To study the process of excitability-induced Golgi fragmentation, we developed a live cell microscopy assay for examining Golgi dispersal in response to changes in membrane excitability induced by nicotine. In the brain, α4β2Rs are the main class of nicotinic receptors with high affinity for nicotine (Albuquerque et al., 2009). In our assay, we expressed α4β2R subunits in non-excitable human embryonic kidney (HEK293) cells (Vallejo et al., 2005) to make these cells nicotine-excitable and then examined the effect nicotine treatment had on Golgi morphology.

When α4β2R-expressing HEK293 cells were exposed to 10 µM nicotine, the Golgi dramatically changed from having a compact perinuclear localization to being distributed among discrete smaller elements dispersed throughout the cytoplasm, as observed by localizing exogenously expressed GFP-tagged β-1,4,galactosyltransferase (GalT-GFP), a late-acting Golgi enzyme (Figure 1A, +nicotine). The changes in Golgi morphology were initiated within 1 hr of nicotine exposure, lasted as long as nicotine was present, and were reversible with nicotine removal (see Figure 1—figure supplement 1). Untreated cells imaged over the same time period showed no Golgi fragmentation, indicating fragmentation was induced by nicotine treatment (Figure 1A, -nicotine).

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Golgi dispersal was initiated at nicotine concentrations as low as 300 nM and saturated at ~1 µM nicotine, as shown in cells expressing GalT-GFP and antibody labeling for the Golgi structural marker, GM130 (Figure 1B). The dose dependence for Golgi fragmentation was similar to the dose dependence for nicotine sensitivity of neurons (Vallejo et al., 2005; Govind et al., 2012). Golgi dispersal by nicotine varied somewhat among α4β2R-expressing cells (Figure 1—figure supplement 2B), most likely due to cell-to-cell differences in α4β2R expression, a feature that increases with cell passage number.

To examine whether different Golgi components redistributed equivalently into Golgi puncta during nicotine treatment, we performed triple-labeling experiments using markers for GM130, sialyltransferase (St3-GFP), and/or galactosyltransferase 2 (GalNAc-T2)(Figure 1C). GM130 is a scaffolding protein peripherally associated with the Golgi and is involved in microtubule organization at the Golgi apparatus (Wu and Akhmanova, 2017). St3 and GalNAc-T2 are both transmembrane Golgi enzymes involved in glycoprotein modifications. We found that some Golgi puncta contained all three Golgi markers, whereas others had only GalNAc-T2 or St3-GFP associated with them and no GM130 labeling (Figure 1C, see arrows in zoomed boxes). The numbers of these puncta were significantly increased by nicotine exposure (Figure 1D). No change in Golgi morphology was observed in HEK293 cells not expressing α4β2Rs after overnight nicotine treatment (Figure 1E), indicating α4β2R expression was required for nicotine-dependent Golgi dispersal.

In neurons, the Golgi is primarily localized in the soma, but small, distinct populations of scattered Golgi elements are also present. One population of scattered Golgi elements are called ‘Golgi outposts’ (Horton and Ehlers, 2003). These structures are enriched in GM130 and other Golgi enzymes, and usually localize only to the most proximal part of the apical dendrite or at dendrite branch points (Horton and Ehlers, 2003). Another population of scattered Golgi elements are called ‘Golgi satellites’ (Mikhaylova et al., 2016). These structures are usually smaller than Golgi outposts and are widely dispersed in dendrites. Golgi satellites lack GM130 and contain glycosylation machinery (Mikhaylova et al., 2016). They have been shown to be near ERESs as well as recycling endosomes, and thus could contribute to the micro-secretory system occurring in dendrites (Mikhaylova et al., 2016). Given this prior work, we tested whether nicotine exposure to α4β2R-expressing neurons led to Golgi dispersal, and if so, whether this led Golgi enzymes to redistribute to dendritic Golgi outposts and/or Golgi satellites, increasing the size and/or abundance of these structures.

We used cultured neurons from rat cortex (unless otherwise specified) to study the effect of nicotine exposure on Golgi localization. Within these cultures, 5–10% of neurons are endogenously expressing α4β2Rs, with their expression largely found in inhibitory neurons (Govind et al., 2012). To ensure the neurons being examined in this system were expressing α4β2Rs and responsive to nicotine, we transfected the neurons with α4 and HA-tagged β2 subunits. We further expressed fluorescent protein (FP)-tagged Golgi enzymes or stained with marker-specific antibodies in order to examine the Golgi in these cells.

