Analysis of Arc/Arg3.1 Oligomerization In Vitro and in Living Cells.

Arc (also known as Arg3.1) is an activity-dependent immediate early gene product enriched in neuronal dendrites. Arc plays essential roles in long-term potentiation, long-term depression, and synaptic scaling. Although its mechanisms of action in these forms of synaptic plasticity are not completely well established, the activities of Arc include the remodeling of the actin cytoskeleton, the facilitation of AMPA receptor (AMPAR) endocytosis, and the regulation of the transcription of AMPAR subunits. In addition, Arc has sequence and structural similarity to retroviral Gag proteins and self-associates into virus-like particles that encapsulate mRNA and perhaps other cargo for intercellular transport. Each of these activities is likely to be influenced by Arc's reversible self-association into multiple oligomeric species. Here, we used mass photometry to show that Arc exists predominantly as monomers, dimers, and trimers at approximately 20 nM concentration in vitro. Fluorescence fluctuation spectroscopy revealed that Arc is almost exclusively present as low-order (monomer to tetramer) oligomers in the cytoplasm of living cells, over a 200 nM to 5 µM concentration range. We also confirmed that an α-helical segment in the N-terminal domain contains essential determinants of Arc's self-association.

Mimicking Protein Kinase C Phosphorylation Inhibits Arc/Arg3.1 Palmitoylation and Its Interaction with Nucleic Acids.

Activity-regulated cytoskeleton-associated protein (Arc) plays essential roles in diverse forms of synaptic plasticity, including long-term potentiation (LTP), long-term depression (LTD), and homeostatic plasticity. In addition, it assembles into virus-like particles that may deliver mRNAs and/or other cargo between neurons and neighboring cells. Considering this broad range of activities, it is not surprising that Arc is subject to regulation by multiple types of post-translational modification, including phosphorylation, palmitoylation, SUMOylation, ubiquitylation, and acetylation. Here we explore the potential regulatory role of Arc phosphorylation by protein kinase C (PKC), which occurs on serines 84 and 90 within an α-helical segment in the N-terminal domain. To mimic the effect of PKC phosphorylation, we mutated the two serines to negatively charged glutamic acid. A consequence of introducing these phosphomimetic mutations is the almost complete inhibition of Arc palmitoylation, which occurs on nearby cysteines and contributes to synaptic weakening. The mutations also inhibit the binding of nucleic acids and destabilize high-order Arc oligomers. Thus, PKC phosphorylation of Arc may limit the full expression of LTD and may suppress the interneuronal transport of mRNAs.

PI4P-Dependent Targeting of ATG14 to Mature Autophagosomes.

Degradation of autophagosomal cargo requires the tethering and fusion of autophagosomes with lysosomes that is mediated by the scaffolding protein autophagy related 14 (ATG14). Here, we report that phosphatidylinositol 4-kinase 2A (PI4K2A) generates a pool of phosphatidylinositol 4-phosphate (PI4P) that facilitates the recruitment of ATG14 to mature autophagosomes. We also show that PI4K2A binds to ATG14, suggesting that PI4P may be synthesized in situ in the vicinity of ATG14. Impaired targeting of ATG14 to autophagosomes in PI4K2A-depleted cells is rescued by the introduction of PI4P but not its downstream product phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Thus, PI4P and PI(4,5)P2 have independent functions in late-stage autophagy. These results provide a mechanism to explain prior studies indicating that PI4K2A and its product PI4P are necessary for autophagosome-lysosome fusion.

Sensitive Detection of Protein Binding to the Plasma Membrane with Dual-Color Z-Scan Fluorescence.

Delicate and transitory protein engagement at the plasma membrane (PM) is crucial to a broad range of cellular functions, including cell motility, signal transduction, and virus replication. Here, we describe a dual-color (DC) extension of the fluorescence z-scan technique, which has proven successful for quantification of peripheral membrane protein binding to the PM in living cells. We demonstrate that the coexpression of a second, distinctly colored fluorescent protein provides a soluble reference species that delineates the extent of the cell cytoplasm and lowers the detection threshold of z-scan PM-binding measurements by an order of magnitude. DC z-scan generates an intensity profile for each detection channel that contains information on the axial distribution of the peripheral membrane and reference protein. Fit models for DC z-scan are developed and verified using simple model systems. Next, we apply the quantitative DC z-scan technique to investigate the binding of two peripheral membrane protein systems for which previous z-scan studies failed to detect binding: human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein and lipidation-deficient mutants of the fibroblast growth factor receptor substrate 2α. Our findings show that these mutations severely disrupt PM association of fibroblast growth factor receptor substrate 2α but do not eliminate it. We further detected binding of HIV-1 MA to the PM using DC z-scan. Interestingly, our data indicate that HIV-1 MA binds cooperatively to the PM with a dissociation coefficient of Kd ∼16 µM and Hill coefficient of n ∼2.

