A distinct trans-Golgi network subcompartment for sorting ... · OCC yields lower numbers than the colocalization coefficient (CC), which depends on the intensities of overlapped

Research Article 735

IntroductionClassical cholinergic neurons and neuroendocrine cells containboth synaptic and peptidergic vesicles. Synaptic vesicle proteinssuch as vesicular acetylcholine transporter (VAChT) andsynaptophysin are sorted at the Golgi into specific transport(constitutive) vesicles for delivery to the active zone at thepresynaptic or plasma membrane (PM), where they are recycledfor the production of synaptic vesicles (De Camilli and Jahn, 1990;Kelly, 1991). Neuropeptides, however, are sorted to large dense-core vesicles (LDCVs), which are also delivered to the release site,but are not fused to the presynaptic membrane or PM under restingconditions (Gondré-Lewis et al., 2006; Park and Loh, 2008). Thusfar, no clear distinction has been made between sortingcompartments and transport routes from the Golgi complex to thepresynaptic membrane or PM for synaptic vesicle and LDCVproteins. One study has reported that VAChT is sorted separatelyfrom LDCV proteins within the Golgi to constitutive vesicles (Liuand Edwards, 1997) in the PC12 neuroendocrine cell line, but thestudy could not distinguish clearly where and when synaptic vesicleversus LDCV proteins are segregated from each other before beingsorted to their target vesicles. Four-dimensional (space and time)dissection of their segregation and sorting is necessary to understandhow neurons and neuroendocrine cells orchestrate spatial andtemporal resolution of sorting of synaptic vesicle and LDCVproteins to the cell periphery for regulated secretion at synapses orinto the bloodstream.

VAChT is a presynaptically localized transmembrane proteinthat imports acetylcholine synthesized by choline acetyltransferase(ChAT) into synaptic vesicles for release at the synaptic cleft(Eiden, 1998; Ferguson et al., 2003). ChAT and VAChT areexpressed in cholinergic neurons in the basal forebrain,hippocampus, hypothalamus, at neuromuscular junctions, and inother sympathetic and parasympathetic nerve terminals (Roghaniet al., 1996; Weihe et al., 1996). Proper in situ expression of ChATand VAChT, which is altered in Alzheimer’s disease (Blusztajn andBerse, 2000), is necessary for efficient sympathetic andparasympathetic neurotransmission. Some cholinergic neurons alsohave LDCVs containing neuropeptides and the vesicularmonoamine transporters (VMATs) that import monoamine intoLDCVs (Schäfer et al., 1997) for later secretion at neurite terminalsduring neurophysiological functions.

Undifferentiated PC12 cells have synaptic-like microvesicles(SLMVs), which contain acetylcholine (Weihe et al., 1996), as wellas LDCVs that contain monoamines and neuropeptides (Liu andEdwards, 1997). Because the content and mechanisms of biogenesisare similar in neuronal synaptic vesicles and SLMVs (Clift-O’Gradyet al., 1990; Mundigl et al., 1993), the PC12 cell is a useful modelsystem for studying how vesicular proteins are sorted to differenttypes of vesicle. VMATs are used as tracking markers for LDCVsin comparison with VAChT in PC12 cells, to distinguish sortingroutes between LDCV and SLMV proteins (Krantz et al., 2000; Liuand Edwards, 1997; Yao et al., 2004). Chromogranin A (CgA), a

SummaryGolgi-to-plasma-membrane trafficking of synaptic-like microvesicle (SLMV) proteins, vesicular acetylcholine transporter (VAChT)and synaptophysin (SYN), and a large dense-core vesicle (LDCV) protein, chromogranin A (CgA), was investigated in undifferentiatedneuroendocrine PC12 cells. Live cell imaging and 20°C block–release experiments showed that VAChT–GFP, SYN–GFP and CgA–RFP specifically and transiently cohabitated in a distinct sorting compartment during cold block and then separated into synapticprotein transport vesicles (SPTVs) and LDCVs, after release from temperature block. We found that in this trans-Golgi subcompartmentthere was colocalization of SPTV and LDCV proteins, most significantly with VAMP4 and Golgin97, and to some degree with TGN46,but not at all with TGN38. Moreover, some SNAP25 and VAMP2, two subunits of the exocytic machinery, were also recruited ontothis compartment. Thus, in neuroendocrine cells, synaptic vesicle and LDCV proteins converge briefly in a distinct trans-Golgi networksubcompartment before sorting into SPTVs and LDCVs, ultimately for delivery to the plasma membrane. This specialized sortingcompartment from which SPTVs and LDCVs bud might facilitate the acquisition of common exocytic machinery needed on themembranes of these vesicles.

Key words: Synaptic vesicles, Large dense-core vesicles, PC12 cells, Protein trafficking

Accepted 26 October 2010Journal of Cell Science 124, 735-744 © 2011. Published by The Company of Biologists Ltddoi:10.1242/jcs.076372

A distinct trans-Golgi network subcompartment forsorting of synaptic and granule proteins in neuronsand neuroendocrine cellsJoshua J. Park1,2,*, Marjorie C. Gondré-Lewis3,*,‡, Lee E. Eiden4 and Y. Peng Loh1,‡

1Section on Cellular Neurobiology, Program in Developmental Neuroscience, Eunice Kennedy Shriver National Institute of Child Health andHuman Development, National Institutes of Health, Bethesda, MD, 20892, USA2Department of Neurosciences, University of Toledo, College of Medicine, Toledo, OH 43614, USA3Department of Anatomy, Howard University College of Medicine, Washington, DC 20059, USA4Molecular Neuroscience Section, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892, USA*These authors contributed equally to this work‡Authors for correspondence ([email protected]; [email protected])

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neuropeptide precursor, is also a good marker for LDCVs in PC12cells (Kim et al., 2001), as well as for peptidergic vesicles in variousneurons of the central and peripheral nervous systems (Schafer etal., 1994). Results on the sorting of SLMVs and LDCVs in PC12cells have been extrapolated to sorting mechanism(s) in neurons.

It has become increasingly evident that there is co-sharing of thesorting routes taken by SLMV and LDCV proteins from the Golgicomplex. A small amount of VAChT was found in LDCVs ofPC12 cells (Liu and Edwards, 1997; Tao-Cheng and Eiden, 1998)and in neurons (Agoston and Whittaker, 1989). Although manygroups have shown that VMATs and CgA are the primary proteinsassociated with LDCVs and not with SLMVs (Liu and Edwards,1997; Tao-Cheng and Eiden, 1998; Weihe et al., 1996), thepossibility exists that LDCV and SLMV proteins co-traffic throughsimilar sorting compartments before segregation.

