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The Journal of Neuroscience, July 16, 2003, 23(15):6188-6199
Previous Article | Next Article
Dual Modes of Endoplasmic Reticulum-to-Golgi Transport in Dendrites Revealed by Live-Cell Imaging
April C. Horton1 andMichael D. Ehlers1,2,3 Departments of 1Neurobiology, 2Cell Biology, and 3Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710
Abstract
Top
Abstract
Introduction
Organelles of the neuronal secretory pathway are critical for the addition of membrane that accompanies neuronal development, as well as for the proper localization of plasma membrane proteins necessary for polarity, synaptic transmission, and plasticity. Here, we demonstrate that two organizations of the secretory pathway exist in neurons: one requiring processing of membrane and lipids in the Golgi complex of the cell body and one in which endoplasmic reticulum (ER)-to-Golgi trafficking is localized to dendrites. Using time-lapse imaging of green fluorescent protein-tagged cargo proteins and compartment markers, we show that organelles of the secretory pathway, including ER, ER exit sites, and Golgi, are present and engage in trafficking in neuronal dendrites. We find that ER-to-Golgi trafficking involves highly mobile vesicular carriers that traffic in both the anterograde and retrograde directions throughout the dendritic arbor. Dendritic Golgi outposts, which appear developmentally during the phase of process outgrowth, are involved in the trafficking of both integral membrane proteins and the secreted neuronal growth factor BDNF. This distributed dendritic Golgi represents an organization of the secretory pathway unique among mammalian cells.
Key words: secretory pathway; protein trafficking; dendrite; local protein synthesis; Golgi; ER exit sites
Introduction
The addition of plasma membrane that accompanies polarization and process outgrowth is a fundamental task of the developing neuron (Futerman and Banker, 1996; Lecuit and Pilot, 2003
In non-neuronal cells, the organelles of the secretory pathway have a highly conserved spatial organization (Hirschberg et al., 1998
Recent studies suggest the presence of local dendritic secretory capacity. For example, trans-Golgi network-derived vesicles undergo calcium-evoked dendritic exocytosis (Maletic-Savatic and Malinow, 1998
In this study, we have used live-cell confocal imaging experiments along with immunocytochemistry in cultured hippocampal neurons to define the secretory pathway in dendrites. We demonstrate that newly synthesized membrane proteins concentrate at punctate immobile ER exit sites present throughout the dendrite, from which they bud into highly mobile carriers. These carriers then traffic either long distances to fuse with somatic Golgi or traffic locally to fuse with numerous distributed dendritic Golgi outposts. These dendritic Golgi outposts are used not only by integral membrane proteins, but also by the secreted neuronal growth factor BDNF. Furthermore, the appearance of dendritic Golgi is developmentally regulated during the period of polarization and process outgrowth. These results reveal a novel distributed organization of the secretory pathway unique among mammalian cells.
Materials and Methods
DNA constructs. cDNA-encoding vesicular stomatitis virus glycoprotein ts045 fused to yellow-fluorescent protein (VSVG-YFP) (Toomre et al., 2000Neuronal culture and transfections. Primary cultures of hippocampal neurons were obtained from rat embryos at embryonic day 18. The entire hippocampus was isolated and dissociated with trypsin, and cells were plated at 60,000 cells/cm 2 in Neurobasal medium (Sigma, St. Louis, MO) supplemented with B27, glutamax I, 5% bovine serum, and 1 µg/ml gentamicin. 5-Fluorouracil deoxyribonucleoside (10 µM) was added 6–7 d after plating, and cells were fed twice weekly thereafter with Neurobasal medium prepared as above but without bovine serum. Hippocampal neurons were grown at 37°C in 5% CO2 on coverslips coated with poly-D-lysine (Sigma). Neurons were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to manufacturer's recommendations, except that 1.5 µg of each DNA in 25 µl Opti-MEM and 1 µl of Lipofectamine 2000 in 25 µl Opti-MEM were mixed and added to coverslips in 12-well plates.
