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First published online 1 March 2005
doi: 10.1242/jcs.01656

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Nectin-like molecule-1/TSLL1/SynCAM3: a neural tissue-specific immunoglobulin-like cell-cell adhesion molecule localizing at non-junctional contact sites of presynaptic nerve terminals, axons and glia cell processes

¹ Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Osaka, Japan
² KAN Research Institute Incorporated, 93 Chudoji-Awatamachi, Shimogyo-ku, Kyoto 600-8815, Japan
³ Department of Anatomy, School of Medicine, Mie University, Tsu 514-8507, Mie, Japan

View larger version (34K): [in a new window]   Fig. 1. Brain-specific expression of Necl-1. (A) Specific recognition of Necl-1 with anti-Necl-1 pAb. Cell lysates (10 µg of protein each) of Necl-1-L, Necl-2-L and wild-type L cells were subjected to SDS-PAGE (10%), followed by western blotting with anti-Necl-1 pAb. (B) Tissue distribution of Necl-1. Homogenates of various mouse tissues (20 µg of protein each) were subjected to SDS-PAGE (10%), followed by western blotting with anti-Necl-1 pAb. (C) Distribution of Necl-1 in the brain. Homogenates of various regions of rat brain (20 µg of protein each) were subjected to SDS-PAGE (10%), followed by western blotting with anti-Necl-1 pAb. (D) Developmental expression of Necl-1 in the brain. Homogenates of various developmental stages of total rat brain (20 µg of protein each) were subjected to SDS-PAGE (10%), followed by western blotting with anti-Necl-1 pAb. The results shown are representative of three independent experiments.

View larger version (113K): [in a new window]   Fig. 2. Localization of Necl-1 in the molecular layer of the cerebellum. (A,B) Immunofluorescence images of mouse cerebellum. Frozen sections of mouse cerebellum were double-stained with anti-Necl-1 pAb and anti-synaptophysin mAb. Aa and Ba, Necl-1 (red); Ab and Bb, synaptophysin (green); Ac and Bc, merge. ML, molecular layer; P, Purkinje cells; GL, granular layer. Bars, 10 µm. The results shown are representative of three independent experiments.

View larger version (129K): [in a new window]   Fig. 3. Localization of Necl-1 at non-junctional cell-cell contact sites in the molecular layer of the cerebellum. Samples were stained with anti-Necl-1 pAb followed by immunogold electron microscopy with the silver-enhanced immunogold method. (A,B) The molecular layer in mouse cerebellum. Open arrowheads, synaptic junctions; red arrows, contact sites between a parallel fiber terminal or axon and a glia cell; green arrows, contact sites of two parallel fiber terminals. (A',B') Schematic drawings of A and B. Pre, parallel fiber terminals or axons of granular cells; Post, Purkinje cell dendrite synapses; Glia, glia cell processes. Bars, 250 nm. The results shown are representative of three independent experiments.

View larger version (110K): [in a new window]   Fig. 4. Localization of Necl-1 in cocultured neurons and glia cells. Immunofluorescence images of mouse hippocampal cocultured neurons and glia cells at 24 days in vitro. (A) Contact sites of neurons. Cells were double stained with anti-Necl-1 pAb and anti-synaptophysin mAb. Aa, Necl-1 (red); Ab, synaptophysin (green); Ac, merge. (B) Contact sites of glia cells. Cells were double stained with anti-Necl-1 pAb and anti-GFAP mAb, a marker for glia cells. Ba, Necl-1 (red); Bb, GFAP (green); Bc, merge. Arrows indicate contact sites of glia cells. (C,D) Contact sites between a neuron and a glia cell. Cells were double-stained with anti-Necl-1 pAb and anti-GFAP mAb, a marker for glia cells. Ca and Da, Necl-1 (red); Cb and Db, GFAP (green); Cc and Dc, merge. Brackets indicate contact sites between a neuron and a glia cell. Inset in C, images at a higher magnification. Bars, 10 µm. The results shown are representative of three independent experiments.

View larger version (149K): [in a new window]   Fig. 5. Localization of Necl-1 at non-junctional cell-cell contact sites in peripheral myelinated nerve fibers. Samples were stained with anti-Necl-1 pAb followed by immunogold electron microscopy with the silver-enhanced immunogold method. (A) Axons with myelin sheath. (B) Nodes of Ranvier. (B') Schematic drawing of B. M, myelin sheath; Ax, axon of peripheral nerve fiber; S, cellular process of Schwann cells. Yellow arrows indicate contact sites of cellular processes of Schwann cells. Bars, 250 nm. The results shown are representative of three independent experiments.

