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. 2011 Mar 1;124(Pt 5):735-44.
doi: 10.1242/jcs.076372.

A distinct trans-Golgi network subcompartment for sorting of synaptic and granule proteins in neurons and neuroendocrine cells

Joshua J Park  1 , Marjorie C Gondré-Lewis, Lee E Eiden, Y Peng Loh
Affiliations

A distinct trans-Golgi network subcompartment for sorting of synaptic and granule proteins in neurons and neuroendocrine cells

Joshua J Park et al. J Cell Sci. .

Abstract

Golgi-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 undifferentiated neuroendocrine 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 synaptic protein transport vesicles (SPTVs) and LDCVs, after release from temperature block. We found that in this trans-Golgi subcompartment there 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 onto this compartment. Thus, in neuroendocrine cells, synaptic vesicle and LDCV proteins converge briefly in a distinct trans-Golgi network subcompartment before sorting into SPTVs and LDCVs, ultimately for delivery to the plasma membrane. This specialized sorting compartment from which SPTVs and LDCVs bud might facilitate the acquisition of common exocytic machinery needed on the membranes of these vesicles.

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Figures

Fig. 1.
Fig. 1.
VAChT–GFP and CgA–RFP pass through a common intermediate compartment. (A) PC12 cells transfected with VAChT–GFP (green) and CgA–RFP (red) under steady state conditions at 37°C were imaged. The magnified insets i–iii and iv show VAChT–GFP and CgA–RFP at the cell periphery and at the cell center, respectively. (B) PC12 cells transfected with VAChT–GFP (green) and CgA–RFP (red) were incubated at 20°C to arrest Golgi-to-PM trafficking and then moved to 37°C to induce it. (C) Overlap coefficient correlations (OCCs) between VAChT–GFP and CgA–RFP were measured at 0, 10, 20, 30 minutes after 20°C block and release. The average OCC ± s.e.m. was calculated from three different experiments (n=30). (D) The real-time OCCs between VAChT–GFP and CgA–RFP for 20 minutes after 20°C block and release were measured (n=6 cells). Average OCC ± s.e.m. at every 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: 5 μm.
Fig. 2.
Fig. 2.
Three-dimensional analysis of the colocalization between VAChT–GFP and CgA–RFP during Golgi-to-PM transport. The colocalization between VAChT–GFP and CgA–RFP was analyzed through the z-axis in cells expressing both proteins. The images taken through the z-axis were combined to 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 30 minutes (D). Inset: magnified merge. Confocal images were used to generate adjacent 3D images. Scale bars: 5 μm.
Fig. 3.
Fig. 3.
Syn–RFP co-migrates with VAChT–GFP and is passed through the CgA–RFP-containing compartment. (A) VAChT–GFP (green) and Syn–RFP (red) co-transfected into PC12 cells were tracked together through 20°C block and release. (B) Syn–GFP (green) and CgA–RFP (red) in PC12 cells were tracked together during 20°C block and release. (C) The OCCs between VAChT–GFP and SYN–RFP were measured at 0, 10, 20 and 30 minutes after release from 20°C block. (D) The OCCs between Syn–GFP and CgA–RFP throughout the temperature block and release were measured. The average OCC ± s.e.m. was calculated from three different experiments (n=30; *P<0.05, compared with the OCC at 10 minutes). Scale bars: 5 μm.
Fig. 4.
Fig. 4.
VAChT–GFP, SYN–GFP and CgA–RFP are not accumulated in p115-containing cis–medial Golgi cisternae after 20°C block and release. The cis and medial Golgi (red) was visualized with a monoclonal antibody against 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 and CgA–RFP with respect to p115 was calculated from three different experiments (n=30). Scale bars: 5 μm.
Fig. 5.
Fig. 5.
SPTV and LDCV proteins are accumulated in a TGN subcompartment that contains Golgin97 and some TGN46, but no TGN38 or calnexin (ER) after 20°C block. (A) The location of the trans-Golgi marker TGN38 (red) tagged with HA visualized by anti-HA polyclonal antibody was compared with that of VAChT–GFP during temperature block and release. The average OCC ± s.e.m. of VAChT–GFP with respect to TfnR was calculated from three different experiments (n=30). SYN–GFP- or CgA–RFP-expressing cells were incubated at 20°C and labeled with primary antibodies against calnexin (ER) (B), Golgin97 (TGN) (C) and TGN46 (D). Scale bars: 5 μm.
Fig. 6.
Fig. 6.
A pool of SPTV proteins, but not LDCV proteins, remains unchanged in recycling endosomes during 20°C block and release. (A) The recycling endosomes were marked with the monoclonal antibody against transferrin receptor (TfnR, red) in cells expressing VAChT–GFP during 20°C block and release. The average OCC ± s.e.m. of VAChT–GFP with respect to TfnR was calculated from three different experiments (n=30). Note that cells of interest are indicated by arrowheads. SYN–GFP- or CgA–RFP-expressing cells were incubated at 20°C and labeled with primary antibodies against EEA1 (EEA, early endosomes) (B) and GGA (C). Scale bars: 5 μm.
Fig. 7.
Fig. 7.
The TGN subcompartment containing SYN–GFP and CgA–RFP recruits the subunits of SNARE complex after 20°C block. (A) SYN–GFP-expressing cells were incubated at 20°C and labeled with antibody against endogenous CgA (eCgA). (B) CgA–RFP-expressing cells incubated at 20°C are labeled with antibody against endogenous synaptophysin (eSYN). Cells expressing either SYN–GFP or CgA–RFP are incubated at 20°C and labeled with primary antibodies against VAMP4 (C), SNAP25 (D) or syntaxin-1 (E). Scale bars: 5 μm.
Fig. 8.
Fig. 8.
Cortical neurons show colocalization of SYN–GFP and CGA–RFP after cold temperature block at 20°C. (A) After cold temperature block, SYN–GFP and CGA–RFP colocalize in the membranous compartment of cortical neurons. (B) The colocalization of SYN–GFP and CGA–RFP is not detectable at steady state at 37°C. (C) The average OCC between SYN–GFP and CGA–RFP in cortical neurons is higher at 20°C than at 37°C (*P<0.05). Scale bars: 5 μm.
Fig. 9.
Fig. 9.
Model of intracellular partitioning of SPTV and LDCV proteins after 20°C block. Percentage overlap of SPTV or LDCV markers within each compartment was estimated from the OCC values that were determined by the area and shape superimposed by two different markers. For example, the OCC=0.3 is converted to 30% overlap. SYN–GFP, a SPTV marker, appears to be divided into central and peripheral populations. The central SYN–GFP population is distributed within the Golgi compartment with overlap with various markers as follows: p115 (10%), TGN38 (VAChT–GFP-based, 10%), TGN46 (30%), Golgin97 (Golgin, 30%), VAMP4 (50%) SNAP25 (20%) and an undefined amount of VAMP2. 40% of the peripheral SYN–GFP overlaps with endosomes containing TfnR (REs: 40%). Conversely, most CgA–RFP, a LDCV marker, is located centrally in the Golgi compartment with overlap with various 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 with syntaxin-1 (Stx)-labeled plasma membrane. None of the CgA–RFP has contact with recycling endosomes. The VAMP4- and Golgin97-positive TGN subcompartment (dark shaded area) appears to harbor the majority of SPTV and LDCV proteins after 20°C block.
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