Journal of Cell Science Accepted manuscript · 6/2/2014 · mEos2 isa bright and photostable photoconvertible fluorescent 143protein that switches from green to red by UV irradiation

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RESEARCH ARTICLE

Transport along the dendritic endoplasmic reticulum mediates thetrafficking of GABAB receptors

José I. Valenzuela1,2, Matı́as Jaureguiberry-Bravo1,2, Daniela A. Salas1,2, Omar A. Ramı́rez1,2,3,Vı́ctor H. Cornejo1,2, Hsiangmin E. Lu4,5, Thomas A. Blanpied4,6 and Andrés Couve1,2,*

ABSTRACT

In neurons, secretory organelles within the cell body are

complemented by the dendritic endoplasmic reticulum (ER) and

Golgi outposts (GOPs), whose role in neurotransmitter receptor

trafficking is poorly understood. c-aminobutyric acid (GABA) type B

metabotropic receptors (GABABRs) regulate the efficacy of synaptic

transmission throughout the brain. Their plasmamembrane availability

is controlled by mechanisms involving an ER retention motif and

assembly-dependent ER export. Thus, they constitute an ideal

molecular model to study ER trafficking, but the extent to which the

dendritic ER participates in GABABR biosynthesis has not been

thoroughly explored. Here, we show that GABAB1 localizes

preferentially to the ER in dendrites and moves long distances within

this compartment. Not only diffusion but also microtubule and dynein-

dependent mechanisms control dendritic ER transport. GABABRs

insert throughout the somatodendritic plasma membrane but dendritic

post-ER carriers containing GABABRs do not fuse selectively with

GOPs. This study furthers our understanding of the spatial selectivity

of neurotransmitter receptors for dendritic organelles.

KEY WORDS: GABA, Dendrite, Endoplasmic reticulum, Golgi

outposts, Neuron, Trafficking

INTRODUCTIONThe control of the availability of neurotransmitter receptors at

appropriate subdomains of the plasma membrane is crucial for

neuronal function and synaptic plasticity. Availability is governed

by mechanisms of intracellular trafficking that deliver newly

synthesized receptors to the plasma membrane and remove

them for storage, recycling or degradation. These mechanisms

determine the activity-dependent strengthening or weakening of

synapses (Ehlers, 2000; Clem and Barth, 2006).

Two major secretory pathways operate in dendrites (Horton and

Ehlers, 2003). In a canonical pathway, proteins are synthesized and

post-translationally modified in the somatic ER, transported to a

perinuclear Golgi for additional modifications and targeted distally

in long-haul post-Golgi vesicles. The distribution of the continuous

ER throughout dendrites (Broadwell and Cataldo, 1983; Spacek

and Harris, 1997), and the localization of functional Golgi

outposts (GOPs), which are defined as discrete Golgi structures

discontinuous with the somatic organelle (Horton and Ehlers,

2003; Horton and Ehlers, 2004; Ye et al., 2007), suggest that local

secretory pathways also exist in neurons. Thus, proteins can be

synthesized and transported by diffusion along the somatodendritic

ER and exported through specialized ER exit sites (ERES) in

dendrites for local Golgi trafficking and delivery to the plasma

membrane (Aridor et al., 2004; Cui-Wang et al., 2012; Chen et al.,

2013). Importantly, glutamate receptors (AMPARs and NMDARs)

are differentially sorted to the perinuclear Golgi or GOPs (Jeyifous

et al., 2009). However, the role of dendritic organelles in the

trafficking of neurotransmitter receptors is not fully understood.

c-aminobutyric acid (GABA) is the major inhibitoryneurotransmitter in the central nervous system, and GABA type

B metabotropic receptors (GABABRs) mediate the slow and

prolonged phase of inhibitory postsynaptic potentials (Bettler

et al., 2004). The receptors are found in both somatodendritic and

axonal compartments, and are heteromers composed of two

subunits, GABAB1 and GABAB2, encoded by the Gabbr1 and

Gabbr2 genes, respectively. GABAB1 contains an arginine-X-

arginine (RXR)-type ER retention motif in the carboxyl-terminus

that is inactivated by GABAB2, resulting in the assembly of a

heterodimer and delivery to the plasma membrane (Margeta-

Mitrovic et al., 2000). In spite of their key function in regulating

synaptic transmission, the mechanisms that control their

availability remain for the most part unexplored, especially the

mechanisms that regulate their exocytic route in dendrites.

Conventional vesicular mechanisms regulate multiple stages of

GABABR trafficking (Pooler et al., 2009; Vargas et al., 2008;

Grampp et al., 2007; Biermann et al., 2010). However, segregated

GABAB1 and GABAB2 subunits are detected by light and electron

microscopy in hippocampal and visual cortex neurons (Gonchar

et al., 2001; Ramı́rez et al., 2009; Biermann et al., 2010). Because

GABAB1 is exported from the ER only upon binding to GABAB2 this

evidence suggests that they utilize an alternative ER transport

pathway into dendrites during the early stages of their biosynthetic

itinerary. However, the roles of dendritic organelles in the trafficking

of GABABRs are not well delineated. Here, we combine fixed and

live-cell confocal and super-resolution imaging of intracellular

GABAB subunits with the use of organelle reporters and trafficking

blockers to explore the contribution of the dendritic ER and GOPs to

the exocytic pathway of GABABRs in cultured hippocampal neurons.

RESULTSGABAB1 colocalizes with the ER in dendritesLocal dendritic trafficking requires newly synthesized membrane

proteins to dwell in, assemble in and exit from the

1Program of Physiology and Biophysics, ICBM, Faculty of Medicine, Universidad deChile, Santiago CP8380453, Chile. 2Biomedical Neuroscience Institute, BNI, Facultyof Medicine, Universidad de Chile, Santiago CP8380453, Chile. 3Program of Anatomyand Development, ICBM, Faculty of Medicine, Universidad de Chile, SantiagoCP8380453, Chile. 4Department of Physiology, University of Maryland School ofMedicine, Baltimore, MD 21201, USA. 5Program in Molecular Medicine, University ofMaryland School of Medicine, Baltimore, MD 21201, USA. 6Program in Neuroscience,University of Maryland School of Medicine, Baltimore, MD 21201, USA.

*Author for correspondence ([email protected])

Received 10 February 2014; Accepted 16 May 2014

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 3382–3395 doi:10.1242/jcs.151092

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somatodendritic ER. Because GABABR assembly is requiredfor the export of the receptor from the ER, the existence of

segregated subunits in dendrites is a good reporter of residence inthe ER (Ramı́rez et al., 2009). To investigate this, we comparedthe colocalization of transfected GABAB1 and GABAB2, the twosubunits of the GABABR, to that of recombinant GABAAa4 andGABAAb3 (encoded by Gabra4 and Gabrb3, respectively),two subunits of the non-related ionotropic GABAA receptor(GABAAR). Colocalization was significantly lower for GABABthan for GABAA subunits (Fig. 1A,B). These observations areconsistent with previous reports (Gonchar et al., 2001; Ramı́rezet al., 2009) and indicate that segregated GABAB subunits are

abundant in dendrites, possibly revealing dendritic ER transportand assembly.

