Macoilin, a conserved nervous system-specific ER membrane protein that regulates neuronal excitability

Abstract

Genome sequence comparisons have highlighted many novel gene families that are conserved across animal phyla but whose biological function is unknown. Here, we functionally characterize a member of one such family, the macoilins. Macoilins are characterized by several highly conserved predicted transmembrane domains towards the N-terminus and by coiled-coil regions C-terminally. They are found throughout Eumetazoa but not in other organisms. Mutants for the single Caenorhabditis elegans macoilin, maco-1, exhibit a constellation of behavioral phenotypes, including defects in aggregation, O₂ responses, and swimming. MACO-1 protein is expressed broadly and specifically in the nervous system and localizes to the rough endoplasmic reticulum; it is excluded from dendrites and axons. Apart from subtle synapse defects, nervous system development appears wild-type in maco-1 mutants. However, maco-1 animals are resistant to the cholinesterase inhibitor aldicarb and sensitive to levamisole, suggesting pre-synaptic defects. Using in vivo imaging, we show that macoilin is required to evoke Ca²(+) transients, at least in some neurons: in maco-1 mutants the O₂-sensing neuron PQR is unable to generate a Ca²(+) response to a rise in O₂. By genetically disrupting neurotransmission, we show that pre-synaptic input is not necessary for PQR to respond to O₂, indicating that the response is mediated by cell-intrinsic sensory transduction and amplification. Disrupting the sodium leak channels NCA-1/NCA-2, or the N-,P/Q,R-type voltage-gated Ca²(+) channels, also fails to disrupt Ca²(+) responses in the PQR cell body to O₂ stimuli. By contrast, mutations in egl-19, which encodes the only Caenorhabditis elegans L-type voltage-gated Ca²(+) channel α1 subunit, recapitulate the Ca²(+) response defect we see in maco-1 mutants, although we do not see defects in localization of EGL-19. Together, our data suggest that macoilin acts in the ER to regulate assembly or traffic of ion channels or ion channel regulators.

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.

Figure 2. maco-1 mutations disrupt macoilin, a polytopic membrane protein with coiled-coil domains that is conserved across Eumetazoa.

(A) maco-1 mutations disrupt D2092.5 which encodes C. elegans macoilin. Arrows indicate locations of the three maco-1 alleles; also indicated is the alternative splicing site. (B–C) Unrooted Neighbor-Joining tree of MACO-1 homologues and 10000 bootstrap replicates analysis and scale bar denoting 0.10 and 0.25 changes per site in (B) and (C), respectively. (B) Branches are grouped by phyla with colored lobes; the average sequence identity with MACO-1 is shown in percentile figures. (C) Zoom-in of the tree of the vertebrate sub-phyla. MACO-2 is a second macoilin gene found in some fish lineages but not in other vertebrates (see text). (D) Multiple alignments of MACO-1 homologues. Predicted transmembrane (TM) and coiled-coil (CC) domains in C. elegans and H. sapiens are shown in red and blue respectively. CE, C. elegans; DM, D. melanogaster; NV, N. vectensis; TR; HS, H. sapiens. A more extensive alignment can be found in Figure S10.

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 µm.

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 µm. 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 µm.

Figure 5. maco-1 mutants are resistant to aldicarb but sensitive to levamisole.

(A) maco-1(db9) animals are resistant to aldicarb but (B) sensitive to levamisole, consistent with a pre-synaptic role for MACO-1. Also plotted are control responses of N2 animals and mutants in the nicotinic acetylcholine receptor subunit unc-29(e193) and the synapse defective gene syd-2(ju37), which encodes alpha liprin.

Figure 6. maco-1 mutants have subtle synaptic morphology defects.

SNB-1::GFP puncta along the ventral (A) and dorsal (C) nerve cords of wild type and maco-1(db9) mutants. (B, D) Quantification of SNB-1::GFP puncta number and area in wild type and maco-1 mutants in ventral (B) and dorsal (D) cords. (E, G) UNC-10::GFP puncta along the ventral (E) and dorsal (G) nerve cords of wild type and maco-1(db9) mutants. (F, H) Quantification of UNC-10::GFP puncta number and area in wild type and maco-1 mutants in ventral (F) and dorsal (H) cords. (I, K) SYD-2::GFP puncta in the ventral (I) and dorsal (K) nerve cords of wild type and maco-1 mutants. (J, L) Quantification of SYD-2::GFP puncta number and area in the ventral (J) and dorsal (L) cords of wild type and maco-1 mutants. The transgenic arrays used are: SNB-1::GFP (juIs1), SYD-2::GFP (hpIs3) and UNC-10::GFP (hpls61). Puncta analysis focussed on a 100 µm interval between motorneurons VD10 and VD12. All images are of 1-day-old adult hermaphrodites. Numbers are mean ± s.e.m. Scale bar, 10 µm. * p<0.05, **p<0.01.

Figure 7. Loss of MACO-1 causes failure of Ca²⁺ transients in O₂-sensing neurons.

Average traces and scatter plots of Ca²⁺ transients in PQR neurons responding to a 21 – 7 – 21 – 7% O₂ cycle as measured by cameleon YC3.60. (A) maco-1 (db9); npr-1 (ad609) worms (n = 13) usually fail to respond to O₂ changes, as opposed to npr-1 (ad609) worms which respond consistently (n = 11; p<0.001). (B) egl-19(ad1006); npr-1 (ad609) worms (n = 11) are also unresponsive to O₂, as compared with npr-1 (ad609) worms (n = 11; p<0.001). (C) nca-1 (gk9); nca-2 (gk5); npr-1(ad609) worms (n = 10) do not significantly (p>0.05) differ in their responses from npr-1(ad609) worms (n = 12). Gray error bars represent s.e.m. The data in Panel C were obtained on a different imaging set-up from those in panels A and B, and so cannot be directly compared.