Roles for Arc in metabotropic glutamate receptor-dependent LTD and synapse elimination: Implications in health and disease.

The Arc gene is robustly transcribed in specific neural ensembles in response to experience-driven activity. Upon induction, Arc mRNA is transported to dendrites, where it can be rapidly and locally translated by activation of metabotropic glutamate receptors (mGluR1/5). mGluR-induced dendritic synthesis of Arc is implicated in weakening or elimination of excitatory synapses by triggering endocytosis of postsynaptic AMPARs in both hippocampal CA1 and cerebellar Purkinje neurons. Importantly, CA1 neurons with experience-induced Arc mRNA are susceptible, or primed for mGluR-induced long-term synaptic depression (mGluR-LTD). Here we review mechanisms and function of Arc in mGluR-LTD and synapse elimination and propose roles for these forms of plasticity in Arc-dependent formation of sparse neural representations of learned experience. We also discuss accumulating evidence linking dysregulation of Arc and mGluR-LTD in human cognitive disorders such as intellectual disability, autism and Alzheimer's disease.

Myristoylation-Dependent Palmitoylation of the Receptor Tyrosine Kinase Adaptor FRS2α.

An early step in signaling from activated receptor tyrosine kinases (RTKs) is the recruitment of cytosolic adaptor proteins to autophosphorylated tyrosines in the receptor cytoplasmic domains. Fibroblast growth factor receptor substrate 2α (FRS2α) associates via its phosphotyrosine-binding domain (PTB) to FGF receptors (FGFRs). Upon FGFR activation, FRS2α undergoes phosphorylation on multiple tyrosines, triggering recruitment of the adaptor Grb2 and the tyrosine phosphatase Shp2, resulting in stimulation of PI3K/AKT and MAPK signaling pathways. FRS2α also undergoes N-myristoylation, which was shown to be important for its localization to membranes and its ability to stimulate downstream signaling events (Kouhara et al., 1997). Here we show that FRS2α is also palmitoylated in cells and that cysteines 4 and 5 account for the entire modification. We further show that mutation of those two cysteines interferes with FRS2α localization to the plasma membrane (PM), and we quantify this observation using fluorescence fluctuation spectroscopy approaches. Importantly, prevention of myristoylation by introduction of a G2A mutation also abrogates palmitoylation, raising the possibility that signaling defects previously ascribed to the G2A mutant may actually be due to a failure of that mutant to undergo palmitoylation. Our results demonstrate that FRS2α undergoes coupled myristoylation and palmitoylation. Unlike stable cotranslational modifications, such as myristoylation and prenylation, palmitoylation is reversible due to the relative lability of the thioester linkage. Therefore, palmitoylation may provide a mechanism, in addition to phosphorylation, for dynamic regulation of FRS2 and its downstream signaling pathways.

Comobility of GABARAP and Phosphatidylinositol 4-Kinase 2A on Cytoplasmic Vesicles.

We previously reported that recruitment of the type IIA phosphatidylinositol 4-kinase (PI4K2A) to autophagosomes by GABARAP, a member of the Atg8 family of autophagy-related proteins, is important for autophagosome-lysosome fusion. Because both PI4K2A and GABARAP have also been implicated in the intracellular trafficking of plasma membrane receptors in the secretory/endocytic pathway, we characterized their interaction in cells under nonautophagic conditions. Fluorescence fluctuation spectroscopy measurements revealed that GABARAP exists predominantly as a cytosolic monomer in live cells, but is recruited to small cytoplasmic vesicles upon overexpression of PI4K2A. C-Terminal lipidation of GABARAP, which is essential for its autophagic activities, is not necessary for its recruitment to these PI4K2A-containing transport vesicles. However, a GABARAP truncation mutant lacking C-terminal residues 103-117 fails to bind to PI4K2A, is not recruited to cytoplasmic vesicles, and does not codistribute with PI4K2A on subcellular organelles. These observations suggest that the PI4K2A-GABARAP interaction plays a role in membrane trafficking both under autophagic and nonautophagic conditions.