To determine whether segregated GABAB1 subunits are enriched

in the dendritic ER, we carried out quantitative colocalization withmarkers of secretory organelles. GABAB1, overexpressed eitheralone or in combination with GABAB2, colocalized preferentiallywith the ER and, to a lesser degree, with the ER-to-Golgi

intermediate compartment (ERGIC) and GOPs (Fig. 1C–E). Inagreement with the role of the GABAB2 subunit in controllingreceptor export from the ER, the colocalization of GABAB1 with

the ER decreased significantly in the presence of GABAB2(GABAB1, 5462%; GABAB1+GABAB2, 4563%; P50.025,Student’s t-test; 6s.e.m.) (Fig. 1D,E). The colocalization ofGABAB1 with the ER was comparable in regions of low andhigh network complexity, such as dendritic shafts and branchingpoints, respectively (Cui-Wang et al., 2012) (shaft, 5266%;branching points, 5064%; P50.758, Student’s t-test) (Fig. 1F).To rule out the possibility of artificial colocalization due tooverexpression, we analyzed neurons derived from GABAB1–

EGFP transgenic mice, which express GABAB1–EGFP atendogenous levels (Casanova et al., 2009). Colocalizationbetween GABAB1 and the ER in this model was comparable to

that observed with overexpressed subunits (Fig. 1G,H). Moreimportantly, blocking ER export in transgenic neurons with Sar1-H79G–HA, a dominant negative form of Sar1 that is locked in a

GTP-bound state and blocks export from the ER (Ward et al.,2001), increased the colocalization of GABAB1 with the ER(control, 3963%; Sar1-H79G–HA, 5064%; P50.04, Student’s t-test) (Fig. 1G,H), indicating that GABAB1 accumulates in the

dendritic ER when export is prevented. These results are consistentwith GABAB1 ER retention and export (Margeta-Mitrovic et al.,2000).

Fig. 1. GABAB1 is enriched in and traffics through the ER in dendrites. (A) Rat hippocampal neurons were co-transfected with GABAB1–Myc (GB1–MYC,green) and GABAB2–FLAG (GB2–FLAG, red) or with the GABAA subunit fusion proteins

pH/BBSb3 (GAb3, green) and RFP-BBSa4 (GAa4, red). Proximal dendritesare shown. Scale bar: 10 mm. (B) Left plot, percentage colocalization of GABAB2 with GABAB1 and of RFP-BBSa4 with pH/BBSb3. Right plot, percentagecolocalization of GABAB1 with GABAB2 or of

pH/BBSb3 with RFP-BBSa4. n54–8 neurons. (C) Neurons were transfected with GABAB1–Myc (green) and labeled withantibodies against Myc and KDEL (ER marker), ERGIC-53 (ERGIC marker) or GM130 (cis-Golgi marker) (red). The top panels are positive controls withGABAB1–Myc and two different secondary antibodies. (D) Same as in C for neurons co-transfected with GABAB1–Myc (red) and GABAB2 (purple).(E) Percentage of colocalization of GABAB1–Myc with KDEL, ERGIC-53 and GM130 (black bars) or of GABAB1–Myc with the same organelle markers in thepresence of GABAB2 (white bars). GABAB1–Myc was used as positive control (hatched bar). n55–7 neurons. (F) Percentage colocalization of GABAB1–Mycwith KDEL in dendritic shafts and branching points (BP). GABAB1–Myc was used as positive control (gray bar). n54–6 neurons. (G) Neurons from transgenicmice expressing GABAB1–GFP (green) were transfected with ER–RFP (red) in the presence or absence of Sar1-H79G–HA (purple). (H) Percentage ofcolocalization of GABAB1–GFP with ER–RFP. n56–7 neurons. All quantitative data show the mean6s.e.m.; *P,0.05; **P,0.01; n.s., non-significant;Student’s t-test.

RESEARCH ARTICLE Journal of Cell Science (2014) 127, 3382–3395 doi:10.1242/jcs.151092

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GABAB1 transports within the ERWe then explored whether the ER functions as a transport

organelle for GABAB1. We first carried out live-cell imaging tocharacterize the dynamic relationship between intracellularGABAB1 and the ER in the soma and dendrites of neurons co-transfected with GABAB1–mRFP or GABAB1–EGFP and ER–

BFP, a vector that encodes a blue fluorescent protein fused to thehuman ER resident protein Sec61b – the encoded fusion proteinstrongly labels the ER (Zurek et al., 2011). Although the spatial

resolution was not sufficient to visualize the tubular networkbecause of its densely packed nature (Spacek and Harris, 1997),punctate and irregular structures containing fluorescent GABAB1were observed in the somatic and dendritic compartments, andtheir distribution patterns changed over time (Fig. 2A,B, toppanels, arrows). Not every puncta of GABAB1 colocalized with

the ER, in agreement with the results presented in Fig. 1, but theoverall structure often rearranged in synchrony. By contrast, thedynamics of the Golgi, labeled with either Golgi–EYFP (EYFPfused to the Golgi-targeted membrane-anchoring signal peptide

from human b-1,4-galactosyltransferase; labels Golgi in thesoma) or Galt2–mCherry (Galt2 is officially known asgalactosyltransferase 2; labels Golgi in the dendrites) and the

ERGIC, labeled with ERGIC-53–YFP (Ward et al., 2001), werenoticeably different from the dynamics of GABAB1 in bothcompartments (Fig. 2A–C).

Next, we explored the diffusion of GABAB1 within thedendritic ER using fluorescence recovery after photobleaching(FRAP, Fig. 2D) on an 8-mm dendritic segment of neuronstransfected with GABAB1–EGFP. ER-retained GABAB1–EGFPwas mobile in dendrites, and lateral mobility in ER tubules wasobserved by high-rate imaging in thick dendrites (Fig. 2E).Prolonged NMDA receptor activation with glutamate produces

reversible ER fission and disrupts the transport of luminal ERproteins (Kucharz et al., 2009; Ramı́rez et al., 2011). Thus, weused this treatment in neurons that were co-transfected with

GABAB1–EGFP and ER–RFP to confirm the ER mobility ofGABAB1–EGFP. As expected, glutamate receptor activationsignificantly altered the structure of the ER and resulted in the

co-rearrangement of ER–RFP and GABAB1–EGFP (Fig. 2F).More importantly, the mobility of GABAB1–EGFP was severelyimpaired (Fig. 2G).

These results demonstrate that GABAB1 localizes to the ER,

which allows the transport of unassembled GABAB1 in dendrites.Because the subunit is not an ER-resident protein but a traffickingcargo we will refer to it as ‘ER-associated GABAB1’ throughout

the rest of the text.