In this study, we have examined the sorting and trafficking ofthe SLMV proteins VAChT and synaptophysin, and the LDCVprotein CgA in undifferentiated PC12 cells to determine wherethese proteins are sorted and when they are segregated into synapticprotein transport vesicles (SPTVs) and LDCVs for delivery to thePM. We adopted a ‘20°C temperature block’ methodology that isused to stop Golgi-to-PM trafficking of lumenal proteins (Griffithset al., 1985; Kuliawat and Arvan, 1992; Simon et al., 1996). Coldtemperature block at 20°C allows coat assembly and budding ofpost-Golgi vesicles whereas it inhibits membrane lipid mobilizationrequired for vesicle fission, resulting in accumulation of secretoryand membrane proteins at the trans-Golgi network (TGN) (Simonet al., 1996). Quantitative live cell imaging and 3D reconstructionsshow that VAChT, synaptophysin and CgA co-traffic for 15 minutesthrough a previously uncharacterized trans-Golgi subcompartmentcontaining Golgin97 and TGN46, but not TGN38, before partingways to SPTVs and LDCVs. Moreover, the trans-Golgisubcompartment recruits VAMP4, and some VAMP2 and SNAP25,the subunits of SNARE complex required for regulated secretion,suggesting that this compartment is the place where the vesicularmembranes of SPTVs and LDCVs are equipped with similarexocytosis machinery. A similar compartment which synaptophysinand CgA cohabit transiently before segregating into SPTVs andLDCVs was also found in rat cortical neurons.

ResultsThe SLMV marker VAChT–GFP and the LDCV marker CgA–RFP partially colocalize in a Golgi subcompartment atsteady stateWe investigated how SLMVs and LDCVs are distributed withrespect to each other in steady state PC12 cells. GFP was taggedto the C-terminus of VAChT (VAChT–GFP) and monomeric RFPwas C-terminally tagged to CgA (CgA–RFP) and they weretransfected into undifferentiated PC12 cells, which are known tohave both SLMVs and LDCVs (Greene and Rein, 1977; Greeneand Tischler, 1976; Melega and Howard, 1981). We examined thedistribution of VAChT–GFP and CgA–RFP 18 hours aftertransfection. VAChT–GFP did not show any colocalization withCgA–RFP at the cell periphery (Fig. 1A, inset i–iii). This suggeststhat VAChT and CgA do not co-exist in the same vesicles at thecell periphery, supporting previous findings that SLMV and LDCVproteins are not targeted to the same vesicles (Krantz et al., 2000;Liu and Edwards, 1997; Yao et al., 2004). However, we found thatVAChT–GFP and CgA–RFP colocalized (Fig. 1A, inset iv) insome perinuclear areas of PC12 cells. The extent of colocalizationbetween VAChT–GFP and CgA–RFP was measured by calculating

the overlap coefficient correlation (OCC), which is determined bysimilarity of shapes or areas between two different color images,but not by intensity merge between them. The formula forcalculating the OCC is described in the Materials and Methods.OCC yields lower numbers than the colocalization coefficient(CC), which depends on the intensities of overlapped area, wholeregion of interest and background. In our measurement ofcolocalization, an OCC of 0.4 is equivalent to a CC of 0.65, whichis regarded as significant colocalization. Therefore, only OCCvalues higher than 0.25 (CC of 0.5) are considered evidence ofsignificant colocalization. The average VAChT and CgA OCCvalues of all PC12 cells at steady state (i.e. at 37°C) was 0.14±0.02

736 Journal of Cell Science 124 (5)

Fig. 1. VAChT–GFP and CgA–RFP pass through a common intermediatecompartment. (A)PC12 cells transfected with VAChT–GFP (green) andCgA–RFP (red) under steady state conditions at 37°C were imaged. Themagnified insets i–iii and iv show VAChT–GFP and CgA–RFP at the cellperiphery and at the cell center, respectively. (B)PC12 cells transfected withVAChT–GFP (green) and CgA–RFP (red) were incubated at 20°C to arrestGolgi-to-PM trafficking and then moved to 37°C to induce it. (C)Overlapcoefficient correlations (OCCs) between VAChT–GFP and CgA–RFP weremeasured at 0, 10, 20, 30 minutes after 20°C block and release. The averageOCC ± s.e.m. was calculated from three different experiments (n30). (D)Thereal-time OCCs between VAChT–GFP and CgA–RFP for 20 minutes after20°C block and release were measured (n6 cells). Average OCC ± s.e.m. atevery fifth shot from the beginning to the end of the time-lapse movie(supplementary material Movie 1) was picked and shown on the line graph(*P<0.05). Scale bars: 5m.

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(mean OCC ± s.e.m.), which indicates little colocalizationthroughout the cell, whereas yellow fluorescence at the peri-Golgiarea indicates extensive colocalization in this area. This resultsuggests that VAChT–GFP and CgA–RFP co-exist in a specificcompartment in the Golgi at steady state.

VAChT–GFP and CgA–RFP transiently colocalize in anintermediate compartment during 20°C block and releaseTo monitor the morphodynamics of trafficking of the secretorymembrane proteins, VAChT–GFP and CgA–RFP, as they exit theGolgi complex, we synchronized the Golgi-to-PM vesicular trafficby incubating PC12 cells at 20°C for 30 hours following a 12 hourtransfection with different vesicular markers. After the 30 hours,the cells were transferred to a 37°C incubator to induce Golgi-to-PM trafficking. First, we examined whether GFP-labeled GPI-anchored protein (GPI–GFP), a protein trafficked between theGolgi and the PM (Lippincott-Schwartz, 2004), was effectivelyaccumulated in the TGN after cold temperature block. In the cellsfixed immediately after incubation at 20°C, there was more GFP–GPI protein found in the p115-positive Golgi compartmentcompared with that in cells maintained at 37°C where the proteinwas mainly at the PM (supplementary material Fig. S1), indicatingthat our cold temperature block was effective in stopping exit ofPM proteins from the Golgi.

