Correlative light-electron microscopy reveals the tubular-saccular ultrastructure of carriers operating between Golgi apparatus and plasma membrane - PubMed (original) (raw)
Correlative light-electron microscopy reveals the tubular-saccular ultrastructure of carriers operating between Golgi apparatus and plasma membrane
R S Polishchuk et al. J Cell Biol. 2000.
Abstract
Transport intermediates (TIs) have a central role in intracellular traffic, and much effort has been directed towards defining their molecular organization. Unfortunately, major uncertainties remain regarding their true structure in living cells. To address this question, we have developed an approach based on the combination of the green fluorescent protein technology and correlative light-electron microscopy, by which it is possible to monitor an individual carrier in vivo and then take a picture of its ultrastructure at any moment of its life-cycle. We have applied this technique to define the structure of TIs operating from the Golgi apparatus to the plasma membrane, whose in vivo dynamics have been characterized recently by light microscopy. We find that these carriers are large (ranging from 0.3-1.7 microm in maximum diameter, nearly half the size of a Golgi cisterna), comprise almost exclusively tubular-saccular structures, and fuse directly with the plasma membrane, sometimes minutes after docking to the fusion site.
Figures
Figure 1
Transport of VSVG–GFP and GPC dynamics in living cells. COS-7 cells expressing VSVG–GFP were incubated for 12 h at 40°C and then shifted to 32°C for observation by time-lapse microscopy. VSVG–GFP fluorescence exhibited a typical ER pattern in cells incubated at 40°C (a). As soon as 10 min after the temperature shift, numerous bright spots (arrows) appeared in the cytoplasm and also in the Golgi area (b), and after 50 min (c; movie 1.2) most fluorescent spots were moving out of the Golgi complex. Three VSVG–GFP-positive GPCs departing from the Golgi were observed (d). The paths of these three GPCs (e) are indicated by their successive positions (recorded every 3 s) with arrows pointing in the direction of motion. Also shown is the velocity (μm/s, on the y-axis) of the three GPCs at different times after exit from the Golgi complex (f). Negative values indicate backward movement. Two GPCs were observed by time-lapse microscopy every 3 s for 60 s after exit from the Golgi complex. Cells were then fixed and immunolabeled for α-tubulin. Images representing successive positions of GPCs (arrowheads) were electronically overlaid to reconstruct the trajectory of the GPCs (g). These trajectories were then electronically superimposed (using CELLocate coordinates) with the α-tubulin pattern (h). The positions of GPCs at any given time coincide with at least one microtubular structure (h). The fluorescence intensity (i, y-axis) of GPC#1 in d at different times after exit from the Golgi complex, was normalized to its fluorescence at the time of exit (i, filled circles). Also shown (i, open circles) are the average values of normalized fluorescence (calculated as described above) relative to the entire population of GPCs in the same cell at different times after formation. GPCs containing VSVG–GFP (arrows) were monitored in living cells after warm-up from 20 to 32°C (j–l). Cells were then fixed while still recording and immunolabeled for cathepsin D (m). Colocalization of cathepsin D (red) and VSVG–GFP (green) was estimated using confocal microscopy. A clear colocalization was found in the Golgi area, whereas no significant colocalization was found in the GPCs (m, arrows). Bar: (a–c) 8 μm; (d, e, g, and h) 3 μm; and (j–m) 3.2 μm.
Figure 2
In vivo dynamics and ultrastructure of individual GPCs studied using correlative light-EM. The GPC in a (arrow) was picked out in vivo by time-lapse confocal microscopy (see movie 2.1). After fixation (see Materials and Methods), cells were immunoperoxidase-labeled and embedded in resin. The structure in b (arrow) was identified as the fluorescent GPC shown in a by electronic superimposition of the two images in a and b using CELLocate coordinates (an example of which is shown in c). As can be seen, the general pattern of immunoperoxidase labeling in b coincided with the fluorescent pattern of VSVG–GFP in a. Serial sections of the cell were then produced (d–f) and the first section displaying the TI (g, arrow) was captured at low magnifications (f and g). Note that the sections contain structures helpful for identification, for example, a protrusion (c–f, arrowheads). h–q represent a series of consecutive 200-nm sections containing the GPC (arrows). The field shown in h–l is the area of the cell identified with a white box in g. 3D reconstruction characterizes the GPC as an elongated sacculus with a short tubular protrusion (r and s). Other GPCs identified using the same approach appeared either as tubules (t, arrow) or sacculus with short tubules (u, arrow). Bar: (a and b) 9 μm; (c) 31 μm; (d–f) 12.2 μm; (g) 6 μm; (h–l) 2.1 μm; (m–q) 700 nm; (r and s) 320 nm; and (t and u) 1.2 μm.
