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doi: 10.1242/10.1242/jcs.00075

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A role for the lysosomal membrane protein LGP85 in the biogenesis and maintenance of endosomal and lysosomal morphology

¹ Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Maidashi 3-1-1, Fukuoka 812-8582, Japan
² Centre for High Resolution Imaging and Processing, University of Dundee, School of Life Sciences, DD1 5EH Dundee, UK
³ Biochemisches Institut, Universität Kiel, Olshausenstrasse 40, D-24098 Kiel, Germany

View larger version (119K): [in a new window]   Fig. 1. Overexpression of LGP85 in COS cells causes large swollen vacuoles. Cultured COS (A,B), HeLa (C,D), or MDCK (E,F) cells were transiently transfected with LGP85 and after 36 hours were fixed and processed for immunofluorescence micrography using anti-LGP85 polyclonal antibody as described in Materials and Methods. Upper panels (A,C,E) show the LGP85 staining and the lower panels (B,D,F) show the corresponding phase-contrast micrographs. Bars, 20 µm.

View larger version (115K): [in a new window]   Fig. 2. The formation of large swollen vacuoles is specific to the overexpression of LGP85. COS cells were transiently transfected with LGP85 (A,B), LGP107 (C,D), or LGP96 (E,F), fixed and stained with each polyclonal antibody. Cells were visualized by immunofluorescence microscopy. Upper columns (A,C,E) show the LGP85, LGP107 and LGP96 staining, respectively, and the lower columns (B,D,F) show the corresponding phase-contrast micrographs. Bars, 20 µm.

View larger version (56K): [in a new window]   Fig. 3. Cytochemical characterization of the LGP85-induced large swollen vacuoles. COS cells were transiently transfected with LGP85, and 36 hours post-transfection were fixed and double-labeled for LGP85 (left) and LAMP-2 (B, middle), LBPA (E, middle), EEA1 (H, middle), TfnR (K, middle), MPR300 (N, middle), and GM130 (Q, middle). Cells were visualized by immunofluorescence microscopy. The right columns show the merged images of LGP85 (red) and each marker (green). Bars, 20 µm.

View larger version (94K): [in a new window]   Fig. 4. Overexpression of LGP85 does not cause enlargement of lysosome, but results in formation and accumulation of the late endosome-lysosome hybrid organelle. To label lysosomes, COS cells were incubated with Texas-Red dextran for 4 hours and chased for 20 hours. After that, cells were transiently transfected with LGP85 and fixed at 12 (A-C), 24 (D-F), and 36 (G-I) hours after transfection, followed by staining for LGP85 (A,D,G, green). Cells were visualized by confocal microscopy. The right columns show the merged images of LGP85 (green) and dextran (red). Bars, 20 µm.

View larger version (105K): [in a new window]   Fig. 5. Ultrastructural analysis of LGP85-induced large vacuoles. COS cells were transiently transfected with LGP85 and, after 36 hours of transfection, were processed for immunoelectron microscopy. Ultra-thin sections were either single-labeled with anti-LGP85, followed by anti-rabbit IgG-10 nm gold (A,B) or double-labeled with anti-LGP85, and anti-LAMP-2 (C, arrowheads), or anti-LGP85 and anti-LBPA (D,E, arrowheads), followed by anti-rabbit IgG-10 nm gold and anti-mouse IgG-5 nm gold. Arrowheads in A show the double limiting membrane structure of LGP85-induced large vacuoles, and a higher magnification image is shown in B. Panel D shows a portion of the limiting membrane of one large LGP85-containing vacuole, with the lumen of the vacuole occupying the upper half of the panel. Note that some LBPA labeling is seen on internal membranes closely associated with the limiting membrane of the vacuole (D, arrows). Bars, 200 nm.

View larger version (77K): [in a new window]   Fig. 6. A subset of LGP85-induced large swollen vacuoles is accessible to transferrin, EGF and dextran added externally. COS cells transfected with LGP85 for 36 hours were incubated with Alexa594-transferrin (red in A-C), Texas Red-EGF (red in D-F), or Texas Red-dextran (red in G-I) as described in Materials and Methods. Cells were then fixed, stained with LGP85 (green in A, D and G), and visualized by confocal microscopy. The right columns (C,F,I) show the merged images of LGP85 (green) and each endocytic marker (red). Bars, 20 µm.

View larger version (54K): [in a new window]   Fig. 7. Overexpression of LGP85 causes impairment of membrane traffic from early endosomes to late endosomes/lysosomes. COS cells transfected with LGP85 for 36 hours were incubated with Alexa594-transferrin (red in C and O), Texas Red-EGF (red in G and S), or Texas Red-dextran (red in K and W) as described in Materials and Methods. Cells were then fixed, stained with LGP85 (blue in left columns) and either EEA1 (green in B, F and J) or LAMP-2 (green in N, R and V), and visualized by confocal microscopy. The right columns (D, H, L, P, T and X) show the merged images of EEA1 (green in D, H and L) or LAMP-2 (green in P, T and X) and each endocytic marker (red). Bars, 20 µm.

View larger version (33K): [in a new window]   Fig. 8. Anti-LGP85 antibodies are internalized to early endosome-like large vacuoles. COS cells transfected with LGP85 for 36 hours were incubated for 1 hour with rabbit polyclonal antibodies against LGP85 at 4°C and chased for 3 hours at 37°C. Cells were then fixed, permeabilized, and incubated with a mouse monoclonal antibody to either EEA1 (A) or LAMP-2 (D). Localization of internalized and bound antibodies revealed by Alexa488-conjugated goat anti-rabbit IgG (green in B and E) and Alexa594-conjugated goat anti-mouse antibody (red in A and D), respectively, was visualized by confocal microscopy. The right columns (C,F) show the merged images of internalized antibodies (green in B and E) and EEA1 (red in A) or LAMP-2 (red in D). Bars, 20 µm.

View larger version (70K): [in a new window]   Fig. 9. Accumulation of cholesterol in LGP85-induced large vacuolar compartments. COS cell cultured in DMEM supplemented with 10% FBS were transiently transfected with LGP85 and fixed after 36 hours after transfection. Cells were immunostained for LGP85 (B) and cytochemically stained with filipin (C) to detect cholesterol. To examine further whether cholesterol accumulated in the LGP85-induced large swollen vacuoles is derived from one containing into cellular membranes or into LDL, COS cells cultured in DMEM supplemented with 10% LPDS instead of 10% FBS for 2 days were transiently transfected with LGP85, fixed at 36 hours after transfection, and stained for LGP85 (F) and cholesterol (G). The right columns (D,H) show the merged images of LGP85 (red) and cholesterol (blue), and the left columns (A,E) show the corresponding phase-contrast micrographs. Bars, 20 µm.

View larger version (70K): [in a new window]   Fig. 10. The dominant-negative form of Rab5b inhibits the formation of LGP85-induced large vacuoles. COS cells were transiently co-transfected with LGP85 and FLAG-tagged wild-type Rab5b or FLAG-tagged Rab5bS34N, and after 36 hours post-transfection fixed and incubated with a rabbit polyclonal antibody to LGP85 (B,D) and a mouse monoclonal antibody to FLAG for labeling of wild-type and mutant Rab5b (A,C). Cells were visualized by confocal microscopy. Bars, 20 µm.