Immunocytochemistry. Immunocytochemistry was performed on hippocampal neurons using monoclonal antibodies against GM130 (Transduction Laboratories, San Diego, CA) and VSVG (a gift from Douglas Lyles, Wake Forest University, Winston-Salem, NC), and polyclonal antibodies against Sec13 (a gift from Wanjin Hong, National University of Singapore, Singapore) and Shank, a marker of excitatory synapses concentrated in dendritic spines (a gift from Eunjoon Kim, Korea Advanced Institute of Science and Technology, Daejeon, Korea). For GM130 staining, coverslips were processed essentially as described previously (Scott et al., 2001
-D-glucopyranoside containing 100 µM bis(sulfosuccinimidyl) suberate (Pierce Chemical, Rockford, IL), for 30 min at room temperature. Cells were then quenched in 0.1% ethylenediamine-HCl, pH 7.5, for 15 min. Coverslips were blocked for 1 hr at 37°C in a buffer of 1% fish gelatin (Sigma, St. Louis, MO), 1% powdered milk, and 1% goat serum in PBS. Cells were then stained in blocking buffer at 4°C overnight. Sec13 antibody was used at a concentration of 0.02 mg/ml. Coverslips were rinsed several times with blocking buffer, and secondary staining was performed in blocking buffer for 1 hr at room temperature. Cells were then rinsed and mounted. For Shank staining, cells were fixed in 4% paraformaldehyde with 4% sucrose, and then permeabilized in 100% ice-cold methanol before blocking and staining.
Live-cell imaging. Twenty-four to 48 hr after transfection, coverslips were imaged in a sealed chamber (Dagan, Minneapolis, MN) filled with imaging buffer consisting of the following (in mM): 120 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2, 10 glucose, and 10 HEPES, pH 7.35, and 250 mOsm to match culture growth medium. Wide-field epifluorescence images were acquired on a Nikon (Tokyo, Japan) TE300 inverted microscope. Confocal images were obtained using a Yokogawa spinning disk confocal (Solamere Technology Group, Salt Lake City, UT), with excitation lines from a 2.5 W Kr-Ar laser (Spectraphysics, Mountain View, CA) selected and shuttered via an acousto-optical tunable filter (Neos Technologies, Melbourne, FL) and emission directed through a filter wheel (Sutter Instruments, Novato, CA) holding bandpass filters (Chroma Technology, Brattleboro, VT). Images were acquired using a 100x or 60x Plan Apochromat objective and analyzed using Metamorph (Universal Imaging, West Chester, PA) with a 12-bit cooled CCD camera (Hamamatsu Inc., Bridgewater, NJ). All live imaging experiments were performed at 32°C. The stage was heated to 32°C using an airstream incubator (Nevtek, Burnsville, VA).
Analysis of carrier trafficking. The locations of VSVG-GFP carriers were tracked through stacks of images by marking the point of maximum intensity within each spot. Coordinates were recorded using the point tracker feature in Metamorph. Trajectories were then plotted using Origin (Microcal Software, Northampton, MA) and overlaid onto the image of the corresponding neuron. Carrier lifetime was defined as the point at which a carrier first appeared to when it was no longer evident, whether because it left the image frame or it merged with another structure.
Analysis of carrier budding. Average fluorescence in a region of constant area was tracked for VSVG-GFP- and Sec24-YFP-labeled puncta. Fluorescence was normalized to the average intensity of a region of the same size in a background region of the same frame.
Analysis of Golgi development. Maximum point projections were created of stacks of confocal images through entire neurons. Regions labeled for GM130 were manually traced. Golgi size was defined as the area of these GM130-labeled regions. Neurons with dendritic Golgi were defined as those that had any GM130 detectable beyond the neuronal soma.
Colocalization of VSVG-YFP and GalTase-CFP. Fluorescence intensity was determined for points along a linear region, five pixels in width, drawn down the length of the dendrite. These intensity values were plotted versus dendritic length, such that peaks on the plot indicate points of fluorescence accumulation. Values were background-subtracted and normalized. The extent of correlation between pixel intensities of each of the two fluorophores, YFP and CFP, was then calculated as a linear regression.
Statistical analysis. Error bars represent SEM. Statistical comparisons were t tests.
