Optogenetics is a powerful tool to precisely manipulate cell signaling in space and time. For example, protein activity can be regulated by several light-induced dimerization (LID) systems. Among them, the phytochrome B (PhyB)–phytochrome-interacting factor (PIF) system is the only available LID system controlled by red and far-red lights. However, the PhyB–PIF system requires phycocyanobilin (PCB) or phytochromobilin as a chromophore, which must be artificially added to mammalian cells. Here, we report an expression vector that coexpresses HO1 and PcyA with Ferredoxin and Ferredoxin-NADP+ reductase for the efficient synthesis of PCB in the mitochondria of mammalian cells. An even higher intracellular PCB concentration was achieved by the depletion of biliverdin reductase A, which degrades PCB. The PCB synthesis and PhyB–PIF systems allowed us to optogenetically regulate intracellular signaling without any external supply of chromophores. Thus, we have provided a practical method for developing a fully genetically encoded PhyB–PIF system, which paves the way for its application to a living animal.
Oda S, Tomioka M, Iino Y. Neuronal plasticity regulated by the insulin-like signaling pathway underlies salt chemotaxis learning in Caenorhabditis elegans. J Neurophysiol 106: 301-308, 2011. First published April 27, 2011 doi:10.1152/jn.01029.2010.-Quantification of neuronal plasticity in a living animal is essential for understanding learning and memory. Caenorhabditis elegans shows a chemotactic behavior toward NaCl. However, it learns to avoid NaCl after prolonged exposure to NaCl under starvation conditions, which is called salt chemotaxis learning. Insulin-like signaling is important for this behavioral plasticity and functions in one of the salt-sensing sensory neurons, ASE right (ASER). However, how neurons including ASER show neuronal plasticity is unknown. To determine the neuronal plasticity related to salt chemotaxis learning, we measured Ca 2ϩ response and synaptic release of individual neurons by using in vivo imaging techniques. We found that response of ASER increased whereas its synaptic release decreased after prolonged exposure to NaCl without food. These changes in the opposite directions were abolished in insulin-like signaling mutants, suggesting that insulinlike signaling regulates these plasticities in ASER. The response of one of the downstream interneurons, AIB, decreased profoundly after NaCl conditioning. This alteration in AIB response was independent of the insulin-like signaling pathway. Our results suggest that information on NaCl is modulated at the level of both sensory neurons and interneurons in salt chemotaxis learning. learning and memory; in vivo imaging LEARNING AND MEMORY ARE CRUCIAL for animals to cope with a constantly changing environment. Previous studies suggested that insulin is involved in learning and memory in mammals (Dou et al. 2005; Zhao et al. 1999). Indeed, studies using mammalian cultured neurons or Xenopus tadpoles suggested that insulin regulates neuronal plasticities such as long-term depression (LTD), internalization of DL-␣-amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA) receptors, and changes in the number of synapses (Chiu et al. 2008;Man et al. 2000). However, how insulin actually regulates learning and memory is still obscure.Caenorhabditis elegans also shows learning and memory such as thermotaxis learning, food-odor associative learning, and salt chemotaxis learning (Mori et al. 2007;Nuttley et al. 2002; Saeki et al. 2001;Tomioka et al. 2006). The molecular mechanisms underlying these behavioral plasticities have been studied well. Insulin-like signaling, for instance, regulates several types of starvation-associated learning (Kodama et al. 2006;Lin et al. 2010;Tomioka et al. 2006). However, the plasticities of neuronal activity underlying these behavioral plasticities are mostly unknown.Salt chemotaxis learning is one of the starvation-associated learning types. In this behavioral plasticity, worms show aversive behavior toward the attractant NaCl after prolonged exposure to NaCl for 10 -60 min without food. However, in the presence of f...
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