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First published online 21 June 2005
doi: 10.1242/jcs.02435

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A unique lysophospholipid acyltransferase (LPAT) antagonist, CI-976, affects secretory and endocytic membrane trafficking pathways

Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA

View larger version (88K): [in a new window]   Fig. 1. CI-976 induces tubule formation from all Golgi compartments. Clone 9 rat hepatocytes were treated with solvent control (A,D,G,J,M) or 20 µM CI-976 (B,C,E,F,H,I,K,L, N, O) for various amounts of time as indicated. Cells were then fixed and stained for immunofluorescence localization of various Golgi marker proteins: GPP130 (A-C), ManII (G-I), TGN38 (J-L) and M6PR (M-O). KDEL-R was visualized by transiently transfecting Clone 9 cells with a mutant form of the KDEL-R coupled to GFP (D-F). After 24 hours, the cells were then treated with 20 µM CI-976 and fixed.

View larger version (69K): [in a new window]   Fig. 2. Tubules can form independently from Golgi subcompartments following CI-976 treatment. Clone 9 rat hepatocytes were treated with solvent control (A,B) or 20 µM CI-976 (C-H) for various amounts of time as indicated. Cells were then fixed and processed for double-immunofluorescence staining of Golgi marker proteins, ManII (A,C,E,G) and TGN38 (B,D,F,H).

View larger version (65K): [in a new window]   Fig. 3. TGN and early endosomes do not fuse. Clone 9 cells were treated with solvent control (A) or 20 µM CI-976 (B) for 50 minutes. Cells were then labeled with 1 mg ml–1 Alexa568-dextran for 5 minutes prior to fixing and staining for immunofluorescence localization of M6PR. The sets of three images (A1-A3 and B1-B3) are from consecutive slices in the Z plane taken using a spinning disc confocal microscope (Perkin-Elmer).

View larger version (93K): [in a new window]   Fig. 4. Endosome tubule formation is stimulated by CI-976. HeLa cells were incubated with Alexa568-Tf for 45 minutes to label endocytic compartments, CI-976 (25 µM) was added, and live cells were imaged immediately by spinning disk confocal microscopy. The time interval between each panel is 6.4 seconds. The arrows point to tubules that formed during the course of these observations. The micrographs are inverted images from the originals.

View larger version (56K): [in a new window]   Fig. 5. CI-976 does not inhibit initial uptake of Tf. TRITC-Tf was pre-bound to the surface of Clone 9 cells at 4°C. Following 15 minutes treatment with either (A) DMSO as a solvent control or (B) 20 µM CI-976 at 4°C, the cells were shifted to 37°C for 5 minutes to allow Tf uptake.

View larger version (152K): [in a new window]   Fig. 6. Tf accumulates in a juxtanuclear cluster in cells treated with CI-976. Clone 9 cells were treated for 10 minutes with (A) DMSO as a solvent control, (B) CI-976 (20 µM), (C) DuP-128 (5 µM) or (D) PKF-058-035 (10 µM). TRITC-Tf was then added (in the continuous presence of compounds indicated above) for 45 minutes at 37°C.

View larger version (48K): [in a new window]   Fig. 7. CI-976 inhibits recycling of Tf. (A-C) Fluorescence micrographs of HeLa cells labeled with TRITC-Tf. (A) Cells incubated with TRITC-Tf for 45 minutes and then fixed. (B) Cells pulse-labeled by TRITC-Tf uptake for 45 minutes followed by a chase without Tf for 25 minutes. (C) Same treatments as B except the chase was in the presence of CI-976 (25 µM). (D) Quantitation of inhibition of Tf recycling in the presence of different concentrations of CI-976; experiments performed as in A-C. Results are expressed as the mean with the error bars representing 1 s.d.

View larger version (123K): [in a new window]   Fig. 8. Tf and TfR accumulate in a juxtanuclear cluster of tubulo-vesicular organelles in HeLa cells treated with CI-976. Cells were treated for 10 minutes with DMSO as a solvent control (A,B) or 20 µM CI-976 (C,D). TRITC-Tf (A,C) was then added for 45 minutes at 37°C. Cells were then fixed and stained for immunofluorescence localization of TfR (B,D).

View larger version (51K): [in a new window]   Fig. 9. Long-term treatment (50 minutes) with CI-976 prevents subsequent uptake of Tf from the cell surface. Clone 9 cells were treated for 50 minutes at 37°C with DMSO as a solvent control (A-C) or 20 µM CI-976 (D-F). Alexa568-Tf (A,D) was then added for 5 minutes at 37°C. Cells were fixed and stained for immunofluorescence localization of M6PRs (B,E). These results are consistent with the conclusion that CI-976 inhibits recycling of TfRs back to the cell surface.

View larger version (122K): [in a new window]   Fig. 10. CI-976 does not qualitatively affect the uptake and delivery of DiI-LDL or Alexa488-EGF to centrally located late endosomal compartments. Clone 9 cells were pre-treated in the presence or absence of 25 µM CI-976 for 15 minutes, pulse labeled with DiI-LDL (5 µg ml–1) and Alexa488-EGF (2 µg ml–1) for 10 minutes, and then chased for either 15 minutes or 1 hour, as indicated on the figure. In treated cells, CI-976 was present throughout the pulse and chase periods.

View larger version (36K): [in a new window]   Fig. 11. Tf accumulates in Rab11-positive recycling endosomes. Clone 9 cells were transiently transfected with GFP-Rab11. After 24 h of transfection, the cells were then treated with (A) DMSO as a solvent control or (B) 20 µM CI-976 for 10 minutes at 37°C prior to labeling with Alexa568-Tf for 45 minutes at 37°C. Arrows indicate juxtanuclear cluster of vesicles that contain both GFP-Rab11 and Alexa568-Tf, as evidenced by the yellow merged images in B1-B3.

View larger version (32K): [in a new window]   Fig. 12. Tf recycling appears to be irreversibly inhibited by CI-976. Clone 9 cells were treated for 10 minutes with DMSO as a solvent control (A,B) or 20 µM CI-976 (C-E). TRITC-Tf was then added for 45 minutes at 37°C. Cells were either fixed (A,C) or were quickly washed in MEM containing 10% Nu-Serum, and subsequently incubated in MEM with 10% Nu-Serum for 1 hour (B,D) or 24 hours (E) at 37°C prior to fixing.

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