Results
Monitoring ER-to-Golgi trafficking in dendrites
To monitor trafficking optically through the early secretory pathway in neuronal dendrites, we used the temperature-sensitive mutant of the vesicular stomatitis virus glycoprotein (ts045), tagged with spectral variants of green fluorescent protein (VSVGGFP) as a representative and classically studied membrane cargo protein (Bergmann, 1989When neurons expressing VSVG-GFP were incubated at 39.5°C to accumulate VSVG in the ER, VSVG-GFP distributed throughout the dendrites and soma (Fig. 1A). This distribution was identical to the widespread ER distribution of both endogenous and exogenous ER resident proteins, including BiP, calnexin, Trap
, and protein disulfide isomerase (data not shown). Moreover, no VSVG-GFP was detectable at the cell surface (data not shown), confirming that at 39.5°C, VSVG was trapped in the neuronal ER. After 2 hr at the permissive temperature of 32°C, surface labeling using an antibody against the luminal epitope of VSVG (Lefrancois and Lyles, 1982
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Figure 1. Secretory cargo exits the neuronal ER via vesiculotubular carriers that traffic bidirectionally in dendrites. A, After incubation for 24 hr at 39.5°C, VSVG-YFP has a diffuse somatodendritic distribution and colocalizes with ER resident proteins (data not shown). Scale bar, 10 µm. B, A confocal slice through the soma of a neuron expressing VSVG-GFP after 2 hr at 32°C. Total VSVG-GFP is shown in green, whereas surface staining using a luminal antibody against VSVG is shown in red. Arrows indicate surface VSVG-GFP. Scale bar, 5 µm. C, Surface labeling of VSVG-GFP in neuronal dendrites after 2 hr at 32°C. Total VSVG-GFP is shown in green, whereas surface VSVG-GFP is shown in red. Scale bar, 5 µm. D, VSVG-GFP distributes diffusely throughout the ER in hippocampal neuron dendrites (15 DIV) after incubation at 39.5°C (top). Five minutes after the switch to 32°C, VSVG-GFP accumulated in punctate, stationary structures in the dendrite (middle, arrowheads). After 15 min at the permissive temperature, VSVG-GFP is present in highly mobile vesiculotubular carriers throughout the dendrite (bottom, arrowheads). In all figures, fluorescence has been inverted so that fluorescent structures appear dark, and time is indicated as minutes:seconds after the switch to 32°C. Scale bar, 1 µm. See supplemental movie S1 (available at www.jneurosci.org) for time lapses. E, Vesicle trajectories plotted on the dendrite of a hippocampal neuron during the first 15 min after release of VSVG-GFP from the 39.5°C block. The highly mobile vesiculotubular carriers trafficked in both the anterograde (yellow tracks) and retrograde (red tracks) directions. The cell soma is at the right. Scale bar, 5 µm. See supplemental movie S2 (available at www.jneurosci.org) for time lapses. F, Successive frames of a time-lapse imaging experiment show a single vesiculotubular carrier (arrowhead) trafficking VSVG-GFP in the direction retrograde to the neuronal soma. The carrier follows a curvilinear path until it reaches the persistent fluorescent structure at the top of the frame (arrow). The carrier then merges with this structure, presumably Golgi. Scale bar, 2.5 µm. See supplemental movie S3 (available at www.jneurosci.org) for time lapses.
In neurons imaged live immediately after release from the 39.5°C block, VSVGGFP had a lacy, diffuse distribution, labeling the ER throughout the dendrites (Fig. 1D, top). Within minutes after switching to 32°C, VSVG-GFP accumulated into scattered, uniformly sized puncta (Fig. 1D, middle). By 15 min after release from the ER, VSVG-GFP was present in many highly mobile tubulovesicular carriers present throughout the dendritic arbor (Fig. 1D, bottom). To track the movement of individual carriers, time-lapse experiments were performed and the resulting trajectories were overlaid on images of dendrites. Such analysis revealed that early post-ER carriers traffic in both anterograde and retrograde directions (Fig. 1E). Figure 1F shows frames from a typical time-lapse experiment of VSVG-GFP in a hippocampal dendrite within the first 15 min after release from the ER. In this example, a carrier (Fig. 1 F, arrowhead) transported VSVG-GFP along a curvilinear path toward the neuronal soma. In the final frame, the carrier merges with the stable, stationary structure in the proximal dendrite (Fig. 1 F, arrow). The total fluorescence of the carrier remained constant, and it moved as a single structure without branching or dividing, suggesting that mobile carriers retain their identity during long-range transport. These data indicate that VSVG-GFP is transported from the ER in carriers that traffic bidirectionally within the dendrites. Such bidirectional traffic of pre-Golgi carriers is inconsistent with a single centralized Golgi destination, and it is in marked contrast to the highly directional centripetal transport of pre-Golgi carriers in nonneuronal cells (Hong, 1998
