Arc/Arg3.1 has an activity-regulated interaction with PICK1 that results in altered spatial dynamics

Abstract

Activity-regulated cytoskeleton-associated protein (Arc; also known as Arg3.1) is an immediate early gene product that is transcribed in dendritic spines and, to date, has been best characterized as a positive regulator of AMPAR endocytosis during long-term depression (LTD) through interaction with endocytic proteins. Here, we show that protein interacting with C terminal kinase 1 (PICK1), a protein known to bind to the GluA2 subunit of AMPARs and associated with AMPAR trafficking, was pulled-down from brain homogenates and synaptosomes when using Arc as immobilized bait. Fluctuation and FLIM-FRET-Phasor analysis revealed direct interaction between these proteins when co-expressed that was increased under depolarizing conditions in live cells. At the plasma membrane, Arc-mCherry oligomerization was found to be concentration dependent. Additionally, co-expression of Arc-mCherry and EGFP-PICK1 followed by depolarizing conditions resulted in significant increases in the number and size of puncta containing both proteins. Furthermore, we identified the Arc binding region to be the first 126 amino acids of the PICK1 BAR domain. Overall, our data support a novel interaction and model where PICK1 mediates Arc regulation of AMPARs particularly under depolarizing conditions.

Arc/Arg3.1 function in long‐term synaptic plasticity: Emerging mechanisms and unresolved issues

“…The molecular basis for Arc‐promoted AMPAR endocytosis and LTD has been largely attributed to the interaction of Arc with components of the endocytic machinery, such as endophilin 2 and 3, dynamin 2 (Chowdhury et al., 2006; Rial Verde et al., 2006) and the clathrin adapter protein AP2 (DaSilva et al., 2016); reviewed in (Wall & Corrêa, 2018). In addition, the association of Arc with protein interacting with C terminal kinase 1 (PICK1) might also be involved (Goo, Sanstrum, Holden, Yu, & James, 2018). PICK1 is a BAR domain protein known to interact with GluA2, and required for NMDAR‐dependent reductions in surface GluA2.…”

ArfGAP1 inhibits mTORC1 lysosomal localization and activation

“…Fluorescence lifetime imaging microscopy (FLIM) and raster imaging correlation spectroscopy (RICS) data were collected using an Alba fluorescence correlation spectrometer (ISS, Champaign, IL), connected to a Nikon TE2000-U inverted microscope (Nikon, Melville, NY) with x-y scanning mirror and a PlanApo VC 60 × 1.2 NA water objective as previously described (Goo et al, 2018;Nguyen et al, 2019). Two-photon excitation was provided by a Chameleon Ultra (Coherent, Santa Clara, CA) tuned to 920 nm for FLIM data collection, while 1,000 nm was used for RICS experiments to obtain optimal excitation of both fluorophores (laser power > 1 mM at plane of excitation).…”

AKAP13 couples GPCR signaling to mTORC1 inhibition

“…Fluorescence Lifetime Imaging Microscopy (FLIM) data were collected using an Alba fluorescence correlation spectrometer (ISS, Champaign, IL), connected to a Nikon TE2000-U inverted microscope (Nikon, Melville, NY) with x-y scanning mirror and a PlanApo VC 60 x 1.2 NA water objective as previously described [49,50]. Two-photon excitation was provided by a Chameleon Ultra (Coherent, Santa Clara, CA) tuned to 900 nm for FLIM data collection to minimize the amount of direct YFP excitation.…”