Neurons expressing α4β2Rs and FP-tagged Golgi markers for St3-GFP, GM130, GalNAc-T2, and/or mannosidase II (Man II) were treated with or without nicotine and the distributions of the Golgi markers were examined (Figure 2A–C). Examining the cell body of these neurons revealed that nicotine treatment caused Golgi structures to fragment into scattered elements (Figure 2Ai–Ci; see boxed areas in images labeled with the different Golgi markers and their corresponding enlarged images). Similar to Golgi dispersal induced by nicotine in non-neuronal cells expressing α4β2Rs, both small and large Golgi puncta appeared over time during nicotine treatment. Large Golgi puncta contained all of the different Golgi markers, whereas small Golgi structures contained mainly Golgi enzymes (i.e., St3, GalNAc-T2, and Man II) without GM130 (Figure 2Ai–Ci).

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We next turned our attention to nicotine’s effect on Golgi-related organelles in the dendrites of these neurons (i.e., Golgi outposts and satellites) (Figure 2A–C; boxed areas in inverted images labeled with [ii] and their corresponding enlarged images). In the absence of nicotine, GM130-labeled Golgi outposts near the apical dendritic zone were present, as were numerous Golgi satellite structures dispersed throughout the dendrite that contained St3, GalNAc-T2, and/or Man II, but lacked GM130 (Figure 2Aii–Cii). Nicotine treatment did not change the low number of GM130-positive Golgi outposts; however, the GM130-negative Golgi satellite structures containing GalNAc-T2, St3, and/or Man II underwent a significant increase in number in response to nicotine treatment (Figure 2Aii–Cii). The increase in Golgi satellites in dendrites during nicotine treatment was also observed in untransfected neurons stained with antibodies to St3 (Figure 2D–E). Additional images are displayed in Figure 2—figure supplements 1 and 2, which tested the specificity of St3 antibody staining in neurons.

To determine whether stimulating neurons by means other than nicotine treatment could also increase Golgi satellite abundance in dendrites, we transfected neurons with St3-Halo to label Golgi satellites and fluorescent Venus to fill the cytoplasm for dendrite identification. The synaptic activity of cortical cultures was then increased by applying either the GABA_A receptor antagonist bicuculline for 18–20 hr or by withdrawing the NMDA receptor antagonist APV for 18–20 hr following 2 days of APV treatment (Figure 2F and G; Thayer et al., 2013). An increase in the number and density of St3-containing puncta per dendrite length was observed for both treatments compared to vehicle control or chronic APV treatment (Figure 2F–H). We thus concluded that changes in neuronal activity alone are sufficient to drive the formation of enzyme-rich Golgi satellites in soma and dendritic processes of neurons.

We next investigated whether newly formed Golgi satellites arising with neuronal excitation participate in secretory trafficking of newly synthesized cargo. To address this question, we assayed the trafficking itinerary of secretory cargo in nicotine-treated cells using the retention using selective hooks (RUSH) system (Boncompain et al., 2012). The RUSH system enables controlled release of newly synthesized ER proteins from the ER into the secretory pathway by breaking a bond between an ER-resident protein and a cargo protein through biotin addition. As regulatable cargo in this system, we used fluorescent mApple-tagged GPI bound to the ER-resident protein KDEL-streptavidin (i.e., RUSH-mApple-GPI).

We introduced RUSH-mApple-GPI into non-neuronal HEK293 cells co-expressing α4β2Rs and St3-GFP and treated these cells with nicotine to fragment the Golgi. RUSH-mApple-GPI was then released from the ER through biotin addition and followed over time (Figure 3A). RUSH-mApple-GPI could be seen quickly passing through St3-GFP-labeled Golgi fragments before arriving at the plasma membrane in time lapse sequences. The kinetics of this trafficking resembled that for RUSH-mApple-GPI in cells not treated with nicotine (Figure 3—figure supplement 1). Thus, dispersed Golgi elements in nicotine-treated cells behave as bona fide units of the secretory pathway.

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Because of technical difficulties in applying the RUSH system to neurons (i.e., there was some ‘leakage’ from ER prior to release of the ER-bound cargo), we used a different system to release bound fluorescent cargo from ER in order to test the role of Golgi satellites in secretory trafficking in neurons. The system used a modified version of a regulatable fluorescent secretory cargo called ESCargo (Casler et al., 2020). ESCargo forms homo-aggregates in the ER lumen when expressed but can be released into the secretory pathway upon addition of a membrane-permeable ligand that dissociates the aggregates (Casler et al., 2020). In neurons expressing ESCargo and Man-GFP, ER aggregates in the soma, dendrites, and axons could be seen prior to release in nicotine-treated cells (Figure 3B). Upon addition of the membrane-permeable ligand, the aggregates of ESCargo disassembled, resulting in soluble ESCargo rapidly filling the ER lumen due to its increased ability to diffuse (Figure 3C). Thereafter, the cargo moved out of the ER into non-motile Man II-containing Golgi puncta distributed along the dendrites. The intensity of ESCargo in many Golgi puncta could be seen to subsequently diminish, consistent with the cargo’s downstream trafficking and delivery to the plasma membrane. These data provide evidence that newly synthesized proteins traffic from the ER through dispersed Golgi satellites en route to the plasma membrane in stimulated neurons.

