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FIGQY phosphorylation defines discrete populations of L1 cell adhesion molecules at sites of cell-cell contact and in migrating neurons

¹ Howard Hughes Medical Institute and Departments of Biochemistry and Cell Biology, Duke University Medical Center, Durham, NC 27710, USA
² Department of Cell and Molecular Physiology, University of North Carolina, Chapel Hill, NC 27599, USA

View larger version (65K): [in a new window]   Fig. 1. The phospho-FIGQY polyclonal antibody is specific for FIGQY-phosphorylated L1 CAMs. (A) Comparison of sequences surrounding the FIGQY tyrosine in neurofascin, L1 and NrCAM, LAD-1 and neuroglian (long (nrg180) and short (nrg167) forms). (B) Peptides corresponding to the sequences shown in A for either neurofascin (NF), L1 and NrCAM (L1/Nr), or LAD-1 in their tyrosine phosphorylated (+) or non-phosphorylated (-) forms were coupled to turkey ovalbumin, electrophoresed and immunoblotted with the phospho-FIGQY polyclonal antibody. Each lane contains approximately 200 ng of coupled peptide. (C) Sections of embryonic (E17) rat forebrain incubated in the presence (+) or absence (-) of the tyrosine phosphatase YOP and immunolabeled with the phospho-FIGQY polyclonal antibody. Scale bars: 25 µm. MZ stands for marginal zone. (D) B104 rat neuroblastoma cells expressing either wild-type neurofascin (Y), neurofascin with the FIGQY tyrosine mutated to phenylalanine (F) or untransfected controls (con) were incubated in the presence (+) or absence (-) of bFGF. Extracts were immunoblotted with either a neurofascin-specific polyclonal antibody (top) or the phospho-FIGQY polyclonal antibody (bottom). (E) Extracts from either E17 rat brain or P6 cerebellum were immunoprecipitated with a polyclonal antibody against L1, neurofascin (NF) or with control IgG (labeled C). Immunoprecipitates were electrophoresed and immunoblotted with the phospho-FIGQY polyclonal antibody demonstrating that both L1 and neurofascin are phosphorylated at their FIGQY tyrosines. (F) Extracts from E17 embryonic brain (E), P10 neonatal brain (N), or adult brain (A) were electrophoresed and immunoblotted with the phospho-FIGQY polyclonal antibody, a previously characterized neurofascin antibody (NF), and an NrCAM antibody. The low molecular weight bands observed with the phospho-FIGQY polyclonal antibody in embryonic brain extract were also observed under control conditions (con) with only 125I-protein A and a control IgG at a similar exposure time. All immunoblots were developed using 125I-protein A except for (B,E), which were developed using enhanced chemiluminescence.

View larger version (37K): [in a new window]   Fig. 2. The phospho-FIGQY polyclonal antibody labels developing nervous system and epithelia in neuroglian-expressing but not neuroglian-null Drosophila embryos. Drosophila heterozygotes (A-D) or neuroglian null mutants (E-H), were examined by triple labeling immunofluorescence with anti-HRP as an antibody penetration control (A,E), anti-neuroglian to distinguish wild type from neuroglian null flies (B,F), and the phospho-FIGQY polyclonal antibody (C,G). Neuroglian nulls exhibited no phospho-FIGQY reactivity. Composite images are shown in (D,H) with HRP signal indicated in blue. Arrowheads mark the nervous system and brackets mark the epidermal epithelium. Scale bar: 25 µm.

View larger version (83K): [in a new window]   Fig. 3. FIGQY phosphorylation is concentrated in paranodal regions of adult sciatic nerve. Sections of adult rat sciatic nerve were labeled with either the phospho-FIGQY polyclonal antibody (A,D,G) or a neurofascin-specific polyclonal antibody (J). Nodes of Ranvier were visualized with the monoclonal antibody against ankyrin G (B,E,H,K). Composite images (C,F,I) demonstrate the minimal overlap between nodal ankyrin G (green) and paranodal phospho-FIGQY (red). The neurofascin-specific polyclonal antibody recognizes both nodes and paranodes (J-L). The phospho-FIGQY polyclonal antibody in G was cytosol cleared, demonstrating no difference in the pattern of immunofluorescence with cytosol clearing (see Materials and Methods for details). White arrowheads indicate the location of nodes of Ranvier. Scale bars: 5 µm in A-I; 2.5 µm in J-L.