Palmitoylation and Membrane Binding of Arc/Arg3.1: A Potential Role in Synaptic Depression.

Activity-regulated cytoskeletal-associated protein (Arc, also known as activity-regulated gene 3.1 or Arg3.1) is induced in neurons in response to salient experience and neural activity and is necessary for activity-induced forms of synaptic plasticity, such as long-term potentiation (LTP) and long-term depression (LTD), cellular substrates of learning and memory. The best-characterized function of Arc is enhancement of the endocytic internalization of AMPA receptors in dendritic spines, a process associated with LTD. Arc has also been implicated in the proteolytic processing of amyloid precursor protein on the surface of endosomes. To mediate these activities, Arc must associate with cellular membranes, but it is unclear whether Arc binds directly to the lipid bilayer or requires protein-protein interactions for membrane recruitment. In this study, we show that Arc associates with pure phospholipid vesicles in vitro and undergoes palmitoylation in neurons, a modification that allows it to insert directly into the hydrophobic core of the bilayer. The palmitoylated cysteines are clustered in a motif, 94CLCRC98, located in the N-terminal half of the protein, which has not yet been structurally characterized. Expression of Arc with three mutated cysteines in that motif cannot support synaptic depression induced by the activity-dependent transcription factor, MEF2 (myocyte enhancer factor 2), in contrast to wild-type Arc. Thus, it appears that palmitoylation regulates at least a subset of Arc functions in synaptic plasticity.

Association of Endophilin B1 with Cytoplasmic Vesicles.

Endophilins are SH3- and BAR domain-containing proteins implicated in membrane remodeling and vesicle formation. Endophilins A1 and A2 promote the budding of endocytic vesicles from the plasma membrane, whereas endophilin B1 has been implicated in vesicle budding from intracellular organelles, including the trans-Golgi network and late endosomes. We previously reported that endophilins A1 and A2 exist almost exclusively as soluble dimers in the cytosol. Here, we present results of fluorescence fluctuation spectroscopy analyses indicating that, in contrast, the majority of endophilin B1 is present in multiple copies on small, highly mobile cytoplasmic vesicles. Formation of these vesicles was enhanced by overexpression of wild-type dynamin 2, but suppressed by expression of a catalytically inactive dynamin 2 mutant. Using dual-color heterospecies partition analysis, we identified the epidermal growth factor receptor on endophilin B1 vesicles. Moreover, a proportion of endophilin B1 vesicles also contained caveolin, whereas clathrin was almost undetectable on those vesicles. These results raise the possibility that endophilin B1 participates in dynamin 2-dependent formation of a population of transport vesicles distinct from those generated by A-type endophilins.

Enhancement of dynamin polymerization and GTPase activity by Arc/Arg3.1.

BACKGROUND: The Activity-regulated cytoskeleton-associated protein, Arc, is an immediate-early gene product implicated in various forms of synaptic plasticity. Arc promotes endocytosis of AMPA type glutamate receptors and regulates cytoskeletal assembly in neuronal dendrites. Its role in endocytosis may be mediated by its reported interaction with dynamin 2, a 100 kDa GTPase that polymerizes around the necks of budding vesicles and catalyzes membrane scission. METHODS: Enzymatic and turbidity assays are used in this study to monitor effects of Arc on dynamin activity and polymerization. Arc oligomerization is measured using a combination of approaches, including size exclusion chromatography, sedimentation analysis, dynamic light scattering, fluorescence correlation spectroscopy, and electron microscopy. RESULTS: We present evidence that bacterially-expressed His6-Arc facilitates the polymerization of dynamin 2 and stimulates its GTPase activity under physiologic conditions (37°C and 100mM NaCl). At lower ionic strength Arc also stabilizes pre-formed dynamin 2 polymers against GTP-dependent disassembly, thereby prolonging assembly-dependent GTP hydrolysis catalyzed by dynamin 2. Arc also increases the GTPase activity of dynamin 3, an isoform of implicated in dendrite remodeling, but does not affect the activity of dynamin 1, a neuron-specific isoform involved in synaptic vesicle recycling. We further show in this study that Arc (either His6-tagged or untagged) has a tendency to form large soluble oligomers, which may function as a scaffold for dynamin assembly and activation. CONCLUSIONS AND GENERAL SIGNIFICANCE: The ability of Arc to enhance dynamin polymerization and GTPase activation may provide a mechanism to explain Arc-mediated endocytosis of AMPA receptors and the accompanying effects on synaptic plasticity.