We next focused on whether dispersed Golgi satellites in dendrites arising under nicotine treatment were localized near ER export sites (ERESs) and recycling endosomes, as reported in nonstimulated neurons (Mikhaylova et al., 2016). This Golgi satellite positioning would minimize the extent that transport vesicles would need to travel to transit cargo from ER to plasma membrane. To identify ERESs in relation to Golgi, nicotine-treated α4β2R-expressing neuronal cultures were co-transfected with St3-GFP to label Golgi membranes, and with the COPII component mCh-Sec23 to label ERESs. To identify endosomes in relation to Golgi, α4β2R-expressing neuronal cultures transfected with St3-GFP were stained with an endosome-specific antibody, anti-EEA1.

St3-GFP-labeled Golgi puncta were often found to reside next to mCh-Sec23 structures in dendrites of nicotine-treated neurons (Figure 4A, see arrows). A line scan analysis across the boxed structure in Figure 4A revealed that the peak intensity of St3-labeled Golgi puncta was ~300 nm from the peak intensity of ERESs marked by Sec23 (Figure 4B). Dendritic Golgi satellites in nicotine-treated neurons were also adjacent to early endosomes labeled with the early endosomal marker EEA1 (Figure 4C, see arrows), with an individual Golgi satellite and endosome within 300 nm of each other’s center (Figure 4D). The number of Golgi satellites closely associated with endosomes was increased by neuronal stimulation (see Figure 8E,F). Additional experiments were performed to examine whether Golgi satellites and endosomes are separate organelles. We found little overlap between St3 staining and other endosomal markers (~25%) while much higher overlap was observed between St3 and other Golgi markers (Figure 4—figure supplements 1 and 2).

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In triple-labeling experiments, in which neurons were transfected with St3-GFP and mCh-Sec23 and then labeled with antibodies to EEA1, we found that many Golgi satellites resided adjacent to both Sec23-labeled and EEA1-labeled puncta (Figure 4E and F). Thus, dendritic Golgi satellites often situate themselves next to sites of ER export and early endosomes, similar to what has been reported for dendritic Golgi satellites in untreated neurons (Mikhaylova et al., 2016).

When microtubules are depolymerized by nocodazole treatment, Golgi enzymes are relocated to ERESs that are still functional for secretory transport (Cole et al., 1996). Because we found that Golgi fragmentation induced by nicotine treatment also leads to Golgi enzymes relocating to transport competent structures near ERESs, we tested whether other features of Golgi fragmentation seen during nicotine treatment were similar to those observed during nocodazole treatment. Golgi fragments induced by either nicotine or nocodazole treatment in non-neuronal cells expressing α4β2Rs had similar size ranges and contents, with some fragments containing both St3 and GM130 (usually larger ones) and others having only St3 (often small ones) (Figure 5A and B; Figure 5—figure supplement 1). In addition, Golgi fragmentation induced by either treatment was reversible upon drug removal (Figure 1—figure supplement 1). Despite these similarities, microtubules (visualized using the microtubule binding protein ensconsin) remained intact when either α4β2R-expressing HEK293 cells or neurons were treated with nicotine, unlike that observed when microtubules were depolymerized with nocodazole (Figure 5C–E). This suggested that Golgi fragmentation in nicotine-treated cells, while sharing similarities to Golgi fragmentation in nocodazole-treated cells, was not due to microtubules being disrupted.

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Because Golgi satellites/fragments in nicotine-treated neurons or non-neuronal cells expressing α4β2Rs contained medial/late-acting Golgi enzymes, including Man II, GalNac-T2 and St3, we asked whether these enzymes could modify glycoprotein receptors, in particular α4β2Rs, presumably by passage through Golgi satellites/fragments. To address this possibility, we used gel electrophoresis and immunoblotting to assay molecular weight changes to the α4 subunit of α4β2Rs residing on the plasma membrane of non-neuronal, α4β2R-expressing cells upon treatment with or without nicotine. The surface pool of α4β2Rs was analyzed in these experiments using surface biotinylation followed by streptavidin pull down and immunoblotting with antibodies to the α4 subunit of α4β2R.