Functional ER exit sites in distal dendrites
Before leaving the ER proper, cargo accumulates at ER exit sites (Hong, 1998
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Figure 2. Functional ER exit sites are present throughout dendrites. A, Dendritic ER exit sites are shown either by staining for the endogenous COPII protein Sec13 (top) or by expressing Sec24-YFP (middle). Colocalizing puncta are yellow in the merged image (bottom), and representative examples are indicated by arrowheads. Scale bar, 5 µm. B, Sec24-YFP marks stable, stationary dendritic ER exit sites in living hippocampal neuron dendrites (15 DIV). Scale bar, 1 µm. See supplemental movie S4 (available at www.jneurosci.org) for time lapses. C, Five minutes after the shift to 32°C from the 39.5°C block, VSVG-CFP (top) accumulates at ER exit sites marked by Sec24-YFP (middle) in a living hippocampal neuron (14 DIV). Arrowheads mark points of colocalization. Scale bar, 2.5 µm. D, Five minutes after the shift from 39.5 to 32°C, VSVG-CFP (left) accumulates at an ER exit site positive for Sec24-YFP (middle). Scale bar, 1 µm. E, Over the next 5 min, VSVG-CFP buds from the ER exit site shown in D. Frames showing the budding event in the VSVG-CFP channel are presented. In each frame, the arrowhead labels the stationary ER exit site, whereas the arrow labels transport vesicles budding from that site and trafficking to the left of the frame, toward the neuronal soma. Frames were acquired every 9 sec. Scale bar, 1 µm. See supplemental movie S5 (available at www.jneurosci.org) for time lapses. F, Quantitative analysis of VSVG-CFP and Sec24-YFP fluorescence intensity at an ER exit site. Although the Sec24-YFP fluorescence merely declines monotonically because of photobleaching (red), the VSVG-CFP fluorescence (green) fluctuates as vesicles repeatedly bud from the ER exit site and additional cargo accumulates. Loss of fluorescence because of budding events (arrows) are interleaved by periods of additional accumulation of VSVG-CFP at the site (arrowheads). The period encompassing the budding event in E is marked by the asterisk.
To determine whether COPII-laden dendritic ER exit sites concentrate cargo, neurons expressing both VSVG-CFP and Sec24-YFP were imaged 5 min after release from 39.5°C block (Fig. 2C). At this time, VSVG-CFP accumulated at discrete spots throughout the dendrite labeled by Sec24-YFP (Fig. 2C), providing strong evidence that these puncta are bona fide dendritic ER exit sites. The functionality of these dendritic ER exit sites was confirmed in two-color confocal time-lapse imaging experiments of neurons coexpressing VSVG-CFP and Sec24-YFP. Within 5 min after the release from the 39.5°C block, VSVG-CFP entered ER exit sites identified by Sec24-YFP (Fig. 2D). In subsequent frames of the time lapse, VSVG-CFP-laden carriers repeatedly budded off from the initial point of concentration and traveled away from the exit site (Fig. 2E). After each budding event, some VSVG-CFP fluorescence remained behind and subsequently accumulated at precisely the same point (Fig. 2E,F). The oscillating changes in fluorescence corresponding to fluorescence loss during budding events and fluorescence increases during cargo accumulation are quantified in Figure 2F. Similar fluorescence fluctuations indicative of budding events were observed at every stable ER exit site examined during the early phase (5–20 min) of secretory trafficking (n = 5 neurons, 20 exit sites). These data indicate that the cargo at an ER exit site does not leave in a single event, but rather is concentrated and available for multiple rounds of export as a series of carriers bud from the site. Furthermore, these experiments show that dendritic ER exit sites are stable, stationary ER subdomains that are functional components of the neuronal secretory pathway. Finally, these findings demonstrate that cargo carriers can originate at ER exit sites dispersed throughout the dendrite.