A mutation associated with centronuclear myopathy enhances the size and stability of dynamin 2 complexes in cells.

BACKGROUND: Dynamin 2 (Dyn2) is a ~100kDa GTPase that assembles around the necks of nascent endocytic and Golgi vesicles and catalyzes membrane scission. Mutations in Dyn2 that cause centronuclear myopathy (CNM) have been shown to stabilize Dyn2 polymers against GTP-dependent disassembly in vitro. Precisely timed regulation of assembly and disassembly is believed to be critical for Dyn2 function in membrane vesiculation, and the CNM mutations interfere with this regulation by shifting the equilibrium toward the assembled state. METHODS: In this study we use two fluorescence fluctuation spectroscopy (FFS) approaches to show that a CNM mutant form of Dyn2 also has a greater propensity to self-assemble in the cytosol and on the plasma membrane of living cells. RESULTS: Results obtained using brightness analysis indicate that unassembled wild-type Dyn2 is predominantly tetrameric in the cytosol, although different oligomeric species are observed, depending on the concentration of expressed protein. In contrast, an R369W mutant identified in CNM patients forms higher-order oligomers at concentrations above 1µM. Investigation of Dyn2-R369W by Total Internal Reflection Fluorescence (TIRF) FFS reveals that this mutant forms larger and more stable clathrin-containing structures on the plasma membrane than wild-type Dyn2. CONCLUSIONS AND GENERAL SIGNIFICANCE: These observations may explain defects in membrane trafficking reported in CNM patient cells and in heterologous systems expressing CNM-associated Dyn2 mutants.

Type II phosphatidylinositol 4-kinase regulates trafficking of secretory granule proteins in Drosophila.

Type II phosphatidylinositol 4-kinase (PI4KII) produces the lipid phosphatidylinositol 4-phosphate (PI4P), a key regulator of membrane trafficking. Here, we generated genetic models of the sole Drosophila melanogaster PI4KII gene. A specific requirement for PI4KII emerged in larval salivary glands. In PI4KII mutants, mucin-containing glue granules failed to reach normal size, with glue protein aberrantly accumulating in enlarged Rab7-positive late endosomes. Presence of PI4KII at the Golgi and on dynamic tubular endosomes indicated two distinct foci for its function. First, consistent with the established role of PI4P in the Golgi, PI4KII is required for sorting of glue granule cargo and the granule-associated SNARE Snap24. Second, PI4KII also has an unforeseen function in late endosomes, where it is required for normal retromer dynamics and for formation of tubular endosomes that are likely to be involved in retrieving Snap24 and Lysosomal enzyme receptor protein (Lerp) from late endosomes to the trans-Golgi network. Our genetic analysis of PI4KII in flies thus reveals a novel role for PI4KII in regulating the fidelity of granule protein trafficking in secretory tissues.

Off-target effects of MEK inhibitors.

The mitogen-activated protein kinases (MAPKs) ERK1/2 regulate numerous cellular processes, including gene transcription, proliferation, and differentiation. The only known substrates of the MAP2Ks MEK1/2 are ERK1/2; thus, MEK inhibitors PD98059, U0126, and PD0325901 have been important tools in determining the functions of ERK1/2. By using these inhibitors and genetically manipulating MEK, we found that ERK1/2 activation is neither sufficient nor necessary for regulated secretion of insulin from pancreatic ß cells or secretion of epinephrine from chromaffin cells. We show that both PD98059 and U0126 reduce agonist-induced entry of calcium into cells in a manner independent of their ability to inhibit ERK1/2. Caution should be used when interpreting results from experiments using these compounds.

Phosphatidylinositol 4-kinase IIα is palmitoylated by Golgi-localized palmitoyltransferases in cholesterol-dependent manner.