After cells transfected with VAChT–GFP and CgA–RFP weresubjected to cold temperature block, some cells were placed infixative immediately to indicate time zero after 20°C block, whereasothers were fixed after 10, 20 or 30 minutes at 37°C. We examinedonly cells with a moderate level of expression of both VAChT–GFPand CgA–RFP. The extent of colocalization between VAChT–GFPand CgA–RFP during early Golgi-to-PM trafficking after releasefrom 20°C block was much higher than that at steady state (Fig. 1B).This suggests that the trafficking of VAChT–GFP and CgA–RFPwas successfully synchronized to maximize the amount of VAChT–GFP and CgA–RFP passing through a common compartment duringearly Golgi-to-PM trafficking. However, the maximum colocalizationwas transient and maintained only until ~10 minutes after releasefrom 20°C block. Then, the OCC values decreased and weresignificantly reduced by 30 minutes. The whole-cell OCC valuebetween VAChT–GFP and CgA–RFP was 0.44±0.02 at 0 minutesand gradually decreased to 0.40±0.03, 0.28±0.02 and 0.19±0.02 at10, 20 and 30 minutes, respectively, post release (Fig. 1C). Next, weimaged live cells to monitor this transient colocalization betweenVAChT–GFP and CgA–RFP and their subsequent trafficking. Ourtime-lapse movies showed that the merging between VAChT–GFPand CgA–RFP was maintained until ~15 minutes and graduallydecreased thereafter (supplementary material Movie 1). OCC valueswere also calculated in real time. The OCC value (Fig. 1D) began at0.47±0.04 at 0 time and ended at 0.22±0.04 after 30 minutes at37°C. These values were similar to the OCC values of fixed cells(Fig. 1C). Based on the analysis of six live-cell movies, it is clearthat the converging and diverging events between VAChT–GFP andCgA–RFP during the Golgi-to-PM trafficking are very dynamicprocesses. These findings suggest that VAChT–GFP and CgA–RFPpass through the common compartment during early Golgi-to-PMtraffic before separating.

VAChT–GFP and CgA–RFP show transient colocalizationin three dimensionsPositional overlap of two different markers through the z-axis cansometimes generate apparent colocalization in two-dimensional

images even though two markers might not actually reside in thesame compartment. Therefore, to obtain more accurate informationabout the colocalization between VAChT–GFP and CgA–RFP, wetook a series of images through the z-axis of fixed cells (~4 mthick) that expressed both VAChT–GFP and CgA–RFP, at aninterval of 0.2 m from the top to the bottom surface. The stacksof images were reconstructed to create 3D images. A typical 3Dimage of a cell incubated at 20°C showed that most of the CgA–RFP was accumulated in the compartment containing VAChT–GFP described above (Fig. 2A). However, some VAChT wasdistributed outside the compartment, suggesting that not allVAChT–GFP accumulated in the compartment upon 20°C block.At 10 minutes after release, the high levels of colocalization weremaintained (Fig. 2B) whereas some CgA–RFP appeared to startbudding from the compartment (Fig. 2B, inset). There were moreelongated tubulovesicular buds of CgA–RFP at 20 minutes afterrelease (Fig. 2C). At 30 minutes after release, CgA–RFP wasaccumulated in LDCVs that were free of VAChT–GFP (Fig. 2D)whereas a minimal level of colocalization between VAChT–GFPand CgA–RFP was still detected, similarly to that seen at steadystate of a cell that has never been exposed to 20°C block. Ouranalysis of the colocalization profiles in three dimensions confirms

737Synaptic and granule protein sorting in PC12 cells

Fig. 2. Three-dimensional analysis of the colocalization between VAChT–GFP and CgA–RFP during Golgi-to-PM transport. The colocalizationbetween VAChT–GFP and CgA–RFP was analyzed through the z-axis in cellsexpressing both proteins. The images taken through the z-axis were combinedto generate a 3D image. The images show 3D colocalization between VAChT–GFP and CgA–RFP at 0 minutes (A), 10 minutes (B), 20 minutes (C) and 30minutes (D). Inset: magnified merge. Confocal images were used to generateadjacent 3D images. Scale bars: 5m.

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that CgA–RFP merges with the VAChT–GFP after 20°C block anddynamically separate into distinct vesicular and tubulovesicularprofiles 10 minutes after release.

SYN–GFP traffics with VAChT–GFP and CgA–RFPWe tested another SLMV marker, synaptophysin, using the sameexperimental paradigm to determine whether its trafficking patternis similar to VAChT–GFP relative to CgA–RFP. We usedsynaptophysin tagged C-terminally with either GFP or monomericRFP (SYN–GFP or SYN–RFP). Both SYN–GFP and SYN–RFPtransfected into PC12 cells cofractionated with endogenous SLMVsin velocity sucrose gradient (data not shown). First, we comparedthe distribution pattern of SYN–RFP with that of VAChT–GFPduring 20°C block and release, and found that SYN–RFP andVAChT–GFP were constantly and significantly colocalized (Fig.3A), yielding OCC values close to 0.8 (Fig. 3C). At 10 minutes,both SYN–RFP and VAChT–GFP were accumulated in anintermediate perinuclear compartment. Later, both were graduallysorted into smaller SPTVs and high OCC values (~0.8) were

always maintained. These data indicate that SYN–RFP andVAChT–GFP pass through the same compartments and vesiclesduring Golgi-to-PM trafficking.

Next, we compared the trafficking of SYN–GFP with that ofCgA–RFP. SYN–GFP partially merged with CgA–RFP after 20°Cblock and the extent of the merge was maintained until ~10minutes after release from the block (Fig. 3B,D), yielding ~0.5 forthe OCC value (Fig. 3D). At 20 minutes, the departure of CgA–RFP from SYN–GFP was more prominent and at 30 minutes,CgA–RFP was clearly observed to be separate from SYN–GFP.The timing of merging and diverging between SYN–GFP andCgA–RFP was very similar to that between VAChT–GFP andCgA–RFP (Fig. 1C,D). These data suggest that the traffickingpattern of SLMV proteins, SYN–GFP and VAChT–GFP, relativeto CgA–RFP is representative of the endogenous SLMV traffickingpathway.

The time-dependent changes of colocalization between SYN–GFP and CgA–RFP were reconstructed in 3D (supplementarymaterial Fig. S2). At 0 minutes, most CgA–RFP was accumulatedin intermediate compartments containing SYN–GFP(supplementary material Fig. S2A). The convergence betweenSYN–GFP and CgA–RFP increased in 3D volume after 10 minutesof release (supplementary material Fig. S2B). At 20 minutes, CgA–RFP appeared to bud off from the common compartment(supplementary material Fig. S2C) and more separation betweenSYN–GFP and CgA–RFP was observed at 30 minutes(supplementary material Fig. S2D). Thus, the 3D analysis ofcolocalization between SYN–GFP and CgA–RFP confirmed thatSYN–GFP transiently merges with and then diverges from CgA–RFP in a similar manner to VAChT–GFP.

