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First published online 28 April 2009
doi: 10.1242/jcs.036103

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Role of syntaxin 18 in the organization of endoplasmic reticulum subdomains

Takayuki Iinuma¹, Takehiro Aoki¹, Kohei Arasaki^1,*, Hidenori Hirose¹, Akitsugu Yamamoto², Rie Samata², Hans-Peter Hauri³, Nagisa Arimitsu¹, Mitsuo Tagaya¹ and Katsuko Tani^1,

¹ School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan
² Department of Bio-science, Nagahama Institute of Bio-science and Technology, Nagahama, Shiga 526-0829, Japan
³ Department of Pharmacology and Neurobiology, Biozentrum, University of Basel, Basel CH-4056, Switzerland

View larger version (68K): [in this window] [in a new window]   Fig. 1. Knockdown of syntaxin 18 induces ER membrane aggregation and disrupts the Golgi complex. (A) HeLa cells were mock-transfected or transfected with lamin A/C siRNA, Syn18(390) or Syn18(770). At 72 hours after transfection, the cells were stained with an antibody against syntaxin 18 (right) or solubilized in phosphate-buffered saline with 0.5% SDS. The lysates (10 µg each) were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies (left). (B) HeLa cells were treated as described in A and stained for Bap31, p115 or -tubulin. The distributions of the proteins investigated were indistinguishable between mock-transfected cells and lamin A/C siRNA-treated cells (data not shown). Scale bars: 10 µm.

View larger version (180K): [in this window] [in a new window]   Fig. 2. Ultrastructure of syntaxin-18-depleted cells. HeLa cells were mock-transfected or transfected with Syn18(390), incubated for 72 hours and processed for electron microscopy. (A) An electron micrograph of a mock-transfected cell. (B-D) Electron micrographs of Syn18(390)-transfected cells. The boxed area in B is shown enlarged in C. G, Golgi complex; M, mitochondria; ER, endoplasmic reticulum; N, nucleus. Arrows, arrowheads and asterisks indicate vesiculated membrane structures, ER patches and dilated ER, respectively. Scale bars: 500 nm.

View larger version (131K): [in this window] [in a new window]   Fig. 3. Immunoelectron microscopy of ER and Golgi proteins in syntaxin-18-depleted cells. HeLa cells were transfected with Syn18(390), incubated for 72 hours and processed for immunoelectron microscopy. Localization of a cis-Golgi marker, p115 (A), a trans-Golgi marker, Vti1a (B), Bap31 (C), CLIMP-63 (D) and Hsp47 (E). N, nucleus. Arrowheads, arrows and asterisks indicate ER, dilated ER and ER patches, respectively. The structures labeled by double asterisks are incompletely fixed ER patches. Scale bars: 500 nm.

View larger version (120K): [in this window] [in a new window]   Fig. 4. Knockdown of syntaxin 18 results in segregation of smooth and rough ER membranes. (A) HeLa cells were mock-transfected (upper panels) or transfected with Syn18(390) (lower panels). At 72 hours after transfection, the cells were double-stained with antibodies against calnexin and Bap31. (B-D) HeLa cells transfected with Syn18(390) were double-stained with the indicated antibodies. A x100 objective lens was used to obtain the images in D (A-C x40). Scale bars: 10 µm.

View larger version (97K): [in this window] [in a new window]   Fig. 5. ER exit sites are disorganized in syntaxin-18-depleted cells. HeLa cells were mock-transfected or transfected with Syn18(390) or Syn18(770), incubated for 72 hours and stained with the indicated antibodies immediately (A) or after incubation with 10 µg/ml nocodazole for 3 hours (B). Scale bars: 10 µm.

View larger version (102K): [in this window] [in a new window]   Fig. 6. ERGIC-53 is not returned to the ER in syntaxin-18-depleted cells. HeLa cells were mock-transfected (A) or transfected with Syn18(390) (B), incubated for 72 hours, double-stained with antibodies against ERGIC-53 and β-COP (top panel) or Bap31 (middle panel), and analyzed by immunofluorescence microscopy using a x100 objective lens (upper two panels; scale bars: 10 µm). Alternatively, the cells were immunolabeled with ERGIC-53 and analyzed by electron microscopy (bottom panel; scale bars: 500 nm). N, nucleus; G, Golgi complex. Arrows indicate ER or dilated ER. Immunoreactivity of ERGIC-53 was observed near the Golgi in mock-transfected cells (A, bottom panel), whereas little, if any, ERGIC-53 immunoreactivity was detected in ER or dilated ER structures in Syn18(390)-transfected cells (B, bottom panel).

View larger version (90K): [in this window] [in a new window]   Fig. 7. Digitonin sensitivity of ERGIC-53 in syntaxin-18-depleted cells. HeLa cells were mock-transfected (A) or transfected with Syn18(390) (B). At 72 hours after transfection, the cells were left untreated or incubated with 10 µM BFA for 30 minutes, permeabilized with digitonin and double-stained with antibodies against ERGIC-53 and Sec61β. Scale bars: 10 µm.

View larger version (43K): [in this window] [in a new window]   Fig. 8. Retrograde transport of VSVG-KDEL-R-YFP to the ER is inhibited by syntaxin 18 depletion. HeLa cells were mock-transfected or transfected with Syn18(390) and treated as described in Materials and Methods. The cells incubated at 32°C were fixed immediately (32°C) or incubated at 40°C for 2 hours before fixation (40°C). (A) VSVG-KDEL-R-YFP was concentrated in the perinuclear region as punctate structures (type I), localized in the ER with some in punctate structures (type II) or distributed as punctate structures throughout the cytoplasm (type III). Scale bar: 10 µm. (B) Quantification of the results. The average of two independent experiments is shown. For each sample, more than 200 cells were evaluated.

View larger version (90K): [in this window] [in a new window]   Fig. 9. Effect of BFA on the distribution of Sec31A and ERGIC-53 in syntaxin-18-depleted cells. HeLa cells were mock-transfected or transfected with Syn18(390). At 72 hours after transfection, the cells were left untreated or incubated with 10 µM BFA for 30 minutes and double-stained with antibodies against ERGIC-53 and Sec31A (A) or Bap31 (B). The boxed areas are shown enlarged in the insets. Scale bars: 10 µm.

View larger version (94K): [in this window] [in a new window]   Fig. 10. BFA induces recovery of the ER architecture in syntaxin-18-depleted cells. (A) HeLa cells transfected with Syn18(390) were left untreated or treated with 10 µM BFA for 30 minutes, and then stained with an antibody against Bap31. The right panels of each pair are enlarged views of the boxed regions. (B) HeLa cells were treated as described in A. BFA was washed out, and the cells were incubated for the indicated times and double-stained with antibodies against Bap31 and PDI. Accumulation of Bap31 at the perinuclear region of mock-transfected cells at 1 hour after BFA washout may reflect its cycling within the ER during BFA recovery, as reported previously (Wakana et al., 2008). Scale bars: 10 µm.

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