Download Supplemental 1

Supplementary Figure S1. IHC of TG worms expressing (b)opsin in neurons and in muscles. a-g,
Representative IHC images of live day 1 TG worms expressing (b)opsin in neurons Alexa-488conjugated 1D4 mAb was injected into pseudocoelom of a worm head (a-d) or middle body section (e-g)
together with Triton X-100 as described in Materials and Methods. a, Bright field (BF) image. b, 1D4
mAb stained many neurons in the head ganglion. c, After 1D4 mAb and Triton X-100 injection,
fluorescence of the co-injected marker DsRed was absent from neurons, indicating that they were
permeabilized. d, A magnified image of b. Neurons stained by 1D4 antibodies were hollow, suggesting
that recombinant (b)opsin was associated with plasma membranes. Two neurons are indicated by
arrowheads in this sample. e, Bright field image. f, 1D4 mAb stained the ventral cord and several motor
neurons, , some with hollow shapes. g, Fluorescence of the co-injected marker DsRed disappeared from
neurons in the injected worm body section after membrane permeabilization with Triton X-100. h-j,
Sample IHC images of a live day 1 TG worm with muscular expression of (b)opsin. Alexa-488conjugated 1D4 mAb was injected into the pseudocoelom of a worm head together with Triton X-100. h,
BF image. i, 1D4 mAb stained several muscle cells in the worm head. j, After antibody and Triton X-100
injection, fluorescence of the co-injected marker DsRed was absent from the injected worm body section,
indicating permeabilization of muscles.
Supplementary Figure S2. In vivo functional analysis of recombinant (h)A 2A R. a, Day 1 TG worms
expressing DsRed only (control) or (h)A 2A R in either neurons ([N](h)A 2A R) or muscles ([M](h)A 2A R)
were transferred onto OP50-seeded NGM plates containing A 2A R agonist CGS21680 or control vehicle
(vehicle). The locomotion velocity of these animals was quantified for 10 min at 30 Hz,but, the first 6 min
of locomotion data were discarded to eliminate the acclimation phase. Locomotion velocities,
representing the averaged speed of animals from 7 to 10 min were computed a, Bar graph of locomotion
velocity in response to 27 µM CGS21680. Error bars indicate S.E.M. **, p<0.01; ***, p<0.001; t-test. n =
12 for the number of animals tested. b, Locomotion velocity of TG worms expressing (h)A 2A R in neurons
in response to different concentrations of CGS21680.
Supplementary Figure S3. GPCR expression levels in worms at different stages of a large-scale
worm culture. a, Worms with muscular (h)A 2A R expression were sampled at the following stages of
large-scale culture: the last day in culture plates (plates), different days (day 1- 3) of the first generation
(G1) in liquid culture, and different days (day 1-4) of the second generation (G2) in liquid culture.
Samples were subjected to immunoblotting against alkaline phosphatase-conjugated 1D4 mAb. Most
worms collected from plates were gravid and the intensity of the corresponding immunoreactivity band
was higher than the corresponding bands in G1 worms. This reflects the fact that worms from plates were
diluted into a large volume of liquid medium. After 3 days culture in liquid, there was a burst of receptor
production such that the intensity of the (h)A 2A R immunoreactivity band had significantly increased at
day 1 of G2, indicating that G2 worms hatched from eggs in liquid medium. After 4days of growth, most
G2 worms matured and there was a progressive increase in receptor production. At day 4 of G2s in liquid,
most worms had reached their young adult stage and crowed the liquid culture medium (panel b). Thus,
we harvested G2worms at day 4 in liquid culture to avoid their starvation and death. b, Shown are bright
field (left panel) and DsRed fluorescence (right panel) images of 2 µl liquid culture of G2 TG worms
expressing (h)A 2A R in muscle at day 4.
Supplementary Figure S4. TG worms expressing (h)A 2A R in muscles. Lysates from TG worm line
expressing (h)A 2A R in muscle were subjected to immunoblotting against alkaline phosphatase-conjugated
1D4 mAb. Molecular markers (Markers) are also shown.