Optical Silencing of C. elegans Cells with Arch Figure 4. Locomotion assay using heat shock-mediated induction of Arch. (A) Scheme showing the schedule of transferring worms to plates with or without all-trans-retinal (ATR) in heat shock induction experiments. i: Animals were cultivated in the presence of ATR throughout the experiments. (Figs. 3B, 4B) ii: Animals were transferred to ATR-supplemented plates 24 h after heat shock (Fig. 4B). iii: Animals were cultivated in the presence of ATR and transferred to ATR-free plates 24 h after heat shock. (B) Time course of light-elicited locomotory paralysis after heat-shock induction of Arch::GFP. Heat-shocked nc3003Ex[hsp-16.2p::Arch::gfp] animals cultivated in the presence of ATR throughout the experiment (circles) (Fig. 4. A(i)) or transferred from ATR-free to ATR-supplemented plates 24 h after heat shock (triangles) (Fig. 4. A(ii)) were examined at each time point. When ATR was present throughout the experiment (circles), paralysis of worms was first noticed 6 h after heat shock. The paralysis rate reached a plateau 12 h after heat shock, and remained constant for 48 h. (mean6SEM; n = 3, Five animals were examined for each trial.) Worms cultivated in the absence of ATR throughout the experiment did not respond to illumination at any time point. When animals were grown and heat shocked in the absence of ATR and then transferred to ATR-supplemented plates 24 h later (triangles), half of them were paralyzed by illumination 1.5 h after transfer, and the paralysis rate reached a plateau within 3 h. (mean6SEM; n = 4, Five animals were examined for each trial.). doi:10.1371/journal.pone.0035370.g004 that worms have ingested with food remains available for Arch when worms are transferred to plates without ATR. ncEx3003[hsp- 16.2p::Arch::gfp] worms raised in the presence of ATR were heat shocked and transferred to a plate without ATR at various time points. When transferred immediately or 3 hours after heat shock, worm locomotion was not interrupted in response to green light for at least 48 hours after transfer. Given that the expression level of Arch 9 hours after heat shock is sufficiently high for half the worms to show a response to illumination when ATR is present, this result indicates that the level of ATR in the body of worms after transfer to an ATR-free plate seems to be reduced to a level insufficient to be incorporated into Arch within 6 hours. We then examined the stability of incorporated ATR. ncEx3003[hsp- 16.2p::Arch::gfp] worms raised in the presence of ATR were heat shocked and transferred to a plate without ATR 24 hours later (Fig. 4A(iii)). When exposed to green light illumination, the worms stopped locomotion even 24 hours after transfer (93%, n = 15), when free ATR should be no longer available for Arch in the body of the worms. Therefore, ATR incorporated into Arch appears to be stable and keep functioning for at least 24 hours. Effects of GFP Segment in Arch::GFP Another possible rate-limiting step in the process whereby Arch acquires a silencing function is translocation to the plasma membrane. Although we did not notice any obvious change in the localization pattern of Arch::GFP around 9 hours after heat shock, a slight change in localization, such as a shift to the plasma membrane from nearby cytoplasm, might nevertheless lead to significant functional differences. To investigate whether removing the GFP segment, whose hydrophilic nature can potentially affect incorpora- tion into membranes, from Arch::GFP might facilitate translocation of Arch to the plasma membrane, we generated transgenes expressing Arch alone via a heat-shock promoter, Ex[hsp-16.2p::Arch]. The dependence of responsiveness to light intensity was almost identical for Ex[hsp16-2p::Arch] and Ex[hsp-16.2p::Arch::gfp] animals, with slightly higher responsiveness for the former at lower light PLoS ONE | www.plosone.org 6 May 2012 | Volume 7 | Issue 5 | e35370