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. 2013 Apr;24(7):1020-9.
doi: 10.1091/mbc.E12-08-0575. Epub 2013 Feb 6.

The Small Molecule Dispergo Tubulates the Endoplasmic Reticulum and Inhibits Export

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Free PMC article

The Small Molecule Dispergo Tubulates the Endoplasmic Reticulum and Inhibits Export

Lei Lu et al. Mol Biol Cell. .
Free PMC article

Abstract

The mammalian endoplasmic reticulum (ER) is an organelle that maintains a complex, compartmentalized organization of interconnected cisternae and tubules while supporting a continuous flow of newly synthesized proteins and lipids to the Golgi apparatus. Using a phenotypic screen, we identify a small molecule, dispergo, that induces reversible loss of the ER cisternae and extensive ER tubulation, including formation of ER patches comprising densely packed tubules. Dispergo also prevents export from the ER to the Golgi apparatus, and this traffic block results in breakdown of the Golgi apparatus, primarily due to maintenance of the constitutive retrograde transport of its components to the ER. The effects of dispergo are reversible, since its removal allows recovery of the ER cisternae at the expense of the densely packed tubular ER patches. This recovery occurs together with reactivation of ER-to-Golgi traffic and regeneration of a functional Golgi with correct morphology. Because dispergo is the first small molecule that reversibly tubulates the ER and inhibits its export function, it will be useful in studying these complex processes.