Figure 3
Dynamics and ultrastructure of individual GPCs as determined by correlative light-EM after release from a 20°C block. A VSVG–GFP-expressing COS-7 cell growing on a CELLocate coverslip was observed after 20–32°C shift in vivo using time-lapse confocal microscopy (movie 3.1; a, arrow and arrowhead). Towards the end of the observation period, the cell was fixed and processed for immunoperoxidase labeling. Two GPCs (b, arrow and arrowhead) were identified as the fluorescent GPCs shown in a on the basis of their coordinates. The general pattern of peroxidase labeling coincided with the fluorescence pattern of VSVG–GFP (compare a and b). To confirm this, the inverted fluorescent image of VSVG–GFP-containing GPCs was overlaid on conventional light images to use intracellular structures (open arrowhead in b, apparently an aggregate of osmiophilic lipid droplets; see also c–h) as reference points in the identification of the GPCs. c–h represent consecutive serial 200-nm sections performed almost tangentially to the basal surface of the cell. Both GPCs (filled arrowhead and filled arrow) were identified in serial sections on the basis of VSVG–GFP labeling and their position relative to the aggregate of lipid droplets (b–h, open arrowhead). 3D reconstruction displayed one GPC as a long tubule (i–k, arrow), whereas the other GPC consisted of globular head and tubular tail (i–k, arrowhead). Bar: (a–c) 10 μm; (c–h) 400 nm; and (i–k) 800 nm.
Figure 4
Fusion of GPCs with PM by correlative light-EM. A VSVG–GFP transfected cell (a–c) was fixed exactly when one of the Golgi-derived fluorescent spots (a and b, arrowheads) started to disappear (b, arrowhead; movie 4.2). The cell surface was then labeled without permeabilization using antibodies against the VSVG ectodomain detected by anti–rabbit Cy3-conjugated antibodies (c, red). Both fuzzy- (c, arrowhead) and sharp-appearing (c, arrow) GPCs were accessible by antibodies, suggesting their contact with the PM. The same approach was used to stain another GPC undergoing fusion (movie 6.3; d–g, arrowhead) using HRP-conjugated secondary antibodies. After resin embedding (f) and sectioning (g), the GPC (f and g, arrowhead) could be easily located using the microvilli (e–g, arrows) as reference marks. Double-labeling of VSVG using immunoperoxidase and immunogold protocols shows patches of both HRP labeling and 10-nm gold particles (h, small arrows) at the GPC fusion site (h, arrow). Bar: (a–c) 9.5 μm; (d–f) 7.5 μm; (g) 930 nm; and (h) 480 nm.
Figure 5
Visualization of a GPC fusion site using scanning EM. Formation, movement, and fusion of GPC (a–e, arrowheads; movie 7.1) was observed under the confocal microscope within a small region (a, black rectangle) of a VSVG–GFP transfected cell. The cell was fixed exactly at the moment of GPC fusion with the PM. The cell surface was then labeled without permeabilization using antibodies against the VSVG ectodomain, and subsequently with protein A conjugated with 30 nm gold, and prepared for scanning EM. The same cell (f, arrows) was identified on the CELLocate grid (the total area shown in a is displayed by the dashed line). g shows the area (indicated by the small white rectangle in f) where GPC fusion occurred. The patch of gold particles lying within the flattened invagination of the cell surface (g, arrowheads) is the GPC fusion site indicated in e (arrowheads). Bar: (a) 9.2 μm; (b–e) 4.2 μm; (f) 10.4 μm; and (g) 410 nm.
Figure 6
Ultrastructure of VSVG–GFP and VSVG containing GPCs. Cells transfected with VSVG–GFP (a and b) or infected with ts-045 VSV (c–g) were fixed and prepared for immunoelectron microscopy using antibodies against the lumenal domain of VSVG. During the 20°C block, VSVG–GFP accumulated in the TGN and in the medial and trans-cisternae of the Golgi stack, whereas the cis compartment remained unstained (a, arrows). Upon warm-up from 20 to 32°C, VSVG–GFP labeling was found not only in the trans-most cisternae of the Golgi (b, arrowheads) and the TGN, but also in tubular structures (b, arrows) detached from the Golgi stack. The PM was also labeled. The same type of labeled tubular structures (arrows) positioned near the PM or Golgi stack were also visible in the cells shifted for 50 min from 40 to 32°C (c) without a 20°C block. Peripheral GPCs appeared both as elongated (d, arrows) or saccular structures (d, arrowhead). To check for colocalization with the endocytic structures, cells were labeled with anti-VSVG antibodies after uptake of WGA-gold conjugate as an endocytosis marker (e). It is evident that no endocytic tracer was found within the GPC (e, arrow), whereas the PM (e, empty arrowheads) and multivesicular body (e, filled arrowheads) were labeled with WGA-gold. Some VSVG-positive tubules (f, arrow) appeared to be connected to the PM. In some cases, an intensely labeled area appeared on the cell surface (g, arrow) presumably in relation to GPC fusion sites. Bar: (a, b, d, e, and g) 500 nm; and (c and f) 400 nm.
Figure 7
3D reconstruction of GPCs. Cells were treated as in Fig. 4. VSVG-labeled structures in 50-nm serial sections were photographed and digitized for 3D reconstruction. a–h show eight consecutive serial sections of the GPC of interest. i and j depict the surface of the GPC (green) and its relationship to the PM (red) at two different angles. Although in each single section the GPC appeared as a tubule (arrows), 3D reconstruction revealed the GPC as a flattened sacculus positioned parallel to the PM. Bar: (a–h) 500 nm; and (i–j) 200 nm.
References
- Alberti S., Miotti S., Stella M., Klein C.E., Fornaro M., Menard S., Colnaghi M.I. Biochemical characterization of Trop-2, a cell surface molecule expressed by human carcinomasformal proof that the monoclonal antibodies T16 and MOv-16 recognize Trop-2. Hybridoma. 1992;11:539–545. - PubMed
- Bergmann J.E. Using temperature-sensitive mutants of VSV to study membrane protein biogenesis. Methods Cell Biol. 1989;32:85–110. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Miscellaneous