As can be seen in Figure 6A, after 17 hr of nicotine treatment, the majority of α4 subunits associated with surface-labeled α4β2Rs had shifted to a higher molecular weight form. The differences in α4 subunit band intensities (-Nic vs. +Nic) were likely due to nicotine treatment changing the conformation and/or expression levels of α4β2Rs, as previously shown (Govind et al., 2012). The α4 subunits could be cleaved to lower molecular, nonglycosylated forms by the glycan-specific cleavage reagents Endo H or PNGase F (Figure 6A). No such shift in molecular weight of surface-labeled α4β2Rs was seen in untreated cells. Incubating cells with swainsonine, a drug that prevents N-glycans on proteins from being converted from high mannose to complex forms, prevented the molecular weight shift in α4 subunits during nicotine treatment (Figure 6B). Further analysis of the sugar modifications on α4 subunits of α4β2Rs in nicotine-treated cells revealed that they were sensitive to neuraminidase (Figure 6C), an enzyme that cleaves off sialic acid from glycans. In addition, α4 subunits could be precipitated by the sialic acid binding lectin SNA (Figure 6D), confirming the glycans on α4 subunits contained sialic acid. The increase in the molecular weight of α4 subunits from sialic acid addition was blocked by ammonium chloride treatment, which increases pH within intracellular organelles (Figure 6E), raising the possibility that low pH-requiring Golgi sialotransferases St3 and/or St6 were involved in the sialic acid addition (Rivinoja et al., 2009).

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To test whether the glycan conversion to complex forms correlated with nicotine-induced Golgi fragmentation, we examined conversion of the surface α4 subunits as a function of time during nicotine treatment (Figure 6F). Cells expressing α4β2R were surface biotinylated at different times after nicotine treatment and biotinylated α4 subunits were then isolated using streptavidin and analyzed by immunoblotting. Within 2 hr of nicotine treatment, α4 subunits associated with α4β2Rs at the cell surface had begun to display complex glycan forms (Figure 6F), with the kinetics of this process (Figure 6G) resembling the timing of Golgi dispersal in these cells (see Figure 1A). Collectively, these data indicate that N-glycans on surface α4β2Rs change from being immature types to mature types containing sialic acid in nicotine-treated cells, with conversion likely occurring as α4β2Rs traffic through dispersed Golgi elements.

The occurrence of a significant shift in the pool of modified sugars on α4 subunits of α4β2Rs within a few hours of nicotine treatment led us to ask whether activity-induced formation of Golgi satellites/fragments in neurons might be responsible for such glycan remodeling through endocytosis and delivery to Golgi satellites. To explore this possibility, neurons co-expressing HA-tagged α4β2Rs and St3-GFP were allowed to bind and take up fluorescently labeled antibodies to HA in the presence or absence of nicotine. Surface-bound Abs were then removed by acid washing so that only endocytosed α4β2Rs were imaged. After this treatment, significant overlap between endocytosed HA antibody-labeled α4β2Rs and small Golgi puncta in somata and dendrites could be seen, with seemingly greater internalization and Golgi delivery under nicotine treatment (Figure 7A). In dendrites, where overlap was easier to quantify, the number of Golgi fragments containing HA antibodies to α4β2Rs increased threefold after nicotine treatment (Figure 7B and C). We also found that endocytosed α4β2Rs co-localized with EEA1-labeled endosomes and that this co-localization increased with nicotine treatment (Figure 7—figure supplement 1). These results demonstrate that endocytosed α4β2Rs can be delivered to Golgi satellites and that nicotine treatment increases this process.

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To address whether α4β2Rs that are endocytosed and delivered to Golgi fragments during nicotine treatment undergo processing by Golgi enzymes, we used α4β2R-expressing HEK293 cells to assay for changes in α4 subunit glycosylation. In these experiments, cells were surface biotinylated at time 0 and biotinylated α4 subunits were then pulled down by streptavidin and analyzed by immunoblotting at different time points in the presence or absence of nicotine. We found that within 2 hr after surface biotinylation and continuing thereafter, the glycans on α4 subunits in nicotine-treated cells had begun being converted to higher molecular weight, complex forms (Figure 7D), consistent with their having been delivered to Golgi fragments from the cell surface. No such conversion was seen in untreated cells.