Characterization of GABAB1 transport within the dendritic ERTo gain a better understanding of ER-associated GABAB1 subunittransport we first used single-particle tracking photoactivatedlocalization microscopy (sptPALM) (Manley et al., 2008; Frost

et al., 2010). This technique is well suited to examine thedynamic properties of single intracellular molecules within smallregions, and allows a spatial resolution of ,10–50 nm (Senguptaet al., 2012). To track receptor subunits, we generated mEos2-

tagged GABAB1 (GABAB1–mEos2). mEos2 is a bright andphotostable photoconvertible fluorescent protein that is switchedfrom green to red fluorescence by UV irradiation (McKinney

et al., 2009). This construct was retained intracellularly andmaintained the characteristic trafficking properties of GABAB1(data not shown). Individual molecular locations within

transfected neurons were recorded in a 20,000-frame series, and

the summation of these positions revealed that GABAB1–mEos2was distributed throughout a complex heterogeneous structure

(Fig. 3A, right panel), similar to that reported for the dendritic ERusing structured illumination or electron microscopy (Cui-Wanget al., 2012; Broadwell and Cataldo, 1983; Spacek and Harris,1997). Single photoswitched GABAB1–mEos2 molecules were

tracked over multiple image frames and were found to be highlymobile, and their motility was not oriented in relation to the axisof the dendritic shaft (Fig. 3A, left panel; Fig. 3B), consistent

with the expected organization of the ER. The molecular velocityspanned a range from nearly immobile to 1 mm/s, with a fractionof the population moving at rates .2 mm/s. Notably, thedistribution of velocities was not different in proximal anddistal segments of a dendrite (Fig. 3B, right panel). Because mostmolecules were tracked for only brief periods over which the

influence of directed transport or confinement is minimized (,4frames or 200 ms), the molecular velocity can be used to estimatean effective diffusion coefficient D, given that the mean squaredmolecular displacement increases as 4DDt, where Dt is theobservation interval. Thus, the modal velocity of 200 nm/sobserved here indicates that a substantial proportion of GABAB1moves within the dendritic ER with a relatively slow effective D,

,0.01 mm2/s, whereas the subpopulation of molecules undergoingrapid motion does so with D surpassing 1 mm2/s. These values areconsistent with the general expectation for motion of

transmembrane proteins in the lipid bilayer (Saxton and Jacobson,1997) and are comparable to those of ER-resident proteins (Bannaiet al., 2004). It should be noted that because each molecule is

tracked only briefly, it is not possible to determine with thisapproach how frequently molecules transition from one rate ofmobility to another, and thus for how long they remain immobile.These observations suggest that two or more subpopulations of

different mobility contribute to the ER transport of GABAB1 indendrites.

We used FRAP to further examine the transport of ER-

associated GABAB1 fused to EGFP or mRFP. We used thepercentage recovery as an indication of the mobile fraction, andrecovery rate (t1/2) was used as an indication of transport velocity

within the ER. The mobile fraction of GABAB1–EGFP was5062% (6s.e.m.) and t1/2 was 7562 s, with minimal interferencefrom acquisition bleaching (Fig. 3C). The percentage recoveryand t1/2 were not affected by the fluorescent tag because the same

results were obtained with GABAB1–mRFP (Fig. 3C). Thetransport of GABAB1 was bidirectional and significantly slowerthan that of ER–RFP (Fig. 3D). The percentage recovery and t1/2were identical in primary, secondary and tertiary branches(percentage recovery of primary branches versus secondary,P50.919; versus tertiary, P50.833; t1/2 of primary branchesversus secondary, P50.242; versus tertiary, P50.275, Student’st-test) (Fig. 3E,F). Significant recovery was observed afterbleaching a large 27-mm stretch of dendritic volume, indicatingthat the ER allows long-distance protein transport (Fig. 3G).

Dendritic transport of ER-associated GABAB1 is controlled bymultiple componentsPrevious studies have suggested that the transport ofneurotransmitter receptors within the dendritic ER is controlledby lateral diffusion (Cui-Wang et al., 2012). Long-distance

transport of ER-associated GABAB1 raises the possibility thatsimple diffusion is not sufficient to explain its mobility. First, toconfirm that our FRAP-based transport assay was not confounded

by the appearance of newly synthesized GABAB1, we blocked


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Fig. 2. GABAB1 is mobile within the neuronal ER. (A) Neurons were co-transfected with GABAB1–mRFP (GB1–RFP, red), ER–BFP (green) and Golgi–EYFP(purple) and were imaged live. A somatic region is shown. Arrows show synchronous rearrangement of GABAB1–mRFP and ER–BFP signals. (B) Neuronswere co-transfected with GABAB1–EGFP (green), ER–BFP (purple) and Galt2–mCherry (red) and were imaged live. A dendrite is shown. Arrows showsynchronous rearrangement of GABAB1–EGFP and ER–BFP signals. (C) Neurons were co-transfected with GABAB1–mRFP (red), ERGIC–YFP (green) andER–BFP (purple), and imaged live. A dendrite is shown. Arrows show the asynchronous rearrangement of GABAB1–mRFP and ERGIC–YFP signals.(D) Diagram illustrating the FRAP experiments carried out for ER proteins in dendrites. (E) The ER mobility of GABAB1–EGFP was evaluated by FRAP in athick dendrite. The image was saturated to allow visualization of the tubular ER structure. (F) Dendrites of neurons transfected with GABAB1–EGFP (green) andER–RFP (red) and imaged live in the absence or presence of 100 mM glutamate (20 min). (G) The mobility of GABAB1–EGFP was evaluated by FRAP (bleachedregion 8 mm) before (left) or after (right) the glutamate treatment. Scale bars: 10 mm.


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protein synthesis by treating neurons with cycloheximide (CHX,0.1 mg/ml, 90 min). Importantly, the percentage recovery and t1/2were unaffected by cycloheximide (Fig. 4A,B, GABAB1–EGFP6CHX percentage recovery, P50.166; t1/2, P50.642,Student’s t-test). Next, taking into account the fact that multiple

cytoskeletal mechanisms drive protein transport in neurons, weexamined their contribution to the ER-mediated transport ofGABAB1 in proximal dendrites. The mobile fraction of GABAB1decreased significantly after high doses of nocodazole (NocoHigh, 100 mM, 4 h), but was unaffected by low doses ofnocodazole (Noco Low, 100 nM, 5–70 min), trichostatin A

(TSA, 125 nM, 30 min) or cytochalasin D (cytoD, 5 mM, 2 h)(Noco High, 3763%, P50.002; Noco Low, 5362%, P50.214;TSA, 4762%, P50.397; cytoD, 5065%, P50.901, Student’s t-test; 6s.e.m.) (Fig. 4C). These results indicate that, in additionto diffusion, a proportion of subunit transport depends onpolymerized microtubules (which are affected by high doses of

nocodazole), but not on dynamic microtubules (which areaffected by low doses of nocodazole), acetylated microtubules

(the levels of which are increased by TSA) or the actincytoskeleton (which is disrupted by cytochalasin D).Interestingly, luminal ER–RFP and the ER membrane protein

cytochrome-b5–EGFP (Cytb5–GFP) were unaffected by highdoses of nocodazole (percentage recovery of ER–RFP6NocoHigh, P50.966; percentage recovery of Cytb5–GFP6NocoHigh, P50.827. t1/2 for ER–RFP6Noco High, P50.361; forCytb5–GFP6Noco High, P50.239, Student’s t-test) (Fig. 4E–H) arguing against a global effect of nocodazole in the transport

of proteins through the ER. To confirm that nocodazole affectedER-retained GABAB1, we preincubated neurons with brefeldinA (BFA), a treatment that results in the dispersal of the Golgiand accumulation of cargo in the ER. Under these conditions,

high doses of nocodazole still decreased the transport ofGABAB1 (control BFA, 6162%; BFA+Noco High, 3266%,

