Optical Silencing of C. elegans Cells with Arch Figure 5. Comparison of light-elicited locomotory paralysis mediated by Arch and Arch::GFP. (A) Dependence of locomotion paralysis of Ex[hsp-16.2p::Arch]animals (black squares) and Ex[hsp-16.2p::Arch::gfp] animals (open circles) to light intensity was measured 12h after heat shock. (B) Time course of light-elicited locomotory paralysis after heat-shock induction of Arch and Arch::GFP. Heat-shocked Ex[hsp-16.2p::Arch] (black squares) and Ex hsp-16.2p::Arch::gfp] (open circles) animals were examined at each time point as shown in Fig. 4. A(i). Three independently isolated lines were used for each transgene: ncEx3002, ncEx3003 and ncEx3004 for Ex[hsp-16.2p::Arch::gfp], and ncEx3039, ncEx3040 and ncEx3041 for Ex[hsp16-2p::Arch]. Shown are the mean6SEM of 9 trials consisting of 3 trials for each line. Five animals were examined for each trial. doi:10.1371/journal.pone.0035370.g005 intensities (Fig. 5A) (p,0.05 Student’s t-test). With Ex[hsp16- 2p::Arch], 2% of the animals became paralyzed at an illumination intensity of 0.5 mW/mm2, half of the animals at 3.0 mW/mm2 and 2 all the animals at 16.1 mW/mm . With Ex[hsp-16.2p::Arch::gfp], 2% 2 of the animals became paralyzed at 0.5 mW/mm , half of the animals at 1.8 mW/mm2, and all the animals at 16.1 mW/mm 2 . The responsiveness of Ex[hsp-16.2p::Arch] animals to light also changed over time in a manner similar to that of Ex[hsp16- 2p::Arch::gfp] animals (Fig. 5B). With Ex[hsp16-2p::Arch] animals, paralysis by green light illumination was first noticed 6 hours after heat shock and the percentage of paralyzed animals increased thereafter. The worms exhibited maximum responsiveness around 12 hours after heat shock, with 95% of the animals stopping locomotion. Thus, deletion of GFP from Arch::GFP does not appear to accelerate the acquisition of the silencing function. The responsiveness to light after 12 hours decreased slightly faster in Ex[hsp-16.22p::Arch] animals (p,0.01 Student’s t-test): 40% of Ex[hsp-16.2p::Arch] animals were paralyzed by green light illumi- nation 48 hours after heat shock, while more than 60% of Ex[hsp- 16.2p::Arch::gfp] animals were paralyzed. It appears that the GFP segment might inhibit the degradation of Arch. Discussion In this paper we report for the first time that Arch derived from archaebacteria can be expressed in C. elegans cells without apparent adverse effects, and can be used to silence activity of neurons and muscles, thereby modifying worm’s behaviors. Arch expressed in neurons and muscles affects worm behavior differently. We found that illumination at a similar level of light intensity affected worms differently depending on whether Arch was expressed in neurons or muscles. An apparently stronger GFP signal in neurons compared with muscle cells in our transgenic lines suggests that the concentration of Arch::GFP is higher in neurons. Since the amplitude of change in membrane potential driven by Arch-mediated proton current is probably proportional to the density of Arch on the cell membrane, illumination with the identical light intensity would promote larger hyperpolarizing effects in neurons than in muscle. Neighboring muscle cells are known to be coupled electrically through gap junctions [24]. With their larger cell size, mosaic expression of Arch and electric coupling of cells in muscle tissues would weaken the polarizing effect of the Arch-mediated proton current. Ex[myo-3p::Arch::GFP] transgenic lines so far established express Arch::GFP in highly mosaic patterns, and we have noticed the presence of dead embryos expressing GFP very intensely in line cultures. Although we have not noticed any morphological damage in Arch-expressing muscle cells, highly abundant expres- sion of Arch or the presence of excessive amounts of the promoter sequence of the myo-3 gene in muscle cells might have a harmful effect on the development or survival of worms. The heat-shock-mediated Arch expression in Ex[hsp- 16.2p::Arch::GFP] worms occurred ubiquitously. While Arch ex- pressed both in neurons and in muscle cells may affect locomotion, PLoS ONE | www.plosone.org 7 May 2012 | Volume 7 | Issue 5 | e35370