In a different set of experiments, we biotinylated the surface of HEK293 cells expressing α4β2R at time 0 and then measured the pool of biotinylated α4 subunits that was resistant to biotin cleavage (by a membrane impermeable cleavage reagent) over time. As shown in Figure 7E, the ‘protected’ (i.e., internalized) pool of biotinylated α4 (i.e., internalized) in nicotine-treated cells increased over time and by 2 hr consisted of α4 subunits with complex-type glycans of increased molecular weight. No such shift was seen in untreated cells. Therefore, nicotine appears to stimulate surface α4β2Rs to traffic to Golgi satellites in HEK293 cells, where their N-glycans are modified to higher molecular weight, complex forms.

We next explored whether the observed nicotine-induced modifications of N-glycans on α4 subunits impacted the overall functioning of α4β2Rs. To address this, we measured nicotine-induced increases in currents in HEK293 cells expressing α4β2Rs and looked to see if they were reduced when swainsonine was added to block glycans on α4β2R from being processed to complex forms. The expressing cells showed significantly enhanced ACh-evoked currents after nicotine treatment (Figure 7F and G, control). Notably, this enhancement was markedly reduced by swainsonine treatment without impacting the low level currents under no nicotine treatment (Figure 7F and G, swainsonine). Other experiments showed that overall levels of surface α4β2Rs did not change during swainsonine treatment (Figure 7—figure supplement 2). These findings thus demonstrate that N-glycan repatterning during nicotine treatment, specifically conversion to complex-type forms containing sialic acid, is an underlying factor in facilitating increased α4β2R activity (i.e., functional upregulation) in nicotine-treated, α4β2R-expressing HEK293 cells.

The above results suggested that Golgi satellites/fragments induced by neuronal excitability can alter the surface glycoproteome through changes in sugar modifications on glycoproteins. To study the overall extent of these changes, we compared N-glycan patterning of glycoproteins on neuronal surfaces before and after increased neuronal activity using two lectins: concanavalin A (Con-A), which recognizes core-glycosylated but not complex-glycosylated glycoproteins, and WGA, which labels glycoproteins with complex-type glycan chains terminated with sialic acid. Prior work using these lectins has revealed that the majority of glycoproteins on the surface of neurons at steady state are significantly core-glycosylated, without complex branched chain elements that include sialic acid (Hanus et al., 2016).

Using WGA to examine glycan patterns on neuronal surfaces, we found that neuronal stimulation by either nicotine or bicuculline treatment led to increased WGA labeling (Figure 8A–C). In live cell imaging experiments, we further found that surface-bound WGA was rapidly internalized into St3-labeled structures upon nicotine treatment of neurons containing α4β2Rs (Figure 8D). Con-A labeling in these same cells, by contrast, did not appear to be significantly endocytosed as indicated by its lack of overlap with St3 structures (Figure 8D), although its staining was clustered, consistent with previous observations of Con-A staining of spines (Hanus et al., 2016). The St3-labeled structures increased twofold in abundance in neurons activated by bicuculline and were closely juxtapositioned to EEA1-labeled endosomes (Figure 8E and F). This efficient endocytosis of WGA in activated neurons could help explain WGA’s widespread use as marker of active neuronal circuits (Broadwell and Balin, 1985).

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Examining dendrites in neurons expressing St3-Halo and HA-tagged α4β2R, in which HA antibodies and WGA were added to the medium, we found that within a short time, WGA and HA antibody-labeled α4β2Rs began appearing in St3-labeled Golgi puncta, with most of the surface pools of WGA and α4β2Rs delivered into the puncta by 4 hr of nicotine treatment (Figure 8G). We also examined WGA endocytosis and delivery to Golgi satellites during bicuculline treatment, comparing it to either untreated cells or nicotine-treated cells. Both nicotine and bicuculline treatment led to increased levels of WGA labeling and delivery to St3-labeled Golgi satellites compared to untreated cells (Figure 8H,I). Taken together, the above results suggest that the extent to which WGA binds to and is taken up by neurons is increased during neuronal activation, presumably because Golgi satellites are now generating glycoproteins with complex-type glycans, which would change the glycan landscape at the cell surface to one enriched in sialic acid.

