TY - JOUR AB - We present two methods for the precise independent focusing of orthogonal linear polarizations of light at arbitrary relative locations. Our first scheme uses a displaced lens in a polarization Sagnac interferometer to provide adjustable longitudinal and lateral focal displacements via simple geometry; the second uses uniaxial crystals to achieve the same effect in a compact collinear setup. We develop the theoretical applications and limitations of our schemes, and provide experimental confirmation of our calculations. AU - Schmid, David AU - Huang, Ting-Yu AU - Hazrat, Shiraz AU - Dirks, Radhika AU - Onur Hosten AU - Quint, Stephan AU - Thian, Dickson AU - Kwiat, Paul G ID - 591 IS - 13 JF - Optics Express TI - Adjustable and robust methods for polarization-dependent focusing VL - 21 ER - TY - JOUR AU - Bernecky, Carrie A AU - Cramer, Patrick ID - 595 IS - 6 JF - EMBO Journal TI - Struggling to let go: A non-coding RNA directs its own extension and destruction VL - 32 ER - TY - JOUR AB - Different interoceptive systems must be integrated to ensure that multiple homeostatic insults evoke appropriate behavioral and physiological responses. Little is known about how this is achieved. Using C. elegans, we dissect cross-modulation between systems that monitor temperature, O2 and CO2. CO2 is less aversive to animals acclimated to 15°C than those grown at 22°C. This difference requires the AFD neurons, which respond to both temperature and CO2 changes. CO2 evokes distinct AFD Ca2+ responses in animals acclimated at 15°C or 22°C. Mutants defective in synaptic transmission can reprogram AFD CO2 responses according to temperature experience, suggesting reprogramming occurs cell autonomously. AFD is exquisitely sensitive to CO2. Surprisingly, gradients of 0.01% CO2/second evoke very different Ca2+ responses from gradients of 0.04% CO2/second. Ambient O2 provides further contextual modulation of CO2 avoidance. At 21% O2 tonic signalling from the O2-sensing neuron URX inhibits CO2 avoidance. This inhibition can be graded according to O2 levels. In a natural wild isolate, a switch from 21% to 19% O2 is sufficient to convert CO2 from a neutral to an aversive cue. This sharp tuning is conferred partly by the neuroglobin GLB-5. The modulatory effects of O2 on CO2 avoidance involve the RIA interneurons, which are post-synaptic to URX and exhibit CO2-evoked Ca2+ responses. Ambient O2 and acclimation temperature act combinatorially to modulate CO2 responsiveness. Our work highlights the integrated architecture of homeostatic responses in C. elegans. AU - Kodama-Namba, Eiji AU - Fenk, Lorenz A. AU - Bretscher, Andrew J. AU - Gross, Einav AU - Busch, K. Emanuel AU - de Bono, Mario ID - 6128 IS - 12 JF - PLoS Genetics SN - 1553-7404 TI - Cross-modulation of homeostatic responses to temperature, oxygen and carbon dioxide in C. elegans VL - 9 ER - TY - JOUR AB - Cas9 is an RNA-guided double-stranded DNA nuclease that participates in clustered regularly interspaced short palindromic repeats (CRISPR)-mediated adaptive immunity in prokaryotes. CRISPR–Cas9 has recently been used to generate insertion and deletion mutations in Caenorhabditis elegans, but not to create tailored changes (knock-ins). We show that the CRISPR–CRISPR-associated (Cas) system can be adapted for efficient and precise editing of the C. elegans genome. The targeted double-strand breaks generated by CRISPR are substrates for transgene-instructed gene conversion. This allows customized changes in the C. elegans genome by homologous recombination: sequences contained in the repair template (the transgene) are copied by gene conversion into the genome. The possibility to edit the C. elegans genome at selected locations will facilitate the systematic study of gene function in this widely used model organism. AU - Chen, Changchun AU - Fenk, Lorenz A. AU - de Bono, Mario ID - 6130 IS - 20 JF - Nucleic Acids Research SN - 1362-4962 TI - Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination VL - 41 ER - TY - JOUR AB - cGMP signaling is widespread in the nervous system. However, it has proved difficult to visualize and genetically probe endogenously evoked cGMP dynamics in neurons in vivo. Here, we combine cGMP and Ca2+ biosensors to image and dissect a cGMP signaling network in a Caenorhabditis elegans oxygen-sensing neuron. We show that a rise in O2 can evoke a tonic increase in cGMP that requires an atypical O2-binding soluble guanylate cyclase and that is sustained until oxygen levels fall. Increased cGMP leads to a sustained Ca2+ response in the neuron that depends on cGMP-gated ion channels. Elevated levels of cGMP and Ca2+ stimulate competing negative feedback loops that shape cGMP dynamics. Ca2+-dependent negative feedback loops, including activation of phosphodiesterase-1 (PDE-1), dampen the rise of cGMP. A different negative feedback loop, mediated by phosphodiesterase-2 (PDE-2) and stimulated by cGMP-dependent kinase (PKG), unexpectedly promotes cGMP accumulation following a rise in O2, apparently by keeping in check gating of cGMP channels and limiting activation of Ca2+-dependent negative feedback loops. Simultaneous imaging of Ca2+ and cGMP suggests that cGMP levels can rise close to cGMP channels while falling elsewhere. O2-evoked cGMP and Ca2+ responses are highly reproducible when the same neuron in an individual animal is stimulated repeatedly, suggesting that cGMP transduction has high intrinsic reliability. However, responses vary substantially across individuals, despite animals being genetically identical and similarly reared. This variability may reflect stochastic differences in expression of cGMP signaling components. Our work provides in vivo insights into the architecture of neuronal cGMP signaling. AU - Couto, A. AU - Oda, S. AU - Nikolaev, V. O. AU - Soltesz, Z. AU - de Bono, Mario ID - 6133 IS - 35 JF - Proceedings of the National Academy of Sciences SN - 0027-8424 TI - In vivo genetic dissection of O2-evoked cGMP dynamics in a Caenorhabditis elegans gas sensor VL - 110 ER -