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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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Hemocytes from mutant alleles of dor and car show block in distinct stages of endolysosomal traffic. Hemocytes from indicated mutants were incubated with LR-Dex (red) and A488-mBSA (green) for 5 min, and the morphology of endosomes was visualized by confocal microscopy in living cells either immediately (A and D) or after indicated chase times (B, C, and E–I). In cells from mutant alleles of dor and car, 5-min endosomes (A and D) contain both probes. Cells from dor1 and dor1car1 do not show endosomal morphological transformation into small sized endosomes even at long chase times (B and H, open arrowheads), whereas endosomes in cells from car1 undergo rapid morphological transition into small dense organelles (E, arrows) but fail to elaborate tubular structures at longer chase times (F and G). Cells from dor4 show a marginal endosomal morphological transformation into small sized compartments (arrow) at longer chase times (C, open arrowhead indicates the large compartment). This defect is completely rescued in cells from dor4/Ydor+ (I, arrowhead, tubular- vesicular compartments). Insets in all panels show magnified view of areas marked by an asterisk. Bars: (shown in A corresponds to A–I) 5 μm; (insets) 1 μm.
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fig6: Hemocytes from mutant alleles of dor and car show block in distinct stages of endolysosomal traffic. Hemocytes from indicated mutants were incubated with LR-Dex (red) and A488-mBSA (green) for 5 min, and the morphology of endosomes was visualized by confocal microscopy in living cells either immediately (A and D) or after indicated chase times (B, C, and E–I). In cells from mutant alleles of dor and car, 5-min endosomes (A and D) contain both probes. Cells from dor1 and dor1car1 do not show endosomal morphological transformation into small sized endosomes even at long chase times (B and H, open arrowheads), whereas endosomes in cells from car1 undergo rapid morphological transition into small dense organelles (E, arrows) but fail to elaborate tubular structures at longer chase times (F and G). Cells from dor4 show a marginal endosomal morphological transformation into small sized compartments (arrow) at longer chase times (C, open arrowhead indicates the large compartment). This defect is completely rescued in cells from dor4/Ydor+ (I, arrowhead, tubular- vesicular compartments). Insets in all panels show magnified view of areas marked by an asterisk. Bars: (shown in A corresponds to A–I) 5 μm; (insets) 1 μm.

Mentions: We next examined the morphology of endosomal compartments accessed by dSR ligands and fluid phase in live hemocytes from dor4, dor1, car1, and dor1car1. Following a similar pulse–chase protocol outlined earlier (Fig. 1), we find that the net internalization of probes was not affected in any of the mutants studied (Fig. 5). The probes are delivered to Rab7-positive (unpublished data) large endosomes in all alleles (Fig. 6) . Distinct from cells from wild-type (Fig. 1 D) and car1 animals (Fig. 6 E), the large endosomes in mutant dor alleles appear blocked in progression to later stages (Fig. 6, A and B, dor1; C, dor4; H, dor1car1). This defect is completely rescued by overexpression of Dor; in cells from dor4/Ydor+ endosomal progression is similar to wild type (Fig. 6 I). Ultrastructure analysis using HRP as a fluid phase probe showed no difference in the morphology and formation of the MVB and the small dense organelle between car1 and Canton-S cells (unpublished data). In the cells from the synthetic lethal mutant dor1car1, after a 2-h chase, many endosomes showed an aberrant distribution of endocytosed probes wherein the dSR ligand is often distributed on the endosomal membrane and the fluid tracer is present in the lumen of exaggerated large sized endosomes (3–4 μm; Fig. 6 H, inset). Cells from car1 animals exhibited a block in transition of small dense vesicles to tubular-vesicular compartments; even after a chase of 2 h (Fig. 6 G compared with Fig. 1 F) or 4 h (data not depicted) only small vesicular structures are observed.


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

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

Hemocytes from mutant alleles of dor and car show block in distinct stages of endolysosomal traffic. Hemocytes from indicated mutants were incubated with LR-Dex (red) and A488-mBSA (green) for 5 min, and the morphology of endosomes was visualized by confocal microscopy in living cells either immediately (A and D) or after indicated chase times (B, C, and E–I). In cells from mutant alleles of dor and car, 5-min endosomes (A and D) contain both probes. Cells from dor1 and dor1car1 do not show endosomal morphological transformation into small sized endosomes even at long chase times (B and H, open arrowheads), whereas endosomes in cells from car1 undergo rapid morphological transition into small dense organelles (E, arrows) but fail to elaborate tubular structures at longer chase times (F and G). Cells from dor4 show a marginal endosomal morphological transformation into small sized compartments (arrow) at longer chase times (C, open arrowhead indicates the large compartment). This defect is completely rescued in cells from dor4/Ydor+ (I, arrowhead, tubular- vesicular compartments). Insets in all panels show magnified view of areas marked by an asterisk. Bars: (shown in A corresponds to A–I) 5 μm; (insets) 1 μm.
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Related In: Results  -  Collection

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fig6: Hemocytes from mutant alleles of dor and car show block in distinct stages of endolysosomal traffic. Hemocytes from indicated mutants were incubated with LR-Dex (red) and A488-mBSA (green) for 5 min, and the morphology of endosomes was visualized by confocal microscopy in living cells either immediately (A and D) or after indicated chase times (B, C, and E–I). In cells from mutant alleles of dor and car, 5-min endosomes (A and D) contain both probes. Cells from dor1 and dor1car1 do not show endosomal morphological transformation into small sized endosomes even at long chase times (B and H, open arrowheads), whereas endosomes in cells from car1 undergo rapid morphological transition into small dense organelles (E, arrows) but fail to elaborate tubular structures at longer chase times (F and G). Cells from dor4 show a marginal endosomal morphological transformation into small sized compartments (arrow) at longer chase times (C, open arrowhead indicates the large compartment). This defect is completely rescued in cells from dor4/Ydor+ (I, arrowhead, tubular- vesicular compartments). Insets in all panels show magnified view of areas marked by an asterisk. Bars: (shown in A corresponds to A–I) 5 μm; (insets) 1 μm.
Mentions: We next examined the morphology of endosomal compartments accessed by dSR ligands and fluid phase in live hemocytes from dor4, dor1, car1, and dor1car1. Following a similar pulse–chase protocol outlined earlier (Fig. 1), we find that the net internalization of probes was not affected in any of the mutants studied (Fig. 5). The probes are delivered to Rab7-positive (unpublished data) large endosomes in all alleles (Fig. 6) . Distinct from cells from wild-type (Fig. 1 D) and car1 animals (Fig. 6 E), the large endosomes in mutant dor alleles appear blocked in progression to later stages (Fig. 6, A and B, dor1; C, dor4; H, dor1car1). This defect is completely rescued by overexpression of Dor; in cells from dor4/Ydor+ endosomal progression is similar to wild type (Fig. 6 I). Ultrastructure analysis using HRP as a fluid phase probe showed no difference in the morphology and formation of the MVB and the small dense organelle between car1 and Canton-S cells (unpublished data). In the cells from the synthetic lethal mutant dor1car1, after a 2-h chase, many endosomes showed an aberrant distribution of endocytosed probes wherein the dSR ligand is often distributed on the endosomal membrane and the fluid tracer is present in the lumen of exaggerated large sized endosomes (3–4 μm; Fig. 6 H, inset). Cells from car1 animals exhibited a block in transition of small dense vesicles to tubular-vesicular compartments; even after a chase of 2 h (Fig. 6 G compared with Fig. 1 F) or 4 h (data not depicted) only small vesicular structures are observed.

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