Antibodies used were either purchased from commercial suppliers or were generous gifts from various researchers. Antibodies against the following antigens were used: GM130 (monoclonal; BD Transduction Laboratories, San Jose, CA), GM130 (polyclonal; Sigma, St. Louis, MO), HA epitope (HA.11, monoclonal, Biolegend, San Diego, CA), St3Gal3 (polyclonal; Abcam, Boston, MA), EEA1 (monoclonal; BD Transduction Laboratories, San Jose, CA), and α4 subunit-specific polyclonal Ab 6964 (gift from Dr S Rogers, University of Utah, Salt Lake City, UT). The fluorescent secondary antibodies and ligands that were used include Alexa Fluor 488‐conjugated goat anti‐rabbit IgG, Alexa Fluor 568‐conjugated goat anti‐mouse IgG, Alexa Fluor 568‐conjugated goat anti‐rabbit IgG, Alexa Fluor 647‐conjugated goat anti‐mouse IgG, Alexa Fluor 647‐conjugated goat anti‐rabbit IgG, Wheat Germ Agglutinin Alexa Fluor 555 and 647, and Concanavilin A Alexa Fluor 647 (Thermo Fisher Scientific, Waltham, MA) and Janelia Fluor 549 Halo ligand (Promega, Madison, WI). Rat α4 and β2 used for generating the stable cell line were provided by Dr Jim Boulter (University of California, Los Angeles, CA). The HA epitope, YPYDVPDYA, and a stop codon were inserted after the last codon of the 3′-translated region of the subunit DNA of the β2 using the extension overlap method as described in Vallejo et al., 2005. Other cDNAs generously provided include: Full-length St3-GFP from Dr Sakari Kellokumpu (University of Oulu, Oulu, Finland) (Rivinoja et al., 2009), GalNAc-T2-mCherry from Dr Dibbyendu Bhattacharya (Tata Memorial Center, Mumbai, India), GalT-3xGFP (amino acid 1–60, Cole et al., 1996), and mCherry-Sec23 from Dr Jennifer Lippincott-Schwartz (HHMI, Janelia Research Campus), GM130-GFP from Dr Christine Suetterlin (University of California, Irvine, CA), pDsRed-ER (Takara Bio, San Jose, CA), RUSH-GPI-mApple, Rush-GPI-Halo, and Ensconsin-mCherry (Addgene, Watertown, MA). For imaging ER bulk flow trafficking to Golgi, a bicistronic plasmid encoding a modified ESCargo (Casler et al., 2020) and Man II-GFP was generated in the laboratory of Benjamin Glick (University of Chicago, Chicago, IL). The Mus musculus Man IIA1 gene was obtained from BioBasic (Toronto, Canada), PCR-amplified, and inserted into the ESCargo plasmid using in-fusion cloning (Takara Bio, San Jose, CA). St3-Halo was generated by replacing the GFP in St3-GFP with Halo tag (GenScript USA Inc, Piscataway, NJ). The following reagents were used: (-)-Nicotine, Nocodazole, Bicuculline (Sigma-Aldrich, St. Louis, MO), DL-APV (Abcam, Boston, MA), EZ link sulfo NHS SS Biotin and ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific, Waltham, MA).

The human embryonic kidney (HEK293T) cell line stably expressing the large T antigen (tSA201 cells) was from Dr J Kyle (University of Chicago, Chicago, IL) and served as our ‘non-neuronal’ cell line. This cell line is not in the list of Database of Cross-Contaminated or Misidentified Cell Lines. Using this parent HEK293T cells, a stable cell line expressing rat α4β2 nAChRs was generated in our lab, expressing untagged α4 and C-terminal, HA epitope-tagged β2 subunits (Vallejo et al., 2005). Both the parent HEK and stable α4β2R HEK cell lines were maintained in DMEM (Gibco, Life Technologies) with 10% calf serum (Hyclone, GE Healthcare Life Sciences, Logan, UT) at 37°C in the presence of 5% CO₂. DMEM was supplemented with Hygromycin (Thermo Fisher Scientific, Waltham, MA) at 0.4 mg/ml for maintaining selection of α4β2R HEK cells. Hoechst staining and immunofluorescent detection were performed periodically to test for mycoplasma contamination. Fresh batches of cells were thawed and maintained only up to 2 months.

HEK cells were grown on 22 mm × 22 mm coverslips (Thermo Fisher Scientific, Waltham, MA) or 35 mm imaging plates (MatTek, Ashland, MA) coated with poly-D-lysine. Stable cells were plated in media without hygromycin for experiments; 75% confluent cultures were transfected with 0.5 µg DNA of indicated constructs with Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, Waltham, MA). After 24 hr of transfection, cells were treated with 10 µM nicotine for 17 hr.

