Limits...
deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster.

Sriram V, Krishnan KS, Mayor S - J. Cell Biol. (2003)

Bottom Line: However, removal of Dor from small sized Car-positive endosomes is slowed, and subsequent fusion with tubular lysosomes is abolished.Overexpression of Dor in car1 mutant aggravates this defect, implicating Car in the removal of Dor from endosomes.This suggests that, in addition to an independent role in fusion with tubular lysosomes, the Sec1p homologue, Car, regulates Dor function.

View Article: PubMed Central - PubMed

Affiliation: National Centre for Biological Sciences, Tata Institute for Fundamental Research, Bangalore 560 065, India.

ABSTRACT
Endosomal degradation is severely impaired in primary hemocytes from larvae of eye color mutants of Drosophila. Using high resolution imaging and immunofluorescence microscopy in these cells, products of eye color genes, deep-orange (dor) and carnation (car), are localized to large multivesicular Rab7-positive late endosomes containing Golgi-derived enzymes. These structures mature into small sized Dor-negative, Car-positive structures, which subsequently fuse to form tubular lysosomes. Defective endosomal degradation in mutant alleles of dor results from a failure of Golgi-derived vesicles to fuse with morphologically arrested Rab7-positive large sized endosomes, which are, however, normally acidified and mature with wild-type kinetics. This locates the site of Dor function to fusion of Golgi-derived vesicles with the large Rab7-positive endocytic compartments. In contrast, endosomal degradation is not considerably affected in car1 mutant; fusion of Golgi-derived vesicles and maturation of large sized endosomes is normal. However, removal of Dor from small sized Car-positive endosomes is slowed, and subsequent fusion with tubular lysosomes is abolished. Overexpression of Dor in car1 mutant aggravates this defect, implicating Car in the removal of Dor from endosomes. This suggests that, in addition to an independent role in fusion with tubular lysosomes, the Sec1p homologue, Car, regulates Dor function.

Show MeSH

Related in: MedlinePlus

Morphological characterization of the endocytic pathway in Drosophila larval hemocytes. (A and B) Phase–contrast (A) and fluorescence (B) images of larval hemocytes incubated with Cy3-mBSA (mBSA, 800 ng/ml; red in B) and F-Dex (Dex, 1 mg/ml; green in B) for 5 min and fixed, obtained using a wide field microscope, show that Dex and mBSA are completely colocalized in endosomes (B, inset). Uptake of Cy3-mBSA is completely competed by inclusion of unlabeled mBSA (A, inset). (C–F) Confocal images of live larval hemocytes incubated with LR-Dex (Dex, red) and A488-mBSA (mBSA, green) for 5 min were obtained either immediately (C) or after a chase period of 15 min (D), 1 h (E), or 2 h (F) without the probes. Insets in B–E show magnified view of areas marked by an asterisk. (C) Endosomes labeled by the 5-min pulse are large (1–2 μm; L, open arrowheads) in size in which Dex labels the lumen of the endosome (middle inset); occasionally intraendosomal membrane staining of mBSA (top inset) can also be seen (bottom inset). (D) In a 15-min chase, Dex (middle inset) and mBSA (top inset) label smaller compartments (0.5–1 μm; S, arrows) where the probes are completely colocalized (bottom inset). (E and F) After a 1- (E) or 2-h (F) chase, both probes remain colocalized, appearing predominantly in tubular-vesicular (T, arrowheads) endosomal compartments (Dex, middle; mBSA, top inset). Bars: (shown in B corresponds to A–F) 5 μm; 1 μm (B–D, insets); 5 μm (A and E, insets). (G) EM of hemocyte incubated with fluid phase HRP for 10 min in the presence of mannan (500 μg/ml) to prevent mannose receptor–mediated uptake of HRP. Cells were fixed and processed for EM either immediately (i–iii) or after a 50-min chase (iv). At 10 min (i and ii), HRP labels MVBs (open arrowhead) and small dense endosomes (iii, arrow). No significant electron-dense structures could be identified in cells that were processed without HRP (unpublished data) or DAB (inset, ia). At higher magnification, the multivesicular nature of the endosome (ii, small arrows, intraendosomal vesicles) and the small densely labeled compartments (iii) are more apparent. Small electron-dense compartments are also visualized by HRP product when 10-min pulse is chased for 50 min (iv, arrowhead) consistent with a fixation- induced fragmentation of tubular vesicular endosomes observed at this time (E). Bars: (G i) 500 nm; (G ia) 1 μm; (G ii–iv) 200 nm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172926&req=5