Post-ER compartments in dendrites differ in mobility and directionality
An analysis of the secretory trafficking events that occurred after release of VSVG-GFP from the 39.5°C block revealed two phases of post-ER trafficking. Within the first 5 min, VSVG-GFP initially present throughout the diffuse ER (Fig. 3A) accumulated at ER exit sites and budded into highly mobile carriers as described above. These carriers trafficked bidirectionally in dendrites (Fig. 1E), with a quantitative bias toward retrograde transport (Fig. 3A,C,F). Within 20 min, VSVGGFP began to accumulate within perinuclear Golgi structures in the cell soma (Fig. 3B), consistent with the trafficking kinetics of VSVG in non-neuronal cells (Presley et al., 1997
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Figure 3. Rapidly transported secretory cargo accumulates at both somatic and dendritic locations after release from the ER. A, During the first 20 min after the release from the 39.5°C block (left), VSVG-GFP is present in vesiculotubular carriers that are highly mobile. Trajectories of moving vesicles are plotted onto an image of a hippocampal neuron. These carriers may traffic in either the retrograde (red) or anterograde direction (yellow), although in this neuron traffic is predominantly inward. In all panels, the time represents the starting point for the time lapse. Scale bar, 10 µm. B, Between 20 and 38 min after release from the 39.5°C block (right) VSVG-GFP has accumulated in stable stationary structures. These structures include perinuclear Golgi structures in the cell soma (white arrows) and more distributed small dendritic structures (green arrows). Although inwardly (red) and outwardly (yellow) moving carriers are still observed during this interval, many more stationary structures (green) are present compared with the early phase of post-ER traffic in A. Scale bar, 10 µm. C, Vesicle lifetime plots show that VSVG-GFP-containing structures are more transient during the early phases of ER-to-Golgi transport. Each horizontal bar represents a single dendritic VSVG-GFP-labeled structure visualized during a live imaging experiment. The length of the bar shows the period during which the VSVG-GFP-labeled structure was evident. D, Vesicle lifetime plots during the later phase of post-ER trafficking shows that between 20 and 40 min after release from 39.5°C, VSVG-GFP is present in more stable, stationary dendritic structures. Bar color code is as in C.E, Mean carrier life times at early and late phases of secretory trafficking. *p < 0.0001; t test. F, Proportion of VSVG carriers that are inwardly moving (red), outwardly moving (yellow), or static (green) during early and late phases of secretory trafficking. During later phases of post-ER traffic, a larger proportion of VSVG-labeled vesicles are stationary.
Thirty-eight carriers from neurons expressing VSVG-GFP were examined during the time window from 0 to 20 min after the release from the 39.5°C block. These early phase carriers exhibited saltatory movement, being stationary for 15–30 sec before moving rapidly (0.5–1.0 µm/sec) along the dendrite. Carrier trajectory plots revealed movement in both the anterograde and retrograde directions, with quantitative bias toward retrograde transport (26 of 38 carriers moved inward) (Fig. 3C,F). Plots of the lifetimes of these carriers, in which the length of the bar represents the lifetime of one carrier and the position of the bar indicates the time over which the carrier was evident, revealed that these carriers form and disappear throughout this early phase of post-ER trafficking (Fig. 3C). Most importantly, the lifetime of early phase carriers was short (Fig. 3E), averaging 2.2 ± 0.2 min, because they frequently merged with larger structures or disappeared into the much brighter neuronal soma.
The behavior of VSVG-GFP-labeled structures within the time window from 20 to 38 min, when VSVG-GFP concentrates within the Golgi, was strikingly different from the carriers appearing earlier. In contrast to the high mobility and shorter lifetime of the early phase ER-to-Golgi carriers (Fig. 3C), later-phase structures were frequently stable and stationary for much longer periods (Fig. 3D). Although only one VSVG-GFP-labeled puncta was entirely stationary during the early phase (Fig. 3C,F), 13 of the 49 carriers followed were stationary during later intervals (Fig. 3D,F). The trajectories of these late-phase carriers revealed a widespread distribution of these stationary accumulations of VSVG-GFP throughout the dendrites (Fig. 3B). Notably, late-phase VSVG-GFP carriers were also much more stable than early phase carriers. On average, the lifetime of these late-phase puncta was more than three times greater than the earlier, more highly mobile carriers (6.5 ± 0.5 min) (Fig. 3E). Thus, post-ER traffic in dendrites consists of at least three kinetically distinguishable populations of vesicles/carriers that differ in both mobility and lifetime. Moreover, these data indicate that secretory cargo accumulates at both somatic and dendritic locations after release from the ER.
