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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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Localization of Deep-orange and Carnation within the late endolysosomal system in larval hemocytes. (A–E) Hemocytes incubated with F-Dex (green) for 15 min were fixed and immunostained (red) for Deep-orange (α-Dor) or Carnation (α-Car) either immediately (A and C) or after the indicated chase times (B, D, and E), and imaged on a confocal microscope. Insets show magnified views of the area marked by an asterisk (left, antibody; middle, F-Dex; right, merge). (F) Histogram shows the percentage of F-Dex–containing endosomes colocalized with α-Dor (orange) or α-Car (purple) at the indicated chase times. Histogram in the inset shows percentage of F-Dex–containing endosomes colocalized with α-Dor at shorter chase times. The results shown represent mean ± SEM from three experiments. Bar: (shown in A corresponds to A–E) 5 μm; (insets) 1 μm.
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fig8: Localization of Deep-orange and Carnation within the late endolysosomal system in larval hemocytes. (A–E) Hemocytes incubated with F-Dex (green) for 15 min were fixed and immunostained (red) for Deep-orange (α-Dor) or Carnation (α-Car) either immediately (A and C) or after the indicated chase times (B, D, and E), and imaged on a confocal microscope. Insets show magnified views of the area marked by an asterisk (left, antibody; middle, F-Dex; right, merge). (F) Histogram shows the percentage of F-Dex–containing endosomes colocalized with α-Dor (orange) or α-Car (purple) at the indicated chase times. Histogram in the inset shows percentage of F-Dex–containing endosomes colocalized with α-Dor at shorter chase times. The results shown represent mean ± SEM from three experiments. Bar: (shown in A corresponds to A–E) 5 μm; (insets) 1 μm.

Mentions: To obtain a mechanistic understanding of Dor and Car in biogenesis of endolysosomes, we examined the localization of Dor and Car on different Rab7-positive endosomal compartments in wild-type cells. F-Dex was pulsed and chased into morphologically distinct stages of the endosomal system as before (Fig. 2), and Dor and Car immunoreactivity associated with corresponding compartments was analyzed. Dor is associated with compartments labeled by a 15-min pulse of F-Dex (Fig. 8 A, inset). However, endosomal compartments accessed by a 15-min pulse followed by a 1-h chase are devoid of Dor immunoreactivity (Fig. 8 B, arrowheads). This is similar to observations made in garland cells wherein dextran beads are first seen in Dor-positive compartments and subsequently in Dor-negative compartments (Sevrioukov et al., 1999). Quantitative analysis shows that immediately after a 15-min pulse of F-Dex, 90% of all F-Dex–containing endosomes are α-Dor positive, whereas almost all endosomes accessed after a 1-h chase are devoid of detectable Dor immunoreactivity (Fig. 8 F). In a more detailed temporal analysis, after a pulse of 5 min wherein a majority of endosomes are large sized and Dor positive, a chase of 45-min results in <20% of F-Dex–containing endosomes retaining Dor immunoreactivity on exclusively small sized endosomes observed at this time (Fig. 8 F, inset).


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

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

Localization of Deep-orange and Carnation within the late endolysosomal system in larval hemocytes. (A–E) Hemocytes incubated with F-Dex (green) for 15 min were fixed and immunostained (red) for Deep-orange (α-Dor) or Carnation (α-Car) either immediately (A and C) or after the indicated chase times (B, D, and E), and imaged on a confocal microscope. Insets show magnified views of the area marked by an asterisk (left, antibody; middle, F-Dex; right, merge). (F) Histogram shows the percentage of F-Dex–containing endosomes colocalized with α-Dor (orange) or α-Car (purple) at the indicated chase times. Histogram in the inset shows percentage of F-Dex–containing endosomes colocalized with α-Dor at shorter chase times. The results shown represent mean ± SEM from three experiments. Bar: (shown in A corresponds to A–E) 5 μm; (insets) 1 μm.
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Related In: Results  -  Collection

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fig8: Localization of Deep-orange and Carnation within the late endolysosomal system in larval hemocytes. (A–E) Hemocytes incubated with F-Dex (green) for 15 min were fixed and immunostained (red) for Deep-orange (α-Dor) or Carnation (α-Car) either immediately (A and C) or after the indicated chase times (B, D, and E), and imaged on a confocal microscope. Insets show magnified views of the area marked by an asterisk (left, antibody; middle, F-Dex; right, merge). (F) Histogram shows the percentage of F-Dex–containing endosomes colocalized with α-Dor (orange) or α-Car (purple) at the indicated chase times. Histogram in the inset shows percentage of F-Dex–containing endosomes colocalized with α-Dor at shorter chase times. The results shown represent mean ± SEM from three experiments. Bar: (shown in A corresponds to A–E) 5 μm; (insets) 1 μm.
Mentions: To obtain a mechanistic understanding of Dor and Car in biogenesis of endolysosomes, we examined the localization of Dor and Car on different Rab7-positive endosomal compartments in wild-type cells. F-Dex was pulsed and chased into morphologically distinct stages of the endosomal system as before (Fig. 2), and Dor and Car immunoreactivity associated with corresponding compartments was analyzed. Dor is associated with compartments labeled by a 15-min pulse of F-Dex (Fig. 8 A, inset). However, endosomal compartments accessed by a 15-min pulse followed by a 1-h chase are devoid of Dor immunoreactivity (Fig. 8 B, arrowheads). This is similar to observations made in garland cells wherein dextran beads are first seen in Dor-positive compartments and subsequently in Dor-negative compartments (Sevrioukov et al., 1999). Quantitative analysis shows that immediately after a 15-min pulse of F-Dex, 90% of all F-Dex–containing endosomes are α-Dor positive, whereas almost all endosomes accessed after a 1-h chase are devoid of detectable Dor immunoreactivity (Fig. 8 F). In a more detailed temporal analysis, after a pulse of 5 min wherein a majority of endosomes are large sized and Dor positive, a chase of 45-min results in <20% of F-Dex–containing endosomes retaining Dor immunoreactivity on exclusively small sized endosomes observed at this time (Fig. 8 F, inset).

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