Macoilin: an ER Protein Regulating Neuron Activity Figure 1. maco-1 mutants exhibit multiple behavioral defects. (A, B) npr-1(ad609) animals aggregate, accumulate on the border of a bacterial lawn, and roam widely in high (21%) ambient O2 but dwell locally in low (11%) O2. maco-1 mutations disrupt these behaviors. (C) maco-1 animals reverse more than wild type in response to a harsh prod to the head (n = 30; t-test). (D, E) maco-1 mutants have swimming defects, with a loss of head swings and increased coiling (n = 15; t-test); (F) maco-1 mutants fail to appropriately suppress egg laying when food is absent (n = 22–29 animals; K-S- test). * equals p,0.05; ** equals p,0.01; *** equals p,0.001. Error bars indicate s.e.m. doi:10.1371/journal.pgen.1001341.g001 is known about this protein family. The only macoilin previously investigated is the mouse homologue, highlighted because it is expressed differentially between wild-type mice and reeler mutants [22],[13]. In situ hybridization indicates that mouse macoilin mRNA is highly expressed in all neuronal differentiation fields from embryonic stage E12.5 to birth [13]. After birth (PG10), expression decreases but remains associated with some neurons such as cerebellar granule cells, olfactory mitral and granule cells, pyramidal neurons in the hippocampus, and granule cells in dentate gyrus [13]. No significant expression of mouse macoilin has been reported outside the nervous system. Comparison of macoilin sequences across phyla highlighted several common features (Figure 2D). All MACO-1 homologs were predicted to have at least three transmembrane domains towards the N-terminus and at least two coiled-coil domains towards the C-terminus. The exact number of transmembrane and coiled-coil domains varied depending on the prediction algorithm and species (data not shown). At the sequence level the transmembrane and coiled-coil domains were the most conserved parts of the protein (Figure 2D). C. elegans macoilin is a pan-neuronally expressed ER protein To determine where maco-1 was expressed, we created transgenic C. elegans that co-expressed maco-1 and gfp coding sequences as a polycistronic message from the maco-1 promoter. These animals first showed GFP fluorescence 6 hours after the first cell division (Figure S3A). By late embryogenesis and in the L1 larva, fluorescence was visible throughout the nervous system (Figure S3A). In adults, the GFP signal remained pan-neuronal, with very occasional expression in other tissues (Figure S3B). Thus, C. elegans MACO-1, like its mouse homologue, is expressed widely and almost exclusively in the nervous system. To study endogenous MACO-1, we raised polyclonal antibodies against the C-terminus of both its protein isoforms. N2 worms stained with the antibody showed bright expression in most or all C. elegans neurons (Figure 3A–3C and Figure S4A). In contrast, maco-1(db9) mutants which have a nonsense mutation that truncates MACO-1 upstream of the epitope sequence showed no signal in the nervous system (Figure 3D, 3E, and Figure S4B, S4C). Interestingly, MACO-1 antibody staining was restricted to the neuronal cell body: no staining was observed in dendrites, in the synapse-rich axon bundles that make up the nerve ring, or in the axons comprising the ventral and dorsal cords (Figure 3A, 3B). To confirm that MACO-1 was absent from synapses, we co- stained worms expressing the synaptic marker SNB-1-GFP in GABAergic neurons (juIs1) with anti-MACO-1 and anti-GFP antibodies: the two markers did not show co-localization (Figure 4A–4D). Moreover, worms co-expressing juIs1 and psnb- 1::maco-1-mcherry showed that transgenic MACO-1-cherry was also restricted to the cell body of neurons, overlapping only with the SNB-1-GFP signal in the cell body of GABAergic neurons PLoS Genetics | www.plosgenetics.org 3 March 2011 | Volume 7 | Issue 3 | e1001341