Fig. 3. Characterization of the ER transport of GABAB1. (A) The mobility of GABAB1–mEos2 was evaluated by using sptPALM in dendrites. Left panel, quiverplots show GABAB1–mEos2 (GB12mEos2) molecules mobilizing in multiple directions (inset is 26magnification). Scale bar: 1 mm. Right panel, GABAB1–mEos2 was imaged in a live neuron for 10 seconds. Scale bar: 2 mm. (B) Net velocity of GABAB1–mEos2 single molecules at initial and distal segments of thedendrite (black and red curves, respectively). (C) FRAP of GABAB1–EGFP (green symbols) and GABAB1–mRFP (red symbols) in dendrites. The uppercurves (empty symbols) correspond to the experimental bleaching without FRAP. n514–28 neurons. (D) FRAP of GABAB1–EGFP (green symbols) and ER–RFP (red symbols) in dendrites. n58 neurons. (E) Percentage recovery of GABAB1–EGFP in primary (1 )̊, secondary (2 )̊ and tertiary (3 )̊ dendriticbranches. n53–5 neurons. (F) t1/2 of recovery of GABAB1–EGFP in primary, secondary and tertiary dendritic branches. n53–5 neurons. All quantitative datashow the mean6s.e.m.; n.s., non-significant. (G) The long-range mobility of GABAB1–EGFP was evaluated by bleaching a 27-mm dendritic region in live (left) orfixed (right) cells. Scale bar: 10 mm.


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P50.016, Student’s t-test) (Fig. 4I,J). Although nocodazolemight be affecting a minor BFA-independent component ofpost-ER COPII-coated carriers, the robust effect of BFA

confirms that microtubule dependence occurs mostly on ER-associated GABAB1. Control experiments demonstrated theeffectiveness of all pharmacological treatments (data notshown).

Interestingly, only TSA decreased t1/2 significantly (t1/2,6862 s; P50.026, Student’s t-test) (Fig. 4D). Although thisexperiment does not directly identify a specific transport

mechanism, tubulin acetylation could decrease GABAB1 confinementby increasing the recruitment of the molecular motors tomicrotubules (Reed et al., 2006; Dompierre et al., 2007; Cai

et al., 2009) or by decreasing ER complexity (Friedman et al.,2010; Cui-Wang et al., 2012). Because the former interpretationinvolves molecular motors and we have demonstrated that a

component of transport is microtubule dependent, we evaluated therole of kinesin-1 and dynein in the transport of dendritic GABAB1.Kinesin-1 co-immunoprecipitates with GABAB1 in brain extracts(Vidal et al., 2007), participates in the axonal localization of

GABAB1 (Valdés et al., 2012) and has been implicated inER dynamics (Woźniak et al., 2009). However, the expressionof a dominant-negative version of kinesin-1 comprising the

cargo-binding domain of Kif5C that interferes with endogenouskinesin–cargo interactions (KIF5CDN–RFP, data not shown)(Falley et al., 2009) had no effect on the GABAB1 mobile

fraction (control RFP, 4463%; KIF5CDN–RFP, 4368%;P50.872, Student’s t-test) or recovery kinetics in dendrites(control RFP, 7164 s; KIF5CDN–RFP, 6963 s; P50.290,Student’s t-test) (Fig. 5A,B).

Because the mixed microtubule orientation in proximaldendrites allows minus-end-directed dynein to drive transportbidirectionally (Kapitein et al., 2010), we used overexpression of

GFP-tagged p50 (also known as dynamitin or DCTN2) (p50–EGFP) to inhibit dynein function (Echeverri et al., 1996;Burkhardt et al., 1997; Presley et al., 1997). Importantly, p50–EGFP overexpression significantly decreased the percentage

recovery of GABAB1 (control EGFP, 5764%; p50–EGFP,4663%; P50.049, Student’s t-test), whereas it had no effect ont1/2 (Fig. 5C,D). GFP-tagged N5-bIII-spectrin (N5-bIII–EGFP),an established blocker of the bIII-spectrin–dynein complex(Devarajan et al., 1997), produced a mild but non-significantdecrease in the percentage recovery (4763%; P50.077,Student’s t-test) and did not alter t1/2 (Fig. 5C,D).

A prediction of our FRAP experiments is that dynein inhibitioncould limit the ER distribution of GABAB1 in dendrites. Thus, we

tested the intracellular distribution of transfected GABAB1–mRFP in the presence of p50–EGFP. As previously reported,dendritic morphology was altered by p50–EGFP (Kapitein et al.,2010) (Fig. 5H). More importantly, the distribution of GABAB1in p50–EGFP-expressing neurons was dramatically different fromthat of control neurons. Specifically, the distribution of ER-associated GABAB1 was restricted to the proximal regions in

dendrites, whereas it was unaltered in axons [GABAB1–mRFPmean intensity (arbitrary units) in dendrites for control EGFP,69.964.8; for p50–EGFP, 39.863.8, P50.00003. In axons forcontrol EGFP, 10.161.3; for p50–EGFP, 14.362.0, P50.095,Student’s t-test] (Fig. 5E,G,H). By contrast, dynein inhibition didnot affect EGFP or MAP2 distribution in dendrites or axons

(MAP2 mean intensity of control EGFP versus p50–EGFP for

Fig. 4. The ER transport of dendritic GABAB1 is controlled by multiple components. (A) Percentage recovery of GABAB1–EGFP for control samples orafter treatment with 0.1 mg/ml cycloheximide (CHX) for 90 min. n54 and 5 neurons, respectively. (B) Same as above for the t1/2. (C,D) Same as in A,B forneurons under control conditions or treated with 100 mM nocodazole for 4 h (Noco High), 100 nM nocodazole for 5–70 min (Noco Low), 125 nM TSA for30 min or 5 mM cytochalasin D for 2 h (Cyto D). n528, 9, 8, 20 and 4 neurons, respectively. At least three independent experiments were performed percondition. (E,F) Same as in A,B for neurons transfected with ER–RFP under control conditions or following treatment with 100 mM nocodazole for 4 h (ER–RFP+Noco). n54 and 7 neurons, respectively. (G,H) Same as in A,B for neurons transfected with Cytb5–GFP under control conditions or followingtreatment with 100 mM nocodazole for 4 h (Cytb5–GFP+Noco). n55 and 6 neurons, respectively. (I,J) Same as in A,B for neurons treated with 0.5 mg/ml BFA for20 h or 0.5 mg/ml BFA for 20 h+100 mM nocodazole for 4 h (BFA+Noco). n53 and 4 neurons, respectively. All data show the mean6s.e.m.; *P,0.05; **P,0.01;n.s., non-significant; Student’s t-test.


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dendrites, P50.309; for axons, P50.144. EGFP mean intensity ofcontrol EGFP versus p50–EGFP for dendrites, P50.334; foraxons, P50.101, Student’s t-test). To further analyze the effect ofp50–EGFP on the intracellular distribution of GABAB1–mRFP,we calculated a polarity index, where an index of .0 or ,0indicates polarization towards dendrites or axons, respectively(Kapitein et al., 2010). The polarity index of ER-associatedGABAB1 in control neurons was 0.7560.03 (Fig. 5F), consistentwith the preferential somatodendritic distribution of GABAB1subunits (Biermann et al., 2010; Valdés et al., 2012). By contrast,upon expression of p502EGFP, the polarity index of GABAB1decreased significantly (0.4760.06; P50.0005, Student’s t-test)(Fig. 5F). As expected, the polarity index of MAP2 and EGFPwere unaffected by dynein inhibition (MAP2, P50.111; EGFP,P50.386, Student’s t-test). Taken together, these datademonstrate that dynein is essential for one component ofdendritic ER transport and for the targeting of GABAB1 subunits.