Phosphatidylinositol 4-kinase IIα (PI4KIIα) is predominantly Golgi-localized, and it generates >50% of the phosphatidylinositol 4-phosphate in the Golgi. The lipid kinase activity, Golgi localization, and "integral" membrane binding of PI4KIIα and its association with low buoyant density "raft" domains are critically dependent on palmitoylation of its cysteine-rich (173)CCPCC(177) motif and are also highly cholesterol-dependent. Here, we identified the palmitoyl acyltransferases (Asp-His-His-Cys (DHHC) PATs) that palmitoylate PI4KIIα and show for the first time that palmitoylation is cholesterol-dependent. DHHC3 and DHHC7 PATs, which robustly palmitoylated PI4KIIα and were colocalized with PI4KIIα in the trans-Golgi network (TGN), were characterized in detail. Overexpression of DHHC3 or DHHC7 increased PI4KIIα palmitoylation by >3-fold, whereas overexpression of the dominant-negative PATs or PAT silencing by RNA interference decreased PI4KIIα palmitoylation, "integral" membrane association, and Golgi localization. Wild-type and dominant-negative DHHC3 and DHHC7 co-immunoprecipitated with PI4KIIα, whereas non-candidate DHHC18 and DHHC23 did not. The PI4KIIα (173)CCPCC(177) palmitoylation motif is required for interaction because the palmitoylation-defective SSPSS mutant did not co-immunoprecipitate with DHHC3. Cholesterol depletion and repletion with methyl-ß-cyclodextrin reversibly altered PI4KIIα association with these DHHCs as well as PI4KIIα localization at the TGN and "integral" membrane association. Significantly, the Golgi phosphatidylinositol 4-phosphate level was altered in parallel with changes in PI4KIIα behavior. Our study uncovered a novel mechanism for the preferential recruitment and activation of PI4KIIα to the TGN by interaction with Golgi- and raft-localized DHHCs in a cholesterol-dependent manner.

The CD3 zeta subunit contains a phosphoinositide-binding motif that is required for the stable accumulation of TCR-CD3 complex at the immunological synapse.

T cell activation involves a cascade of TCR-mediated signals that are regulated by three distinct intracellular signaling motifs located within the cytoplasmic tails of the CD3 chains. Whereas all the CD3 subunits possess at least one ITAM, the CD3 ε subunit also contains a proline-rich sequence and a basic-rich stretch (BRS). The CD3 ε BRS complexes selected phosphoinositides, interactions that are required for normal cell surface expression of the TCR. The cytoplasmic domain of CD3 ζ also contains several clusters of arginine and lysine residues. In this study, we report that these basic amino acids enable CD3 ζ to complex the phosphoinositides PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,5)P(2), and PtdIns(3,4,5)P(3) with high affinity. Early TCR signaling pathways were unaffected by the targeted loss of the phosphoinositide-binding functions of CD3 ζ. Instead, the elimination of the phosphoinositide-binding function of CD3 ζ significantly impaired the ability of this invariant chain to accumulate stably at the immunological synapse during T cell-APC interactions. Without its phosphoinositide-binding functions, CD3 ζ was concentrated in intracellular structures after T cell activation. Such findings demonstrate a novel functional role for CD3 ζ BRS-phosphoinositide interactions in supporting T cell activation.

Molecular brightness analysis reveals phosphatidylinositol 4-Kinase IIß association with clathrin-coated vesicles in living cells.

Mammalian cells express two classes of phosphatidylinositol 4-kinase (PI4K), designated as Types II and III, that phosphorylate phosphatidylinositol to generate PI4P. A number of studies have indicated that these enzymes are important for Golgi trafficking and both early and late stages of endocytosis. In this study, we focus on PI4KIIß, a protein that is evenly distributed between membrane and soluble fractions, and is believed to participate in stimulus-dependent phosphoinositide signaling. Using molecular brightness analysis, we found that EGFP-tagged PI4KIIß exists as two distinct species in the cytoplasm: a soluble monomer and a high-order complex enriched with multiple copies of PI4KIIß. This observation was confirmed by an autocorrelation analysis that identified two species with distinct mobilities. We further demonstrate that the high-order complex enriched with PI4KIIß is sensitive to inhibition of palmitoylation, indicating that it is associated with membranes, very likely vesicles. Indeed, we show that the high-order PI4KIIß complex is sensitive to expression of dynamin 2 (K44A), a dominant-negative inhibitor of endocytosis. Using dual-color heterospecies partition analysis, we directly detected that PI4KIIß comoves with clathrin light chain on vesicles. This analysis allows us to isolate the comobile species in the presence of strong background contribution from the monomeric pool of PI4KIIß. Our results strongly suggest that PI4KIIß is involved in an early stage of endocytosis and is associated with clathrin-coated vesicles. Moreover, we establish molecular brightness as a powerful tool for characterizing cellular cytosolic vesicles that are otherwise difficult to characterize by other techniques.