View larger version (62K): [in a new window]   Fig. 4. FIGQY-phosphorylation occurs at the vertebrate neuromuscular junction. ACh receptors at the rat neuromuscular junction as labeled by bungarotoxin (A,D) are confined to the crests of junctional folds while ankyrin G (B) is confined to the deep regions of these folds in muscle cell plasma membrane. The two distributions do not overlap (C, arrows indicate identical position in all three images). FIGQY-phosphorylation (E) is strictly confined to the crests of junctional folds with ACh receptors (D) but has a more punctate distribution than ACh receptors. Composite images are shown in C,F. Scale bar: 2 µm. N, location of nerve terminals; M, muscle cell.

View larger version (79K): [in a new window]   Fig. 5. FIGQY phosphorylated neuroglian is present at the developing neuromuscular junction and at sites of muscle-tendon attachment in Drosophila embryos. (A) The developing neuromuscular junction of Drosophila embryos is shown as marked by Fas II labeling (white arrowhead). FIGQY-phosphorylated neuroglian is present at the neuromuscular junction (B, C for composite). (D-F) Embryos were labeled with an anti neuroglian antibody (D) and the phospho-FIGQY polyclonal antibody (E). A composite image is shown in F, with the developing neuromuscular junction marked by a white arrowhead. The phospho-FIGQY polyclonal antibody also labels muscle-tendon attachment sites (B-I, yellow arrowheads) as indicated by overlap with PS2 (G) (yellow arrowheads). Composite image shown in (L). Neuroglian is also present at these sites (D-F, yellow arrowheads) although it is not dramatically enriched. Scale bars: 10 µm. A, axon of presynaptic nerve cell; M, muscle cell.

View larger version (154K): [in a new window]   Fig. 6. FIGQY-phosphorylated L1 CAMs are localized to adherens junctions in vertebrate and Drosophila epithelial tissues. Drosophila embryos at stage 12-15 were labeled with a neuroglian antibody (B), demonstrating the presence of neuroglian in the basolateral domain of the hindgut. Labeling the same embryo with the phospho-FIGQY polyclonal antibody (A) revealed FIGQY-phosphorylated neuroglian concentrated in apicolateral regions. Composite image is shown in C. Embryos were also labeled with the adherens junction marker armadillo (E) and the phospho-FIGQY polyclonal antibody (D). A composite image is shown in F, with areas of overlap in yellow. (G) A section of E15 rat lung was labeled with the phospho-FIGQY polyclonal antibody demonstrating labeling at adherens junctions. A higher magnification image of the boxed area in G is shown in H. (I) The phospho-FIGQY polyclonal antibody signal is concentrated apically in the developing midgut of the E15 rat. Adherens junctions could not be resolved in these sections. NrCAM shows a similar distribution (J). Scale bars: 10 µm in A-C,G,I,J; 5 µm in D-F.

View larger version (150K): [in a new window]   Fig. 7. FIGQY tyrosine phosphorylation occurs in regions of neuroblast generation and migration and regions of axon extension. (A) A low magnification image of an E17 rat brain labeled with the phospho-FIGQY polyclonal antibody. The anterior region of developing cortex in E18 brain sections was labeled with the phospho-FIGQY polyclonal antibody (B), a neurofascin polyclonal antibody (C) or an L1 polyclonal antibody (D) showing overlap between expression of these L1 CAMs and phospho-FIGQY. A similar pattern of phospho-FIGQY labeling in E15 embyros (E) was displaced when the phospho-FIGQY polyclonal antibody was pre-incubated in the presence of a fivefold molar excess of tyrosine phosphorylated nfFIGQY peptide (F) but not in the presence of a fivefold molar excess of tyrosine phosphorylated AngII peptide (G). Sections of P8 cerebellum were labeled with the phospho-FIGQY polyclonal antibody (H), a polyclonal antibody against neurofascin FNIII domains (I) or a polyclonal antibody against L1 (J) showing an overlapping distribution of phospho-FIGQY and L1 CAMs. (K-M) The presence of FIGQY phosphorylation in adult brain in neuroblasts migrating in the rostral migratory stream from the lateral ventricle to the olfactory bulb is shown. The phospho-FIGQY polyclonal antibody label partially overlaps with the early neuronal marker ß III tubulin in the subventricular zone (L) and (M) for overlap. CP, cortical plate; EGL, external germinal layer; IGL, internal granule cell layer; IZ, intermediate zone; LV, lateral ventricle; ML, molecular layer; MZ, marginal zone; VZ, ventricular zone. Scale bars: 100 µm in A; 25 µm in B-M.