Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

Article Title: Neuroligin 1 is dynamically exchanged at postsynaptic sites

doi: 10.1523/JNEUROSCI.0896-10.2010

Figure Lengend Snippet: Dynein inhibition (through dynamitin overexpression), a competing PSD-95 peptide or a NLG1 C-terminal mutant specifically interfere with NLG1 internalization in neurons. A, B, Neuronal internalization assay using myc-NLG1. Expression of EGFP, dynamitin-EGFP or the competitive peptide GFP-PSD-95 (256–398) (green). Surface membrane myc-NLG1 (blue), internalized myc-NLG1 (red). High sucrose prevents myc-NLG1 internalization. Blockade of dynein-mediated transport through dynamitin-EGFP overexpression, competitive interference with the NLG1 C-terminal PDZ binding motif through GFP-PSD-95 (256–398) expression or deletion of the C-terminal 6 amino acids in NLG1 (NLGΔC-term.) significantly reduce myc-NLG1 internalization in neurons (p<0.001). Values reflect numbers of fluorescent signals of internalized myc-NLG1, analysed using the Metamorph software. Scale bar: 8 μm. Error bars represent the SEM. (***): p<0.001. C, D, Internalized myc-NLG1 colocalizes with EEA1, a marker of the endocytic pathway. Neuronal internalization assay using myc-NLG1 (control experiment to A, B) with subsequent immunodetection of the early endosomal marker protein EEA1. As a transfection control, cells were cotransfected with EGFP. For a better visualization of merged puncta, EEA1 (as originally detected in the blue channel) is shown in a false color (green). Myc-NLG1 is shown in red. EGFP is shown in white. C, EGFP channel. D, EEA1 and myc-NLG1 channels as a magnification of the boxed area in C. Note that 16.45±1.74% of NLG1-myc is internalized in the time period analysed (compare with B). Yellow colocalized puncta indicate that internalized myc-NLG1 enters the endocytic pathway (arrows). Green puncta represent early endosomes that are negative for myc-NLG1. Red puncta represent internalized myc-NLG1-positive structures other than early endosomes (e.g. late endosomes, recycling endosomes, lysosomes). E, Quantification of D. 64.51%±4.08% of internalized myc-NLG1 signals colocalize with EEA1 (yellow). 38.0%±10.58% of EEA1-positive puncta do not colocalize with internalized myc-NLG1 (green). 35.49%±7.64% of internalized myc-NLG1 puncta do not colocalize with EEA1 (red). Values reflect numbers of fluorescent signals of internalized myc-NLG1, analysed using the Metamorph software. Error bars represent the SEM. (***): p<0.001.

Article Snippet: Antibodies The following antibodies were used for immunoprecipiation and Western blotting: anti-actin (1:2,000, SIGMA); anti-arp1 ( LaMonte et al., 2002 ), anti-pan-Cadherin (1:2,000, Abcam); anti-Calbindin-D-28K (SIGMA, 1:100), anti-dynamitin (IP1097) ( LaMonte et al., 2002 ), anti-dynein intermediate chain (1:2,000, Chemicon); anti-EEA1 (1:100, BD Bioscience), anti-Flag-M2 (1:250, SIGMA), anti-GluR2 (1:50, Millipore), anti-KIF5C (1:2,000, ABR); anti-Kinesin 1 (1:1000, Chemicon), anti-NLG1 (1:2,000, Synaptic Systems); anti-NLG1/NLG3 (1:2,000, Synaptic Systems); anti N-Cadherin (1:1,000, SIGMA); anti nNOS (1:500, CST); anti-p22 ( LaMonte et al., 2002 ), anti-p62 ( LaMonte et al., 2002 ), anti-p150glued ( LaMonte et al., 2002 ), anti-PSD-95 (1:250, BD Bioscience); anti-PSD-95 (1:2,000, ABR); anti-SAP97 (1:1,000, ABR); mouse unspecific IgG (SIGMA); rabbit unspecific IgG (SIGMA).

Techniques: Inhibition, Over Expression, Mutagenesis, Expressing, Binding Assay, Software, Marker, Immunodetection, Transfection