Primary cultures of rat cortical neurons were prepared as described (Govind et al., 2012) using Neurobasal Media (NBM), 2% (v/v) B27, and 2 mM L-glutamine (all from Thermo Fisher Scientific, Waltham, MA). Dissociated cortical neurons from E18 Sprague Dawley rat pups were plated on slips or plates coated with poly-D-lysine (Sigma, St. Louis, MO). For live imaging, neurons were plated in 35 mm glass bottom dishes (MatTek, Ashland, MA). Cells were plated at a density of 0.25 × 10⁶ cells/ml on 35 mm dishes or per well in a six-well plate. Neuronal cultures were transfected at DIV 10 with the Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, Waltham, MA) according to manufacturer’s recommendations. Neurons were transfected with cDNAs of α4, β2_HA and various Golgi markers, ER/ERES markers or RUSH constructs; 0.5 µg of each DNA up to a total of 2 µg were used per 35 mm imaging dish or per well of a six-well plate. Twenty-four hours after transfection, neurons were treated with 1 μM nicotine for 17 hr. Bicuculline and DL-APV treatments were performed as described previously (Thayer et al., 2013). Briefly, 1 day prior to transfection, neurons were treated with either 200 µM DL-APV or vehicle. After 2 days of treatment, media-containing drug was replaced with conditioned media lacking drug in one group of neurons (‘APV withdrawal’), while the second group of neurons that had been treated were left unchanged (‘chronic APV’). One day later, all three groups were fixed and stained.

Cultured neurons grown on coverslips were fixed with 4% paraformaldehyde and 4% sucrose for 10 min, then incubated with blocking solution (2% glycine, 1% BSA, 0.2% gelatin, 0.5 M NH₄Cl, PBS) for 1 hr prior to primary and secondary antibody incubations in blocking solution at room temperature (RT). Primary antibody incubations were for 1 hr and secondary antibodies for 45 min unless otherwise mentioned. Surface expressed α4β2 receptors were imaged by live-labeling the cells with mouse anti-HA antibody (1:500) for 40 min prior to fixation. Cells were permeabilized with 0.1% Triton X-100 followed by fluorescent secondary antibody.

For internalized staining (antibody-feeding assay), primary antibody was added into the culture medium and incubated at 37°C for 40 min, then cells were washed twice with acid wash buffer (0.5 M NaCl, 0.5% acetic acid, pH 2) for 15 s each, and fixed. Cells were then incubated with blocking solution and secondary antibody. For permeabilized staining, after fixation, the cells were permeabilized in 0.1% Triton/PBS for 5–10 min, blocked and stained with primary and secondary antibodies. The stained coverslips were mounted to glass slides with ProLong Gold (Life Technologies) mounting media and left in dark to harden overnight.

Fluorescence images were acquired using a Leica SP5 Tandem Scanner Spectral 2-Photon scanning confocal microscope (Leica Microsystems), or Marianas Yokogawa type spinning disk confocal microscope with back-thinned EMCCD camera. Images were processed and analyzed using ImageJ/Fiji (US National Institutes of Health).

α4β2R cells were grown on 6 cm plates. Cells were treated with or without 10 μM nicotine for 17 hr and washed thrice with PBS (containing 1 mM MgCl₂ and 1 mM CaCl₂), and incubated with 0.5 mg/mL Sulfo-NHS-SS-Biotin at RT for 30 min. Cells were washed thrice with 10 mM Tris HCl, pH 7.4 containing 1 mM CaCl₂, and 1 mM MgCl₂. Cells were lysed immediately to isolate biotinylated surface proteins/receptors using lysis buffer (150 mm NaCl, 5 mm EDTA, 50 mm Tris, pH 7.4, 0.02% NaN3, plus 1% Triton X-100) containing protease inhibitors (2 mm phenylmethylsulfonyl fluoride, 2 mm N-ethylmaleimide, and chymostatin, pepstatin, leupeptin, and tosyllysine chloromethyl ketone at 10 μg/ml). To isolate internalized biotinylated proteins, cells were maintained in DMEM at 37°C following surface biotinylation for the indicated time intervals. At each time points, Sulfo-NHS-SS-Biotin on the cell surface was cleaved with glutathione. Cells were then harvested and lysed; biotinylated proteins were pulled down using streptavidin agarose beads (MilliporeSigma, Burlington, MA) and were analyzed on Western blot.

Deglycosylation experiments were performed on biotinylated surface receptors expressed in α4β2R cells. Surface biotinylated proteins immobilized on streptavidin agarose beads were denatured in Denaturing Buffer (New England Biolabs) at 100°C for 10 min in accordance with the manufacturer’s protocol. For peptide-N-glycosidase F (PNGase F) treatment, 10× G7 reaction buffer, 10% (v/v) Nonidet P-40, and 500 units of PNGase F were added. For endoglycosidase H (Endo H) treatment, 10× G5 reaction buffer and 1000 units of Endo H were added. For Neuraminidase treatment 10× GlycoBuffer and 50 units of Neuraminidase enzyme was added. The samples were incubated at 37°C either for 2 hr, then heated for 3 min at 80°C in 2× sodium dodecyl sulfate (SDS) loading buffer.