Distributed Golgi outposts in distal dendrites
The presence of VSVG-GFP in punctate structures throughout the dendrites at times when VSVG-GFP is known to accumulate in the Golgi (Presley et al., 1997
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Figure 4. Distributed Golgi outposts in distal dendrites. A, Staining for GM130 reveals the presence of Golgi structures within the neuronal soma (top, arrow) and immediately proximal dendrite (top, arrowhead) (14 DIV). Examination of distal dendrites revealed numerous tubular and punctate GM130-positive structures (lower, arrowheads). B, Dendritic Golgi labeled for GM130 (red) do not colocalize with Shank (green). C, Expression of GalTase-CFP shows the distribution of Golgi in the soma (top, arrow), proximal dendrite (top, arrowheads), and distal dendrite (bottom, arrowheads) in a living 14 DIV hippocampal neuron. Scale bars, 5 µm.
To test whether the distributed outposts of Golgi matrix in dendrites were associated with enzyme-containing Golgi membranes, we expressed galactosyltransferase, a well characterized resident Golgi enzyme, tagged with cyan fluorescent protein (GalTase-CFP) (Cole et al., 1996
Dendritic Golgi outposts receive early secretory cargo
Small Golgi fragments are sufficient to produce a functional secretory system (Pelletier et al., 2000
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Figure 5. Dendritic Golgi function in early secretory trafficking. A, Twenty minutes after release from 39.5°C block, VSVG-GFP (green) collects into fragmented Golgi in both the soma and the dendrites of expressing hippocampal neurons (14 DIV) (top). Both somatic (left) and dendritic (right, arrows) Golgi stain for GM130 (red). Scale bars, 5 µm. B, A live neuron (17 DIV) expressing both VSVG-YFP (red) and GalTase-CFP (green) imaged 20 min after the release from the 39.5°C block shows VSVG-YFP present in somatic (left) and dendritic (right) structures positive for GalTase-CFP. Scale bars, 5 µm. C, Immediately after the 39.5°C block, VSVG-YFP (red) is distributed diffusely throughout the ER of the dendrite of a living hippocampal neuron (15 DIV). GalTase-CFP (green) marks punctate dendritic Golgi outposts (arrows) in the same neuron. Scale bar, 5 µm. D, The same dendrite as in C imaged 20 min after the release from the 39.5°C block. At this point, VSVG-YFP (red) has accumulated in GalTase-CFP-labeled structures (green). Note that the positions of dendritic Golgi outposts (arrows), as marked by GalTase-CFP, remain in a relatively fixed position over the 20 min. E, Time-lapse frames illustrating fusion of a mobile post-ER carrier (arrowhead) with a stationary dendritic Golgi outpost (arrow). Scale bar, 1 µm. See supplemental movie S6 (available at www.jneurosci.org) for time lapses. F, Line-scan analysis along the dendrite shows no initial spatial relationship between VSVG-YFP (red) and GalTase-CFP (green) at 0 min after the release from the 39.5°C block. G, Line-scan analysis 20 min after the release from the 39.5°C block shows that regions of accumulated VSVG-YFP fluorescence (red peaks) correspond to regions of GalTase-CFP fluorescence (green peaks). H, Correlation plot of VSVG-YFP and GalTase-CFP pixel values shows little correlation between initial distributions of fluorescence (0 min). The linear fits to the data (red line) and the correlation coefficients (r) are shown in H and I. I, Twenty minutes later, VSVG-YFP and GalTase-CFP fluorescence values are much more highly correlated as VSVG-YFP traffics into GalTase-CFP labeled structures.
To visualize cargo transport into dendritic Golgi directly, two-color time-lapse imaging was performed on hippocampal neurons expressing VSVG-YFP and GalTase-CFP. After prolonged incubation at 39.5°C, VSVG-YFP localized to diffuse distributed ER in dendrites, whereas GalTase-CFP labeled numerous round or elongated Golgi outposts throughout the dendritic length (Fig. 5C). After 20 min at 32°C, the distribution of GalTase-CFP was essentially unchanged (Fig. 5D, bottom, arrows). However, the distribution of VSVG-YFP changed dramatically over this period, becoming concentrated in the structures labeled positive for GalTase-CFP (Fig. 5D, top, arrows). Furthermore, rapid time-lapse imaging frequently captured the merging of mobile VSVG-laden transport carriers with stationary collections of cargo indicative of dendritic Golgi (Fig. 5E).