View larger version (96K): [in a new window]   Fig. 6. Homophilic cell-cell adhesion activity of Necl-1. (A) Cell aggregation activity of Necl-1 when a single-cell suspension was rotated for 10 minutes. Aa, wild-type L cells; Ab, Necl-1-L cells. Bars, 100 µm. (B) Localization of Necl-1 at cell-cell contact sites of Necl-1-L cells. Necl-1-L cells were stained with anti-FLAG mAb. Arrow indicates cell-cell contact sites. Bar, 10 µm. (C) Homo-cis-dimer formation of Necl-1. A single-cell suspension of Necl-1-L cells was incubated in the presence or absence of BS3. The lysates (20 µg of protein each) were subjected to SDS-PAGE (10%), followed by western blotting with anti-FLAG mAb. Arrow, monomer; arrowhead, dimer; double-arrowhead, multimer. The results shown are representative of three independent experiments.

View larger version (90K): [in a new window]   Fig. 7. Heterophilic cell-cell adhesion activity of Necl-1 when a single-cell suspension was rotated for 10 minutes. Heterophilic cell-aggregation activity between Dil-labeled Necl-1-L cells and (A) Necl-2-L cells, (B) Necl-5-L cells, (C) nectin-1-L cells, (D) nectin-2-L cells and (E) nectin-3-L cells. (F) Heterophilic cell aggregation activity between nectin-1-L and nectin-3-L cells. Bars, 100 µm. The results shown are representative of three independent experiments.

View larger version (44K): [in a new window]   Fig. 8. Inability of Necl-1 to bind afadin. (A) Yeast two-hybrid assay. Yeast transformants with the indicated plasmids were streaked on synthetic complete medium lacking adenine to score the ADE2-reporter activity and incubated at 30°C for 3 days. (B) Coimmunoprecipitation assay. FLAG-tagged Necl-1 was immnunoprecipitated with anti-FLAG mAb from the extract of Necl-1-L cells. For the control experiments, nectin-2 was immunoprecipitated with anti-nectin-2 mAb from the extract of nectin-2-L cells. Immunoprecipitates were then subjected to SDS-PAGE (10% polyacrylamide gel), followed by western blotting with anti-FLAG, anti-nectin-2 or anti-afadin mAbs. (C) Affinity chromatography. GST-Necl-1-CP or GST-nectin-3-CP (2 nmol of protein each) was applied to amylose resin beads on which MBP-afadin-PDZ (200 pmol of protein each) was immobilized. After the beads were extensively washed, the bound proteins were subjected to SDS-PAGE (13%), followed by protein staining with Coomassie brilliant blue. The results shown are representative of three independent experiments.

View larger version (64K): [in a new window]   Fig. 9. Binding of Dlg3, Pals2 and CASK to Necl-1. (A) Yeast two-hybrid analysis of the binding of Dlg3, Pals2 and CASK to Necl-1 and Necl-2. Yeast transformants with the indicated plasmids were streaked on synthetic complete medium lacking adenine to score the ADE2-reporter activity and incubated at 30°C for 3 days. pGAD10-Dlg3 (lacking the N-terminal 1-5 aa) was a positive clone which was isolated by yeast two-hybrid screening of pGBD-C1-Necl-1-EC from a rat brain library. (B) Coimmunoprecipitation of Dlg3, Pals2 or CASK with Necl-1 or Necl-2. HEK293 cells were co-transfected with the indicated plasmids and FLAG-tagged Necl-1, Necl-1-C, Necl-2 or Necl-2-C were immunoprecipitated with anti-FLAG mAb from the extract of co-transfected HEK293 cells, respectively. The immunoprecipitates were then subjected to SDS-PAGE (10%), followed by western blotting with anti-FLAG, anti-HA or anti-Myc mAbs. (C) Coimmunoprecipitation of Necl-1 with Dlg3 from mouse cerebellum. Dlg3 was immunoprecipitated with anti-Dlg3 antiserum from an extract of mouse cerebellum. Non-immune rabbit serum was used for the control experiments. The immunoprecipitates were subjected to SDS-PAGE (10%), followed by western blotting with affinity-purified anti-Dlg3 or anti-Necl-1 pAbs. (D) Necl-1-dependent recruitment of Dlg3, Pals2 or CASK to cell-cell contact sites. Necl-1-L (a,c,e) or Necl-1-C-L cells (b,d,f) were transfected with pCMV-HA-Dlg3 (a,b), pCMV-HA-Pals2 (c,d) or pEFBOS-Myc-CASK (e,f), and double-stained with anti-FLAG mAb (green, a1-f1) and anti-HA mAb (red, a2-d2), or anti-Myc pAb (red, e2,f2). Arrows indicate cell-cell contact sites. Bars, 10 µm. The results shown are representative of three independent experiments.

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