1. Neuroligin 1 Is Dynamically Exchanged at Postsynaptic Sites

저자: Inga U. Schapitz,Bardo Behrend, Yvonne Pechmann, Corinna Lappe-Siefke, Silas J. Kneussel, Karen E. Wallace, A. Vanessa Stempel, Fritz Buck, Seth G. N. Grant, Michaela Schweizer, Dietmar Schmitz, Ju¨rgen R. Schwarz, Erika L. F. Holzbaur, and Matthias Kneussel

저널: J Neurosci. 2010 Sep 22;30(38):12733-44.

사용한 제품: Dynabeads Streptavidin MyOne C1 (Cat# 65001, 65002)

Abstract

Neuroligins are postsynaptic cell adhesion molecules that associate with presynaptic neurexins. Both factors form a transsynaptic connection, mediate signaling across the synapse, specify synaptic functions, and play a role in synapse formation. Neuroligin dysfunction impairs synaptic transmission, disrupts neuronal networks, and is thought to participate in cognitive diseases.

Here we report that chemical treatment designed to induce long-term potentiation or long-term depression (LTD) induces neuroligin 1/3 turnover, leading to either increased or decreased surface membrane protein levels, respectively. Despite its structural role at a crucial transsynaptic position, GFP-neuroligin 1 leaves synapses in hippocampal neurons over time with chemical LTD-induced neuroligin internalization depending on an intact microtubule cytoskeleton. Accordingly, neuroligin 1 and its binding partner postsynaptic density protein-95 (PSD-95) associate with components of the dynein motor complex and undergo retrograde cotransport with a dynein subunit.

Transgenic depletion of dynein function in mice causes postsynaptic NLG1/3 and PSD-95 enrichment. In parallel, PSD lengths and spine head sizes are significantly increased, a phenotype similar to that observed upon transgenic overexpression of NLG1 (Dahlhaus et al., 2010). Moreover, application of a competitive PSD-95 peptide and neuroligin 1 C-terminal mutagenesis each specifically alter neuroligin 1 surface membrane expression and interfere with its internalization. Our data suggest the concept that synaptic plasticity regulates neuroligin turnover through active cytoskeleton transport.

제품 사용법

Surface biotinylation assay

A surface biotinylation assay was performed to assess the relative number of neuroligin molecules in the plasma membrane as a measure of Western blot signal intensities. For biotinylation of cell surface proteins, cells were washed with PBS, incubated with 1 mM biotinylation reagent (biotinamidohexanoic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt, Sigma) for 20 min at 4°C and quenched twice with 100mM glycine in HEPES buffer for 20 min at 4°C. Cells were harvested with PBS/1% Triton X-100, centrifuged at 1000   g for 5 min, and then loaded on streptavidin beads (Dynabeads MyOne Streptavidin C1, Invitrogen) followed by an incubation period of 3 h at 4°C, with subsequent washing and resuspension in 4  SDS sample buffer.