Number and brightness analysis of LRRK2 oligomerization in live cells.

Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein that contains enzymatically functional GTPase and kinase domains. Several noncoding LRRK2 gene polymorphisms have been associated with susceptibility to Parkinson's disease (PD), Crohn's disease, and leprosy. Many LRRK2 coding polymorphisms have been associated with or causally linked to PD. The G2019S point mutation within the LRRK2 kinase domain is the most common cause of familial PD. The G2019S mutation appears to alter LRRK2 kinase activity. Some but not all studies have reported that LRRK2 kinase activity is dependent upon LRRK2 dimerization and membrane localization. It is important to define the oligomeric state(s) of LRRK2 in living cells, which to date have only been characterized in vitro. Here we use confocal and total internal reflection microscopy coupled with number and brightness analysis to study the oligomeric states of LRRK2 within the cytosol and on the plasma membrane of live CHO-K1 cells. Our results show, for the first time to our knowledge, that LRRK2 is predominantly monomeric throughout the cytosol of living cells, but attains predominately higher oligomeric states in the plasma membrane.

Stabilization of phosphatidylinositol 4-kinase type IIbeta by interaction with Hsp90.

Mammalian cells express two isoforms of type II phosphatidylinositol 4-kinase: PI4KIIα and PI4KIIß. PI4KIIα exists almost exclusively as a constitutively active integral membrane protein because of its palmitoylation (Barylko, B., Gerber, S. H., Binns, D. D., Grichine, N., Khvotchev, M., Südhof, T. C., and Albanesi, J. P. (2001) J. Biol. Chem. 276, 7705-7708). In contrast, PI4KIIß is distributed almost evenly between membranes and cytosol. Whereas the palmitoylated membrane-bound pool is catalytically active, the cytosolic kinase is inactive (Wei, Y. J., Sun, H. Q., Yamamoto, M., Wlodarski, P., Kunii, K., Martinez, M., Barylko, B., Albanesi, J. P., and Yin, H. L. (2002) J. Biol. Chem. 277, 46586-46593; Jung, G., Wang, J., Wlodarski, P., Barylko, B., Binns, D. D., Shu, H., Yin, H. L., and Albanesi, J. P. (2008) Biochem. J. 409, 501-509). In this study, we identify the molecular chaperone Hsp90 as a binding partner of PI4KIIß, but not of PI4KIIα. Geldanamycin (GA), a specific Hsp90 inhibitor, disrupts the Hsp90-PI4KIIß interaction and destabilizes PI4KIIß, reducing its half-life by 40% and increasing its susceptibility to ubiquitylation and proteasomal degradation. Cytosolic PI4KIIß is much more sensitive to GA treatment than is the integrally membrane-associated species. Exposure to GA induces a partial redistribution of PI4KIIß from the cytosol to membranes and, with brief GA treatments, a corresponding increase in cellular phosphatidylinositol 4-kinase activity. Stimuli such as PDGF receptor activation that also induce recruitment of the kinase to membranes disrupt the Hsp90-PI4KIIß interaction to a similar extent as GA treatment. These results support a model wherein Hsp90 interacts predominantly with the cytosolic, inactive pool of PI4KIIß, shielding it from proteolytic degradation but also sequestering it to the cytosol until an extracellular stimulus triggers its translocation to the Golgi or plasma membrane and subsequent activation.

Differential Mobility and Self-Association of Arc/Arg3.1 in the Cytoplasm and Nucleus of Living Cells.

Arc, also known as Arg3.1, is an activity-dependent immediate-early gene product that plays essential roles in memory consolidation. A pool of Arc is located in the postsynaptic cytoplasm, where it promotes AMPA receptor endocytosis and cytoskeletal remodeling. However, Arc is also found in the nucleus, with a major portion being associated with promyelocytic leukemia nuclear bodies (PML-NBs). Nuclear Arc has been implicated in epigenetic control of gene transcription associated with learning and memory. In this study, we use a battery of fluorescence nanoimaging approaches to characterize the behavior of Arc ectopically expressed in heterologous cells. Our results indicate that in the cytoplasm, Arc exists predominantly as monomers and dimers associated with slowly diffusing particles. In contrast, nuclear Arc is almost exclusively monomeric and displays a higher diffusivity than cytoplasmic Arc. We further show that Arc moves freely and rapidly between PML-NBs and the nucleoplasm and that its movement within PML-NBs is relatively unobstructed.