For the SNA lectin pull down experiments, lysates from α4β2R cells were incubated with agarose-linked Sambucus Nigra lectin, SNA (100 μl; Vector Laboratories) with rotation at 4°C overnight. The samples were washed three times with lysis buffer containing 0.1% (w/v) Triton X-100 in Tris-buffered saline (pH 7.4), and then heated for 5 min at 100°C in 2× SDS loading buffer (75 μl). The proteins were separated by electrophoresis in a 7.5% SDS-polyacrylamide gel (SDS-PAGE), transferred to a polyvinylidene difluoride membrane, then incubated with the primary (polyclonal anti-α4 antibody) and HRP-conjugated anti-rabbit secondary antibodies. The signal was detected using enhanced chemiluminescence with Amersham hyperfilm (Sigma, St. Louis, MO). Inhibition of α-Man II was carried out by treating α4β2R cells with 4 μg/ml swainsonine for 17 hr in the presence or absence of 10 μM nicotine. Inhibition of the activity of all Golgi associated glycosyl transferases was carried out by raising the intracellular pH. Cells were incubated with 10 mM ammonium chloride (NH₄Cl). Surface-expressed α4 subunits were analyzed by biotinylation/streptavidin agarose pull down and Western blot analysis.

St3 punctal densities were quantified using maximum z projections of background-subtracted images. Thresholded punctal size and density was analyzed using the Analyze Particle function in Fiji/ImageJ. Analysis of the co-localization or overlap between two fluorescent punctal signals was carried out by background subtracting and thresholding image fields so that only puncta that were twofold greater than background were selected. Co-localizing puncta were evaluated using the Analyze Particle function in Fiji/ImageJ. Results are expressed as mean ± SEM of n samples unless stated otherwise. Experiments were conducted from a minimum of two independent culture preparations, with 5–10 neurons per experimental group. Statistical comparisons were made using two-tailed Student’s t-tests or ANOVA/Tukey’s post hoc analysis as indicated. Statistical graphs were generated with StatPlus software.

HEK cells stably expressing α4β2 receptors maintained as described above were plated at low density on glass coverslips before treating for 15–18 hr with media-containing nicotine or other drugs. Cells were voltage-clamped in whole-cell configuration at a holding potential of −70 mV using an Axopatch 200B amplifier running pClamp 10 (Molecular Devices; Sunnyvale, CA). Currents were elicited from cells lifted from the coverslip by the fast application of 1 mM ACh using a piezo-ceramic bimorph system with a solution exchange time of ~1 ms. External solution consisted of (in mM): 150 NaCl, 2.8 KCl, 1.8 CaCl₂, 1.0 MgCl₂, 10 glucose, and 10 HEPES, adjusted to pH 7.3. Internal pipette solution was (in mM): 110 CsF, 30 CsCl, 4 NaCl, 0.5 CaCl₂, 10 HEPES, and 5 EGTA, adjusted to pH 7.3. Peak amplitudes and time courses of desensitization were determined by post hoc analysis using Clampfit 10. Statistical differences were tested for using one-way ANOVAs with Tukey’s multiple comparison test in GraphPad (La Jolla, CA).

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

This work was financially supported by NIH RO1 DA035430, DA044760, and DA043361 (WNG) R01 GM104010 (BSG), T32 GM007183 (FV), and Peter F McManus Foundation (WNG). We thank Vytas Bindokas (University of Chicago) and Louie Kerr (Marine Biological Laboratory), Abhishek Kumar, Panagiotis Chandris, and Hari Shroff (NIH/NIBIB) for technical support on microscopy; Luke Lavis (Janelia research campus) for Halo dyes and Dibbyendu Bhattacharya (Tata Memorial Center), Sakari Kellokumpu (University of Oulu), Jim Boulter (University of California), Steven Standley (Western University of Health Sciences) for cDNA constructs used in this study. We thank U of C student, Briana Turner for assistance. The authors declare that they have no competing interests. We especially thank Dr Karl Matlin for his thorough reading of the manuscript and many insightful comments and suggestions.

All animal procedures were approved by the University of Chicago Institutional Animal Care and Use Committee (protocol #72016) and are in accordance with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association. Strict adherence to AVMA guidelines was followed to prevent pain and suffering of animals.

© 2021, Govind et al.

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