GABABRs traffic through canonical and local secretoryroutesTransport through a subcompartment of the dendritic ER

determines the specific sorting of NMDARs to GOPs (Jeyifouset al., 2009). Likewise, the localization and transport of nascentGABAB1 subunits within the dendritic ER might result in local

trafficking through GOPs. To examine whether GABABRs trafficthrough GOPs, we co-transfected GABAB1–Myc and GABAB2–

FLAG, which assemble and traffic to the plasma membrane,together with ARF1-Q71I–HA, a constitutively active mutant ofthe small GTPase ARF1 – involved in COPI recruitment – that

prevents export from the Golgi and produces the accumulation ofcargo in the somatic Golgi and GOPs in neurons (Zhang et al.,1994; Jeyifous et al., 2009). Under steady-state conditions,

GABAB1 was mostly excluded from the Golgi in the perinuclearregion and in dendrites (Fig. 6A,B). Upon expression of ARF1-Q71I–HA, GABAB1 accumulated significantly in both somaticand dendritic Golgi structures (P50.0061 and P50.0001,respectively, Student’s t-test) (Fig. 6A,B,E). These resultsindicate that newly synthesized receptors traffic through theperinuclear Golgi and GOPs.

It has been suggested that dwell times and diffusion within theER define the exploration volumes of nascent proteins and,therefore, spatially determine ER export domains (Herpers and

Rabouille, 2004; Cui-Wang et al., 2012). Thus, we reasoned thatGABAB1 subunits with long ER residency would preferably feedGOPs, whereas rapidly exported subunits might preferentially use

the perinuclear Golgi. To test this hypothesis, we used GABAB1-DC–Myc, a mutant subunit that contains a deletion of 104 aminoacids in its carboxyl terminus. This mutant protein lacks the ER

Fig. 5. Dendrite-specific ERtransport and targeting of GABAB1depends on dynein. (A) Percentagerecovery of GABAB1–EGFPfluorescence for neurons transfectedwith RFP or KIF5DN–RFP. n517 and8 neurons, respectively. Threeindependent experiments wereperformed per condition. (B) Same asin A for the t1/2. (C,D) Same as in A,Bfor neurons co-transfected withGABAB1–mRFP and EGFP,dynamitin (p50–EGFP) or N5-bIII-spectrin (N5-bIII-EGFP). n513–26neurons. Four independentexperiments were performed percondition. (E) Quantification ofGABAB1–mRFP fluorescenceintensities in axons and dendrites ofneurons transfected with controlEGFP or p50–EGFP. n516 neuronsper condition. (F) Polarity index ofGABAB1–mRFP for neuronstransfected with EGFP or p50–EGFP.n516 neurons per condition. Allquantitative data show themean6s.e.m.; *P,0.05; ***P,0.001;n.s., non-significant; Student’s t-test.(G,H) Representative images ofneurons co-transfected withGABAB1–mRFP (GB1–mRFP, red)and a double dose of control EGFPplasmid (EGFP/EGFP, green) (G) orp50–EGFP plus EGFP (p50–EGFP/EGFP, green) (H) and labeled with thedendritic marker MAP2 (purple).Dendritic segments outlined in whiteare enlarged below the main image.Arrowheads mark the axon. Scalebars: 10 mm.


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retention domain, does not need GABAB2 for ER export and isefficiently transported to the plasma membrane (Restituitoet al., 2005). Unexpectedly, even under basal conditions, the

colocalization of this mutant subunit with GOPs was 70% higherthan that of full-length GABAB1 (percentage colocalizationwith Golgi–EYFP for GABAB1–Myc+GABAB2, 2468%; forGABAB1-DC–Myc, 6668%; P50.0002, Student’s t-test)(Fig. 6C–E). These results indicate that dendritic ER transportdoes not restrict the sorting of GABABRs to GOPs.

To further explore the ER-to-Golgi trafficking of GABABRs,we used a recently described pulse-chase system based on thefusion of four repeats of mutant FKBP12 (each repeat is referredto as an FM domain) to a transmembrane protein of interest. This

fusion results in ER retention due to aggregation of the FMdomains (Al-Bassam et al., 2012). Addition of the rapamycinanalog D/D solubilizer allows disaggregation of the FM domains,

release from the ER and visualization of a synchronous pool oftrafficking cargo. We engineered the FM domains at the N-terminus of GABAB1-DC (FM–GABAB1-DC–EGFP), which isexposed to the ER lumen during trafficking (Fig. 7A). Afteraddition of D/D solubilizer, FM–GABAB1-DC–EGFP wasefficiently released from the ER and reached the plasma

membrane (data not shown). Importantly, in neurons, FM–GABAB1-DC–EGFP was released from the ER after the additionof D/D solubilizer and subsequently condensed in the somaticGolgi and in GOPs (Fig. 7B). Individual carriers of ER-released

GABAB1-DC–EGFP traveled bidirectionally in dendrites with amild retrograde bias and merged with stationary GOPs that wereat variable distances from their site of appearance (the percentage

of carriers that merged with GOPs that traveled ,2.5 mm or.2.5 mm was 60% and 40%, respectively. Retrograde transport,70%; anterograde transport, 30%; six neurons, three independent

experiments) (Fig. 7C). Instantaneous velocities of post-ERcarriers were in the range of those reported for carriers of theERGIC (Appenzeller-Herzog and Hauri, 2006) (Fig. 7D). Taken

together, these results indicate that GABAB1 traffics through boththe perinuclear Golgi and GOPs, and that their use is notdetermined by ER residency times.

Finally, we speculated that the use of dendritic organelles

might influence the site of delivery at the neuronal plasmamembrane. To spatially characterize the insertion of newlysynthesized GABABRs, the kinetics of de novo insertion were

evaluated by quantifying the appearance of plasma membranebgtx-Myc–GABAB1a, a modified version of the GABAB1a subunit(one of the two alternative isoforms of GABAB1) containing an a-bungarotoxin-binding site engineered at the extracellular N-terminus. De novo exocytosed bgtx-Myc–GABAB1a subunitswere specifically and irreversibly labeled with fluorescent a-bungarotoxin (Bgtx-594) after saturating the recycling pool(Bgtx-646) as reported previously (Wilkins et al., 2008;Terunuma et al., 2010). We measured the mean intensity ofBgtx-594 at the outline of the soma or dendrite to estimate the

Fig. 6. GABABRs traffic throughsatellite GOPs. (A) Neurons were co-transfected with GABAB1-Myc (GB1–MYC, red) and Golgi–EYFP (green), orwith GABAB1–Myc (red), Golgi–EYFP(green) and ARF1-Q71I–HA (purple) toblock exit from the Golgi. GABAB2–FLAGis not shown. (B) Same as in A forproximal dendrites and GOPs. (C) Somasof neurons co-transfected with GABAB1-DC–Myc (GB1-DC–MYC, red) and Golgi–EYFP (green). (D) Same as in C forproximal dendrites and GOPs.(E) Percentage of colocalization ofGABAB1–Myc with Golgi–EYFP in theabsence (white bars) or presence ofARF1-Q71I–HA (black bars) or GABAB1-DC–Myc (gray bars) in the soma (left) anddendrites (right). Data show themean6s.e.m.; n54–8 neurons; **P,0.01;***P,0.001; Student’s t-test. Scale bars:10 mm.


