Visualization of TGN to endosome trafficking through fluorescently labeled MPR and AP-1 in living cells - PubMed (original) (raw)
Visualization of TGN to endosome trafficking through fluorescently labeled MPR and AP-1 in living cells
Satoshi Waguri et al. Mol Biol Cell. 2003 Jan.
Abstract
We have stably expressed in HeLa cells a chimeric protein made of the green fluorescent protein (GFP) fused to the transmembrane and cytoplasmic domains of the mannose 6-phosphate/insulin like growth factor II receptor in order to study its dynamics in living cells. At steady state, the bulk of this chimeric protein (GFP-CI-MPR) localizes to the trans-Golgi network (TGN), but significant amounts are also detected in peripheral, tubulo-vesicular structures and early endosomes as well as at the plasma membrane. Time-lapse videomicroscopy shows that the GFP-CI-MPR is ubiquitously detected in tubular elements that detach from the TGN and move toward the cell periphery, sometimes breaking into smaller tubular fragments. The formation of the TGN-derived tubules is temperature dependent, requires the presence of intact microtubule and actin networks, and is regulated by the ARF-1 GTPase. The TGN-derived tubules fuse with peripheral, tubulo-vesicular structures also containing the GFP-CI-MPR. These structures are highly dynamic, fusing with each other as well as with early endosomes. Time-lapse videomicroscopy performed on HeLa cells coexpressing the CFP-CI-MPR and the AP-1 complex whose gamma-subunit was fused to YFP shows that AP-1 is present not only on the TGN and peripheral CFP-CI-MPR containing structures but also on TGN-derived tubules containing the CFP-CI-MPR. The data support the notion that tubular elements can mediate MPR transport from the TGN to a peripheral, tubulo-vesicular network dynamically connected with the endocytic pathway and that the AP-1 coat may facilitate MPR sorting in the TGN and endosomes.
Figures
Figure 1
Distribution of the GFP-CI-MPR chimeric protein. (A) HeLa cells stably expressing GFP-CI-MPR were fixed and examined by fluorescence microscopy. (B and C) HeLa cells stably expressing GFP-CI-MPR (green) and expressing transiently markers of the trans-Golgi network, namely gpI (B) or a VSVG epitope-tagged sialyltransferase (ST; C). The cells were fixed and immunolabeled with a mAb against the VSVG epitope or the SG1 anti-gpI mAb followed by a Texas Red–conjugated goat anti-mouse antibody (red). (D) HeLa cells expressing GFP-CI-MPR (green) were fixed and labeled with a polyclonal antibody against the luminal domain of CI-MPR (red). (E) These cells were also allowed to internalize Alexa594-labeled transferrin for 10 min at 37°C (Tf-Alexa594; E). (B–D) The GFP signal in the Golgi region; (E) GFP signal in the periphery of the cell. Merged images are presented in the right column. Arrows indicate long tubules emanating from the perinuclear region. Arrowheads in E indicate examples of the overlap between both signals. Bars, 10 μm. (F) Thawed thin sections of HeLa cells stably expressing GFP-CI-MPR and transiently expressing the VSVG epitope-tagged sialyltransferase were double immunolabeled with a polyclonal anti-GFP antibody (5 nm colloidal gold, arrow) and a monoclonal anti-VSVG antibody (10 nm colloidal gold). Bar, 0.2 μm.
Figure 2
Formation of tubular elements from the TGN. (A) HeLa cells expressing the GFP-CI-MPR were examined by videomicroscopy. Images were taken at 2-s time intervals. Inverted images at the indicated time intervals are displayed. Arrowheads or squares indicate the tips of TGN-tubules, the arrows indicate the detachment of long tubular elements from the TGN and the breaking points into smaller tubular elements. The short line indicates a more intense GFP labeling that moves along the tubule toward the tip and then detaches from it. (B) Movement of TGN-derived tubules toward the cell periphery. Images were collected at 1-s intervals for 1 min. Paths of different TGN-derived tubules were traced as colored lines. The circles indicate fusion with GFP-CI-MPR containing structures. Bars, 10 μm.