SLMVs and LDCVs converge in a Golgin97-positive trans-Golgi subcompartmentFirst, we examined the possibility that the incubation at 20°Cdisrupts the Golgi complex, thereby causing accumulation ofvesicular proteins in a non-Golgi structure. Thus, we quantitativelycompared Golgi morphology at 37°C and 20°C. The comparisonshowed that there was no disruptive effect on Golgi structure whencells were incubated at 20°C (supplementary material Fig. S3).Given that 20°C block should prevent the exit of secretory and PMproteins from the Golgi compartments (Simon et al., 1996), weexpected that VAChT–GFP, SYN–GFP and CgA–RFP wouldaccumulate in one of the Golgi compartments, such as the TGN.We fixed cells expressing those constructs at different time pointsafter release from the 20°C block before immunolabeling themwith the antibodies against p115, a marker for cis- and medial-Golgi cisternae. VAChT–GFP, SYN–GFP and CgA–RFP did notaccumulate in the p115-containing compartment, but rather in thearea around the cis- and medial-Golgi cisternae (Fig. 4A–C). After20 minutes, most CgA had diffused away from the peri-p115 area.VAChT–GFP and CgA–RFP did not show significant levels ofcolocalization with p115 during cold block and release (Fig. 4D,OCCVAChT–GFP0.19±0.06, OCCCgA–RFP0.12±0.04, OCCSYN–GFP0.3±0.02), whereas a small pool of SYN–GFP appeared to associatewith the cis and medial Golgi. Therefore, the SPTV and LDCVproteins seem to have accumulated in a p115-negative compartmentwith similarity to that described by Lippincott-Schwartz(Lippincott-Schwartz, 2004).

To further characterize this compartment, we examined whetherVAChT–GFP was accumulated in a TGN38-containing trans-Golgi compartment upon 20°C block, but found that VAChT–GFP


Fig. 3. Syn–RFP co-migrates with VAChT–GFP and is passed through theCgA–RFP-containing compartment. (A)VAChT–GFP (green) and Syn–RFP (red) co-transfected into PC12 cells were tracked together through 20°Cblock and release. (B)Syn–GFP (green) and CgA–RFP (red) in PC12 cellswere tracked together during 20°C block and release. (C)The OCCs betweenVAChT–GFP and SYN–RFP were measured at 0, 10, 20 and 30 minutes afterrelease from 20°C block. (D)The OCCs between Syn–GFP and CgA–RFPthroughout the temperature block and release were measured. The averageOCC ± s.e.m. was calculated from three different experiments (n30; *P<0.05,compared with the OCC at 10 minutes). Scale bars: 5m.

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was neither accumulated in, nor even passed through theTGN38 compartment during 20°C block and release (Fig. 5A;OCCVAChT–GFP for 30 minutes0.15±0.04). Consistent with otherstudies using the 20°C block strategy, we confirmed that none ofthe proteins accumulated in the endoplasmic reticulum (ER) bymeasuring colocalization with the ER marker, calnexin (Fig. 5B;OCCSYN–GFP0.06±0.01 and OCCCgA–RFP0.04±0.02). Given thatthe TGN is heterogeneous with respect to its lipid and proteincomposition, we hypothesized that there might be a non-TGN38trans-Golgi subcompartment where VAChT–GFP, SYN–GFP andCgA–RFP were accumulated upon 20°C block. We labeled cellsexpressing SYN–GFP and CgA–RFP with antibodies againstGolgin97 and TGN46, which are other well-characterized TGNmarkers. Both SYN–GFP and CgA–RFP colocalized with Golgin97(OCCSYN–GFP0.36±0.04 and OCCCgA–RFP0.34±0.05), whereas

SYN–GFP, but not CgA showed significant colocalization withTGN46 (OCCSYN–GFP0.32±0.04 and OCCCgA–RFP0.19±0.04)(Fig. 5C,D). Thus, the Golgin97-positive trans-Golgisubcompartment is the place where most of SYN–GFP and CgA–RFP is accumulated upon 20°C block, whereas the TGN46-positivetrans-Golgi subcompartment appears to retain SYN–GFP andVAChT–GFP, but not CgA–RFP.

A subpopulation of synaptic vesicle proteins remainsassociated with recycling endosomes during 20°C blockand releaseRecycling endosomes located close to the PM are known to housesome of the SLMV proteins recycled from the PM even attemperatures as low as 18°C (Desnos et al., 1995; Schmidt et al.,1997). Generation of SLMVs by endocytosis from the PM hasbeen demonstrated by tracking VAMP (Desnos et al., 1995) andsynaptophysin (Schmidt et al., 1997) trafficking, and by assayingfor recycling endosomes containing transferrin receptor (Desnos etal., 1995; Grote and Kelly, 1996; Lichtenstein et al., 1998;Wiedenmann and Franke, 1985). These studies showed that matureSLMVs come from recycling endosomes, and are not necessarilydirectly derived from the Golgi. Therefore, we hypothesized thatsome of the VAChT–GFP arriving at the PM before 20°C blockmight be present in the recycling endosomes. The recyclingendosomes were visualized with antibodies against transferrinreceptor (TfnR). The majority of the TfnR-labeled recyclingendosomes highly overlapped with VAChT–GFP from thebeginning (time 0) to the end of the 30 minute incubation at 37°C(Fig. 6A) yielding OCC values that were consistently above 0.4.In addition to recycling endosomes just beneath the PM, a traceamount of TfnR was found inside the cell that did not significantlyoverlap with VAChT–GFP (Fig. 6A). These results suggest that thepool of VAChT–GFP that is TfnR positive was neither mobilizedby 20°C block and release, nor re-routed to the newly describedsorting Golgin97-positive TGN, but rather remained in the recyclingendosome pool apposed to the PM. The time-dependent changesof distribution of SYN–GFP and CgA–RFP with respect to theTfnR-positive recycling endosomes were also examined(supplementary material Fig. S4). As with VAChT, a subpopulationof SYN–GFP remained within the recycling endosomes during20°C block and release (supplementary material Fig. S4A), yieldingan OCC range of 0.38–0.50 with TfnR (supplementary materialFig. S4C), whereas the other SYN–GFP pool remained centrally inthe cell. By contrast, CgA–RFP never contacted the TfnR-positiverecycling endosomes (supplementary material Fig. S4B) asevidenced by OCC values of CgA–RFP with TfnR lower than 0.1


Fig. 4. VAChT–GFP, SYN–GFP and CgA–RFP are not accumulated inp115-containing cis–medial Golgi cisternae after 20°C block and release.The cis and medial Golgi (red) was visualized with a monoclonal antibodyagainst p115 and compared with VAChT–GFP (A), SYN–GFP (B) and CgA–RFP (C). (D)The average OCC ± s.e.m. of VAChT–GFP, SYN–GFP andCgA–RFP with respect to p115 was calculated from three differentexperiments (n30). Scale bars: 5m.

Fig. 5. SPTV and LDCV proteins are accumulated in aTGN subcompartment that contains Golgin97 andsome TGN46, but no TGN38 or calnexin (ER) after20°C block. (A)The location of the trans-Golgi markerTGN38 (red) tagged with HA visualized by anti-HApolyclonal antibody was compared with that of VAChT–GFP during temperature block and release. The averageOCC ± s.e.m. of VAChT–GFP with respect to TfnR wascalculated from three different experiments (n30). SYN–GFP- or CgA–RFP-expressing cells were incubated at20°C and labeled with primary antibodies against calnexin(ER) (B), Golgin97 (TGN) (C) and TGN46 (D). Scale bars:5m.