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number of newly inserted receptors. In neurons transfected with

bgtx-Myc–GABAB1a and GABAB2–EGFP, de novo insertion wasevident after 15 min of Bgtx-594 labeling, and graduallyincreased over 60 min (Fig. 8A). The timecourses of de novo

insertion of bgtx-Myc–GABAB1a in the soma and in dendriteswere remarkably similar (Fig. 8B). Indeed, the ratio ofsomatic:dendritic insertion revealed only a mild positive slope

(Fig. 8C). Overall these data do not support an early exocytosisevent in the cell body followed by subsequent diffusion andredistribution to dendrites (Rosenberg et al., 2001), but rather the

simultaneous delivery of newly synthesized receptors to the entiresomatodendritic compartment. These data are in agreement withthe use of the entire ensemble of exocytic organelles.

DISCUSSIONThe large size and morphological complexity of neuronsintroduce significant challenges to the delivery of membrane

cargoes, which might be difficult to convey exclusively throughlong-haul post-Golgi vesicles. Thus, local trafficking involvingthe dendritic ER and GOPs provides an alternative means to

regulate the supply of membrane components, but its role in thedelivery of neuronal proteins is still poorly understood (Hortonand Ehlers, 2003). Here, we demonstrate that GABAB1 transport

in dendrites is independent of COPI vesicle formation and takes

place within the dendritic ER. The transport of ER-associatedGABAB1 is not controlled by diffusion alone, but also by anactive mechanism involving microtubules and dynein. GABABRs

traffic through both the perinuclear Golgi and GOPs, and insertat the plasma membrane throughout the somatodendriticcompartment. We conclude that GABABRs utilize a long-

distance ER transport mechanism and both perinuclear andlocal secretory routes prior to reaching the plasma membrane.Our observations argue against the view that dendritic ER

transport restricts the trafficking of neurotransmitter receptors toGOPs, and they are consistent with the hypothesis that ERassembly of GABABRs is the rate-limiting step for receptortrafficking.

ER localization of GABAB1 in dendritesWe demonstrate that the ER retention of GABAB1 produces a

broad intracellular ER-enriched distribution in dendrites, with asmall proportion localizing at the ERGIC and Golgi, possiblyrevealing an ER–ERGIC–Golgi retrieval mechanism (Ramı́rez

et al., 2009; Biermann et al., 2010; Brock et al., 2005). Otherneurotransmitter receptors and ion channels are also enriched inthe ER and display a variety of ER retention signals (Ramı́rez and

Fig. 7. GABAB1 carriers merge with GOPs after ER export. (A) Diagram of the FM-based pulse-chase system implemented for a GABAB1 construct withEGFP fused to the C-terminus and four FM domains fused to the N-terminus (FM4x4). (B) Neurons were transfected with FM4-GB1-DC–EGFP (green) and fixedbefore or after a 60–120-min treatment with D/D solubilizer (DD). The Golgi was stained with GM130 (red). Arrows point to Golgi structures that show littlecolocalization before the addition of D/D solubilizer (0 min), but precise colocalization after incubation with D/D solubilizer (60–120 min). Scale bar: 10 mm.(C) Neurons were co-transfected with FM4-GB1-DC–EGFP (green) and Galt2–mCherry (red) to visualize GOPs in dendrites (upper panels). After 12 h ofexpression, protein synthesis was inhibited with cycloheximide and neurons were incubated with D/D solubilizer for 30 min at 37˚C and imaged live. Lowerpanels show a time series of the boxed dendritic segment. Arrows indicate moving carriers that merge with GOPs. Arrowheads show a static GABAB1 structure.Scale bar: 5 mm. (D) Diagram of the trajectory of the moving carrier visualized in C. The graph shows velocities in each interval.


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Couve, 2011) but it is still unclear to what extent ER retention is

related to local dendritic checkpoints and how these regulate latertrafficking stages.

ER cargoes in neurons might be confined by regions of high

network complexity, especially those at dendritic branching points(Cui-Wang et al., 2012). However, the continuity of the ER allowsproteins to disperse throughout the organelle. In our experiments,the time of expression was sufficient to distribute GABAB1 through

regions of low and high complexity and to localize the subunitintracellularly within the entire somatodendritic compartment. Thisphenomenon was not an artifact of overexpression, because the

colocalization with the ER in dendrites was similar in transgenicneurons that expressed GABAB1–EGFP at endogenous levels andincreased after blocking ER export. By contrast, NMDARs

accumulate exclusively in the soma after blockade of ER export,providing evidence that multiple ER subcompartments control thetrafficking of different neurotransmitter receptors (Jeyifous et al.,2009).

ER transport in dendritesThe intracellular velocities of GABAB1 are comparable to those

reported for ER resident proteins (GFP–KDEL, RFP–KDEL, GFP–SERCA2a and GFP–IP3R1) (Bannai et al., 2004). Although diffusionhas been elegantly characterized for other neurotransmitter receptors

– namely, ER-retained AMPA subunits (Cui-Wang et al., 2012) –lateral diffusion alone is not sufficient to explain the ER transport of

GABAB1 in neurons. Three pieces of evidence strongly support this

conclusion: the microtubule-dependent transport of GABAB1, themicrotubule dependence even after BFA-induced ER retention, andthe dynein-dependent transport and localization of ER-associated

GABAB1.Regarding the underlying transport mechanism, our results show

that a component of ER-associated GABAB1 transport is impairedafter depolymerization of microtubules. However, our data do not

support the idea that microtubule-dependent ER remodelingcontrols the mobility of GABAB1, because the transport of ER–RFP and Cytb5–GFP were unchanged by nocodazole. Others have

proposed that vesicular-like carriers mediate a motor-assistedcomponent of ER transport in dendrites, potentially identifying anactive mechanism (Bannai et al., 2004; Jeyifous et al., 2009). With

our available data we cannot conclude whether the mobility ofGABAB1 is mediated by a vesicular-like component or by transportalong the continuous ER network. Although it is unusual toenvisage a motor dragging cargo within the plane of the bilayer, we

favor this view, and we speculate that the diffusion ofneurotransmitter receptors within the plane of the ER membraneprovides a basal dynamics that might be suitable for modification

by molecular motors. Supporting this notion, microtubule andmotor-driven motility of ERES occurs independently of themovement of ER membranes (Gupta et al., 2008).

We also speculate that the active component of ER-retainedGABAB1 introduces an additional mechanism for the confinement

Fig. 8. GABAB1 insertion is somatodendritic. (A) Neurons were co-transfected with bgtx-Myc–GABAB1a (GB1-Bgtx) and GABAB2–EGFP (GB2–EGFP,green). The population of recycling receptors was saturated with Bgtx-646 (blue). De-novo-inserted receptors were visualized by binding to Bgtx-594 (red)between 0 and 60 min. Scale bar: 20 mm. (B) The intensity of red fluorescence (de novo insertion) was measured at the somas (black) and proximal dendrites(white) and plotted against time. A.U., arbitrary units. n53 independent experiments; data show the mean6s.e.m.; 17–26 neurons; *P,0.05; ***P,0.001;Student’s t-test. (C) The ratio of somatic versus dendritic intensity was calculated from the data in B.