fig1: Morphological characterization of the endocytic pathway in Drosophila larval hemocytes. (A and B) Phase–contrast (A) and fluorescence (B) images of larval hemocytes incubated with Cy3-mBSA (mBSA, 800 ng/ml; red in B) and F-Dex (Dex, 1 mg/ml; green in B) for 5 min and fixed, obtained using a wide field microscope, show that Dex and mBSA are completely colocalized in endosomes (B, inset). Uptake of Cy3-mBSA is completely competed by inclusion of unlabeled mBSA (A, inset). (C–F) Confocal images of live larval hemocytes incubated with LR-Dex (Dex, red) and A488-mBSA (mBSA, green) for 5 min were obtained either immediately (C) or after a chase period of 15 min (D), 1 h (E), or 2 h (F) without the probes. Insets in B–E show magnified view of areas marked by an asterisk. (C) Endosomes labeled by the 5-min pulse are large (1–2 μm; L, open arrowheads) in size in which Dex labels the lumen of the endosome (middle inset); occasionally intraendosomal membrane staining of mBSA (top inset) can also be seen (bottom inset). (D) In a 15-min chase, Dex (middle inset) and mBSA (top inset) label smaller compartments (0.5–1 μm; S, arrows) where the probes are completely colocalized (bottom inset). (E and F) After a 1- (E) or 2-h (F) chase, both probes remain colocalized, appearing predominantly in tubular-vesicular (T, arrowheads) endosomal compartments (Dex, middle; mBSA, top inset). Bars: (shown in B corresponds to A–F) 5 μm; 1 μm (B–D, insets); 5 μm (A and E, insets). (G) EM of hemocyte incubated with fluid phase HRP for 10 min in the presence of mannan (500 μg/ml) to prevent mannose receptor–mediated uptake of HRP. Cells were fixed and processed for EM either immediately (i–iii) or after a 50-min chase (iv). At 10 min (i and ii), HRP labels MVBs (open arrowhead) and small dense endosomes (iii, arrow). No significant electron-dense structures could be identified in cells that were processed without HRP (unpublished data) or DAB (inset, ia). At higher magnification, the multivesicular nature of the endosome (ii, small arrows, intraendosomal vesicles) and the small densely labeled compartments (iii) are more apparent. Small electron-dense compartments are also visualized by HRP product when 10-min pulse is chased for 50 min (iv, arrowhead) consistent with a fixation- induced fragmentation of tubular vesicular endosomes observed at this time (E). Bars: (G i) 500 nm; (G ia) 1 μm; (G ii–iv) 200 nm.

Mentions: In a recent study (Guha et al., 2003), we have shown that fluorescently conjugated maleylated BSA (Fl-mBSA) binds specifically to endogenously expressed dSR at the cell surface of hemocytes (Fig. 1 A) and is then internalized via a dynamin (shibire)-dependent pathway into early sorting endosomes marked by the small GTPase Rab5. On the other hand, majority of the fluid phase is internalized via a shibire-independent pathway into distinct Rab5-negative endosomes. Similar to a recently described pathway in mammalian cells (Sabharanjak et al., 2002), both these populations of endosomes were not labeled by the late endosomal marker Rab7; subsequent incubation in the absence of fluorescently labeled probes for 5 min (a chase of 5 min) results in dSR ligand and fluid phase probes becoming extensively colocalized as observed in fixed cells (Fig. 1 B).


deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster.

