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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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Eye color mutants affect the removal of Dor and Car from Rab7-positive endosomes. (A–F) Hemocytes from mutants of dor and car, synthetic lethal dor1car1, and dor4/Ydor+ were incubated for 15 min with F-Dex (green) and fixed after indicated chase times, immunostained (red) for Deep-orange (α-Dor), and imaged on a confocal microscope. Insets in A–D show magnified view of areas marked by an asterisk (top, antibody; middle, F-Dex; bottom, merge). Bold arrows (E and F) indicate antibody-stained structures lacking endocytic probes. (G and H) Histograms show the percentage of F-Dex–containing endosomes colocalized with α-Dor (G) or α-Car (H) at the indicated chase times in wild-type (blue), dor1 (green), dor4 (yellow), dor1car1(red), and car1 (gray). The results shown represent the mean ± SEM from two experiments. Bars: (shown in F corresponds to A–F) 5 μm; (inset) 1 μm.
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fig9: Eye color mutants affect the removal of Dor and Car from Rab7-positive endosomes. (A–F) Hemocytes from mutants of dor and car, synthetic lethal dor1car1, and dor4/Ydor+ were incubated for 15 min with F-Dex (green) and fixed after indicated chase times, immunostained (red) for Deep-orange (α-Dor), and imaged on a confocal microscope. Insets in A–D show magnified view of areas marked by an asterisk (top, antibody; middle, F-Dex; bottom, merge). Bold arrows (E and F) indicate antibody-stained structures lacking endocytic probes. (G and H) Histograms show the percentage of F-Dex–containing endosomes colocalized with α-Dor (G) or α-Car (H) at the indicated chase times in wild-type (blue), dor1 (green), dor4 (yellow), dor1car1(red), and car1 (gray). The results shown represent the mean ± SEM from two experiments. Bars: (shown in F corresponds to A–F) 5 μm; (inset) 1 μm.

Mentions: In cells from dor mutants (including dor1car1), levels of Dor immunostaining are lower than those observed in wild-type hemocytes (unpublished data; Sevrioukov et al., 1999). However, Dor (Fig. 9 , A–C and G) and Car (Fig. 9 H) immunoreactivity are persistent on the Rab7-positive large sized endosomes compared with wild-type cells. This defect is completely rescued in cells from dor4/Ydor+ animals (Fig. 9 F). These results strongly suggest that mutations in dor prevent normal release of Dor and Car proteins from endosomal membranes.


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

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

Eye color mutants affect the removal of Dor and Car from Rab7-positive endosomes. (A–F) Hemocytes from mutants of dor and car, synthetic lethal dor1car1, and dor4/Ydor+ were incubated for 15 min with F-Dex (green) and fixed after indicated chase times, immunostained (red) for Deep-orange (α-Dor), and imaged on a confocal microscope. Insets in A–D show magnified view of areas marked by an asterisk (top, antibody; middle, F-Dex; bottom, merge). Bold arrows (E and F) indicate antibody-stained structures lacking endocytic probes. (G and H) Histograms show the percentage of F-Dex–containing endosomes colocalized with α-Dor (G) or α-Car (H) at the indicated chase times in wild-type (blue), dor1 (green), dor4 (yellow), dor1car1(red), and car1 (gray). The results shown represent the mean ± SEM from two experiments. Bars: (shown in F corresponds to A–F) 5 μm; (inset) 1 μm.
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fig9: Eye color mutants affect the removal of Dor and Car from Rab7-positive endosomes. (A–F) Hemocytes from mutants of dor and car, synthetic lethal dor1car1, and dor4/Ydor+ were incubated for 15 min with F-Dex (green) and fixed after indicated chase times, immunostained (red) for Deep-orange (α-Dor), and imaged on a confocal microscope. Insets in A–D show magnified view of areas marked by an asterisk (top, antibody; middle, F-Dex; bottom, merge). Bold arrows (E and F) indicate antibody-stained structures lacking endocytic probes. (G and H) Histograms show the percentage of F-Dex–containing endosomes colocalized with α-Dor (G) or α-Car (H) at the indicated chase times in wild-type (blue), dor1 (green), dor4 (yellow), dor1car1(red), and car1 (gray). The results shown represent the mean ± SEM from two experiments. Bars: (shown in F corresponds to A–F) 5 μm; (inset) 1 μm.
Mentions: In cells from dor mutants (including dor1car1), levels of Dor immunostaining are lower than those observed in wild-type hemocytes (unpublished data; Sevrioukov et al., 1999). However, Dor (Fig. 9 , A–C and G) and Car (Fig. 9 H) immunoreactivity are persistent on the Rab7-positive large sized endosomes compared with wild-type cells. This defect is completely rescued in cells from dor4/Ydor+ animals (Fig. 9 F). These results strongly suggest that mutations in dor prevent normal release of Dor and Car proteins from endosomal membranes.

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