Another possibility that would account for the delay is that newly synthesized Arch might undergo a conformation change, or post- translational modifications before becoming effective. Our finding that ATR can be incorporated into preexisting Arch within 1.5 hours argues against the idea that the delay represents a period needed for incorporation of ATR. A previous in vitro study showing that ATR does not affect the rate-limiting step of bacteriorhodopsin folding [25] also supports this notion. We have also shown that Arch, once having incorporated ATR, remains functional in the absence of ambient ATR. This is consistent with the fact that retinoids keep binding to bacteriorhodopsin after light-driven isomerization [26]. Comparison with NpHR NpHR, an inward chloride pump, is currently the most widely used optogenetic tool for silencing neurons. A previous in vitro study showed that Arch can silence neurons at a lower light intensity than NpHR [11]. In a previous study, the green light with an intensity of 4.4 mW/mm2 or 10.2 mW/mm2 were used for silencing of C. elegans body wall muscle cells expressing NpHR [7]. With Arch driven by the same myo-3 promoter, we found that the light intensity of 8.3 mW/mm2 was sufficient to stop locomotion of about half of the animals. While we have not compared the light dependence of silencing effects of these two pumps directly, Arch appears to have hyperpolarizing effects comparable to NpHR on C. elegans cells. The resistance to inactivation during prolonged illumination makes Arch a useful alternative to NpHR in optogenetic studies of C. elegans. Materials and Methods C. elegans Culture Conditions Worm strains expressing Arch were grown on Nematode Growth Medium (NGM) plates [27] seeded with solution of Escherichia coli OP50 and 500 mM all-trans-retinal (ATR) (Sigma- Aldrich, USA). Animals were maintained at 20uC in the dark unless indicated otherwise. C. elegans wild-type strain N2 and CB307 unc-47(e307) III were used. Plasmid Construction Arch::gfp cDNA was amplified from AAV-FLEX-Arch-GFP [11] (Addgene, Cambridge, MA, USA) by PCR using 59AAGGTA- CCGGTAGAAAAAATGGACCCCATCGCTCTGAGG3’ and 59TTGGTACCTTACTTGTACAGCTCGTCCA3’ as primers, and was cloned into pBluescriptII (pBS, Strategene, USA) with restriction enzymes KpnI/SacI. To generate hsp-16.2p::Arch::gfp (pOKA020), Arch::gfp cDNA cut out from pBS cDNA was cloned into the KpnI/SacI site of pPD49.78 containing the hsp-16.2 promoter [28]. The other plasmids were made using the TOPO cloning kit and the Gateway system (Invitrogen, USA). To generate the entry clone pENTR/D carrying Arch::gfp (pOKA002), Arch::gfp cDNA was PCR- amplified by using 59GGTACCGGTAGAAAAAATGGACC- CCTCGCTCTGCAGGCTGG3’ and 59ATGGACCCCTCGC- TCTGC3’, and 59 AAGGTACCTTACTTGTACAGCTCGTC3’, and then was cloned into pENTR/D by TOPO reaction to generate pOKA001 and pOKA002, respectively (following the manufacturer’s instructions). pOKA002 was recombined by LR reaction into destination vectors pDEST-F25B3.3p and pDEST-aex-3p [29], and pOKA051 was recombined into pDEST-myo-3p [29] (gift from Hidehito Kuroyanagi). To make pENTR/D Arch (pOKA003), Arch cDNA was amplified using 59GGTACCGGTAGAAAAAATG- GACCCCTCGCTCTGCAGGCTGG3’ and 59TTATGCTAC- TACCGGTCGGT3’, and then was cloned into pENTR/D by Optical