Macoilin: an ER Protein Regulating Neuron Activity Figure 4. MACO-1 resides in the endoplasmic reticulum (ER). Confocal optical sections of neurons in C. elegans co-stained with anti-MACO-1 and anti-GFP antibodies and expressing different markers: (A–D) the synaptic marker synaptobrevin-1-GFP (juIs1) (SNB-GFP); (E–I) an extrachromosomal array expressing the general ER marker YFP-Phosphatidylinositol synthase (YFP-PIS); (J–M) an extrachromosomal array expressing the rough ER marker Translocating chain-associating membrane protein (YFP-TRAM). Staining: A, F, J, anti-GFP antibodies; B, H, L, DAPI; C, G, K, anti- macoilin antibody. Neurons correspond to (A–D) the ventral cord motor-neurons between the gonad posterior reflex and the pre-anal ganglia, (F–I) the retrovesicular ganglia and (J–M) head. In C and D arrows indicate MACO-1 signal in the neuronal cell bodies and arrowheads indicate synapses. In I and M arrowheads indicate co-localisation between the MACO-1 and the ER markers PIS and TRAM. Scale bars represent (A–D) 2, (E) 5 and (F–M) 1 mm. N. MACO-1-mCherry fails to localize to synapses. Shown is a merge of green and red fluorescence for a section of the ventral cord from animals that express psnb-1::maco-1-mcherry (psnb-1 drives expression in all neurons) and juIs1, punc-25::snb-1-GFP. The arrows indicate cell bodies; arrowheads indicate synapses. Scale bar is 5 mm. doi:10.1371/journal.pgen.1001341.g004 wild-type animals, average puncta area in the ventral nerve cord 2 was 1.5860.13 mm , with an average of 23.2860.79 puncta per 100 mm (n = 22 animals). maco-1 mutants had fewer puncta that tended to be larger: the average size in the ventral nerve cord was 2.6260.54 mm2 with an average of 19.8761.18 mm puncta per 100 mm (n = 23) (Figure 6A–6D). These data suggest that maco-1 influences pre-synaptic structure. We next investigated whether maco-1 mutants exhibit active zone defects, using SYD-2::GFP [29] and UNC-10::GFP [30] as markers (Figure 6E–6L). Both fusion proteins were expressed in the GABAergic VD and DD motorneurones from the unc-25 promoter. Wild-type animals carrying the punc-25::syd-2::gfp transgene hpIs3 have regularly-sized and spaced puncta along the dorsal and ventral nerve cords, with an average punctal area of 0.3260.017 mm2 and on average 36.7861.96 (n = 17) puncta per 100 mm. In maco-1(db9) adult animals the number of SYD-2::GFP puncta along the dorsal nerve cord was increased but their size was similar to wild-type (Figure 6I–6L). The average area of puncta in maco-1 mutants was 0.3160.014 mm2, with an average of 45.4461.51 (n = 23) puncta per 100 mm. Wild-type animals carrying the punc-25::unc-10::gfp transgene hpIs61 have uniformly shaped and evenly distributed puncta along the ventral and dorsal cord. In maco-1(db9) adult animals, there was a reduced number of puncta but they were larger than in wild-type animals in the PLoS Genetics | www.plosgenetics.org 6 March 2011 | Volume 7 | Issue 3 | e1001341