The time-dependent increase in colocalization between newly released VSVG-YFP and GalTase-CFP was evident when the fluorescence intensity along the length of the dendrite was plotted for each fluorophore (Fig. 5F,G). Initially, there was little coincidence of intensity peaks for VSVG-YFP and GalTase-CFP (Fig. 5F). However, 30 min later, VSVG-YFP fluorescence appeared in peaks threefold to fourfold more intense than the diffuse pattern at time 0 (Fig. 5G), indicating that VSVG-YFP had accumulated into specific dendritic regions over time. A comparison of the VSVG-YFP and GalTase-CFP plots after 20 min showed that nearly every point of VSVG-YFP accumulation corresponded to a position along the dendrite at which GalTase-CFP was localized (Fig. 5G). Spatial pixel intensity correlation plots revealed that, although initially little correlation existed between VSVG-YFP and GalTase-CFP pixel intensity values (Fig. 5H), after 20 min the correlation between the two proteins increased significantly (Fig. 5I). Finally, although the relative sizes of the GalTase-CFP peaks varied somewhat over time, the position of these peaks did not (Fig. 5, compare F and G). This is consistent with the observed stationary nature of dendritic Golgi over time (Fig. 5C,D). In summary, these findings demonstrate that the requisite machinery exists for ER-to-Golgi trafficking in neuronal dendrites and that this machinery is competent for cargo transport.
The secreted neurotrophic factor BDNF uses both somatic and dendritic Golgi
The dendritic release of soluble growth factors is thought to mediate local signaling and morphological regulation (Yacoubian and Lo, 2000
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Figure 6. BDNF-GFP traffics to somatic and dendritic Golgi. A, BDNF-GFP expressed in a hippocampal neuron (15 DIV). BDNF-GFP is present throughout the neuronal soma (top) and dendrites (bottom) in punctate vesicular structures. B, After 3 hr at 20°C, BDNF-GFP (green) has accumulated into somatic Golgi structures positive for GM130 (red). C, Incubation at 20°C also causes accumulation of BDNF-GFP into dendritic Golgi, identified by staining for GM130. Areas of colocalization are indicated by arrows. Scale bars, 10 µm.
Developmental expansion and partitioning of neuronal Golgi
Although distal dendritic Golgi outposts were present in a subset of neurons, they were not a universal phenomenon in all neurons or all dendrites. To determine the origin of dendritic Golgi outposts, we performed a study of Golgi morphology in developing neurons by staining for GM130 in cultured neurons of various ages in vitro. The growth of neurons in culture is a well described phenomenon and is known to proceed through stages analogous to neuronal growth in vivo (Dotti et al., 19889 µm 2) perinuclear structures with a compact morphology (Fig. 7A, top left, C,D), very similar to the Golgi complex of typical non-neuronal cell types (Shorter and Warren, 2002
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Figure 7. Expansion and partitioning of the Golgi during neuronal development. A, Golgi visualized by staining for GM130 in neurons at early stages of development. At 1 DIV the Golgi complex is small, compact, and exclusively somatic, but subsequently grows along with the neuron (3 DIV). By 4 DIV, Golgi extends far into the dendrites as discrete Golgi outposts (bottom, arrows). Scale bar, 5 µm. B, Golgi in a mature neuron (27 DIV) marked by staining for GM130. Note the presence of dendritic Golgi outposts (arrows). Scale bar, 5 µm. C, Traces of Golgi, labeled by GM130 staining, from neurons of various ages shows the developmentally regulated growth and extension of Golgi into proximal and middle neuronal dendrites. Distal Golgi structures are present but not shown here because of the small size and scale. Scale bar, 5 µm. D, Quantitative analysis of Golgi growth during neuronal maturation. Data are means ± SEM of Golgi area (n = 130 total neurons, with at least 11 for each time point). Normalized histogram of the proportion of neurons with dendritic Golgi during neuronal maturation.