Figures

FIGURE 1:
FIGURE 1:
Dispergo inhibits the traffic of VSVGts-GFP from the ER to the plasma membrane. (A) Structures of synthetic dispergo and the related natural product carpanone. (B) Inhibitory effect of dispergo on the arrival of VSVGts-GFP to the cell surface. BSC1 cells were transduced with adenovirus to express VSVGts-GFP overnight at 40°C. Cells were subsequently incubated with DMSO (control), dispergo, or carpanone at the nonpermissive temperature (40°C) for 1 h, followed by incubation at the permissive temperature (32°C) for 3 h. VSVGts at the cell surface was detected by incubation by the 8G5 monoclonal antibody specific for the ectodomain of VSVG. Scale bar, 60 μm. (C) The effect of dispergo on the arrival of VSVGts-GFP to the cell surface was quantified by determining for each cell the ratio of the surface 8G5 fluorescence signal corrected by background to the total VSVGts-GFP fluorescence signal. Data are presented as a semilog plot for results obtained from ∼1500 cells imaged in three independent experiments. Values for each point are mean ± SD; the IC50 (∼6.8 μM) was calculated as the concentration at which 50% of the VSVG signal was present at the cell surface.
FIGURE 2:
FIGURE 2:
Dispergo inhibits the traffic of VSVGts-GFP from the ER to the Golgi apparatus. BSC1 cells stably expressing GalT-tomato were transduced with adenovirus to express VSVGts-GFP overnight at 40°C. Cells were then treated with DMSO (control) or dispergo for 40 min at 40°C and subsequently shifted to 32°C for 20 min. (A) Control cells treated with DMSO only. (B–D) Cells treated with dispergo. Note in B the redistribution of VSVGts-GFP into patches in cells kept at 40°C during the dispergo treatment. (C) Under these experimental conditions, a significant amount of GalT-tomato colocalized with Giantin, validating the use of GalT-tomato as a Golgi marker. Endogenous Giantin was identified by immunostaining. (D) Colocalization of VSVGts-GFP patches with endogenous TRAPα, an ER marker (arrowheads) visualized by immunostaining in cells treated with dispergo. All images were from fixed BSC1 cells acquired using wide-field fluorescence microscopy. Scale bar, 10 μm.
FIGURE 3:
FIGURE 3:
Effect of dispergo on the dynamics and morphology of the ER. (A) The effects of dispergo are reversible. Two-dimensional time-lapse series of BSC1 cells stably expressing GFP-Sec61β treated with dispergo for 1 h, showing the gradual appearance of ER patches (top, solid arrowheads; see Supplemental Movie S1). After a further 2-h incubation with dispergo, the compound was removed (bottom). The time-lapse series shows the gradual disappearance of the ER patches (bottom, open arrowheads; see Supplemental Movie S2). (B, C) Two-dimensional time-lapse series from BSC1 cells expressing mRFP1-Sec61β (B) or GFP-Sec61β (C). The images in B highlight the disappearance of ER cisternae (arrowheads) and appearance of ER patches upon dispergo treatment. The images in C are from an enlarged region of A, bottom, and show the reversal of these effects upon removal of dispergo. Scale bars, 10 μm. (D) Rapid exchange of GFP-Sec61β within ER patches and with adjacent ER. The images are from a FRAP experiment conducted on an ER patch (green box) in a BSC1 cell stably expressing GFP-Sec61β pretreated with dispergo for 3 h (see Supplemental Movie S3). The ER patch within the unbleached region (red box) was used as a FRAP control. Scale bar, 10 μm. (E) Quantification of the mean fluorescence intensity within the boxed regions of the FRAP experiment. (F) Example of electron microscopy images obtained from BSC1 cells treated with dispergo for 3 h in the absence of ectopic expression of proteins. Left, ER patches highlighted with solid arrowheads. N, nucleus. Scale bar, 2 μm. Right, enlarged highlights of the abundance of relatively circular small membrane profiles contained within the ER patches; membrane profiles ∼100 nm in width extending away from the ER patches are highlighted with open arrowheads. Scale bar, 500 nm. (G) Dispergo induces ER tubules and patches in the absence of microtubules. BSC1 cells stably expressing GFP-Sec61β were first treated with nocodazole for 2 h (left), followed by nocodazole and dispergo for an additional 2 h. Scale bar, 10 μm. All fluorescence images were acquired live using spinning disk confocal microscopy.
FIGURE 4:
FIGURE 4:
Dispergo blocks the recruitment of VSVGts-GFP to the ERES. BSC1 cells were transduced with adenovirus to express VSVGts-GFP overnight at 40°C. Cells were treated with either DMSO (control) or dispergo for 40 min (A, C). Subsequently, the temperature was shifted to 32°C for 10 min (40°C→32°C; B, D) before fixation and immunofluorescence detection of endogenous Sec31a. Images were acquired using wide-field fluorescence microscopy. The enlarged boxed regions in the merge images are shown in the bottom left of the corresponding composite images. Arrowheads highlight the colocalization of VSVGts-GFP with Sec31a in control. Scale bar, 10 μm.
FIGURE 5:
FIGURE 5:
Disappearance of the Golgi apparatus and retention of its components in the ER induced by dispergo. (A–C) Cells were treated for 3 h with DMSO (control) or dispergo before immunofluorescence labeling to follow the distributions of several ER (TRAPα, GFP-Sec61β, and Sec31a) and Golgi apparatus (VAMP4-GFP, GM130, ERGIC53-GFP, and GPP130) markers. (A) NRK cells stably expressing Vamp4-GFP immunostained with an anti-TRAPα antibody. Arrowheads highlight the colocalization of Vamp4-GFP and TRAPα in ER patches. (B) BSC1 cells transiently expressing GFP-ERGIC53 immunostained with antibodies specific for GM130 and Sec31a. The arrowheads in the enlarged boxed region highlight colocalization of GFP-ERGIC53 and GM130 at the ERES marked by Sec31a. Empty arrowheads indicate the ERGIC53-positive ER patches induced by dispergo. (C) BSC1 cells stably expressing GFP-Sec61β labeled with anti-GPP130 antibody. The images highlight lack of enrichment of GPP130 in ER patches. (D) Golgi markers had different kinetics of relocation upon dispergo treatment. NRK cells stably expressing Vamp4-GFP without or with dispergo treatment for 20 min were processed for immunofluorescence labeling of GM130. This example highlights that the redistribution of Golgi markers to the ER can occur at different rates (in this case GM130 was faster than Vamp4-GFP). All images were acquired by a wide-field microscope. Scale bars, 10 μm.
FIGURE 6:
FIGURE 6:
Relocalization of the Golgi marker GalT-tomato to the ER induced by dispergo. (A) BSC1 cell transiently expressing GalT-tomato and GFP-Sec61β were treated with dispergo and imaged by spinning disk confocal microscopy (see Supplemental Movie S4). Arrowheads highlight colocalization of GalT-tomato and GFP-Sec61β at ER patches marked by GFP-Sec61β. Scale bar, 10 μm. (B) Time dependence for the loss of GalT-tomato from the perinuclear region corresponding to the Golgi apparatus induced by dispergo in BSC1 cells stably expressing GalT-tomato. The fluorescence intensity of GalT-tomato at the perinuclear region determined before dispergo addition was used to normalize the data. Plot shows mean values and SD for each time point (n = 13 cells). Golgi t1/2 represents the mean time ± SD that it took for the signal of GalT-tomato to be reduced by 50%. “ER patches onset” marks the time at which the appearance of ER patches labeled with GFP-Sec61β was first detected (n = 10 cells).

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