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of ER cargoes that might counteract the dispersing tendency ofdiffusion. As a matter of fact, our data suggest that microtubule

acetylation increases the recruitment of a higher proportion offaster molecular motors that decrease the confinement ofGABABRs.

Are canonical and local trafficking in dendritesmutually exclusive?Our data show that GABAB1 localizes to the dendritic ER evenafter co-transfection with GABAB2, that GABABRs accumulate

in GOPs and that they are delivered to the plasma membranethroughout the somatodendritic compartment. Thus, the evidencesuggests that a local secretory route operates for GABABRs.

Other receptors also employ local routes, but differences in howthese routes are utilized are already apparent. For instance,endogenous and overexpressed NMDARs accumulate exclusively

on GOPs upon expression of ARF1-Q71I–HA (Jeyifous et al.,2009), whereas GABABRs accumulate in the somatic Golgi andin GOPs. Although the intrinsic limitations of overexpression

should be considered, the differences between GABABRs andNMDARs suggest that distinct neurotransmitter receptors displaydifferent degrees of spatial selectivity for organelles.

ER export kinetics and lateral diffusion determine the spatial

range over which export occurs (Herpers and Rabouille, 2004;Cui-Wang et al., 2012). Although most of our findings areconsistent with this proposal, our results with GABAB1-DCindicate to the contrary that an improved ability to exit the ERincreases trafficking through GOPs. One interpretation of theseobservations is that post-ER carriers fuse indiscriminately with

the perinuclear Golgi and GOPs, which act in concert to increasethe trafficking capacity of the neuron. Interestingly, upon ERrelease, the temperature-sensitive VSVG mutant or BDNF use

both the perinuclear Golgi and GOPs (Horton and Ehlers, 2003).Our experiments using GABAB1 are consistent with this view ofnon-selective processing and suggest that, for a subset of neuronalcargoes, ER transport does not restrict sorting to GOPs and that

selected proteins use the complete ensemble of Golgi structures.In this context, the absence of dendritic Golgi in ,30% ofneurons (Horton and Ehlers, 2003; Horton et al., 2005) and the

presence of GOPs in only ,18% of mature dendrites (Hortonet al., 2005), in a scenario where all dendrites containneurotransmitter receptors, suggest that local trafficking cannot

be exclusive. However, metabotropic glutamate receptor(mGluR) activity confines ER transport (Cui-Wang et al., 2012)and enhances the formation of ERES containing NMDARs(Aridor et al., 2004). Therefore, it is still unknown how, or if, the

balance of canonical and local trafficking is modified by activity,and whether preferential use of local routes influences the localavailability of receptors at synapses in response to activity.

Local supply of GOPsThe dendritic distribution of the ER and GOPs suggests thatintermediate transport carriers might operate locally. Indeed,VSVG carriers that merge with GOPs have been reported (Horton

and Ehlers, 2003). Additionally, packets containing VSVGbecome concentrated at branching points, presumably GOPs,after local ER export (Chen et al., 2013). However, a relevant

issue that remains to be addressed is to what extent the local ERcontributes to supply nearby GOPs. We have observed post-ERcarriers merging with GOPs after both long and short trajectories,

suggesting that dendritic ER export is not necessarily coupled tolocal trafficking. Therefore, although our results indicate that the

dendritic ER is a crucial component for the transport of GABABsubunits, they do not support the notion that GOPs feed

exclusively on the local ER, presenting a more complexscenario of local trafficking in dendrites.

MATERIALS AND METHODSAnimalsAdult pregnant female Sprague-Dawley rats were purchased from the

Central Animal Facility at Universidad Católica de Chile and were

euthanized by asphyxia in a CO2 chamber according to the Guide for

Care and Use of Laboratory Animals of The National Research Council

(1996). GABAB1–EGFP mice were kindly provided by Bernhard Bettler

(University of Basel, Switzerland). They carry a bacterial artificial

chromosome containing GABAB1-EGFP in a homozygous knockout

background for GABAB1. The construct is expressed under control of

GABAB1 promoter elements (Schuler et al., 2001; Casanova et al., 2009).

CellsCOS-7 cells were transfected by electroporation (Couve et al., 1998).

Primary hippocampal neurons were cultured from embryonic day (E)18

rats or E18 GABAB1–EGFP transgenic mice (Goslin and Banker, 1991)

and transfected by using Ca2+ phosphate at 7–15 days in vitro (Valdés

et al., 2012; Jiang and Chen, 2006). Endogenous subunits do not

assemble with recombinant subunits until 4–5 days post-transfection

(Valdés et al., 2012); therefore, all experiments were analyzed between

12 and 48 h post-transfection.

Reagents and DNA plasmidsBFA, cycloheximide, cytochalasin D, TSA and nocodazole were

purchased from Sigma (St Louis, MO). D/D Solubilizer was purchased

from Clontech (Mountain View, CA). Myc–GABAB1, FLAG–GABAB2,

HA–GABAB2, Myc–GABAB1-DC, GABAB1a–EGFP, GABAB2–EGFP,GABAB1a–mRFP and Bgtx-Myc–GABAB1 were as described previously

(Couve et al., 1998; Calver et al., 2001; Couve et al., 2002; Restituito

et al., 2005; Ramı́rez et al., 2009; Terunuma et al., 2010). To generate

GABAB1a–mEos2, rat GABAB1a was subcloned into pmEos2-N1

(between the SpeI and BamHI sites). mEos2, kindly provided by Sean

McKinney (Howard Hughes Medical Institute, Janelia Farm Research

Campus, MD), is fused to the intracellular carboxyl terminus of

GABAB1a. To generate FM–GABAB1-DC–EGFP, four FM domainswere fused to the N-terminus of GABAB1-DC by amplifying GABAB1-DC using the primers 59-ATCTCGAGGAGCAGAAGCTGATCTCAG-39 and 39-CTTTTGTCCTAGTAGGTGGTCTTAAGCG-59, andsubcloning the resulting amplicon into the Type I FM4-EGFP vector

(Thuenauer et al., 2004) between the XhoI and EcoRI sites.

The GABAA subunitsRFP-BBSa4 and pH/BBSb3 have been described

previously (Abramian et al., 2010; Bogdanov et al., 2006). pDsRed-C1

(RFP), pEYFP-Golgi, pEYFP-ER and pDsRed2-ER (ER-RFP) were

obtained from Clontech (Mountain View, CA). Galt2–mCherry was

kindly provided by Alfredo Cáceres (Instituto de Investigación Médica

Mercedes y Martı́n Ferreyra, Argentina). Cytb5–EGFP was kindly

provided by Claudio Hetz (Universidad de Chile, Chile). BFP–Sec61b(ER–BFP), ARF1-Q71I–HA and Sar1-H79G–HA have been described

previously (Ward et al., 2001; Jeyifous et al., 2009; Zurek et al., 2011).

Kif5CDN–RFP corresponds to amino acids 678–955 of KIF5C

(accession number NM_001107730). This construct is also referred to

as DN2 (Falley et al., 2009). Dynamitin subunit p50–EGFP and N5-bIII-spectrin were kindly provided by Anna Akhmanova (Erasmus Medical

Center, The Netherlands) and Michael Stankewich (Yale University,

New Haven, CT), respectively (Devarajan et al., 1997; Splinter et al.,

2012). All manipulations and the fidelity of DNA constructs were

verified by sequencing.

