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First published online 6 December 2006
doi: 10.1242/dev.02725

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Neuronal polarity is regulated by a direct interaction between a scaffolding protein, Neurabin, and a presynaptic SAD-1 kinase in Caenorhabditis elegans

Samuel Lunenfeld Research Institute, Mount Sinai Hospital and Department of Microbiology and Medical Genetics, University of Toronto, Ontario, M5G 1X5, Canada.

View larger version (38K): [in this window] [in a new window]   Fig. 1. Loss of sad-1 and nab-1 functions lead to polarity defects in various neurons. (A) L1-stage sad-1 and nab-1 animals have DD polarity defects. Arrowheads indicate the ectopic dorsal SNB-1::GFP (juIs1) or UNC-49B::GFP (oxIs22) signal. A schematic diagram of the normal connectivity of DD neurons in L1 is shown on the left of the images. (B) sad-1 and nab-1 mutations lead to a decreased number of DD synapses in adult-stage C. elegans. The number of juIs1 puncta on the dorsal nerve cord of nab-1;lin-5, lin-5;sad-1 and nab-1;lin-5;sad-1 animals was compared with lin-5 animals (n>15 animals, P< 0.001 by Tukey-Kramer multiple comparison test). (C) Polarity defects in a DA8 cholinergic motoneuron of sad-1 and nab-1 shown by SNB-1::GFP (wdIs20). sad-1 and nab-1 animals show SNB-1::GFP puncta in the dendritic region of the neuron (arrowheads). *DA8 cell body. (D) ASI chemosensory neurons are visualized using the Pstr-3 SNB-1::GFP vesicle marker (kyIs105). Wild-type animals shows discrete vesicle clusters along the axon, but none in the dendritic process (arrowhead). Both sad-1 and nab-1 animals show puncta in the dendritic and axonal processes. *ASI-neuron cell body. Scale bar: 5 µm in C.

View larger version (62K): [in this window] [in a new window]   Fig. 2. sad-1 and nab-1 mutants fail to restrict axonal fate in VD neurons. (A) GABAergic synapses along the dorsal cord in wild-type, sad-1- and nab-1-mutant young adults visualized by juIs1 marker. nab-1 mutants show no synaptic morphology defects. After unc-30 RNAi treatment to block juIs1 expression in DD neurons, ectopic synaptic-vesicle clusters in VD neurons were detected in both nab-1 and sad-1 mutants. Arrowhead shows the dorsal nerve cord. (B) nab-1 mutants have a reduced number of synapses along the axon of VD neurons before and after unc-30 RNAi treatment. Arrow shows VD neuron cell body. (C) nab-1 and sad-1 mutants display ectopic dorsal SYD-2::GFP, an active-zone marker, in VD neurons. Arrowhead shows the dorsal nerve cord. (D) juIs1 phenotypes in wild-type (wt, left upper panels) or nab-1 (left lower panels) animals were analyzed with MathLab software (developed by C. Mok, University of Toronto, Canada). The intensity and width of individual fluorescent punctum, as well as the distance between puncta (inter-punctal width), was calculated from juIs1 images of wild-type and nab-1 animals. Left panels; a graphical representation and the corresponding juIs1 image. Average values of the punctal intensity (upper right panel), punctal width (lower right panel) and interpunctal width (lower right panel) were plotted and shown. No significant difference was found between wild-type and nab-1 values (n=13, P>0.05). Scale bar: 5 µm in A,C.

View larger version (34K): [in this window] [in a new window]   Fig. 3. SAD-1 and NAB-1 interact in vitro and in vivo. (A) Yeast two-hybrid assays to determine the NAB-1-interacting domain of SAD-1. Upper left panel; schematic representation of SAD-1 deletions used to map the region for NAB-1 interaction. Lower left panel; Y274 yeast strain was cotransformed with a NAB-1-AD prey plasmid and various LexA-SAD-1-deletion bait plasmids, and tested for ß-gal activity on X-gal plates. Blue color indicates interaction. Right panels; yeast transformed with different bait- and prey-plasmid combinations (as indicated) were grown in trp- leu- SD media, and color development with X-gal allowed to occur. LexA-SAD-1 amino acids 280-914 (SAD-1) or LexA-SAD-1 amino acids 280-911 (SAD-1DKV) were used as bait and NAB-1 as prey. The lacZ reporter was expressed in only yeast strains carrying both full-length SAD-1 and NAB-1. (B) GST-pull-down assays showed that GST-NAB-1 (or the PDZ domain) selectively interacts with the 110 kD isoform of SAD-1. Upper panels; GST, GST-NAB-1 PDZ domain and GST-full-length NAB-1 precipitated FLAG-tagged SAD-1 from the total-protein lysate from C. elegans strains carrying an integrated, fully functional SAD-1::FLAG array. Lysate input and GST input are shown at the bottom. Lower panel; GST, GST-SAI-2 and GST-NAB-1 PDZ precipitated endogenous SAD-1 from wild-type C. elegans lysate. SAI-2, another SAD-1-interacting protein identified from the yeast two-hybrid screen, pulled-down both isoforms of SAD-1, whereas NAB-1 precipitated only the 110 kD isoform. (C) Co-immunoprecipitation experiments showed that SAD-1 and NAB-1 interact in vivo. C. elegans lysates prepared from wild type, hpIs66 or hpIs66; sad-1 were immunoprecipitated with either anti-SAD-1 or anti-GFP antibody (for NAB-1::GFP), probed with anti-GFP antibody and then stripped and re-probed with anti-SAD-1 antibody, or vice versa. (D) Two alternatively spliced forms of sad-1. Schematic representation of the two splice variants encoded by the sad-1 gene is shown. We sequenced all the existing cDNA clones of sad-1 and discovered that two clones (yk134f11 and yk238h3) in which an additional exon was present in the C-terminal region of the clone, which leads to an earlier stop than the predicted SAD-1 coding region. The corresponding amino acid sequence truncated in this short isoform is shown.

