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First published online 31 August 2004
doi: 10.1242/jcs.01370

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Role for Rab7 in maturation of late autophagic vacuoles

¹ Institute of Biochemistry, University of Kiel, Olshausenstr. 40, 24098 Kiel, Germany
² Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Universita degli Studi di Lecce, 73100 Lecce, Italy
³ Department of Biochemistry, Juntendo University School of Medicine, 2-1-1, Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan

View larger version (57K): [in a new window]   Fig. 2. Rab7 localised in the membranes of autophagic vacuoles. (A) Subcellular fractionation of rat liver. Rab7 was detected by western blotting in both autophagic vacuolar membranes and lysosomal/late endosomal membranes. The characterisation of the fractions has previously been published (Kabeya et al., 2000). LC3II only is shown, as no LC3I was detected in the membrane fractions. (B-F) Localisation of GFP-Rab7 by immunoelectron microscopy. HeLa cells were transfected with pEGFP-Rab7 and grown for 1 day. The cells were starved of amino acids for 2 hours and prepared for immunogold labelling with anti-GFP (10 nm gold) and anti-Lamp1 (5 nm gold) antibodies. (B) Western blotting was used to show that all GFP was still connected to Rab7 in these conditions. Lane 1, cells transfected with GFP, and lane 2, cells transfected with GFP-Rab7. Early autophagic vacuoles (AVi) showed labelling for GFP-Rab7 in the outer and inner membranes (C). (D-F) Late autophagic vacuoles (AVd) with their limiting membranes labelled for GFP-Rab7. Some GFP-Rab7 was also present in the contents, in agreement with the association of GFP-Rab7 with both the outer and inner limiting membranes of the early vacuoles (C). Some AVd were also weakly labelled for Lamp1 (E and F, arrowheads), or Lamp1 was present in vesicles close to them (F, arrow). The AVd in D contains a partially degraded mitochondrion (m). PM, plasma membrane.

View larger version (147K): [in a new window]   Fig. 1. LC3 and Rab7 distributions changed during amino acid starvation. Triple immunofluorescence staining of LC3, Rab7 and lysobisphosphatidic acid (LBPA) in HeLa cells. The cells were grown on coverslips and either fixed without treatment (0 h) or incubated in serum and amino acid free medium for 1 to 4 hours as indicated on the left. The cells were fixed with 4% paraformaldehyde and permeabilised with saponin. Optical sections are shown. Overlay of LC3 (green) and Rab7 (red) is shown in the right column. Arrows and inserts in the 2 h and 4 h rows indicate large LC3-positive structures, which started to appear after 2 hours starvation. These structures were also positive for Rab7, as indicated by the arrows in the Rab7 column and yellow colour in the merge column. At later time points, LBPA was also present in these structures, as indicated by small arrowheads, and yellow colour in the inserts, in the LBPA column (LC3 is green, LBPA red). LC3 and Rab7 also colocalised in small vesicles that probably represent newly formed autophagosomes (large arrowheads in 1 h and 2 h cells). Bar, 10 µm.

View larger version (84K): [in a new window]   Fig. 3. Autophagic vacuoles were frequently detected in the perinuclear area close to the Golgi apparatus. HeLa cells were starved for 2 hours to induce autophagy and prepared for immunogold labelling of LC3 (A) or Lamp1 (B) followed by a secondary antibody coupled to 5 nm gold (arrowheads). Autophagic vacuoles (AV) were frequently observed to accumulate close to the nucleus (Nu) and the Golgi stack (G), where endo/lysosomal vacuoles (LE) were also present. Some autophagic vacuoles were observed to fuse with the Lamp1-positive endo/lysosomal vacuoles (B).