Figure 3
Effect of brefeldin A. (A) Images of GFP-CI-MPR–expressing cells were taken at 2-s time intervals during BFA treatment. Inverted images at the indicated time intervals (min:sec) are presented. The first frame (0:00) shows the cell immediately after adding BFA. Bar, 10 μm. (B) Growth of tubular elements in the absence (BFA−) or the presence (BFA+) of brefeldin A. Four examples of growing TGN tubules are shown.
Figure 4
Fate of tubular elements detaching from the TGN. (A) TGN-derived tubular elements mix with GFP-labeled peripheral structures. An area of a GFP-CI-MPR–expressing HeLa cell was selected as indicated in the left panel and examined by videomicroscopy. Images were taken at a high speed (3 frames per second). Inverted images are presented. Sequences of images taken at the indicated time intervals are displayed in B. The arrow indicates a tubular element detaching from a TGN tubule and fusing with a peripheral compartment labeled with the GFP. (B) GFP-labeled, peripheral structures are highly dynamic. Cells were examined as in A. The arrow and the asterisk indicate different peripheral structures containing the GFP-CI-MPR. (C) Mixing of GFP-CI-MPR–positive structures with endocytic compartments. GFP-CI-MPR–expressing cells were incubated at 4°C with Alexa594-transferrin for 30 min and then washed and reincubated at 37°C for 10–15 min. The cells were examined by confocal microscopy. Images were recorded every second. The arrow indicates the overlap between the two fluorescent markers. Bar, 2 μm.
Figure 5
Distribution of endogenous coat components. (A–C) HeLa cells expressing GFP-CI-MPR (green) were fixed, labeled with either the 100.3 mAb against γ-adaptin (red; AP-1; A and C) or a polyclonal antibody against clathrin (red; B), and examined by laser confocal microscopy. (A and B) the signals in the Golgi region; (C) signals in the periphery of the cell. Note that AP-1 or clathrin signals appear as concentrated spots along the TGN tubules (arrowheads).
Figure 6
Steady state distribution of YFP-tagged γ-adaptin. (A and B) HeLa cells expressing both CFP-CI-MPR (green) and YFP-γ-adaptin (red; YFP-AP1) were treated without (A) or with BFA (B) for 10 min and fixed. (C) Hela cells expressing YFP-γ-adaptin (green) were also labeled with antibodies against clathrin (red). (D) HeLa cells expressing GFP-γ-adaptin (green; GFP-AP1) were allowed to internalize Alexa594-labeled transferrin for 10 min at 37°C (red; Tf-Alexa594). Merged images are presented in the middle column. Bars, 10 μm
Figure 7
Dynamics of YFP-AP-1 and CFP-CI-MPR. HeLa cells expressing both CFP-CI-MPR (green) and YFP-γ-adaptin (red; YFP-AP1) were observed by time-lapse laser scanning microscopy. Images were taken every 1.5 s with multitrack mode. A selected area indicated in A is displayed in B and C at the time intervals as indicated. Merged images are shown at the bottom. (B) A TGN-derived tubular element (arrow) without any AP1-coat moves toward the periphery and acquires an AP1-coat. (C) Another tubular element with an AP1-coat (arrow) detaches from the TGN and appears to fuse with the peripheral compartment (asterisk) that has been formed in B. Bars, 10 μm.
Figure 8
Schematic representation of MPR transport. In this model, tubular elements, containing the GFP-CI-MPR and partly coated with AP-1 and clathrin patches, detach from the TGN, and fuse with peripheral tubulo-vesicular structures also containing this marker. These structures rapidly fuse with each others probably forming a highly dynamic network whose various separate elements make contact with early endocytic structures. Typical clathrin-coated vesicles, as purified using classical methods, could represent a subpopulation of these tubulo-vesicular transport intermediates selected for their size and high density. It is also possible that these tubulo-vesicular transport intermediates are fragile and yield vesicles upon cell breakage.
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