View larger version (24K): [in this window] [in a new window]   Fig. 4. Gene structure of nab-1 and the domain structures of its predicted isoforms. (A) Predicted multiple isoforms encoded by the nab-1 gene. The expected molecular weight of each isoform is listed. Grey boxes; exons. Lines; introns. hpIs66 transgenic animals carry an integrated array of a construct where GFP sequence was inserted at the 3' end of the nab-1 gene just before the stop codon. The genetic lesion of the two nab-1-deletion mutants, gk164 and ok943, are shown. (B) The predicted isoforms of NAB-1 contain multiple protein motifs, except isoform c. The domain structures of the predicted isoforms of NAB-1 are shown schematically. The shortest isoform, C43E11.6c, does not contain any known protein motif.

View larger version (44K): [in this window] [in a new window]   Fig. 5. NAB-1::GFP is expressed in epithelia and in the nervous system. (A,B) Embryos carrying NAB-1::GFP from its own promoter (hpIs66) are shown with fluorescence (A) or DIC (B) microscopy. (C-E) hpIs66 animals express NAB-1::GFP in the nervous system (nerve ring, and dorsal and ventral nerve cord) and excretory canal. By the L4 stage, NAB-1::GFP is also seen in the developing vulva (E). (F,G) Enlarged portions of the dorsal nerve cord from D, and ventral cord from E are shown. Arrowheads show punctate expression pattern of NAB-1::GFP. Scale bar: 5 µm. ec, excretory canal; nr, nerve ring; vc, ventral cord; dc, dorsal cord.

View larger version (78K): [in this window] [in a new window]   Fig. 6. NAB-1 is a presynaptic protein and partially co-localizes with SAD-1. (A) hpIs66 animals were co-stained with anti-GFP antibody (green) and either anti-SNT-1 (red, left panels) or anti-UNC-10 (red, right panels). (B) Wild-type animals co-stained with anti-SAD-1 antibody (red) and either anti-SNT-1 (green, left panels) or anti-UNC-10 (green, right panels). (C) hpIs66 animals were co-stained with anti-GFP (green) and anti-SAD-1 antibodies (red). (D) Young adult animals co-expressing SNB-1::mRFP (red) and NAB-1::GFP (green, left panels) or UNC-10::mRFP (red) and NAB-1::GFP (green, right panels) in GABAergic neurons. (E) Young adult animals co-expressing SAD-1::mRFP (red) and SNB-1::GFP (green) (left panels) or SAD-1::mRFP (red) and UNC-10::GFP (green) (right panels) in GABAergic neurons. (F) Wild-type animals co-expressing SAD-1::mRFP (red) and NAB-1::GFP (green) in GABAergic neurons. Last panels of each column show the enlarged image of areas indicated by white boxes. Scale bar: 5 µm.

View larger version (27K): [in this window] [in a new window]   Fig. 7. Neuronal expression of NAB-1 is sufficient to rescue nab-1 polarity defects. (A) Wild type, nab-1 mutants and nab-1 mutants expressing NAB-1 from various promoters, and nab-1 mutants expressing the NAB-1-deletion constructs were subjected to unc-30 RNAi treatment. Images of the dorsal SNB-1::GFP vesicle clusters are shown. Diagram (bottom of A) shows NAB-1 deletions used. (B) Quantification of the number of ectopic dorsal SNB-1::GFP puncta per animal (n=15). P<0.001 (nab-1, Pmyo-3 NAB-1 and Punc-25 NAB-1204-378 versus wild-type). Scale bar: 5 µm in A.

View larger version (38K): [in this window] [in a new window]   Fig. 8. SAD-1 PDZ-binding site is required for neuronal polarity but not for synaptogenesis. (A) SAD-1 lacking the PDZ-binding site rescues synaptic morphology defects. juIs1 puncta along the dorsal nerve cord of wild-type animals, sad-1 mutants and sad-1 animals expressing either SAD-1 (sad-1+SAD-1) or SAD-1 lacking PDZ binding site (sad-1+SAD-1DKV) from pan-neuronal Punc-115 are shown. (B) SAD-1DKV site fails to rescue sad-1 polarity defects in VD neurons. Pictures of dorsal synapse morphology after unc-30 RNAi in wild-type animals, sad-1 mutants and sad-1 animals carrying the SAD-1 rescuing construct are shown. (C) Quantification of ectopic dorsal-puncta number in animals treated with unc-30 RNAi (n=15, P<0.001 all versus wild type). (D) Both SAD-1-long (sad-1+SAD-1(l)) and -short (sad-1+SAD-1(s)) isoform cDNAs expressed from Punc-25 rescue synapse morphology defects. Morphology of juIs1 puncta in the dorsal nerve cords of L4-stage animals is shown. (E) Only SAD-1-longisoform cDNA rescued the VD polarity defects of sad-1 mutation. Pictures of the dorsal synapses in animals carrying the same expression arrays as in D after unc-30 RNAi. (F) Quantification of ectopic dorsal synapse number by VD neurons of animals treated with unc-30 RNAi in E (n=15, P<0.001 versus wild type). Scale bar: 5 µm in A,D.