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at any time point (supplementary material Fig. S4D). This indicatesthat CgA–RFP is never routed to the recycling endosomes.

In addition to TfnR, possible colocalization of SYN–GFP andCgA–RFP with EEA1-containing early endosomes after 20°C blockwas examined. This analysis yielded an OCCSYN–GFP0.20±0.02and OCCCgA–RFP0.04±0.01 for SYN–GFP and CgA–RFP relativeto EEA1, respectively, suggesting that some SYN–GFP, but noCgA reached EEA1-positive early endosomes during 20°C block(Fig. 6B). We then examined whether adaptor proteins involved inclathrin-dependent endocytosis are recruited to the recyclingendosomes using antibodies against adaptor protein-2 (AP-2) orGolgi-associated -adaptin ADP-ribosylation factor binding protein(GGA). Anti-AP2 antibodies yielded poor immunostaining.However, using anti-GGA antibody, we found that GGA did notcolocalize with SYN–GFP, whereas it appeared to surround CgA–RFP-positive compartments (Fig. 6B; OCCSYN–GFP0.09±0.04 andOCCCgA–RFP0.20±0.04).

The trans-Golgi subcompartment containing SYN–GFP andCgA–RFP recruits VAMP4 and some SNAP25 and VAMP2Given that both SLMVs and LDCVs share similar exocytosismachinery (De Camilli and Jahn, 1990; Eaton et al., 2000; Kelly,1991; Kim et al., 2006), it is possible that this newly identifiedTGN subcompartment where both synaptic and LDCV proteinsare accumulated upon 20°C block might recruit SNARE proteinssuch as VAMP2, VAMP4, SNAP25 and syntaxin-1, which areinvolved in exocytosis. We examined this possibility afterconfirming that the TGN subcompartment when transfected withSYN–GFP, can recruit endogenous CgA and conversely that CgA–RFP-containing TGN can recruit endogenous synaptophysin (Fig.7A,B; OCCSYN–GFP and OCCCgA–RFP reached 0.57 and 0.64,respectively). Among the SNARE subunits, VAMP4, a vesicularSNARE protein (v-SNARE), showed the highest levels ofcolocalization with both SYN–GFP and CgA–RFP (Fig. 7C;OCCSYN–GFP0.56±0.04 and OCCCgA–RFP0.41±0.05). VAMP2 wasalso found in the new TGN subcompartment after 20°C block butshowed large variations in the degree of its colocalization withinthe subcompartment in different cells (data not shown). SNAP25partly colocalized with SYN–GFP (OCC0.20±0.05) but not withCgA–RFP (OCC0.06±0.02) (Fig. 7D). However, the t-SNAREsyntaxin-1, which aligned along the PM, colocalized with neitherSYN–GFP nor CgA–RFP (Fig. 7E). Based on these results, wededuce that the TGN subcompartment in which synaptic and LDCVproteins cohabit occurs before post-Golgi trafficking and recruitsSNARE proteins to equip both SPTVs and LDCVs with similarexocytosis machinery for regulated secretion.

Both VAChT–GFP and SYN–GFP exhibit partialcolocalization with CgA–RFP at steady stateThus far, we have demonstrated that there is a newly described trans-Golgi subcompartment where the colocalization of CgA–RFP withVAChT–GFP and SYN–GFP is prominent after 20°C block and canbe maintained up to 15 minutes after release. The larger VAChT–GFP and CgA–RFP colocalization subcompartment observed after20°C block is probably an expanded form of the smallsubcompartment seen at steady state where VAChT–GFP and CgA–RFP colocalized (Fig. 1A). The steady state colocalization betweenVAChT–GFP and CgA–RFP was confirmed by 3D reconstitution(supplementary material Fig. S5A). We also examined the steadystate colocalization between VAChT–GFP and SYN–RFP; andbetween SYN–GFP and CgA–RFP in parallel. The 3D image showedan overall colocalization between VAChT–GFP and SYN–RFPthroughout the cell (supplementary material Fig. S5B). Conversely,the cells expressing SYN–GFP and CgA–RFP only showed partialoverlap in the cytoplasm (supplementary material Fig. S5C). Incolocalization analysis, VAChT–GFP and SYN–RFP extensivelyoverlapped in the same compartment whereas VAChT–GFP versusCgA–RFP and SYN–GFP versus CgA–RFP showed only partialcolocalization (supplementary material Fig. S5D). This suggests thatin this special sorting subcompartment VAChT–GFP, SYN–GFP andCgA–RFP are co-sorted transiently, even at steady state.


Fig. 6. A pool of SPTV proteins, but not LDCVproteins, remains unchanged in recycling endosomesduring 20°C block and release. (A)The recyclingendosomes were marked with the monoclonal antibodyagainst transferrin receptor (TfnR, red) in cells expressingVAChT–GFP during 20°C block and release. The averageOCC ± s.e.m. of VAChT–GFP with respect to TfnR wascalculated from three different experiments (n30). Notethat cells of interest are indicated by arrowheads. SYN–GFP- or CgA–RFP-expressing cells were incubated at20°C and labeled with primary antibodies against EEA1(EEA, early endosomes) (B) and GGA (C). Scale bars:5m.

Fig. 7. The TGN subcompartment containing SYN–GFP and CgA–RFPrecruits the subunits of SNARE complex after 20°C block. (A)SYN–GFP-expressing cells were incubated at 20°C and labeled with antibody againstendogenous CgA (eCgA). (B)CgA–RFP-expressing cells incubated at 20°Care labeled with antibody against endogenous synaptophysin (eSYN). Cellsexpressing either SYN–GFP or CgA–RFP are incubated at 20°C and labeledwith primary antibodies against VAMP4 (C), SNAP25 (D) or syntaxin-1 (E).Scale bars: 5m.

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We compared the steady state distribution of VAChT–GFP,SYN–GFP and CgA–RFP with that of the cis–medial Golgi (p115).Both VAChT–GFP and SYN–GFP partially overlapped with theGolgi (supplementary material Fig. S6A,B), yielding OCC valuesof 0.27±0.05 and 0.32±0.06, respectively (supplementary materialFig. S6G), whereas CgA–RFP never showed any overlap with theGolgi marker (OCC0.02±0.02; supplementary material Fig.S6C,G). We also compared their steady state distribution with therecycling endosomes using TfnR as a marker. Both VAChT–GFPand SYN–GFP were highly enriched in the recycling endosomes(supplementary material Fig. S6D,E) as they were after 20°C blockand release, suggesting that the population of VAChT–GFP andSYN–GFP in the recycling endosomes is constantly refilled at37°C and 20°C and distinct from the population in the sortingsubcompartment. The OCC values of VAChT–GFP or SYN–GFPwith TfnR were 0.37±0.06 and 0.46±0.06, respectively(supplementary material Fig. S6H). By contrast, no CgA–RFP atsteady state was found in the TfnR-containing compartment(supplementary material Fig. S6F), yielding OCC values close to0.03 (supplementary material Fig. S6H). This indicates that CgA–RFP is never recycled or trafficked to the TfnR-positive recyclingendosomes at either 37°C or 20°C.

