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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.

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Endosomal degradation in hemocytes from wild-type and eye color mutant animals. (A) Hemocytes from wild-type (top) and dor1car1 (bottom) animals were incubated with Cy3-mBSA for 5 min, fixed either immediately (left) or after a 2-h chase (right), and imaged on a wide field microscope. Note the loss in endosomal fluorescence in hemocytes from wild-type compared with dor1car1 after a 2-h chase. (B) Histogram shows quantification of total cell- associated fluorescence of Cy3-mBSA at indicated chase times normalized to cell-associated fluorescence at the 5-min time point. (C) Histogram shows quantification of total cell-associated fluorescence of Cy3-mBSA at 2 h of chase normalized to cell-associated fluorescence at the 5-min time point on treating wild-type cells with a protease inhibitor cocktail or in different alleles as indicated. The differences observed in endosomal degradation between cells from Canton-S and all other alleles were significant (P < 0.0001). (D) Histogram shows relative extent of endosomal acidification in cells from indicated mutants, incubated with F-Dex for 5 min, and imaged live, either immediately (0) or after a 2-h chase period (2) on a wide field microscope. Extent of endosomal acidification is expressed as a ratio of FITC fluorescence before neutralization of endosomal pH normalized to FITC fluorescence after neutralization with nigericin. The results shown represent the mean ± SD from two experiments. Bar: 5 μm.
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fig5: Endosomal degradation in hemocytes from wild-type and eye color mutant animals. (A) Hemocytes from wild-type (top) and dor1car1 (bottom) animals were incubated with Cy3-mBSA for 5 min, fixed either immediately (left) or after a 2-h chase (right), and imaged on a wide field microscope. Note the loss in endosomal fluorescence in hemocytes from wild-type compared with dor1car1 after a 2-h chase. (B) Histogram shows quantification of total cell- associated fluorescence of Cy3-mBSA at indicated chase times normalized to cell-associated fluorescence at the 5-min time point. (C) Histogram shows quantification of total cell-associated fluorescence of Cy3-mBSA at 2 h of chase normalized to cell-associated fluorescence at the 5-min time point on treating wild-type cells with a protease inhibitor cocktail or in different alleles as indicated. The differences observed in endosomal degradation between cells from Canton-S and all other alleles were significant (P < 0.0001). (D) Histogram shows relative extent of endosomal acidification in cells from indicated mutants, incubated with F-Dex for 5 min, and imaged live, either immediately (0) or after a 2-h chase period (2) on a wide field microscope. Extent of endosomal acidification is expressed as a ratio of FITC fluorescence before neutralization of endosomal pH normalized to FITC fluorescence after neutralization with nigericin. The results shown represent the mean ± SD from two experiments. Bar: 5 μm.

Mentions: To determine the extent of degradation of endocytosed protein ligands in the endolysosomal system in wild-type (Canton-S strain) cells, Cy3-mBSA was pulsed into cells for 5 min (Fig. 5 A) and chased for different times. Quantification of total cell (Fig. 5 B) and endosome-associated (unpublished data) fluorescence at the end of each chase time showed a dramatic reduction (∼80–85%) between 1 and 2 h. At this time, the endocytosed probes are in tubular, Rab7-positive endosomes (Figs. 1–3), indicating this compartment as a major site for endosomal degradation. The reduction in fluorescence is inhibited by a protease-inhibitor cocktail (Fig. 5 C), confirming that loss in fluorescence is a measure of protein degradation in these endosomal compartments.


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

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

Endosomal degradation in hemocytes from wild-type and eye color mutant animals. (A) Hemocytes from wild-type (top) and dor1car1 (bottom) animals were incubated with Cy3-mBSA for 5 min, fixed either immediately (left) or after a 2-h chase (right), and imaged on a wide field microscope. Note the loss in endosomal fluorescence in hemocytes from wild-type compared with dor1car1 after a 2-h chase. (B) Histogram shows quantification of total cell- associated fluorescence of Cy3-mBSA at indicated chase times normalized to cell-associated fluorescence at the 5-min time point. (C) Histogram shows quantification of total cell-associated fluorescence of Cy3-mBSA at 2 h of chase normalized to cell-associated fluorescence at the 5-min time point on treating wild-type cells with a protease inhibitor cocktail or in different alleles as indicated. The differences observed in endosomal degradation between cells from Canton-S and all other alleles were significant (P < 0.0001). (D) Histogram shows relative extent of endosomal acidification in cells from indicated mutants, incubated with F-Dex for 5 min, and imaged live, either immediately (0) or after a 2-h chase period (2) on a wide field microscope. Extent of endosomal acidification is expressed as a ratio of FITC fluorescence before neutralization of endosomal pH normalized to FITC fluorescence after neutralization with nigericin. The results shown represent the mean ± SD from two experiments. Bar: 5 μm.
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Related In: Results  -  Collection

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fig5: Endosomal degradation in hemocytes from wild-type and eye color mutant animals. (A) Hemocytes from wild-type (top) and dor1car1 (bottom) animals were incubated with Cy3-mBSA for 5 min, fixed either immediately (left) or after a 2-h chase (right), and imaged on a wide field microscope. Note the loss in endosomal fluorescence in hemocytes from wild-type compared with dor1car1 after a 2-h chase. (B) Histogram shows quantification of total cell- associated fluorescence of Cy3-mBSA at indicated chase times normalized to cell-associated fluorescence at the 5-min time point. (C) Histogram shows quantification of total cell-associated fluorescence of Cy3-mBSA at 2 h of chase normalized to cell-associated fluorescence at the 5-min time point on treating wild-type cells with a protease inhibitor cocktail or in different alleles as indicated. The differences observed in endosomal degradation between cells from Canton-S and all other alleles were significant (P < 0.0001). (D) Histogram shows relative extent of endosomal acidification in cells from indicated mutants, incubated with F-Dex for 5 min, and imaged live, either immediately (0) or after a 2-h chase period (2) on a wide field microscope. Extent of endosomal acidification is expressed as a ratio of FITC fluorescence before neutralization of endosomal pH normalized to FITC fluorescence after neutralization with nigericin. The results shown represent the mean ± SD from two experiments. Bar: 5 μm.
Mentions: To determine the extent of degradation of endocytosed protein ligands in the endolysosomal system in wild-type (Canton-S strain) cells, Cy3-mBSA was pulsed into cells for 5 min (Fig. 5 A) and chased for different times. Quantification of total cell (Fig. 5 B) and endosome-associated (unpublished data) fluorescence at the end of each chase time showed a dramatic reduction (∼80–85%) between 1 and 2 h. At this time, the endocytosed probes are in tubular, Rab7-positive endosomes (Figs. 1–3), indicating this compartment as a major site for endosomal degradation. The reduction in fluorescence is inhibited by a protease-inhibitor cocktail (Fig. 5 C), confirming that loss in fluorescence is a measure of protein degradation in these endosomal compartments.

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