Figure 3.

Effects of isotopic labeling on the expression of (b)opsin in [M,N](b)opsin TG worms and the functionality of (b)isorhodopsin in [N](b)opsin and [M,N](b)opsin TG worms. U, unlabeled; L, triply isotopically labeled (¹³C, ¹⁵N,~100%; ²H, ~70%) worms. A) Representative Alexa-488-conjugated 1D4 mAb fluorescent images of adult [M,N](b)opsin worms without (U, left panel) and with isotope labeling (L, right panel). 1D4 mAb stained many neurons in the head ganglion, tail ganglion (white arrows), and was also detected in body wall muscles (orange arrows), as well as in muscles of the neck and head. B) Representative immunoblotted gel (left panel) of opsin expression in unlabeled and isotopically labeled [M,N](b)opsin worms together with quantification of the immunoblotting results (right panel). Worms collected from three independent experiments (Exp1-3) were sonicated and centrifuged to remove debris. Worm supernatants of 0.33 μl together with 0.75 ng of (b)rhodopsin purified from bovine retina were used. Error bars indicate means ±SE. *P<0.05; Students t test. C) Light-responsive motor behavior of triple-labeled worms. Unlabeled and labeled L4 [N](b)opsin and [M,N](b)opsin TG worms were preincubated with either vehicle, 10 μM all-trans-retinal or 10 μM 9-cis-retinal overnight and then transferred onto unseeded NGM tracking plates. Vigorously crawling TG animals were then exposed to blue light (1000 lux, 488 ±20 nm) for 1 s. Light-responsive motor behaviors of these animals were recorded and scored according to the response index (see Materials and Methods). Data were derived from 3 independent experiments with 5–10 animals each. Error bars indicate means ±SE. *P<0.05; Student’s t test.