Elsevier

Brain Research

Volume 1153, 11 June 2007, Pages 20-33
Brain Research

Research Report
Activity-regulated cytoskeleton-associated protein Arc/Arg3.1 binds to spectrin and associates with nuclear promyelocytic leukemia (PML) bodies

https://doi.org/10.1016/j.brainres.2007.03.079Get rights and content

Abstract

Activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) is an immediate early gene, whose expression in the central nervous system is induced by specific patterns of synaptic activity. Arc is required for the late-phase of long-term potentiation (LTP) and memory consolidation, and has been implicated in AMPA receptor trafficking. Since Arc's molecular function remains incompletely understood, we have determined its subcellular localization in cultured hippocampal neurons and HEK 293T cells. Fluorescence microscopy experiments revealed that both endogenous and exogenous Arc protein was primarily found in the nucleus, where it concentrated in puncta associated with promyelocytic leukemia (PML) bodies, proposed sites of transcriptional regulation. Arc co-localized and interacted with the βIV spectrin splice variant βSpIVΣ5, a nuclear spectrin isoform associated with PML bodies and the nuclear matrix. A small region of Arc containing the coiled-coil domain is also restricted to β-spectrin-positive puncta, while the isolated spectrin homology domain is diffusely localized. Finally, Arc and βSpIVΣ5 synergistically increased the number of PML bodies. These results suggest that Arc functions as a spectrin-binding protein, forming a complex that may provide a role at sites of transcriptional regulation within the nucleus.

Introduction

Arc, also known as Arg3.1, is an immediate early gene (IEG) whose expression is strongly induced by neuronal activity patterns that elicit long-term alterations in synaptic strength (Link et al., 1995, Lyford et al., 1995). Arc is unique among IEGs because its mRNA is rapidly transported to distal dendrites and selectively localizes at activated synapses, where it has the potential to be locally translated (Steward and Worley, 2001, Steward et al., 1998). Arc plays a critical role in the late-phase of long-term potentiation (LTP) and is required for the consolidation of long-term memory. Induction of LTP in the hippocampus increases Arc protein expression, while blocking Arc expression by antisense oligonucleotide infusion or gene knockout causes defects in both late-phase LTP and memory tasks (Guzowski et al., 2000, Rodriguez et al., 2005, Plath et al., 2006). In addition, novel sound or taste stimuli, as well as spatial exploration of novel environments, rapidly induce Arc expression in a subset of neurons in the cortex and hippocampus (Guzowski et al., 1999, Montag-Sallaz et al., 1999, Bock et al., 2005, Chawla et al., 2005, Ramirez-Amaya et al., 2005).

It has been hypothesized that locally translated Arc interacts with existing cytoskeletal proteins, leading to specific modifications of synapses that are undergoing activity-dependent remodeling. Recently, Arc was proposed to regulate AMPA receptors trafficking (Chowdhury et al., 2006, Plath et al., 2006, Rial Verde et al., 2006, Shepherd et al., 2006). In addition, a number of findings suggest that Arc associates with the cytoskeleton. Arc interacts with CaMKII, leading to an increase in neurite length mediated by cytoskeletal remodeling (Donai et al., 2003). Arc expression also reduces the immunoreactivity of microtubule-associated protein 2 (MAP2), a protein associated with the actin cytoskeleton (Fujimoto et al., 2004). Finally, Arc co-sediments with actin in crude cell extracts and has been proposed to share homology with α-spectrin (Lyford et al., 1995). However, Arc has not yet been shown to directly interact with a component of the cytoskeleton. In addition to its synaptic localization, Arc protein has been found in the cell body and nucleus (Irie et al., 2000), which may contain a cytoskeletal structure known as the nuclear matrix (Tsutsui et al., 2005). While a role for Arc in the dendritic compartment has recently been defined, Arc's role in the nucleus remains poorly understood.

In order to further study the function of Arc protein, its subcellular localization was determined in cultured hippocampal neurons and HEK 293T cells. Arc was found primarily in the nucleus and concentrated in puncta associated with promyelocytic leukemia (PML) bodies, which are proposed sites of transcriptional regulation (Wang et al., 2004). Arc co-localized and directly interacted with βSpIVΣ5, a spectrin associated with PML bodies and the nuclear matrix (Tse et al., 2001). Finally, co-expression of Arc and βSpIVΣ5 increased the number of PML bodies in HEK 293T cells, suggesting a role for this complex in PML body function.

Section snippets

Results

In earlier experiments using rats that received electric stimulation, Arc protein was localized by antibody staining to the cell body, nucleus, and dendrites of neurons in hippocampal slices (Steward and Worley, 2001). Here we have re-examined the subcellular localization of Arc in cultured hippocampal neurons, which provide superior resolution.

Discussion

The subcellular localization of Arc protein was investigated in detail using HEK 293T cells and hippocampal neurons. In both cell types, Arc was found to accumulate in the nucleus, where it formed a complex with a β-spectrin isoform. Furthermore, we found that Arc and β-spectrin associated with PML bodies, both individually and as a complex.

Cell culture

Hippocampal rat brain tissue (E18) was obtained from Brainbits, Inc. (Springfield, IL) and was cultured as previously described (Van de Ven et al., 2005). HEK 293T cells were obtained from the Duke University Cell Culture Facility, and were cultured in high glucose DMEM (Sigma-Aldrich, St. Louis, MO) with 10% fetal bovine serum (FBS) (Invitrogen Corporation, Carlsbad, CA). Like the neurons, these cells were plated on the poly-d-lysine coated glass-bottom dishes for imaging experiments. In

Acknowledgments

We thank Dr. William Tse, Children's Memorial Research Center, Northwestern University, Chicago, IL for SpβIVΣ5-GFP in pcDNA4/HisMaxA, Dr. Tim Ley, Washington University School of Medicine, Department of Medicine, St. Louis, MO for EGFP-PML in EGFPC1, and Dr. Roger Tsien, University of California, Department of Pharmacology, La Jolla, CA for mCherry in pRSET-B. We also thank Dr. Donald McDonnell and Dr. Ching-yi Chang, Duke University, Department of Pharmacology, Durham, NC for their

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