The dynamic regulation of PICK1 BAR domain dimerisation during synaptic plasticity

Abstract

PICK1 is a uniquely structured protein because it contains both a BAR domain and a PDZ domain. The PDZ domain mediates a plethora of protein-protein interactions, including with the GluA2 subunit of AMPA receptors. Through the BAR domain, PICK1 is capable of recognising and binding lipid membranes, although there is no evidence indicating a curvature induction mechanism for this protein. PICK1 has been shown to readily assemble into dimers and higher order oligomers in solution, but investigations looking at PICK1 dimerisation in a cellular environment are limited. PICK1 is mainly expressed in neurons and has been shown to participate in the internalisation of GluA2-containing AMPARs in response to synaptic plasticity. Additionally, PICK1 has been defined as a calcium-binding protein. In this study, I aim to investigate how PICK1 dimerisation is regulated in response to calcium and the potential consequences this has for PICK1 function.
I present novel findings in support of a membrane tubulating mechanism for PICK1. This is the first demonstration of a role for PICK1 in the active generation of membrane curvature, as well as the first evidence showing stimulus-induced dimerisation of BAR domains. PICK1 dimerisation is increased by approximately two-fold in the presence of calcium when overexpressed in HEK293 cells and using purified protein. Furthermore, this is a biphasic effect which suggests that calcium-induced PICK1 dimerisation is a direct regulatory mechanism for PICK1 function, mediated through acidic regions within its structure. Additionally, FLIM-FRET imaging of neurons showed that PICK1 dimerisation was upregulated immediately after LTD only. Cryo-EM imaging and COS-7 tubulation assays demonstrated that PICK1 can form tubules only in the presence of calcium. By replacing the 271DDDE274 region with alanine which abolishes this calcium binding pocket, I was able to generate a mutant that shows impaired dimerisation in response to calcium. When tested in the COS-7 tubulation assay, this mutant was severely impaired in its ability to produce tubules. Taken together, these results provide a potential mechanism for PICK1 function where calcium stimulation during synaptic plasticity promotes PICK1 dimerisation which in turn leads to membrane remodelling and the potential internalisation of GluA2-containing AMPA receptors.

Date of Award	28 Sept 2021
Original language	English
Awarding Institution	University of Bristol
Supervisor	Krasimira Tsaneva-Atanasova (Supervisor) & Jonathan G Hanley (Supervisor)