QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 4 January 2005
doi: 10.1242/jcs.01615

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ben-Tekaya, H. Articles by Hauri, H.-P. Search for Related Content PubMed PubMed Citation Articles by Ben-Tekaya, H. Articles by Hauri, H.-P. Social Bookmarking                         What's this?

Live imaging of bidirectional traffic from the ERGIC

¹ Department of Pharmacology and Neurobiology, Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland
² Department of Cell Biology and Biophysics, European Molecular Biology Laboratory Heidelberg, 69117 Heidelberg, Germany

View larger version (96K): [in a new window]   Fig. 1. GFP-ERGIC-53 localizes and oligomerizes like endogenous ERGIC-53. (A) HeLa cells stably expressing GFP-ERGIC-53 were treated with sodium butyrate overnight, fixed with paraformaldehyde, and stained for ß-COP, Sec31 or transferrin receptor. Cells were observed with a confocal fluorescence microscope. Insets show higher magnifications. (B) GFP-ERGIC-53 cells treated with sodium butyrate or non-transfected cells were pulsed with [35S]methionine and chased for the indicated times. Proteins were immunoprecipitated either with anti-ERGIC-53 for the control cells or with anti-GFP for the stable clone. Immunoprecipitates were analyzed by non-reducing SDS-PAGE and visualized by fluorography. During the time course, dimers (lower bands) disappear as hexamers (higher bands) form until a steady state is reached when each species is represented as 50%. GFP-ERGIC-53 dimers appear as two bands corresponding to homodimers and heterodimers (lower arrowhead) with endogenous ERGIC-53. These dimers are converted to hetero-hexamers (upper arrowheads) and homo-hexamers. (C) Quantification of dimer and hexamer formation during the chase period. 100% is the sum of homo-dimers and homo-hexamers at a given chase time. A representative experiment is shown. Bar, 5 µm.

View larger version (63K): [in a new window]   Fig. 2. Live imaging of GFP-ERGIC-53 reveals two types of structures with different dynamics. (A) A whole cell and its schematic representation where paths of stationary structures imaged every 10 seconds have been traced (see supplementary material Movie 2). Note that these structures undergo very short range to no movement. Lower panel shows image series of the outlined portion in the upper panel. Arrowheads point two peripheral ERGIC structures fusing with each other. Arrows indicate elongated ERGIC structures. (B) Representative images from supplementary material Movie 3. GFP-ERGIC-53-expressing HeLa cells were imaged in the xyz directions every 10 seconds. Arrows indicate long-lasting structures that can persist for up to 31 minutes. (C) Data taken from supplementary material Movie 4 imaged with an interval of 0.2 second. Representative frames show three peripheral ERGIC structures (arrowheads) that hardly move during the entire imaging interval. The arrow points to a peripheral stationary spot shooting out an elongated structure. Lower panel shows the whole cell and a schematic representation of paths that GFP-ERGIC-53 fast-moving structures followed during the imaging time. Note that the movement does not follow a defined direction, and often seems to connect two peripheral stationary structures. Bar, 5 µm. Time is shown as minutes.seconds.

View larger version (145K): [in a new window]   Fig. 3. ERGIC stationary structures in the periphery and in the Golgi area are equivalent. (A) Time points from supplementary material Movie 5 showing the relocalization of GFP-ERGIC-53 to the ER when cells are treated with H89. The arrow points to a peripheral ERGIC structure that loses fluorescence and disappears without moving to the Golgi area. (B) Time series from supplementary material Movie 6 showing the recovery of the same cell after H89 washout. The arrow points to a peripheral ERGIC structure that gets brighter during recovery but does not move to the Golgi area (arrowhead) which is refilled at the same time as peripheral ERGIC structures appear. (C) NEM blocks H89-induced recycling of GFP-ERGIC-53 to the ER. HeLa cells stably expressing GFP-ERGIC-53 were treated with H89 for 20 minutes in the presence or absence of NEM or left untreated (control), fixed with paraformaldehyde, stained for Sec13 (red), and analyzed by confocal microscopy. Insets show higher magnifications. Bar, 5 µm. Time is indicated as minutes.seconds.