AntibodiesAntibodies against Myc and ERGIC-53 were purchased from Sigma.

Antibodies against KDEL and GM130 were purchased from StressGen

(Ann Arbor, MI). Antibodies against HA were purchased from Roche

(Indianapolis, IN). Anti-GFP antibodies (ab6556) were purchased from


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Abcam (Cambridge, UK). Fluorescently labeled secondary antibodies

were purchased from Jackson Immuno Research Laboratories (West

Grove, PA).

Immunofluorescence, time-lapse microscopy and FRAPImmunofluorescence was performed in permeabilized neurons (Ramı́rez

et al., 2009). Fixed and live-cell imaging was performed on a spectral

confocal microscope Olympus FluoView FV1000 (Center Valley, PA)

with a UPLSAPO 606/1.35 objective and 36 digital zoom, in Tyrodemedium (124 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2,

30 mM D-glucose and 25 mM HEPES pH 7.4) in an equilibrated

microscopy suite at 23 C̊. For FRAP experiments, dendritic regions of

interest (ROIs) of 60660 pixels (small ROI) or 2006200 pixels (largeROI), ,15 mm away from the soma, were bleached for 2 s using thetornado mode with the argon 473-nm laser at 100% power for EGFP-

tagged proteins or with the argon 405-nm and 559-nm laser at 100%

power for mRFP-tagged proteins. Fluorescence recovery was measured

every 10 s at 20% laser power using KALMAN2 by imaging the entire

field. Boxes of 8 mm2 were selected within the photo-bleached ROI toquantify recovery using ImageJ. Individual FRAPs were fitted to a first-

order exponential decay curve using established criteria, except for those

for Cytb5–EGFP, which were best fitted to an asymptotic type I curve

(Snapp et al., 2003), and selected by fitting (R2§0.95).The average intensity of the fluorescent signals in dendrites and axons

was determined using ImageJ (method modified from Kapitein et al.,

2010). Briefly, confocal images were acquired with a UPLSAPO 606/1.35 objective using sequential acquisition settings and KALMAN2 at

204862048 pixel resolution. Each image was a z-series of 5–10 imagesof which the maximum projection was used. Lines of width 1 pixel were

traced along axons and the major dendrite. Dendrites and axons were

identified based on their morphology and by immunostaining for MAP2.

Average pixel intensities were determined along the traced lines and the

background was subtracted. The average dendrite (Id) and axonal (Ia)

intensities were used to calculate the polarity index (PI) using

PI5(Id2Ia)/(Id+Ia).

ColocalizationColocalization analysis was performed as described previously (Ramı́rez

et al., 2010). All parameters were kept constant for a particular type of

experiment. Two-channel fluorescent image stacks [intensity I(x,y,z),

(0,255), voxel size Dx/Dy/Dz5137/137/250 nm] were recorded in themulti-track mode and KALMAN4. Four optical slices per neuron were

selected and deconvolved by Huygens Scripting software (Scientific

Volume Imaging, Hilversum, The Netherlands) using an algorithm based

on the Classic Maximum Likelihood Estimator. Image processing routines

for segmentation, visualization, calculation of colocalization coefficients

and statistical validation were based on Interactive Data Language (ITT,

Boulder, CO). Percentage colocalization for Manders coefficients (M1 and

M2) was calculated using the CDA (confined displacement algorithm)

plugin for ImageJ, followed by interpolation of the Manders coefficients

using the formula: Percentage colocalization5[M(0)2M(random)/M(100)2M(random)]6100, where M(0) is Manders at the original position,M(100) is the theoretical maximum (1), and M(random) is the

corresponding value for the random. Statistical validation was performed

using CDA.

sptPALMsptPALM was carried out by modifying a previously reported approach

(Frost et al., 2010). Briefly, imaging was conducted on an Olympus IX81

inverted microscope with a 1006/1.45 total internal reflectionfluorescence (TIRF) oil-immersion objective. Output from a set of

diode lasers (Coherent, Santa Clara, CA) was directed to the rear of the

microscope using a custom optical path. Molecules were simultaneously

photoconverted and excited using 405-nm (,100 mW) and 561-nm(20 mW) light through oblique illumination (near total internal

reflection) to reduce background fluorescence. A total of 10,000–

20,000 frames were collected at 5 Hz with 10-ms laser pulses to

minimize receptor motion during the frame (Frost et al., 2012).

Fluorescence was detected by an iXon+ 897 EM-CCD camera (Andor

Technology, South Windsor, CT) placed after a 1.66magnifying optic,resulting in a pixel size of 100 nm. Following acquisition, molecules

were localized by fitting a two-dimensional elliptical Gaussian function

to a 969 pixel array surrounding the peak, and locations were assembledinto tracks using available algorithms (Manley et al., 2008). Molecules

were segregated into those that were tracked over multiple frames and

those that appeared in a single frame. The distribution of tracked

molecules was calculated based on the density of molecules within a

search radius of 500 nm. Local net velocities were calculated using the

first and last points of tracks originating within this radius. Velocities and

vectors were mapped only in pixels with at least three tracks originating

within the search radius.

a-bungarotoxin labelingNeurons were co-transfected with bgtx-Myc–GABAB1a and GABAB2–

EGFP. At 48 h post-transfection, live neurons were incubated for

30 min with 13 mM (+)-tubocurarine chloride hydrate (Sigma). Theywere then incubated with a-bungarotoxin (Invitrogen, Eugene, OR)conjugated to Alexa FluorH 646 for 120 min at 37 C̊, washed with PBS

and incubated with a-bungarotoxin conjugated to Alexa FluorH 594 fordifferent periods of time at 37 C̊. Neurons were fixed and imaged on an

Olympus BX61WI upright microscope, an Olympus disk-scanning unit

and an UPlanFLN 606/1.25. Mean intensity was quantified usingImageJ in a 261-mm ROI at the outline of the somatic or dendritic(20 mm from the soma) regions to estimate the fluorescence associatedwith the plasma membrane.

AcknowledgementsWe thank Richard Saliba (Tufts University, Boston, USA), Viviana Valdés(Universidad Mayor, Santiago, Chile), José Cánovas and Marı́a A. Juricic (bothUniversidad de Chile, Santiago, Chile) for technical assistance and for criticalreading of the manuscript.

Competing interestsThe authors declare no competing interests.

Author contributionsA.C, J.I.V. and T.A.B. conceived and designed the experiments. J.I.V. and M.J-Bperformed the experiments and analyzed the data. J.I.V., A.C. and T.A.B.interpreted the data. H.L. and O.R. contributed image analysis routines. V.H.C.and D.S. generated reagents. J.I.V. and A.C. wrote the manuscript.

FundingThis work was supported by Iniciativa Cientı́fica Milenio [grant number P09-015-Fto A.C. and J.I.V.]; National Institutes of Health [grant number MH80046 to H.L.and T.A.B.]; Fondo Nacional de Desarrollo Cientı́fico y Tecnológico [grant number3110157 to O.R., 1100137 and 1140617 to A.C. and M.J.-B.]. D.S., J.I.V. andV.H.C. were supported by Comisión Nacional de Investigación Cientı́fica yTecnológica. Deposited in PMC for release after 12 months.

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