Sriram V, Krishnan KS, Mayor S - J. Cell Biol. (2003)

Morphological characterization of the endocytic pathway in Drosophila larval hemocytes. (A and B) Phase–contrast (A) and fluorescence (B) images of larval hemocytes incubated with Cy3-mBSA (mBSA, 800 ng/ml; red in B) and F-Dex (Dex, 1 mg/ml; green in B) for 5 min and fixed, obtained using a wide field microscope, show that Dex and mBSA are completely colocalized in endosomes (B, inset). Uptake of Cy3-mBSA is completely competed by inclusion of unlabeled mBSA (A, inset). (C–F) Confocal images of live larval hemocytes incubated with LR-Dex (Dex, red) and A488-mBSA (mBSA, green) for 5 min were obtained either immediately (C) or after a chase period of 15 min (D), 1 h (E), or 2 h (F) without the probes. Insets in B–E show magnified view of areas marked by an asterisk. (C) Endosomes labeled by the 5-min pulse are large (1–2 μm; L, open arrowheads) in size in which Dex labels the lumen of the endosome (middle inset); occasionally intraendosomal membrane staining of mBSA (top inset) can also be seen (bottom inset). (D) In a 15-min chase, Dex (middle inset) and mBSA (top inset) label smaller compartments (0.5–1 μm; S, arrows) where the probes are completely colocalized (bottom inset). (E and F) After a 1- (E) or 2-h (F) chase, both probes remain colocalized, appearing predominantly in tubular-vesicular (T, arrowheads) endosomal compartments (Dex, middle; mBSA, top inset). Bars: (shown in B corresponds to A–F) 5 μm; 1 μm (B–D, insets); 5 μm (A and E, insets). (G) EM of hemocyte incubated with fluid phase HRP for 10 min in the presence of mannan (500 μg/ml) to prevent mannose receptor–mediated uptake of HRP. Cells were fixed and processed for EM either immediately (i–iii) or after a 50-min chase (iv). At 10 min (i and ii), HRP labels MVBs (open arrowhead) and small dense endosomes (iii, arrow). No significant electron-dense structures could be identified in cells that were processed without HRP (unpublished data) or DAB (inset, ia). At higher magnification, the multivesicular nature of the endosome (ii, small arrows, intraendosomal vesicles) and the small densely labeled compartments (iii) are more apparent. Small electron-dense compartments are also visualized by HRP product when 10-min pulse is chased for 50 min (iv, arrowhead) consistent with a fixation- induced fragmentation of tubular vesicular endosomes observed at this time (E). Bars: (G i) 500 nm; (G ia) 1 μm; (G ii–iv) 200 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172926&req=5

