Macoilin: an ER Protein Regulating Neuron Activity Figure 3. Endogenous MACO-1 is localised to the cell body of neurons. Immunohistochemical staining of N2 larvae (A) and of N2 and maco- 1 adult worms (B–E) using affinity purified anti-MACO-1 antibodies. Arrowheads indicate staining in neuronal cell bodies. A, B and D show fluorescence images whereas C and E show DIC images. Arrows indicate absence of staining in the nerve ring. maco-1(db9) mutants, which bear a premature stop codon truncating MACO-1 before the epitope recognised by the anti-MACO-1 antibodies, exhibit no neuronal staining (see also Figure S4). Scale bars represent 20 mm. doi:10.1371/journal.pgen.1001341.g003 C. elegans macoilin mutants have wild-type neuronal cell morphology, axon guidance, and axon polarity The broad neuronal expression patterns of C. elegans and mouse macoilins suggest that this protein family has a general role in the development or function of the nervous system. To elucidate this role, we first examined the anatomy of the nervous system in maco- 1(db9) mutants using neuron-specific GFP reporters. We examined mechanosensory neurons, chemosensory neurons, and GABAergic motor neurons. We detected no overt abnormalities in the cell bodies, axons, dendrites or cilia of any neuron we examined (Figure S7A–S7C and data not shown). We next asked if maco-1 regulates neuronal polarity (i.e. the placement of synapses) or axonal trafficking of synaptic vesicles. To test this we visualized synaptic vesicles in live animals using a fluorescently-tagged synaptic vesicle marker, synaptobrevin-GFP (SNB-1::GFP). We saw no defects either in the GABAergic DD motor neurons or in the URX O2- sensing neurons (Figure S7D–S7G and data not shown). These results suggest that MACO-1 is not required for correct establish- ment or maintenance of neuron polarity. Precursor vesicles containing synaptic proteins are generated at the cell body and transported to synapses by microtubule-based motor proteins [23]. In C. elegans, this transport requires the KIF1A kinesin homologue unc-104 [23]. In unc-104 mutants tagged synaptobrevin expressed from the punc-25::snb-1::gfp transgene is retained in the cell bodies of the DD and VD motor neurons [24]. Macoilins have been proposed to function in axonal traffic [13]; however, in adult maco-1(db9) worms expressing the punc-25::SNB-1::GFP marker, the tagged synaptobrevin was still localized along the ventral and dorsal nerve cords (Figure 6A and 6C). This suggests that MACO-1 is not essential for transport of synaptic vesicles. maco-1 mutants are resistant to aldicarb but sensitive to levamisole C. elegans synaptic function can be assayed by studying responses to the acetylcholinesterase inhibitor aldicarb and the acetylcholine receptor agonist levamisole [20,25]. Aldicarb inhibits acetylcho- linesterase (AChE), leading to accumulation of acetylcholine at the neuromuscular junction (NMJ), overstimulation of acetylcholine receptors, and paralysis of wild-type animals. Mutants defective in synaptic release have reduced acetylcholine accumulation and are therefore resistant to aldicarb [20]. However these mutants retain sensitivity to levamisole, which directly activates post-synaptic acetylcholine receptors. In contrast, mutants defective in postsyn- aptic responses to ACh are resistant to both aldicarb and levamisole [26]. maco-1(db9) mutants were resistant to aldicarb but sensitive to levamisole (Figure 5A, 5B), suggesting they have presynaptic defects in neurotransmission. maco-1 mutants show subtle synapse morphology defects The aldicarb resistance of maco-1 mutants prompted us to examine synapse structure in these animals more closely. GABAergic type D motor neurons form neuromuscular junctions (NMJs) with ventral and dorsal body wall muscles [27]. We visualized the presynaptic terminals of these neurons using the punc-25::snb-1::gfp transgene juls1 [28]. Wild-type animals bearing juIs1 have SNB-1::GFP puncta of uniform shape and size distributed evenly along the ventral and dorsal nerve cord. These puncta correspond to the presynaptic termini of the 13 VD and 6 DD neurons, respectively. We measured puncta size and number along a 100 mm section of the ventral nerve cord (Figure 6). In PLoS Genetics | www.plosgenetics.org 5 March 2011 | Volume 7 | Issue 3 | e1001341