SYN–GFP partially colocalizes with CgA–RFP in ratcortical neurons after cold temperature blockGiven that CgA also exists in cortical neurons (Adams et al.,1993), the distinct sorting compartment where CgA and synapticvesicle protein cohabit in PC12 cells is expected to also exist inthese neurons. To test this possibility, E18 primary cortical neuronswere transfected with SYN–GFP and CgA–RFP and then incubatedat 20°C or 37°C before being fixed for colocalization analysis.SYN–GFP was found in small compartments where CgA–RFP

accumulated in cortical neurons after cold block (Fig. 8A).Conversely, little to no colocalization between SYN–GFP andCgA–RFP was observed at 37°C (Fig. 8B). The average OCCvalue of ~0.26 (CC~0.5) at 20°C was much higher than ~0.07 at37°C (Fig. 8C). Thus, there appears to be a neuronal compartmentin primary cortical neurons where synaptic vesicle and LDCVproteins cohabit transiently, which is similar to those in PC12 cells.

DiscussionPeptidergic neurons and neuroendocrine cells simultaneouslyoperate two regulated secretory systems comprising synapticvesicles and LDCVs to respond to different physiologicalrequirements. Synaptic vesicles (and SLMVs in PC12 cells)package and release neurotransmitters such as acetylcholine, -aminobutyric acid and glutamate, to mediate crosstalk betweenneurons (Blusztajn and Berse, 2000; Parsons et al., 1993; Vizi etal., 1989; Weihe et al., 1996) and between neuroendocrine cells(Bauerfeind et al., 1993), whereas LDCVs secrete monoaminesand various neuropeptides for neurotransmission and to modulateneurophysiological homeostasis. Nonetheless, it is thought thatsynaptic vesicles and SLMVs share many components (e.g.synaptotagmin and VAMP2) of the exocytosis machinery withLDCVs (De Camilli and Jahn, 1990; Eaton et al., 2000; Kelly,1991; Kim et al., 2006). The commonality in the usage of exocytosismachinery components suggests a shared sorting compartmentwhere SLMVs and LDCVs converge and acquire similar proteinsnecessary for exocytosis. In this study, we report the existence ofsuch a compartment. We discovered a previously uncharacterizedTGN subcompartment where SLMV and LDCV proteins cohabittransiently during transport from the Golgi to the PM. The TGNsubcompartment contains Golgin97, some TGN46, but littleTGN38, and recruits VAMP4, and some VAMP2 and SNAP25,subsequently. We propose that this compartment is the final sortingcompartment to generate SPTVs and LDCVs, to allow moreefficient acquisition of their common exocytosis machinery.

Cold temperature block has been used to stop the exit of secretoryand transmembrane proteins from the ER at 15°C (Milgram andMains, 1994) and the Golgi at 20°C in different cell lines (Griffithset al., 1985; Kuliawat and Arvan, 1992; Milgram and Mains, 1994;Simon et al., 1996). Incubation of cells at 20°C has been reportedto have minimal effect on the rate of protein synthesis (Griffiths etal., 1985) and clathrin coat formation for vesicle budding (Simonet al., 1996), but has significant effects on retarding the mobilityand metabolism of membrane lipids required for generation ofmembrane curvature and fission, resulting in co-accumulation ofsecretory and Golgi resident proteins at the TGN (Simon et al.,1996).

In our studies, cold temperature block of PC12 cells resulted inthe accumulation at the Golgi of a GFP-tagged GPI-anchoredprotein, which traffics between the Golgi and the PM (Lippincott-Schwartz, 2004). By contrast, SYN–GFP, a SPTV marker that co-migrates with VAChT–GFP during post-Golgi transport, showedhigh levels of colocalization with CgA–RFP at 20°C in a TGNsubcompartment containing Golgin97 and some TGN46, but littleTGN38 and p115 (cis–medial Golgi). From these results, weconclude that this Golgin97-positive TGN subcompartment iswhere SPTV and LDCV proteins transiently cohabit for morerefined sorting of these proteins into separate vesicles. In corticalneurons, which are known to have synaptic and peptidergic vesicles,as well as a regulated secretory pathway, we found a TGNsubcompartment that was similar to that in PC12 chromaffin cells.


Fig. 8. Cortical neurons show colocalization of SYN–GFP and CGA–RFPafter cold temperature block at 20°C. (A)After cold temperature block,SYN–GFP and CGA–RFP colocalize in the membranous compartment ofcortical neurons. (B)The colocalization of SYN–GFP and CGA–RFP is notdetectable at steady state at 37°C. (C)The average OCC between SYN–GFPand CGA–RFP in cortical neurons is higher at 20°C than at 37°C (*P<0.05).Scale bars: 5m.

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SLMV-targeted proteins, such as VAChT and synaptophysin,are constantly transported to the PM in SPTVs and then recycleddirectly to SLMVs, or to the recycling endosomes where SLMVsare generated (Desnos et al., 1995; Grote and Kelly, 1996;Lichtenstein et al., 1998; Wiedenmann and Franke, 1985).Consistent with the latter, we observed a pool of SYN–GFP andVAChT–GFP, which stayed in the TfnR-positive recyclingendosomes, and was not significantly affected by 20°C block. Bycontrast, LDCV proteins such as the neuropeptide precursor,chromogranin A (CgA), are sorted and packaged at the inner faceof the TGN at cholesterol- and sphingolipid-rich membranemicrodomains that bud off to form LDCVs (Dhanvantari and Loh,2000; Gondré-Lewis et al., 2006; Kim et al., 2006). These LDCVsare transported to secretion sites and stored until stimulated release,without recycling at the PM to recycling endosomes. Since SLMVs(or SVs) are separated from LDCVs in density gradientfractionation (Liu and Edwards, 1997; Yao et al., 2004), it isthought that SLMVs (or SVs) and LDCVs do not cross eachother’s traffic route. However, under normal conditions, someLDCV proteins are found in SLMVs (or SVs) and vice versa, inboth neuroendocrine cells (De Camilli and Jahn, 1990; Kelly,1991; Liu and Edwards, 1997; Yao et al., 2004) and neurons(Agoston and Whittaker, 1989; Nirenberg et al., 1997; Nirenberget al., 1995), suggesting that the idea of complete separation oftheir sorting and trafficking routes might not be entirely accurate.

