Optical Silencing of C. elegans Cells with Arch Figure 3. Defects in the locomotory behavior caused by silencing subsets of motor neurons. (A) A crawling track of an ncEx2351[unc- 47p::Arch::gfp] animal. When the freely moving animal was illuminated with green light for 1 minute, it performed loopy movement (dotted square). After turning off the green light, it resumed normal sinusoidal movement. Scale bar = 500 mm (B-D) Locomotory behavior of worms expressing Arch in motor neurons subsets. Arch::GFP was expressed in D-type (VD, DD), A-type (VA, DA), B-type (VB, DB) and VA +VB motor neurons in ncEx2351[unc- 47p::Arch::gfp], ncEx2371[acr-5p::Arch::gfp], ncEx3068[unc-4p::Arch::gfp] and ncEx2365[del-1p::Arch::gfp] animals, respectively. Locomotion of unc- 47(e307) animals was also scored. For all transgenic strains, animals behaviors under green light illumination (ON = open box) and those without illumination (OFF = filled box) differed significantly (p,0.001, Fisher’s exact test for the locomotion assay under the freely moving condition and Student’s t test for the touch response assay). Exceptions are forward movement of ncEx3068[unc-4p::Arch::gfp] animals, which did not change significantly under illumination, and forward movement of ncEx2365[del-1p::Arch::gfp] animals elicited by gentle posterior touch (p = 0.012). Error bars indicate 6SEM. (B) Forward (F) and backward movement (B) was scored in worms moving freely. Percentage of animals exhibiting the ‘‘Class 3 (severe)’’, ‘‘Class2 (mild)’’ and ‘‘Class1 (no)’’ phenotype in locomotory behaviors when they were illuminated with green light is shown. Defects were classified as ‘‘Class 30 when no corresponding movement or response was observed. Other abnormalities, such as retardation or decrease in the extent of movement, are classified as ‘‘Class2’’. (C) Forward movement (F) to gentle posterior touch and backward (B) movement to gentle anterior touch were scored. Responses out of five touches are shown. (D) Forward (F) and backward movement (B) to harsh touch was scored, and was shown additively in each bar. Responses out of five touches are shown. doi:10.1371/journal.pone.0035370.g003 Uptake and Stability of ATR in Arch The heat-shock induction experiments described above sug- gested that Arch acquire silencing function fully only 12 hours after heat shock induction. The unexpectedly long period that is needed before Arch acquires silencing function prompted us to examine the possibility that Arch may need to undergo maturation processes that might require several hours to complete. To investigate whether ATR, an essential cofactor of Arch, might be involved in the posited maturation processes, we examined the incorporation and stability of ATR in Arch. We examined whether the time required for incorporation of ATR into Arch caused the observed delay of several hours before Arch becomes functional following its expression (Fig. 4). ncEx3003[hsp-16.2p::Arch::gfp] worms raised in the absence of ATR were heat shocked and were transferred to a plate containing ATR 24 hours later (Fig. 4A(ii)). The worms raised without ATR expressed GFP similarly to those raised with ATR. About half of the animals stopped locomotion in response to green light illumination 1.5 hours after transfer, and more than 80% of the animals stopped locomotion under green light 3 hours after transfer (Fig. 4B). Therefore, ATR seems to be incorporated into the preexisting Arch that was synthesized in the absence of ATR, and function there. The results also indicate that Arch is stable in the absence of ATR. Importantly, the relatively short 1.5-hour period that worms required before becoming responsive to green light illumination following transfer to ATR-containing plates indicates that incorpo- ration of ATR into Arch occurs rather rapidly, and this alone probably cannot explain the observed delay of several hours. We also examined how long ATR keeps functioning after being incorporated into Arch. First, we estimated how long the ATR PLoS ONE | www.plosone.org 5 May 2012 | Volume 7 | Issue 5 | e35370