fig1: Morphological characterization of the endocytic pathway in Drosophila larval hemocytes. (A and B) Phase–contrast (A) and fluorescence (B) images of larval hemocytes incubated with Cy3-mBSA (mBSA, 800 ng/ml; red in B) and F-Dex (Dex, 1 mg/ml; green in B) for 5 min and fixed, obtained using a wide field microscope, show that Dex and mBSA are completely colocalized in endosomes (B, inset). Uptake of Cy3-mBSA is completely competed by inclusion of unlabeled mBSA (A, inset). (C–F) Confocal images of live larval hemocytes incubated with LR-Dex (Dex, red) and A488-mBSA (mBSA, green) for 5 min were obtained either immediately (C) or after a chase period of 15 min (D), 1 h (E), or 2 h (F) without the probes. Insets in B–E show magnified view of areas marked by an asterisk. (C) Endosomes labeled by the 5-min pulse are large (1–2 μm; L, open arrowheads) in size in which Dex labels the lumen of the endosome (middle inset); occasionally intraendosomal membrane staining of mBSA (top inset) can also be seen (bottom inset). (D) In a 15-min chase, Dex (middle inset) and mBSA (top inset) label smaller compartments (0.5–1 μm; S, arrows) where the probes are completely colocalized (bottom inset). (E and F) After a 1- (E) or 2-h (F) chase, both probes remain colocalized, appearing predominantly in tubular-vesicular (T, arrowheads) endosomal compartments (Dex, middle; mBSA, top inset). Bars: (shown in B corresponds to A–F) 5 μm; 1 μm (B–D, insets); 5 μm (A and E, insets). (G) EM of hemocyte incubated with fluid phase HRP for 10 min in the presence of mannan (500 μg/ml) to prevent mannose receptor–mediated uptake of HRP. Cells were fixed and processed for EM either immediately (i–iii) or after a 50-min chase (iv). At 10 min (i and ii), HRP labels MVBs (open arrowhead) and small dense endosomes (iii, arrow). No significant electron-dense structures could be identified in cells that were processed without HRP (unpublished data) or DAB (inset, ia). At higher magnification, the multivesicular nature of the endosome (ii, small arrows, intraendosomal vesicles) and the small densely labeled compartments (iii) are more apparent. Small electron-dense compartments are also visualized by HRP product when 10-min pulse is chased for 50 min (iv, arrowhead) consistent with a fixation- induced fragmentation of tubular vesicular endosomes observed at this time (E). Bars: (G i) 500 nm; (G ia) 1 μm; (G ii–iv) 200 nm.
Mentions: In a recent study (Guha et al., 2003), we have shown that fluorescently conjugated maleylated BSA (Fl-mBSA) binds specifically to endogenously expressed dSR at the cell surface of hemocytes (Fig. 1 A) and is then internalized via a dynamin (shibire)-dependent pathway into early sorting endosomes marked by the small GTPase Rab5. On the other hand, majority of the fluid phase is internalized via a shibire-independent pathway into distinct Rab5-negative endosomes. Similar to a recently described pathway in mammalian cells (Sabharanjak et al., 2002), both these populations of endosomes were not labeled by the late endosomal marker Rab7; subsequent incubation in the absence of fluorescently labeled probes for 5 min (a chase of 5 min) results in dSR ligand and fluid phase probes becoming extensively colocalized as observed in fixed cells (Fig. 1 B).

Bottom Line: However, removal of Dor from small sized Car-positive endosomes is slowed, and subsequent fusion with tubular lysosomes is abolished.Overexpression of Dor in car1 mutant aggravates this defect, implicating Car in the removal of Dor from endosomes.This suggests that, in addition to an independent role in fusion with tubular lysosomes, the Sec1p homologue, Car, regulates Dor function.

View Article: PubMed Central - PubMed

Affiliation: National Centre for Biological Sciences, Tata Institute for Fundamental Research, Bangalore 560 065, India.

ABSTRACT
Endosomal degradation is severely impaired in primary hemocytes from larvae of eye color mutants of Drosophila. Using high resolution imaging and immunofluorescence microscopy in these cells, products of eye color genes, deep-orange (dor) and carnation (car), are localized to large multivesicular Rab7-positive late endosomes containing Golgi-derived enzymes. These structures mature into small sized Dor-negative, Car-positive structures, which subsequently fuse to form tubular lysosomes. Defective endosomal degradation in mutant alleles of dor results from a failure of Golgi-derived vesicles to fuse with morphologically arrested Rab7-positive large sized endosomes, which are, however, normally acidified and mature with wild-type kinetics. This locates the site of Dor function to fusion of Golgi-derived vesicles with the large Rab7-positive endocytic compartments. In contrast, endosomal degradation is not considerably affected in car1 mutant; fusion of Golgi-derived vesicles and maturation of large sized endosomes is normal. However, removal of Dor from small sized Car-positive endosomes is slowed, and subsequent fusion with tubular lysosomes is abolished. Overexpression of Dor in car1 mutant aggravates this defect, implicating Car in the removal of Dor from endosomes. This suggests that, in addition to an independent role in fusion with tubular lysosomes, the Sec1p homologue, Car, regulates Dor function.

Show MeSH
Related in: MedlinePlus