View larger version (90K): [in a new window]   Fig. 4. GFP-ERGIC-53 tubule formation during rewarming from 16°C. GFP-ERGIC-53-expressing HeLa cells were incubated for 3 hours at 16°C, shifted to 37°C for 1 minute 53 seconds and imaged every 0.6 second (see supplementary material Movie 8). 0 second indicates the time at which imaging started. Extensive tubular structures appear from peripheral ERGIC spots and move with no preference in different directions. A single ERGIC structure can simultaneously extend tubules in opposite directions without being consumed (arrow). Several rounds of tubule emission from the same ERGIC spot do not consume it (arrowhead). Bar, 5 µm. Time, minutes.seconds.

View larger version (22K): [in a new window]   Fig. 5. GFP-ERGIC-53 and VSV G-GFP take different routes from the ERGIC. Quantification of the flow rate direction of GFP-ERGIC-53 (A) and VSV-G-GFP (B) in cells rewarmed from 16°C to 37°C or 15°C to 32°C, respectively. The protein flow rate is plotted in arbitrary units (normalized to 1) against the direction of movement. Note that VSV-G-GFP is directed toward the Golgi whereas GFP-ERGIC-53 is directed away from the Golgi. These differences are statistically significant (Student's t-test, P0.05). Bars show the mean±s.d. (GFP-ERGIC-53, n=67; VSV-G-GFP, n=73); No, nondirectional movement.

View larger version (155K): [in a new window]   Fig. 6. Sorting of GFP-ERGIC-53 and ssDsRed in peripheral ERGIC structures. Data from cells co-expressing GFP-ERGIC-53 and ssDsRed rewarmed after a 3-hour 16°C block. (A) Time series (taken every 10 seconds) from supplementary material Movie 9. 3.30 indicates the time after rewarming to 37°C. The filled arrowhead indicates a stationary GFP-ERGIC-53 structure from which ssDsRed segregates. Note that even in the proximity of the Golgi, the ERGIC stationary structure does not move toward this area. The empty arrowhead indicates another stationary structure from which ssDsRed segregates. 11 minutes 50 seconds later, this same structure receives new ssDsRed material that is shot toward the Golgi at time point 19.50 (red arrowhead). Note that the Golgi (asterisk) becomes redder as the rewarming proceeds. Time, minutes.seconds. (B) Cell imaged with an interval of 0.2 second, 12 minutes after shifting the temperature from 15°C to 37°C. 0.00 indicates the time at which the imaging started. The filled arrowhead points to a stationary structure that segregates ssDsRed as in A. Some GFP-ERGIC-53 structures are positive for ssDsRed and move together towards (arrow) the Golgi (asterisk) or toward other ERGIC structures (empty arrowhead). Often, moving structures carrying both GFP-ERGIC-53 and ssDsRed cross several GFP-ERGIC-53 stationary structures (long arrows). Time, seconds.milliseconds. Bar, 5 µm.

View larger version (30K): [in a new window]   Fig. 7. Model of the organization of the early secretory pathway and sorting of anterograde and retrograde traffic in the ERGIC. The cycling protein GFP-ERGIC-53 (green) and anterograde cargo, such as VSV-G-GFP or ssDsRed (red), leave the ER at ER-exit sites (ERES) of the transitional elements of the rough ER (TE) and are transported to the ER-Golgi intermediate compartment (ERGIC) by a microtubule-independent process. The ERGIC is a collection of stationary tubulovesicular clusters exhibiting short range, non-directional, but microtubule-dependent movement. The clusters can split or fuse with one another (dashed arrow). Sorting in the ERGIC leads to anterograde carriers (AC), mediating transport of ssDsRed to the cis-Golgi and to retrograde carriers (RC) mediating transport of GFP-ERGIC-53 back to the ER. Both pathways are dependent on intact microtubules. Individual ERGIC-clusters are connected by fast-moving carriers (FC) that carry both markers and are transported along microtubules. MT, microtubules.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter