Journal:

Article Title: Late endosome motility depends on lipids via the small GTPase Rab7

doi: 10.1093/emboj/21.6.1289

Figure Lengend Snippet: Fig. 1. Motility and cholesterol accumulation. (A–C) HeLa cells were transfected with CD63–GFP (A) or co-transfected with CD63–GFP and either dynamitin-myc–GFP (B), or a head-deleted mutant of KHC (C). Dynamitin-myc–GFP remained exclusively cytosolic, and was not detected after 100–200 ms exposure times; co-expression was verified by indirect immunofluorescence using anti-myc antibodies for dynamitin, or the Suk4 anti-kinesin antibody for the KHC mutant (endogenous and overexpressed KHC were easily distinguished by the intensity of the signal). Images were collected at 1 s interval over a time period of 25 s and then all images were stacked; arrows point to the cell periphery. Then, a moving object appears as a series of closely associated spots that reveals its track, as in the control cell in (A). (A′–C′) Initial and final positions were color-coded in red and green, respectively: a moving object is both red and green, and an immobile object yellow. (A′′–C′′) Traces of individual elements. Control (D and F) or NPC (E) fibroblasts were incubated at 37°C for 13 h with Alexa568-labeled antibodies against CD63 without (D and E) or with (F) 3 µg/ml U18666A, and analyzed as above (A′–C′). Bars: (A–C) 2 µm; (D–F) 3.7 µm.

Article Snippet: Plasmids The plasmid containing CD63–GFP was obtained after cloning CD63 into a modified pEGFP-C1 (Clontech, Palo Alto, USA) vector with the CMV promoter replaced by the bos promoter (from G.Griffiths, School of Pathology, Oxford, UK).

Techniques: Transfection, Mutagenesis, Expressing, Immunofluorescence, Incubation, Labeling

Journal:

Article Title: Late endosome motility depends on lipids via the small GTPase Rab7

doi: 10.1093/emboj/21.6.1289

Figure Lengend Snippet: Fig. 2. Other organelles. (A) HeLa cells transfected with the mitochondrial marker ECFP (CFP-mito) or with CD63–GFP were treated or not with U18666A. Frames were captured every 12 s to limit light damage, over 5 min to visualize better mitochondrial motility, and analyzed as in Figure 1A′–C′. (B) HeLa cells expressing NAGT1–GFP treated or not with U18666A and then with brefeldin A for 30 min, as indicated, were labeled with anti-Lamp1 antibodies and processed for microscopy. Bars: (A) 3.9 µm; (B) 6.2 µm.

Article Snippet: Plasmids The plasmid containing CD63–GFP was obtained after cloning CD63 into a modified pEGFP-C1 (Clontech, Palo Alto, USA) vector with the CMV promoter replaced by the bos promoter (from G.Griffiths, School of Pathology, Oxford, UK).

Techniques: Transfection, Marker, Expressing, Labeling, Microscopy

Journal:

Article Title: Late endosome motility depends on lipids via the small GTPase Rab7

doi: 10.1093/emboj/21.6.1289

Figure Lengend Snippet: Fig. 5. Motors. Cells were transfected with CD63–GFP alone (A), or co-transfected with CD63–GFP and either dynamitin-myc–GFP (B), KHC (C) or Kif2β-myc (D). (As in Figure 1, overexpressed dynamitin remained cytosolic; co-expression was verified in all cases.) Cells were then treated with U18666A for 13 h. Motility analysis was as in Figure 1. Bars: 4 µm. (E and F) The bird’s eye distances (not the trajectory) between initial and final positions were quantified after 25 s. Control, control without U18666A; U18, U18666A as in (A); U18 + Dynamitin, dynamitin and U18666A; motility was partially restored as in (B) (1), but stopped at the cell periphery (2); U18 + KHC, KHC and U18666A as in (C); U18 +Kif2β, Kif2β and U18666A as in (D).

Article Snippet: Plasmids The plasmid containing CD63–GFP was obtained after cloning CD63 into a modified pEGFP-C1 (Clontech, Palo Alto, USA) vector with the CMV promoter replaced by the bos promoter (from G.Griffiths, School of Pathology, Oxford, UK).

