.L and H.M.D Abbreviations: CARS, coherent anti-Stokes Raman scattering; RME-2, receptor-mediated endocytosis 2; SREBP-1, sterol regulatory element binding protein 1; TPEF, two-photon excited fl uorescence.
The bimolecular fluorescence complementation (BiFC) assay is a powerful tool for visualizing and identifying protein interactions in living cells. This assay is based on the principle of protein-fragment complementation, using two nonfluorescent fragments derived from fluorescent proteins. When two fragments are brought together in living cells by tethering each to one of a pair of interacting proteins, fluorescence is restored. Here, we provide a protocol for a Venus-based BiFC assay to visualize protein interactions in the living nematode, Caenorhabditis elegans. We discuss how to design appropriate C. elegans BiFC cloning vectors to enable visualization of protein interactions using either inducible heat shock promoters or native promoters; transform the constructs into worms by microinjection; and analyze and interpret the resulting data. When expression of BiFC fusion proteins is induced by heat shock, the fluorescent signals can be visualized as early as 30 min after induction and last for 24 h in transgenic animals. The entire procedure takes 2-3 weeks to complete.
Protein interactions are essential components of signal transduction in cells. With the progress in genome-wide yeast two hybrid screens and proteomics analyses, many protein interaction networks have been generated. These analyses have identified hundreds and thousands of interactions in cells and organisms, creating a challenge for further validation under physiological conditions. The bimolecular fluorescence complementation (BiFC) assay is such an assay that meets this need. The BiFC assay is based on the principle of protein fragment complementation, in which two nonfluorescent fragments derived from a fluorescent protein are fused to a pair of interacting partners. When the two partners interact, the two non-fluorescent fragments are brought into proximity and an intact fluorescent protein is reconstituted. Hence, the reconstituted fluorescent signals reflect the interaction of two proteins under study. Over the past six years, the BiFC assay has been used for visualization of protein interactions in living cells and organisms, including our application of the BiFC assay to the transparent nematode Caenorhabditis elegans. We have demonstrated that BiFC analysis in C. elegans provides a direct means to identify and validate protein interactions in living worms and allows visualization of temporal and spatial interactions. Here we provide a guideline for the implementation of BiFC analysis in living worms and discuss the factors that are critical for BiFC analysis.
Fos and Jun are components of activator protein-1 (AP-1) and play crucial roles in the regulation of many cellular, developmental, and physiological processes. Caenorhabditis elegans fos-1 has been shown to act in uterine and vulval development. Here, we provide evidence that C. elegans fos-1 and jun-1 control ovulation, a tightly regulated rhythmic program in animals. Knockdown of fos-1 or jun-1 blocks dilation of the distal spermathecal valve, a critical step for the entry of mature oocytes into the spermatheca for fertilization. Furthermore, fos-1 and jun-1 regulate the spermathecalspecific expression of plc-1, a gene that encodes a phospholipase C (PLC) isozyme that is rate-limiting for inositol triphosphate production and ovulation, and overexpression of PLC-1 rescues the ovulation defect in fos-1(RNAi) worms. Unlike fos-1, regulation of ovulation by jun-1 requires genetic interactions with eri-1 and lin-15B, which are involved in the RNA interference pathway and chromatin remodeling, respectively. At least two isoforms of jun-1 are coexpressed with fos-1b in the spermatheca, and different AP-1 dimers formed between these isoforms have distinct effects on the activation of a reporter gene. These findings uncover a novel role for FOS-1 and JUN-1 in the reproductive system and establish C. elegans as a model for studying AP-1 dimerization.
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