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Updated: May 1, 2026
Super-Resolution Imaging to Study Co-Localization of Proteins and Synaptic Markers in Primary Neurons
Published on: October 31, 2020
Nanoscopic spine localization of Norbin, an mGluR5 accessory protein
Linda Westin, Matthias Reuss, Maria Lindskog
1Department of Women's and Children's Health, Science for Life Laboratory, Karolinska Institutet, 17165 Solna, Sweden. brismar@kth.se.
Background:
Norbin is a neuron-specific, cytosolic protein that interacts with the metabotropic glutamate receptor 5 (mGluR5) and has a profound impact on mGluR5 signaling. Yet, little is known about its synaptic distribution.
Results:
Here we have analyzed the spatial relationship between Norbin, postsynaptic density protein 95 (PSD-95), actin and mGluR5 in spines using super-resolution microscopy. Norbin was found to have a high degree of colocalization with actin and a lower degree of colocalization with PSD-95. Co-immunoprecipitation studies confirmed that interaction occurs between Norbin and actin, but not between Norbin and PSD-95. Norbin was also found to have a high degree of colocalization with the perisynaptically located mGluR5. Findings based on structured illumination microscopy (3D-SIM) of exogenous expressed Norbin-GFP were confirmed by stimulated emission depletion microscopy (STED) of immunolabeled endogenous Norbin.
Conclusions:
Norbin associates with actin rather than with PSD-95 in dendritic spines. Results regarding protein localization and colocalization performed with conventional confocal microscopy must be interpreted with great caution. The now available super-resolution microscopy techniques provide more accurate information about sub-cellular protein localization than previously was possible.