Indeed, our studies show that even at steady state, there exists acompartment where VAChT–GFP and CgA–RFP colocalizeadjacent to the Golgi (Fig. 1A). This compartment might be similarto structures described in previous studies as large immaturegranules or immobile vesicular compartments in the cell soma(Arvan and Castle, 1998; Rudolf et al., 2001; Santos et al., 2001;Thoidis and Kandror, 2001). However, an additional function ofthose structures as a sorting compartment where synaptic vesicleand LDCV proteins converge was never attributed to them. Usinga temperature block at 20°C and release at 37°C, we were able toclearly visualize induced accumulation and flow of VAChT–GFPand CgA–RFP through this compartment. The OCC values andthree-dimensional reconstitution of VAChT–GFP or SYN–GFPand CgA–RFP after temperature block and release demonstratethat a significant amount of VAChT–GFP or SYN–GFP and CgA–RFP pass through this sub-TGN sorting compartment for about 15minutes after release from 20°C block. Later, VAChT–GFP orSYN–GFP and CgA–RFP are split into SPTVs and LDCVs,respectively. We tried to separate this intermediate compartmentfrom other floating membranous compartments, such as PM andcis- and medial-Golgi cisternae, by subcellular fractionation, butcould not (data not shown), suggesting that it has similar buoyancyto those cell components.

Why would the cell sort synaptic and LDCV proteins into acommon compartment? Synaptic vesicles, SLMVs and LDCVsappear to use a similar set of exocytic machinery proteins (e.g.SNAP25, VAMP proteins, etc.) for regulated exocytosis. Forexample, VAMP2/synaptobrevin2, an exocytic machinery proteinin SLMVs or synaptic vesicles (Pennuto et al., 2003) and LDCVs(Eaton et al., 2000; Kim et al., 2006; Nevins and Thurmond, 2005)mediates exocytosis for both. Indeed, we found that the Golgin97-positive TGN subcompartment where both SPTV and LDCVproteins transiently cohabit contained a significant amount ofVAMP4. VAMP2 also appears to surround the TGNsubcompartment, although the extent of its colocalization with thecompartment varied considerably in different cells. SNAP25 was

recruited to SPTVs later, but not so much to LDCVs. Thisobservation substantiates our idea that the newly found Golgin97TGN subcompartment is the additional sorting station where asimilar set of exocytosis machinery proteins are conferred on thesurface of SLMVs and LDCVs. Mechanisms have been describedin endocrine cells for budding off of ‘immature’ LDCVs from theTGN at cholesterol- and sphingolipid-rich membrane microdomains(‘lipid rafts’), which might also be enriched with components ofthe exocytosis machinery (Dhanvantari and Loh, 2000; Zhang etal., 2003). To overcome the great heterogeneity of proteinsassociated with the Golgi that renders it more difficult for SPTVsto specifically obtain a similar set of vesicular membrane proteinsas LDCVs, we propose that both SPTV and LDCV proteins are co-accumulated in this TGN subcompartment, which recruits SNAREmachinery subunits simultaneously at its cytoplasmic surface.

We provide a model of the intracellular distribution of SPTVand LDCV proteins in undifferentiated PC12 cells after 20°C block(Fig. 9). We estimate the percentage of intracellular partition ofSPTV and LDCV proteins after 20°C block based on the principleof OCC values that are determined by the extent of similarity ofshapes and areas overlapped by two different markers. At 20°C,SPTV proteins appear to reside in two major compartments: the


Fig. 9. Model of intracellular partitioning of SPTV and LDCV proteinsafter 20°C block. Percentage overlap of SPTV or LDCV markers within eachcompartment was estimated from the OCC values that were determined by thearea and shape superimposed by two different markers. For example, theOCC0.3 is converted to 30% overlap. SYN–GFP, a SPTV marker, appears tobe divided into central and peripheral populations. The central SYN–GFPpopulation is distributed within the Golgi compartment with overlap withvarious markers as follows: p115 (10%), TGN38 (VAChT–GFP-based, 10%),TGN46 (30%), Golgin97 (Golgin, 30%), VAMP4 (50%) SNAP25 (20%) andan undefined amount of VAMP2. 40% of the peripheral SYN–GFP overlapswith endosomes containing TfnR (REs: 40%). Conversely, most CgA–RFP, aLDCV marker, is located centrally in the Golgi compartment with overlap withvarious markers as follows: p115 (10%), TGN38 (not determined: N.D.),TGN46 (10%), Golgin97 (30%), VAMP4 (40%), VAMP2 (30%), GGA (20%)and SNAP25 (10%). Neither SYN–GFP nor CgA–RFP is associated withsyntaxin-1 (Stx)-labeled plasma membrane. None of the CgA–RFP has contactwith recycling endosomes. The VAMP4- and Golgin97-positive TGNsubcompartment (dark shaded area) appears to harbor the majority of SPTVand LDCV proteins after 20°C block.

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VAMP4-positive TGN subcompartment (~50%) and TfnR-containing recycling endosomes (~40%). The VAMP4-positiveTGN subcompartment shares SPTV proteins with the compartmentscontaining TGN46 (~30%), Golgin97 (~30%), SNAP25 (~20%)and some VAMP2. The TfnR-positive recycling endosomes appearto contain a pool of SPTV proteins in early endosomes (~20%).However, the majority of LDCV proteins appear to accumulate inthe VAMP4 (~40%)-positive TGN subcompartments, which partlyoverlap with those containing Golgin97 (~30%), SNAP25 (~10%),GGA (~20%) and VAMP2. No LDCV proteins associated with theTfnR recycling endosomes. The residual population of LDCVproteins might be scattered throughout other Golgi compartments(p115, TGN38, TGN46). Based on the highest OCC values shownby both SYN–GFP and CgA–RFP, the Golgin97-positive TGNsubcompartment is where SPTV and LDCV proteins are co-sortedfor acquisition of SNARE subunits for the regulated exocytosis.

In conclusion, we show that in undifferentiated PC12 cells,SLMV and LDCV proteins are initially sorted and move togetherinto a previously uncharacterized Golgin97 trans-Golgisubcompartment where they are later packaged separately intoSPTVs and LDCVs. Unknown factors might contribute to theseparation, which is worthy of future study. Our study does notsupport co-packaging of SLMV or synaptic vesicle proteins intoLDCVs for delivery to the cell surface. Sorting of SLMV (or SV)and LDCV proteins into this newly identified trans-Golgisubcompartment before parting ways allows for an economic andeffective means for SPTVs and LDCVs to acquire some exocytosismachinery, such as VAMP4, VAMP2 and SNAP25. A similar routeexists in cortical neurons. This implies a role for this compartmentin CNS neuronal function.