Techniques: Transfection, Expressing

Journal:

Article Title: Late endosome motility depends on lipids via the small GTPase Rab7

doi: 10.1093/emboj/21.6.1289

Figure Lengend Snippet: Fig. 3. Antibodies against LBPA and pulse–chase. (A and B) HeLa or (C) BHK cells transfected with CD63–GFP were incubated for 24 h without (A) or with the monoclonal antibody against LBPA (B; 6C4) or Lamp1 (C; 4A1). (We used BHK cells because large amounts of 4A1 were available; cholesterol also accumulates in BHK cells after 6C4 treatment; Kobayashi et al., 1999.) Motility was analyzed as in Figure 1. (D) Outline. HeLa cells treated for 10 h with U18666A (the drug remained present throughout the experiment), were labeled with a 15 min pulse of endocytosed Oregon green–dextran (Dextran OG). After a 45 min chase, labeled endosomes were clearly motile (+/+). Motility was low after a second 45 min chase (+/–) and abolished after a third 1 h chase (–). Then, cells were labeled with a second pulse of rhodamine–dextran (Dextran Rh), and the chase protocol was repeated. Samples were fixed and analyzed by triple-channel fluorescence microscopy after the first (E) and second (F) wave. Bars: (A–C) 1.5 µm; (E) 4 µm; (F) 2.4 µm.

Article Snippet: Plasmids The plasmid containing CD63–GFP was obtained after cloning CD63 into a modified pEGFP-C1 (Clontech, Palo Alto, USA) vector with the CMV promoter replaced by the bos promoter (from G.Griffiths, School of Pathology, Oxford, UK).

Techniques: Pulse Chase, Transfection, Incubation, Labeling, Fluorescence, Microscopy

Journal:

Article Title: Late endosome motility depends on lipids via the small GTPase Rab7

doi: 10.1093/emboj/21.6.1289

Figure Lengend Snippet: Fig. 4. Microtubules. (A) Control or NPC fibroblasts were labeled with the indicated antibodies. (B and C) HeLa cells transfected with CD63–GFP were treated for 13 h with U18666A, and the drug remained present throughout the experiment. Cells were then treated for 1 h with nocodazole (B; U18 + Noc) and then re-incubated for 90 min without nocodazole (C; U18 + Post-Noc). Motility analysis was as in Figure 1. Bars: (A) 8 µm; (B and C) 4 µm.

Article Snippet: Plasmids The plasmid containing CD63–GFP was obtained after cloning CD63 into a modified pEGFP-C1 (Clontech, Palo Alto, USA) vector with the CMV promoter replaced by the bos promoter (from G.Griffiths, School of Pathology, Oxford, UK).

Techniques: Labeling, Transfection, Incubation

Journal:

Article Title: Late endosome motility depends on lipids via the small GTPase Rab7

doi: 10.1093/emboj/21.6.1289

Figure Lengend Snippet: Fig. 6. Rab7 and cholesterol. (A) Co-transfection with CD63–GFP and Rab5-myc (co-expression was verified by immunofluorescence using anti-myc antibodies). (B) Transfection with Rab7–GFP. (C) Co-transfection with CD63–GFP and Rab7N125I–GFP (Rab7N125I–GFP remained cytosolic and was not detected after 100–200 ms exposure; co-expression was verified after longer exposures). (D) After transfection with Rab7N125I–GFP, late endocytic vesicles were labeled with a 15 min pulse of rhodamine–dextran (DexRh) followed by a 45 min chase, as in Figure 3D, to reveal better the partial redistribution to the periphery. (E) Cells co-transfected with Rab7–GFP and KHC were treated with U18666A for 13 h. (F and G) Cells co-transfected with CD63–GFP and Rab7N125I–GFP were treated with U18666A for 13 h (F), and distances between initial and final positions were quantified (G) as in Figure 5. Motility analysis was as in Figure 1. Arrows point to the cell periphery. Bars: (A, D and E) 4 µm; (B and C) 2 µm; (F) 2.8 µm.

Article Snippet: Plasmids The plasmid containing CD63–GFP was obtained after cloning CD63 into a modified pEGFP-C1 (Clontech, Palo Alto, USA) vector with the CMV promoter replaced by the bos promoter (from G.Griffiths, School of Pathology, Oxford, UK).

Techniques: Cotransfection, Expressing, Immunofluorescence, Transfection, Labeling