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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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Rab7 labels endolysosomes in larval hemocytes. (A–C) Hemocytes incubated with F-Dex for 15 min were fixed either immediately (A) or, after a 1- (B) or 2-h (C) chase period in the absence of endocytic probe, immunostained for Rab7 and imaged on a confocal microscope. Images show that Rab7 (red; middle insets) labels different stages of the endosomal system accessed by endocytosed F-Dex (green, left insets) after indicated chase times. Insets show a magnified view of areas marked by an asterisk. (D) Histogram shows the percentage of F-Dex– containing endosomes per cell colocalized with immunolocalized Rab7. The results shown represent the mean ± SEM obtained from two experiments. Bars: (shown in C corresponds to A–C) 5 μm; (insets) 1 μm.
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fig3: Rab7 labels endolysosomes in larval hemocytes. (A–C) Hemocytes incubated with F-Dex for 15 min were fixed either immediately (A) or, after a 1- (B) or 2-h (C) chase period in the absence of endocytic probe, immunostained for Rab7 and imaged on a confocal microscope. Images show that Rab7 (red; middle insets) labels different stages of the endosomal system accessed by endocytosed F-Dex (green, left insets) after indicated chase times. Insets show a magnified view of areas marked by an asterisk. (D) Histogram shows the percentage of F-Dex– containing endosomes per cell colocalized with immunolocalized Rab7. The results shown represent the mean ± SEM obtained from two experiments. Bars: (shown in C corresponds to A–C) 5 μm; (insets) 1 μm.

Mentions: After a 5-min pulse, increasing chase times (Fig. 1, D–F) result in major morphological changes in endosomal compartments accessed by Fl-mBSA and LR-Dex, clearly distinguishing them from MVBs observed at 5 min. After a 15-min chase period, endosomes appear smaller (0.5–1.0 μm) and show complete colocalization of Fl-mBSA and fluid tracer within the endosome (Fig. 1 D, arrows and inset). EM analysis confirms the formation of small HRP-filled electron-dense structures (Fig. 1 G iii). After a 1-h chase, endosomal morphology is tubular-vesicular (Fig. 1 E), and this persists for 2 (Fig. 1 F) to 4 h (data not depicted). This tubular morphology is sensitive to osmotic stress (unpublished data), analogous to tubular lysosomes observed in mammalian bone marrow–derived macrophages labeled with the fluid tracer lucifer yellow (Knapp and Swanson, 1990) and conventional fixing protocols. Tubules are not observed by fluorescence (Fig. 3 C) and EM analyses (Fig. 1 G iv) of fixed samples. Comparison of the morphology of endosomes as a function of chase times in live cells versus those obtained in fixed cells is shown in Fig. 2 .


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

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

Rab7 labels endolysosomes in larval hemocytes. (A–C) Hemocytes incubated with F-Dex for 15 min were fixed either immediately (A) or, after a 1- (B) or 2-h (C) chase period in the absence of endocytic probe, immunostained for Rab7 and imaged on a confocal microscope. Images show that Rab7 (red; middle insets) labels different stages of the endosomal system accessed by endocytosed F-Dex (green, left insets) after indicated chase times. Insets show a magnified view of areas marked by an asterisk. (D) Histogram shows the percentage of F-Dex– containing endosomes per cell colocalized with immunolocalized Rab7. The results shown represent the mean ± SEM obtained from two experiments. Bars: (shown in C corresponds to A–C) 5 μm; (insets) 1 μm.
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fig3: Rab7 labels endolysosomes in larval hemocytes. (A–C) Hemocytes incubated with F-Dex for 15 min were fixed either immediately (A) or, after a 1- (B) or 2-h (C) chase period in the absence of endocytic probe, immunostained for Rab7 and imaged on a confocal microscope. Images show that Rab7 (red; middle insets) labels different stages of the endosomal system accessed by endocytosed F-Dex (green, left insets) after indicated chase times. Insets show a magnified view of areas marked by an asterisk. (D) Histogram shows the percentage of F-Dex– containing endosomes per cell colocalized with immunolocalized Rab7. The results shown represent the mean ± SEM obtained from two experiments. Bars: (shown in C corresponds to A–C) 5 μm; (insets) 1 μm.
Mentions: After a 5-min pulse, increasing chase times (Fig. 1, D–F) result in major morphological changes in endosomal compartments accessed by Fl-mBSA and LR-Dex, clearly distinguishing them from MVBs observed at 5 min. After a 15-min chase period, endosomes appear smaller (0.5–1.0 μm) and show complete colocalization of Fl-mBSA and fluid tracer within the endosome (Fig. 1 D, arrows and inset). EM analysis confirms the formation of small HRP-filled electron-dense structures (Fig. 1 G iii). After a 1-h chase, endosomal morphology is tubular-vesicular (Fig. 1 E), and this persists for 2 (Fig. 1 F) to 4 h (data not depicted). This tubular morphology is sensitive to osmotic stress (unpublished data), analogous to tubular lysosomes observed in mammalian bone marrow–derived macrophages labeled with the fluid tracer lucifer yellow (Knapp and Swanson, 1990) and conventional fixing protocols. Tubules are not observed by fluorescence (Fig. 3 C) and EM analyses (Fig. 1 G iv) of fixed samples. Comparison of the morphology of endosomes as a function of chase times in live cells versus those obtained in fixed cells is shown in Fig. 2 .

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