Materials and MethodsDNA constructs and antibodiesVAChT–GFP, synaptophysin–GFP (Syn–GFP), and synaptophysin–RFP (Syn–RFP)were given by Zu-Hwang Sheng (NINDS, NIH). CgA–RFP was made in ourlaboratory. HA-tagged TGN38 (HA-TGN38) GPI-anchored protein–GFP and GalT–GFP were from Jennifer Lippincott-Schwartz (NICHD, NIH). The mouse antibodiesagainst p115, SNAP25 and transferrin receptor were obtained from BD Bioscience(San Diego, CA). The rabbit antibodies against HA tag, calnexin, GGA and EEA1were from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit antibody againstCgA (HL6013) was made in our laboratory. The rabbit antibodies against VAMP4,Golgin97, GGA, mouse antibodies against syntaxin-1, synaptophysin, TGN46, andchicken antibodies against VAMP2 were purchased from AbCam (Cambridge,MA).

Cell culture, transfection and immunocytochemistryPC12 cells were grown in DMEM medium (GIBCO-BRL, Life Technologies, GrandIsland, NY) supplemented with 10% FBS (GIBCO-BRL) and 5% horse serum(GIBCO-BRL). 2�104 cells were seeded on 25�25 mm coverslips in 35 mm dishesor six-well plates and grown for 18 hours in growth medium before subjecting themto transfection and immunocytochemistry procedures. Transfection of DNA constructswas performed using LipofectamineTM 2000 according to the manufacturer’s protocol(Invitrogen, Carlsbad, CA). DNA (4 g) plus lipofectamine (10 l) in OPTI-MEMI (GIBCO-BRL) and 1.5 ml DMEM medium were added to the cells, followed byincubation for 18 hours.

For 20°C block and release, PC12 cells grown on coverslips in six-well plateswere transfected with DNA constructs and incubated at 37°C and 5% CO2, 95% airfor 12 hours to allow active protein synthesis. Cells were incubated at 20°C, 5%CO2, 95% air for 20°C block of protein trafficking. After 30 hours, cells on one six-well plate were fixed immediately (0 minutes after release from 20°C block). Theother plates were incubated at 37°C for 10, 20, 30 minutes before fixation, equivalentto 10, 20, 30 minutes after release from cold (20°C) block. We minimized the timefor transfer of cells from 20°C to 37°C by placing both 20°C and 37°C incubatorsclose together.

E18 rat cortical neurons were purchased from GenLantis (San Diego, CA) andtransfected with 8 g of SYN–GFP and CgA–RFP (4 g each) using AmaxaNucleofector (program: O-003). The neurons were incubated in 10% FBS plusDMEM for 2 days for recovery and in B27 neurobasal medium (Genlantis, SanDiego, CA) for two more days for differentiation. The transfected cortical neurons

were incubated for 30 hours either at 20°C or 37°C with 5% CO2, 95% air and thenfixed.

For immunocytochemistry, all steps were performed at room temperature unlessotherwise noted. Cells were rinsed with PBS, fixed in 3.5% formaldehyde in PBSfor 30 minutes, and permeabilized in 0.1% Triton X-100 in PBS for 30 minutes.Cells were then blocked in TTBS (TBS, 0.1% Tween-20 and 2% BSA), incubatedfor 30 minutes in primary antibodies, washed in TTBS (5 minutes, three times), andincubated in Alexa Green (488 nm) or Alexa Red (568 nm) secondary antibody(Molecular Probes, Eugene, OR) for 15 minutes. Samples were washed again andmounted on slides in GEL/MOUNT (Biomeda, Foster City, CA) for analysis.

MicroscopyThe expression of VAChT–GFP, Synaptophysin–GFP/RFP (Syn–GFP/RFP), andCgA–RFP in PC12 cells was determined by visual inspection under a Nikon uprightLABPHOT microscope (Nikon, Kanagawa, Japan). Immunofluorescence microscopywas performed at room temperature using a Zeiss Axiovert 200 M invertedmicroscope (Carl Zeiss, Thornwood, NY) equipped with 100� Zeiss alpha planfluor oil, 1.45 NA, DIC objectives. Images were acquired by a Meta detector forspectral imaging (Carl Zeiss) and digitized using ‘LSM 510 Meta’ software version3.5 (Carl Zeiss). The LSM510 Meta software was also used to calculate the OverlapCoefficient Correlation (OCC) using an absolute frequency of moderate (50 arbitraryunits) intensity pixels in the red and green channels. Region of Interest was limitedto one cell per image.

According to the developer of the software, OCC�i[Sc1i � Sc2 i]/SQRT(�i[S1 i]2

� �i[S2 i]2), where i is ‘i’th pixel in the region of interest, Sc1 is signal intensity ofpixel involved in colocalization in the first channel, Sc2 the signal intensity of pixelinvolved in colocalization in the second channel, S1 the signal intensity of pixel,regardless of colocalization, in the first channel, S1 is the signal intensity of pixel,regardless of colocalization, in the second channel and SQRT represents square root.

For time-lapse imaging, PC12 cells expressing VAChT–GFP or Syn–RFP alongwith CgA–RFP were maintained in Phenol-Red-free DMEM plus 10% FBS with 5%horse serum in a temperature-controlled (37°C) Bioptechs Delta T live-cellenvironmental chamber (Bioptechs, Butler, PA) and imaged using the Zeiss Axiovert200M inverted microscope and ‘LSM 510 Meta’ software (Carl Zeiss) at 1.57second exposure per image acquisition for 1500 acquisitions for 30 minutes. Imageswere converted to 8-bit movies using ‘LSM 510 Meta’ software.

We thank the lab members in SCN, NICHD for technical assistanceand helpful discussions. We thank Zu-Hang Sheng (NINDS, NIH) forthe synaptophysin constructs and Jennifer Lippincott-Schwartz(NICHD, NIH) for the GPI–GFP construct and her helpful discussions.We thank Vincent Schram and Chip Dye in the NICHD MicroscopyImaging Core for technical support. This research was supported bythe Intramural Research Program of the NICHD, NIH. J.J.P. issupported by NICHD K22 and ARRA grants. M.G.L. is supported bythe Howard University Medical Alumni Association (HUMAA)Endowed Chair in the Basic Sciences, an intramural SEED grant, andNINDS/NIH Grant # NS065385. Deposited in PMC for release after12 months.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/124/5/735/DC1

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A distinct trans-Golgi network subcompartment for sorting ... · OCC yields lower numbers than the colocalization coefficient (CC), which depends on the intensities of overlapped

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