Microsoft Excel for statistical analyses using Student’s two-tailed t- test. Behavioral and pharmacological assays Aggregation assays were done as previously described [14]. Egg- laying assays followed [59] with the following modifications. Worms were synchronized and young adults picked to unseeded plates to remove adhering food before transfer to plates seeded with 50 ml of E. coli OP50 or to un-seeded plates. Rings of 100 ml of 4M D-Fructose were painted on no-food plates to trap animals. Worms were left for one hour, then removed and eggs counted. Plates in which worms could not be found were discarded (around 10% in plates without food). Harsh touch was assayed by poking animals with a platinum wire pick. To analyze swimming defects, single worms were transferred to M9 media and left to equilibrate for a minute; head swings were counted during 10 second intervals; for coiling we counted the number of times the worm’s nose touched the mid body in one minute. The results of the behavioral assays were analyzed using a two-tailed t-test. Aldicarb and levamisole assays were done as described [25]. Briefly, sensitivity to 1 mM aldicarb (Chem Services) or 0.4 mM levamisole was determined by assaying the time course of the onset of paralysis following acute exposure of a population of animals to these drugs. In each experiment, 25 worms were placed on drug plates and prodded every 10 min over a 2 h period to determine if they retained the ability to move. Worms that failed to respond at all to the harsh touch were classified as paralyzed. Each experiment was repeated five times. Ca2+ imaging Ca2+ responses of PQR neurons to O2 stimuli were imaged as described previously (Persson et al 2009) on an inverted microscope (Axiovert, Zeiss), using a 406 C Apochromat lens and Metamorph acquisition software (Molecular Devices). To measure Ca2+ we used the ratiometric FRET sensor YC3.60 [35]. Briefly, worms were glued to agarose pads using Nexaband glue (WPI Inc) and placed under the stem of a Y-chamber microfluidic device. Photobleaching was limited by using a 2.0 optical-density filter and a shutter to limit exposure time to 100 ms per frame. An excitation filter (Chroma) restricted illumination to the cyan channel. A beam splitter (Optical Insights) was used to seperate the cyan and yellow emission light. The ratio of the background- subtracted fluorescence in the CFP and YFP channels was calculated with Jmalyze [60]. Fluorescence ratio (YFP/CFP) plots and measurements of mean baseline ratios and mean peak ratios were made in Matlab (The MathWorks). Movies were captured at 2 frames per second. Average Ca2+ traces were compiled from at least six recordings per condition made across two or more days. Statistics Whenever the data fitted a normal distribution (p,0.05, Kolmogorov-Smirnov) a two-sample (unranked) t-test was used. For non-normally distributed data, the non-parametric Kolmo- gorov-Smirnov (K-S) -test was used. Supporting Information Figure S1 maco-1 mutants exhibit robust attraction to AWA and AWC-sensed odors, and strong avoidance of high osmotic tension. (A) maco-1(db1); npr-1(ad609) and maco-1(db9); npr-1(ad609) mutants chemotax strongly to a 1/1000 dilution of diacetyl, an odor detected by the AWA neurons, and (B) a 1/200 dilution of benzaldehyde, an odor detected by AWC neurons. N2 and npr- 1(ad609) animals are shown as positive controls. osm-9(ky10) Macoilin: an ER Protein Regulating Neuron Activity mutants are defective in a TRPV like channel required in AWA for diacetyl sensing; tax-4(p678) mutants are defective in a cGMP- gated ion channel required in AWC for benzaldehyde responses. For A and B each bar represents the average of 4 assays. (C) maco-1 worms exhibit strong avoidance of regions with high osmotic tension. Each data point represents the average response of at least 400 animals in 40 assays. For all panels error bars indicate s.e.m. (D) maco-1(db9) mutants do not aggregate or border. (E, F) The maco-1 locomotory defects increase with age. Shown are number of head swings made per second by swimming animals the first and fourth day of adulthood (E). (F) Plots the ratio of head swings on day 1 compared to day 4 for swimming npr-1 and maco-1; npr-1 animals. (G) maco-1(db9); npr-1(ad609) and maco-1(db9)/qDf16; npr- 1(ad609) animals have similar phenotypes. Shown is the reversal frequency after harsh touch as a percentage of the response of npr- 1(ad609) animals. Found at: doi:10.1371/journal.pgen.1001341.s001 (0.26 MB PDF) Figure S2 The D2092.5 gene rescues maco-1 associated phenotypes. (A) Aggregation and bordering deficits of maco- 1(db9); npr-1(ad609) animals are substantially but not completely rescued by a fosmid containing D2092.5, as well as by a PCR fragment spanning the D2092.5 gene. The graph compares the behavior of animals bearing the transgene, as indicated by the GFP co-injection marker, and those that do not (n = 3; D-test). The co-injection markers are myo-2::GFP (for the PCR fragment) and unc-122::GFP (for the fosmid). (B) A PCR product spanning the D2092.5 gene suppresses the excessive reversals of maco-1(db9); npr- 1(ad609) animals induced by a harsh prod (n = 28–30; t-test). (C) A fosmid containing D2092.5 partially rescues the swimming defects of maco-1(db9); npr-1(ad609) animals (n = 40–70; D-test). (D) A PCR product spanning the D2092.5 gene restores suppression of egg-laying in maco-1(db9); npr-1 animals when food is absent (n = 23–28). * equals p,0.05; ** equals p,0.01; *** equals p,0.001. Errors indicate s.e.m. Found at: doi:10.1371/journal.pgen.1001341.s002 (0.66 MB PDF) Figure S3 maco-1 is expressed in neurons from early stages. Confocal projections of worms transgenic for pmaco-1::maco-1::gfp, an operon that expresses soluble GFP in tandem with MACO-1. (A) Early developmental stages and (B) adult worms. Time indicates approximate number of hours after first division. Scale bars represent (A) 10 and (B) 40 mm. Found at: doi:10.1371/journal.pgen.1001341.s003 (4.07 MB PDF) Figure S4 Endogenous MACO-1 is localized in the cell body of neurons. Immunohistochemical staining using affinity purified anti-MACO-1 antibodies. Shown are the tail regions of (A) N2 and (B) maco-1(db9) adults, and (C) maco-1(db9) larvae. The stop codon associated with the db9 allele truncates the epitope used to generate the anti-MACO-1 antibody. Arrowheads indicate antibody signal. Scale bars represent 20 mm. Found at: doi:10.1371/journal.pgen.1001341.s004 (2.04 MB PDF) Figure S5 MACO-1 is excluded from neurites and localizes to ER-like structures at embryonic stages. Embryos at (A) approx- imately the 100 cell stage, (B) the comma-stage, (C) the three-fold stage, and (D) a L1 larvae emerging from egg-shell stained with anti-MACO-1 polyclonal antibodies; panel D also shows DAPI staining. Scale bars represent 5 mm. Found at: doi:10.1371/journal.pgen.1001341.s005 (1.78 MB PDF) Figure S6 MACO-1 is an ER resident protein. Confocal optical sections showing neuronal cell bodies in worms bearing extra- chromosomal arrays that express either the YFP-tagged nuclear envelope marker emerin (A–D), or the YFP-tagged Golgi marker mannosidase (E–H), and co-stained with anti-MACO-1 antibodies PLoS Genetics | www.plosgenetics.org 11 March 2011 | Volume 7 | Issue 3 | e1001341