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The protein tyrosine kinase Hck is located on lysosomal vesicles that are physically and functionally distinct from CD63-positive lysosomes in human macrophages

Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique UMR 5089, 205 Route de Narbonne, 31077 Toulouse, France

View larger version (93K): [in a new window]   Fig. 1. Hck vesicles belong to the endocytic pathway. Adherent MDMs were incubated with the endocytic tracer, rhodamine-dextran (Rho-dextran). After a chase of five hours, cells were fixed and labeled with a monoclonal anti-CD63 Ab or polyclonal anti-Hck Abs revealed by FITC-conjugated Abs. Colocalization of Rho-dextran with CD63 (compare B with A) or Hck (compare E with D) appears in yellow in the merged picture (C and F). Data are from one experiment that is representative of four independent experiments. Bar, 7 µm.

View larger version (96K): [in a new window]   Fig. 2. Chronological acquisition of CI-MPR, CD63 and Hck during the maturation of phagosomes containing zymosan. MDMs were infected with zymosan under synchronized conditions. At different post-infection times (5 (a,b), 10 (c,d) or 30 (e,f) minutes), cells were fixed and permeabilized in methanol. Double immunostaining was performed using Abs directed against CD63 and CI-MPR or Hck. We focused on phagosomes, and the presence of the markers at the phagosomal membrane was determined by a fluorescent ring (see arrows). (A) CI-MPR, a marker of late endosomes (a,c,e) was delivered to phagosomes before CD63 (b,d,f) (arrows). (B) CD63 (a,c,e) was delivered to phagosomes before Hck (b,d,f) (arrows). Occasionally Hck and CD63 were found on the same phagosome (arrowhead). Data are from one experiment that is representative of two to four independent experiments. Bar, 7 µm.

View larger version (53K): [in a new window]   Fig. 3. Phagosomal translocation of p61Hck lysosomal isoform in transfected CHO-CR3. CHO-CR3 cells transiently expressing the lysosomal p61Hck-GFP (A) or cytosolic p61G2AHck-GFP (B) were incubated with serum-opsonized zymosan. Cells were fixed and non-permeabilized, extracellular zymosan particles were seen with primary Abs revealed by secondary TRITC-conjugated Abs. Translocation of p61Hck-GFP on phagosomes is visualized by a green fluorescent ring (A, arrow) although no fluorescent ring was seen on the phagosomal membrane (black hole) in cells expressing the cytosolic form of Hck (B, arrowhead) as observed by confocal microscopy. Bar, 7 µm.

View larger version (100K): [in a new window]   Fig. 4. Sucrose induces osmotic swelling of CD63-positive lysosomes without affecting Hck vesicles. Sucrosomes were formed by incubating MDMs in culture medium supplemented with 0.05 M sucrose for 20 hours. After a chase of five hours in sucrose-free culture medium, cells were fixed and permeabilized in methanol. Double immunolabeling was performed using Abs directed against CD63 and CI-MPR or Hck. Sucrose induced the formation of large vacuoles surrounded by CD63 (B,D) but devoid of CI-MPR (A) and Hck (C) (arrows). For A and C, sucrosomes are seen as black holes surrounded by cytoplasm without specific immunolabeling. Data are from one experiment that is representative of three independent experiments. Bar, 7 µm.

View larger version (20K): [in a new window]   Fig. 5. Effects of nocodazole on the fusion of Hck and CD63 lysosomes with phagosomes containing zymosan. MDMs in contact with zymosan for 20 minutes at 4°C were further incubated for 10 minutes with nocadazole (10 µM) or control buffer. Phagocytosis was initiated by transferring the cells to 37°C for 30 minutes in the absence or in the presence of nocodazole. Double immunolabeling was then performed with anti-CD63 and anti-Hck Abs. The percentage of positive phagosomes for each marker in the absence (white bar) or in the presence of nocodazole (black bar) was determined by counting 100 phagosomes from at least 10 different fields in duplicate samples. Data are means± s.d. of three experiments.

View larger version (63K): [in a new window]   Fig. 6. Role of phagocytic receptors in the fusion of phagosomes with Hck and CD63 lysosomes. MDMs were incubated with latex beads coated with mannosylated-BSA (Man-BSA-latex) or human IgG (IgG-latex), and phagocytosis was performed under synchronized conditions. Two hours post-infection, cells were fixed and permeabilized with methanol, and double immunostaining was performed using Abs directed against CD63 or Hck. Mannosylated-latex beads resided in phagosomes negative for Hck (A) and positive for CD63 (B) (arrows). IgG-coated latex beads resided in phagosomes positive for Hck (C) and CD63 (D) (arrows). Bar, 8 µm. (E) Recruitment of Hck and CD63 to phagosomes containing IgG-latex beads was quantified as a percentage of the phagosomes positive for each marker at different time points. Data are from one experiment that is representative of four independent experiments.

View larger version (77K): [in a new window]   Fig. 7. Hck lysosomes fuse with phagosomes containing M. kansasii only when mycobacteria are serum-opsonized. MDMs were infected with non-opsonized or immune-serum-opsonized FITC-M. kansasii under synchronized conditions. Two hours post-infection, cells were fixed and immunolabeled for CD63 or Hck. Secondary Abs were TRITC-conjugated. We focused on phagosomes. Phagosomes containing non-opsonized or serum-opsonized mycobacteria (A,C) were positive for CD63 (B,D) (arrows). Some non-opsonized mycobacteria (A) were also found in CD63-negative phagosomes (B) (arrowheads). Phagosomes containing non-opsonized mycobacteria (E) were negative for Hck (F) (arrows) although phagosomes harboring serum-opsonized mycobateria (G) were positive for Hck (H) (arrows). Data are from one experiment that is representative of three independent experiments. Bar, 5.5 µm.