Discussion
Here we report dual modes of early secretory traffic in neurons. Figure 8 summarizes these results in a model of the neuronal secretory pathway. (1) In all neurons, cargo buds from the ER at specialized ER exit sites, located in both the soma and dendrites, and traffics to the neuronal Golgi. (2) In a subset of neurons, Golgi is present only in the neuronal soma, necessitating long-range inward trafficking of pre-Golgi carriers (long-range ER-to-Golgi transport). (3) Other neurons have, in addition to somatic Golgi, dendritic Golgi structures that function in secretory trafficking (local ER-to-Golgi transport). Dendritic ER exit sites and Golgi compartments function in secretory trafficking in parallel with the somatic Golgi, and are thus positioned to serve particular dendritic regions or sets of synapses.
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Figure 8. Proposed model of ER-to-Golgi trafficking in neuronal dendrites. An illustration of the dual organization of early secretory pathway components in neuronal dendrites. See text for details.
Dendritic Golgi outposts are novel components of the secretory pathway
A principal finding from the current study is the observation that many neurons possess both somatic Golgi and discrete, discontinuous Golgi-type structures located far into the dendrites. These dendritic Golgi outposts possess both matrix and enzyme components, and function similarly to the somatic Golgi in the transport of VSVG and BDNF. In other mammalian cells, the Golgi consists of a series of interconnected, flattened cisternae that are located in the perinuclear region (Rambourg and Clermont, 1990Previous ultrastructural studies have reported membranes analogous to Golgi cisternae within the spines of distal dendrites (Pierce et al., 2000
Elaboration of a dendritic arbor and extension of an axon define neuronal shape and are the key morphological features of neuronal maturation (Bradke and Dotti, 2000
The distribution of neuronal Golgi is regulated by development
Here we have shown that dendritic Golgi outposts appear during early periods of neuronal differentiation. Golgi outposts may form as fragments derived from somatic Golgi that expand into dendrites as the dendrites themselves grow. Alternatively, Golgi outposts may form de novo from transitional ER elements in a manner similar to that observed in the unicellular yeast Pichia pastoris (Rossanese et al., 1999Secretory organelles and dendritic synthesis of membrane proteins and secreted factors
Much attention has been given to the possibility of local protein synthesis as a mechanism for synapse-specific modifications underlying synaptic plasticity (Jiang and Schuman, 2002Long-range secretory trafficking to and from the soma
A remarkable feature of the current study is that, although many neurons possess components of the secretory pathway allowing dendritic ER-to-Golgi trafficking autonomous of the neuronal soma, other neurons have a Golgi compartment that is entirely somatic. In these neurons, all cargo exiting the ER in the dendrites must traffic in a manner retrograde to the somatic Golgi. Indeed, even in neurons possessing dendritic Golgi outposts, the bulk of ER-to-Golgi traffic is long-range toward the soma. This raises important questions about protein targeting in these neurons and suggests that pre-Golgi carriers retain specific identities over long distances. In other cells, chaperone proteins cycling between the ER and Golgi may fulfill this function (Klumperman, 2000
Footnotes
Received Jan. 10, 2003; revised May. 9, 2003; accepted May. 9, 2003.This work was supported by grants from the National Institutes of Health (NIH), the American Heart Association, the Alzheimer's Association, the McKnight Foundation, the Klingenstein Fund, the Muscular Dystrophy Association, the Spinal Cord Research Foundation, the National Alliance for Research on Schizophrenia and Depression, the Sloan Foundation, the North Carolina Biotechnology Center, and the Ellison Medical Foundation (M.D.E.) and fellowships from the NIH Medical Scientist Training Program and the Ruth K.Broad Foundation (A.C.H.). We thank Kai Simons for VSVG3-SP-YFP, Jean-Pierre Paccaud for Sec24-YFP, Bai Lu for BDNF-GFP, Wanjin Hong for Sec13 antibody, Eunjoon Kim for Shank antibody, and Douglas Lyles for VSVG antibody. We also thank Chi Zhang and Haiwei Zhang for culturing neurons and Tom Blanpied, Juliet Hernandez, and Derek Scott for helpful comments on this manuscript.
Correspondence should be addressed to Dr. Michael D. Ehlers, Department of Neurobiology, Duke University Medical Center, Box 3209, Durham, NC 27710. E-mail: ehlers{at}neuro.duke.edu.
Copyright © 2003 Society for Neuroscience 0270-6474/03/236188-12$15.00/0
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