title: Shank-cortactin interactions control actin dynamics to maintain flexibility of neuronal spines and synapses.
abstract: The family of Shank scaffolding molecules (comprising Shank1, 2 and 3) are core components of the postsynaptic density (PSD) in neuronal synapses. Shanks link surface receptors to other scaffolding molecules within the PSD, as well as to the actin cytoskeleton. However, determining the function of Shank proteins in neurons has been complicated because the different Shank isoforms share a very high degree of sequence and domain homology. Therefore, to control Shank content while minimizing potential compensatory effects, a miRNA-based knockdown strategy was developed to reduce the expression of all synaptically targeted Shank isoforms simultaneously in rat hippocampal neurons. Using this approach, a strong (>75%) reduction in total Shank protein levels was achieved at individual dendritic spines, prompting an approximately 40% decrease in mushroom spine density. Furthermore, Shank knockdown reduced spine actin levels and increased sensitivity to the actin depolymerizing agent Latrunculin A. A SHANK2 mutant lacking the proline-rich cortactin-binding motif (SHANK2-ΔPRO) was unable to rescue these defects. Furthermore, Shank knockdown reduced cortactin levels in spines and increased the mobility of spine cortactin as measured by single-molecule tracking photoactivated localization microscopy, suggesting that Shank proteins recruit and stabilize cortactin at the synapse. Furthermore, it was found that Shank knockdown significantly reduced spontaneous remodelling of synapse morphology that could not be rescued by the SHANK2-ΔPRO mutant. It was concluded that Shank proteins are key intermediates between the synapse and the spine interior that, via cortactin, permit the actin cytoskeleton to dynamically regulate synapse morphology and function.
authors.affiliation: Department of Physiology, University of Maryland School of Medicine, Baltimore, MD, 21201, USA.|Program in Neuroscience, University of Maryland School of Medicine, Baltimore, MD, 21201, USA.|Cell Biology, Department of Biology, Faculty of Science, Utrecht University, 3584, CH Utrecht, the Netherlands.
authors.forename: Harold D
authors.initials: HD
authors.lastname: MacGillavry
authors.affiliation: Department of Physiology, University of Maryland School of Medicine, Baltimore, MD, 21201, USA.|Program in Neuroscience, University of Maryland School of Medicine, Baltimore, MD, 21201, USA.
authors.forename: Justin M
authors.initials: JM
authors.lastname: Kerr
authors.affiliation: Department of Physiology, University of Maryland School of Medicine, Baltimore, MD, 21201, USA.
authors.forename: Josh
authors.initials: J
authors.lastname: Kassner
authors.affiliation: Department of Physiology, University of Maryland School of Medicine, Baltimore, MD, 21201, USA.|Program in Neuroscience, University of Maryland School of Medicine, Baltimore, MD, 21201, USA.|Department of Neurology, University of California San Francisco, San Francisco, CA, USA.
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Frost
authors.affiliation: Department of Physiology, University of Maryland School of Medicine, Baltimore, MD, 21201, USA.|Program in Neuroscience, University of Maryland School of Medicine, Baltimore, MD, 21201, USA.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Blanpied
mesh_terms: D000818:Animals; D051356:Cortactin / Q000378:metabolism*; D049229:Dendritic Spines / Q000378:metabolism*; D005260:Female; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D008297:Male; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D051381:Rats; D013569:Synapses / Q000378:metabolism*
chemical_list: D051356:Cortactin; C495870:Cttn protein, rat; D009419:Nerve Tissue Proteins; C120531:Shank1 protein, rat; C485686:Shank2 protein, rat; C120532:Shank3 protein, rat
keywords: F-actin; dendritic spine; postsynaptic density; rat; single-molecule tracking