in only a small subset of C. elegans neurons [48] and some of these are clearly functional in maco-1 mutants (e.g. AWC); by contrast MACO-1 is expressed throughout the nervous system, not only in C. elegans but also in mouse. The L-type VGCC a1 subunit EGL-19 is also required for O2- evoked responses in PQR, and is expressed widely both in the nervous system and in muscle (this work; [44]. Loss-of-function mutants of egl-19 have much more severe phenotypes than maco-1 mutants: egl-19(null) mutants die as embryos. This discrepancy in phenotype makes it unlikely that MACO-1 is critical for function of all EGL-19 containing channels. Consistent with this, mutations in maco-1 do not appear to disrupt localization of GFP- EGL-19 either in muscle or in neurons. However it remains possible that MACO-1 regulates assembly of particular subtypes of EGL-19– containing channels. L-type VGCC are composed of multiple subunits and it is the precise combination of subunits that determines the channel’s regulatory properties; additionally egl-19 mRNA itself is alternatively spliced close to its C-terminus, in a region implicated in Ca2+ feedback regulation [49]. An alternative scenario is that MACO-1 regulates an as yet undiscovered pathway that helps amplify the depolarization initiated by cGMP-gated ion channel activation. Identifying proteins that interact with macoilin or mutants that recapitulate the maco-1 phenotypes will allow these hypotheses to be tested to help further unravel the function of this novel family of nervous system proteins. Materials and Methods Genetics Strains used were maintained as described previously [50] and are listed in Text S1). db1 and db9 were isolated as suppressors of aggregation from a screen of 20,000 haploid genomes; details of the screen will be described elsewhere. db129 was isolated in a non- complementation screen using the db1 allele. The db1 mutation was mapped to a 30 kb interval at the centre of Chromosome I between the SNP markers in cosmids F48A9 and D2092 using a combination of three-factor mapping and SNP genotyping [51]. Sequence mining and phylogenetics PSI-Blast was used to search for Macoilin protein sequences, using human Macoilin as probe, at the NCBI (www.ncbi.nlm.nih. gov), Joint Genome Institute (www.jgi.doe.gov), ENSEMBL (www. ensembl.org), and the Sanger Institute (www.sanger.ac.uk). From approximately 100 sequences retrieved (e-value . e-10), a subset was obtained after removing splice variants, and redundant sequences. The amino acid sequences were aligned using various programs run under the umbrella of the M-Coffee server [52]. The multiple alignment was visually inspected and curated using BioEdit [53] (Figure S10). Un-rooted phylogenetic trees were generated using a Neighbour-Joining method [54]; the robustness of the nodes was verified with 10000 bootstrap replicates using the program Phylo-Win [55]. Molecular biology The cDNA sequence of maco-1 was obtained by sequencing clone yk1296a05 from the Kohara collection and by using RT- PCR. Briefly, N2 mixed stage animals were extracted with Trizol, and 1–5 mg of purified total RNA reverse transcribed using an oligo-d(T) primer and SUPER RTase at 42 uC for 1 h. Primers specific for maco-1 exons and the SL1 spliced leader sequence were used to amplify maco-1 cDNA, and the PCR products sequenced. The maco-1 expression construct was generated using the Gateway system (Invitrogen) [56]. The Destination vector included 4 kb of Macoilin: an ER Protein Regulating Neuron Activity the sequence upstream of the maco-1 start site but omitted 318 bp between the trans-splice site and the initiation codon. The Entry vector places maco-1 cDNA plus 9 bp of the sequence upstream in an artificial operon with gfp [34]. This construct was sequenced and injected at 50 ng/ml with lin-15(+) as the co-injection marker. Transgenic rescue: The fosmid WRM0640bE08, containing D2092.5, was injected into the strain AX129, maco-1(db9);npr- 1(ad609) at a concentration of 2 ng/ml, with 50 ng/ml of punc- 122::gfp as a co-injection marker. Further transgenic rescue experiments were carried out using a PCR amplified genomic DNA fragment containing the D2092.5 gene, including 4 kb upstream of the initiation codon and 1 kb after the stop codon. This PCR product was injected into the AX59, maco-1(db9); npr- 1(ad609) strain at a concentration of 2 ng/ml, using pmyo-2::gfp as a co-injection marker (4 ng/ml) and 1 kb-ladder (96 ng/ml) as carrier. Sub-cellular markers (a kind gift of Melissa M. Rolls, Penn State University) were used as described [4]. The plasmids used were C24F3.1a (pglr-1::yfp-TRAM), Y46G5a.5 (pglr-1::yfp-PIS), F55- 8H1.1 (pglr-1::yfp-MANS) and M01D7.6 (pglr-1::yfp-EMERIN). These plasmids were individually injected at 4 ng/ml into AX206, lin-15(n765ts) animals with a lin-15(+) co-injection marker (40 ng/ml). All primer sequences used are available upon request. The pgcy-32::tax-4-gfp transgene was made using Gateway; tax-4 cDNA was tagged at the 3’end with gfp and injected (at 10 ng/ul) with a lin-15(+) co-injection marker (40 ng/ul) into npr-1(ad609) lin-15(n765ts) animals. A fosmid containing the full-length egl-19 gene was modified by recombineering so as to express N- terminally GFP tagged EGL-19 from its endogenous control sequences. The recombineered fosmid was co-injected at 5 ng /ul with a pgcy-32::mcherry co-injection marker (25 ng/ul) and carrier DNA (DNA ladder at 70 ng/ul). Immunostainings Mix-staged worms were stained following the modified Ruvkun and Finney method [57]. Primary antibodies were used at dilutions of 1/50 and 1/500 for mouse monoclonal anti-GFP antibody (clones 7.1 and 13.1; Roche, Germany) and rabbit polyclonal anti-MCL, respectively. Antibodies were incubated at 4 uC for 16 hrs with gentle mixing. The secondary antibodies Alexa Fluor 546 nm goat anti-rat IgG (H+L) (Invitrogen, UK) and AlexaFluor 488 nm goat anti-mouse (Invitrogen, UK) were used at a final dilution of 1/500 (4 mg/ml) and 1/250 (10 mg/ml), respectively; DAPI was added to a final concentration of 5 mM. After an incubation of 2 hrs at room temperature, worms were thoroughly washed with AbB Buffer (the details of buffer composition can be found in the Text S1), mounted in agarose and imaged. Fluorescent microscopy and quantification Live animals were anaesthetized with 10 mM sodium azide, mounted on 2 % agarose pads, and examined under epifluores- cence using a Zeiss Axioskop fluorescent microscope. Confocal images were taken using a Radiance Plus Confocal Scanning System (Bio-Rad). The images were processed and analyzed with LaserSharp2000 software (Bio-Rad). The different GFP markers were visualized in the different backgrounds as described previously [29],[58]. Measurements of GFP puncta were per- formed on confocal images. Briefly, confocal images were projected into a single plane using the maximum projection method and exported as a tiff file with a scale bar. Fluorescence intensity, number of puncta, total fluorescence and punctum area were measured in ImageJ. These numbers were exported to PLoS Genetics | www.plosgenetics.org 10 March 2011 | Volume 7 | Issue 3 | e1001341