View larger version (97K): [in a new window]   Fig. 5. Effect of Rab7 RNAi on LC3 labelling. The cells were treated as in Fig. 4 and prepared for immunofluorescence with anti-LC3 (green) and anti-Lamp1 (red). Optical sections are shown. Arrowheads indicate large ring-like LC3-positive structures, which typically showed labelling for Lamp1 in the centre of the ring (arrowheads and inserts in B). These structures were only rarely seen in the Rab7 RNAi cells (C,D). Quantitation of the large LC3- and Lamp1-positive structures per cell is shown in E. Statistical significance was estimated using t-test. Bar, 10 µm.

View larger version (33K): [in a new window]   Fig. 4. Effect of transfection with the Rab7 RNAi construct on Rab7 and Rab5 levels. HeLa cells were transiently transfected with empty pSUPER or pSUPER containing the Rab7 RNAi insert. Two parallel dishes were prepared for each construct. After 3 days the cells were prepared for western blotting with anti-Rab7 and anti-Rab5. Equal amounts of protein were loaded in each lane. Notice that Rab7 was heavily downregulated, whereas Rab5 was not affected.

View larger version (37K): [in a new window]   Fig. 6. Downregulation of Rab7 expression with RNAi caused accumulation of late autophagic vacuoles. HeLa cells were transfected with empty pSUPER or pSUPER Rab7 RNAi and grown for 3 days. Three parallel dished were kept in full culture medium (FCS), another three dishes were starved of amino acids for 2 hours (EBS), and another three dishes were first starved of amino acids for 2 hours and then shifted back to full medium for 2 hours (EBS - FCS). (A) Expression levels of Rab7 and LC3 were controlled by western blotting. Equal amounts of cell extract (40 µg) were loaded in each lane, and Ponceau staining was used to confirm equal transfer of proteins to each lane on the PVDF membrane (not shown). (B) Accumulation of early (Avi) and late (Avd) autophagic vacuoles was estimated by quantitative electron microscopy. The P values indicate statistical significance of the difference between the Rab7 RNAi and the pSUPER sample (t-test). Error bars indicate s.e.m.

View larger version (106K): [in a new window]   Fig. 7. Effect of Rab7 constructs on localisation of endogenous LC3 in HeLa cells. The cells were transiently transfected with pEGFP-Rab7 wild-type (WT), Q67L (constitutively active) or T22N (dominant negative) as indicated on the left, and grown for 1 day. The cells were starved of serum and amino acids for 2 hours, fixed in cold methanol, and LC3 and Lamp1 were stained by immunofluorescence. Optical sections are shown. Overlay of LC3 (green) and Lamp1 (red) is shown in the third column. The fourth column shows GFP fluorescence from the Rab7 constructs. Notice the large ring-shaped LC3-positive structures in Rab7 WT and Q67L expressing cells, indicated by arrows in the LC3 and merge columns. The centre of the rings typically showed labelling for Lamp1 as indicated by the arrows and inserts in the merge column. These structures were not observed in cells expressing GFP-Rab7 T22N. Bar, 20 µm.

View larger version (146K): [in a new window]   Fig. 8. Late autophagic vacuoles were observed in cells expressing GFP-Rab7 T22N. HeLa cells were transfected with GFP-Rab7 T22N. One day later the cells were starved of amino acids for 2 hours and prepared for immunogold labelling with anti-GFP and 10 nm gold-coupled secondary antibodies. Cells expressing GFP-Rab7 T22N accumulated late autophagic vacuoles (AVd).

View larger version (55K): [in a new window]   Fig. 9. Recruitment of Rab7 to autophagic vacuoles was retarded in mouse fibroblasts deficient in Lamp1 and Lamp2. Control (A) and Lamp1/Lamp2-deficient (B) cells were transiently transfected with GFP-Rab7 (green), grown for 1 day, and starved of serum and amino acids (EBS) for 1 hour (A, B) or 2 hours (not shown). The cells were fixed and prepared for immunofluorescence staining with anti-LC3 (red). Optical sections are shown. Colocalisation of LC3 and Rab7 (arrowheads) was quantitated as the number of yellow dots per cell (C). The P values indicate statistical significance compared to the control cells (t-test). Bar, 20 µm.

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