Silencing of C. elegans Cells with Arch TOPO reaction. pOKA003 was recombined into pDEST-hsp-16.2p [29] by LR reaction (gift from Hidehito Kuroyanagi). Destination vectors containing neuron-subset-specific promoters were constructed by inserting the PCR-amplified genomic fragments into the SphI site of pDEST-PL (gift from Hidehito Kuroyanagi) using the following primers: for pDEST-del-1p, 59AAGCATGCTCAGTATCATTGATTATTATCATTAGT- TCG3’ and. 59AAGCATGCACCCCATCTTTTCAAAAACTTCGGCTT- C3’; for pDEST-unc-47p, 59AAGCATGCCTGGGTAAACCTATCATACGAAACTGC3’ and. 59AAGCATGCTCTAGACTGTAATGAAATAAATGTGAC- GCTGTCG3’; for pDEST-acr-5p, 59AAGCATGCGGTATACTTATCGGTGAGTTGAATATG- CACC3’ and. 59AAGCATGCTCTAGAGCTGAAAATTGTTTTTAAAGC- ATTGAAACTGG3’. To generate del-1p::Arch::gfp, unc-47p::Arch::gfp, acr-5p::Arch::gfp, and unc-4p::Arch::gfp, pOKA002 was recombined into pDEST-del- 1p, pDEST-unc-47p, pDEST- acr-5p, and pDEST-unc-4p (gift from Hidehito Kuroyanagi) by LR reaction respectively. Transgenic Strains Transgenic animals were generated by microinjection of DNA into the distal arms of gonads of N2 hermaphrodites as previously described [30]. pOKA049 (myo-3p::Arch::gfp, 70 ng/ml) and pRF4 (rol- 6d, 400 ng/ml), which is a transgenic marker conferring the Roller phenotype, were injected together into N2 worms to create the line ncEx3031[pOKA049 (myo-3p::Arch::gfp), pRF4 (rol-6d)]; pOKA052 (F25B3.3p::Arch::gfp, 170 ng/ml) and pRF4 (600 ng/ml) to create ncEx3034[pOKA052(F25B3.3p::Arch::gfp), pRF4 (rol-6d)]; pOKA048 (aex-3p::Arch::gfp, 160 ng/ml) and pRF4 (600 ng/ml) to create ncEx3026[pOKA048 (aex-3p::Arch::gfp), pRF4 (rol-6d)]; pOKA020 (hsp- 16.2p::Arch::gfp, 75 ng/ml) and pRF4 (125 ng/ml) to create ncEx3002, ncEx3003 and ncEx3004[pOKA020 (hsp-16.2p::Arch::gfp), pRF4 (rol-6d)]; pOKA056 (hsp-16.2p::Arch, 110.8 ng/ml), pPD49.78 GFP (hsp16- 2p::gfp, 140 ng/ml) and pRF4 (230 ng/ml) to create ncEx3039, ncEx3040 and ncEx3041[pOKA056 (hsp-16.2p::Arch), pPD49.78 gfp (hsp16-2p::gfp), pRF4 (rol-6d)]. Non-roller transgenic animals were generated by using pCFJ90 (myo-2p::mCherry) which drives expression of mCherry in the pharyngeal muscles, as a transgenic marker. We injected into N2 worms pOKA073 (del-1p::Arch::gfp, (200 ng/ml), pCFJ90 (10 ng/ml) and pBluescript (500 ng/ml) to create ncEx2365[pO- KA073(del-1p::Arch::gfp), pCFJ90 (myo-2p::mCherry)]; pOKA074 (unc-47p::Arch::gfp, (200 ng/ml), pCFJ90 (10 ng/ml) and pBluescript (500 ng/ml) to create ncEx2351[pOKA074(unc- 47p::Arch::gfp), pCFJ90 (myo-2p::mCherry)]; pOKA075 (acr-5p::Arch::gfp, (200 ng/ml), pCFJ90 (10 ng/ml) and pBluescript (500 ng/ml) to create ncEx2371[pOKA075(acr-5p::Arch::gfp), pCFJ90 (myo-2p::mCherry)]; and pOKA068 (unc-4p::Arch::gfp, (200 ng/ ml), pCFJ90 (10 ng/ml) and pBluescript (500 ng/ml) to create ncEx3068[pOKA068(unc-4p::Arch::gfp), pCFJ90 (myo-2p::mCherry)]. Since all the strains expressing Arch::GFP used in this study carried a transgene in the form of an extra-chromosomal array, the expression level of Arch::GFP varied among animals of a single transgenic line. It also varied among cells in a transgenic animal. Thus we did not observe the expression of Arch::GFP in all body wall muscle cells of an ncEx3031[myo-3p::Arch::gfp] animal, nor in PLoS ONE | www.plosone.org 9 May 2012 | Volume 7 | Issue 5 | e35370