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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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Mutant alleles of dor fail to deliver Golgi-derived hydrolase to endosomes. Hemocytes from wild-type (A and B), car1 (C), dor1 (D), and dor1car1 (E) incubated with F-Dex (green) for 5 (A) or 15 (B–E) min were fixed and immunostained with antiserum against pro–cathepsin L (α-proCathepsin L; red) either immediately (A) or after 2 h (B–E) and imaged on a confocal microscope. Insets in A–E show magnified views of areas marked by an asterisk (α-proCathepsin L, top; F-Dex, middle inset). Note the accumulation of large ring-like organelles containing pro–cathepsin L in the dor alleles (D and E, bold arrows). Histogram in F shows the percentage of F-Dex–containing endosomes colocalized with pro–cathepsin L at the indicated chase times in different alleles. Note the complete rescue of defect in fusion of Golgi-derived vesicles with endosomes in hemocytes from dor4/Ydor+. The results represent the mean ± SEM derived from two experiments. Bars: (shown in E corresponds to A–E) 5 μm; (insets) 1 μm.
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fig7: Mutant alleles of dor fail to deliver Golgi-derived hydrolase to endosomes. Hemocytes from wild-type (A and B), car1 (C), dor1 (D), and dor1car1 (E) incubated with F-Dex (green) for 5 (A) or 15 (B–E) min were fixed and immunostained with antiserum against pro–cathepsin L (α-proCathepsin L; red) either immediately (A) or after 2 h (B–E) and imaged on a confocal microscope. Insets in A–E show magnified views of areas marked by an asterisk (α-proCathepsin L, top; F-Dex, middle inset). Note the accumulation of large ring-like organelles containing pro–cathepsin L in the dor alleles (D and E, bold arrows). Histogram in F shows the percentage of F-Dex–containing endosomes colocalized with pro–cathepsin L at the indicated chase times in different alleles. Note the complete rescue of defect in fusion of Golgi-derived vesicles with endosomes in hemocytes from dor4/Ydor+. The results represent the mean ± SEM derived from two experiments. Bars: (shown in E corresponds to A–E) 5 μm; (insets) 1 μm.

Mentions: The effects of mutant alleles of dor characterized thus far do not provide an explanation for reduced endosomal degradation observed in cells from mutant animals. To determine whether defects in endosomal degradation observed in cells from dor alleles are due to alterations in specific trafficking steps, we monitored the delivery of Golgi-derived hydrolases to the endolysosomal system. Cysteine proteinases like cathepsin L are important constituents of the lytic system in lysosomes (Turk et al., 2001; Zwad et al., 2002) and are delivered directly from the Golgi to MVBs and lysosomes in the pro form where they undergo cleavage to yield the mature proteinase (Turk et al., 2001). To detect the delivery of Golgi cargo we have used antiserum generated against Sacrophaga peregrina pro–cathepsin L that cross-reacts with Drosophila cathepsin L–like enzyme encoded by cp1 (Tryselius and Hultmark, 1997) and monitored the time course of intersection of endocytosed F-Dex with this immunoreactivity. At the earliest time monitored (5 min), a fraction (∼20%) of large sized F-Dex–containing endosomes is labeled by antiserum against pro–cathepsin L in cells from wild-type (Fig. 7 A, open arrowhead) and car1 animals (unpublished data). This suggests that at least some of Golgi-derived pro–cathepsin L is delivered to the 5 min, Rab7-positive large sized endosomes. At this time, endosomes in cells from dor alleles and dor1car1 cells do not show any detectable pro–cathepsin L staining (unpublished data). When a 15-min pulse of F-Dex is chased for longer times in cells from wild-type and car1 animals an increasing fraction of F-Dex–containing endosomes shows staining for pro–cathepsin L as seen qualitatively in Fig. 7, B and C, and quantitatively in Fig. 7 F. However, in none of the dor alleles, is significant pro–cathepsin L staining detected even at late times (Fig. 7, D–F; dor4, unpublished data). This defect is completely rescued in cells overexpressing Dor (dor4/Ydor+; Fig. 7 F and Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.20010166/DC1).


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

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

Mutant alleles of dor fail to deliver Golgi-derived hydrolase to endosomes. Hemocytes from wild-type (A and B), car1 (C), dor1 (D), and dor1car1 (E) incubated with F-Dex (green) for 5 (A) or 15 (B–E) min were fixed and immunostained with antiserum against pro–cathepsin L (α-proCathepsin L; red) either immediately (A) or after 2 h (B–E) and imaged on a confocal microscope. Insets in A–E show magnified views of areas marked by an asterisk (α-proCathepsin L, top; F-Dex, middle inset). Note the accumulation of large ring-like organelles containing pro–cathepsin L in the dor alleles (D and E, bold arrows). Histogram in F shows the percentage of F-Dex–containing endosomes colocalized with pro–cathepsin L at the indicated chase times in different alleles. Note the complete rescue of defect in fusion of Golgi-derived vesicles with endosomes in hemocytes from dor4/Ydor+. The results represent the mean ± SEM derived from two experiments. Bars: (shown in E corresponds to A–E) 5 μm; (insets) 1 μm.
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Related In: Results  -  Collection

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fig7: Mutant alleles of dor fail to deliver Golgi-derived hydrolase to endosomes. Hemocytes from wild-type (A and B), car1 (C), dor1 (D), and dor1car1 (E) incubated with F-Dex (green) for 5 (A) or 15 (B–E) min were fixed and immunostained with antiserum against pro–cathepsin L (α-proCathepsin L; red) either immediately (A) or after 2 h (B–E) and imaged on a confocal microscope. Insets in A–E show magnified views of areas marked by an asterisk (α-proCathepsin L, top; F-Dex, middle inset). Note the accumulation of large ring-like organelles containing pro–cathepsin L in the dor alleles (D and E, bold arrows). Histogram in F shows the percentage of F-Dex–containing endosomes colocalized with pro–cathepsin L at the indicated chase times in different alleles. Note the complete rescue of defect in fusion of Golgi-derived vesicles with endosomes in hemocytes from dor4/Ydor+. The results represent the mean ± SEM derived from two experiments. Bars: (shown in E corresponds to A–E) 5 μm; (insets) 1 μm.
Mentions: The effects of mutant alleles of dor characterized thus far do not provide an explanation for reduced endosomal degradation observed in cells from mutant animals. To determine whether defects in endosomal degradation observed in cells from dor alleles are due to alterations in specific trafficking steps, we monitored the delivery of Golgi-derived hydrolases to the endolysosomal system. Cysteine proteinases like cathepsin L are important constituents of the lytic system in lysosomes (Turk et al., 2001; Zwad et al., 2002) and are delivered directly from the Golgi to MVBs and lysosomes in the pro form where they undergo cleavage to yield the mature proteinase (Turk et al., 2001). To detect the delivery of Golgi cargo we have used antiserum generated against Sacrophaga peregrina pro–cathepsin L that cross-reacts with Drosophila cathepsin L–like enzyme encoded by cp1 (Tryselius and Hultmark, 1997) and monitored the time course of intersection of endocytosed F-Dex with this immunoreactivity. At the earliest time monitored (5 min), a fraction (∼20%) of large sized F-Dex–containing endosomes is labeled by antiserum against pro–cathepsin L in cells from wild-type (Fig. 7 A, open arrowhead) and car1 animals (unpublished data). This suggests that at least some of Golgi-derived pro–cathepsin L is delivered to the 5 min, Rab7-positive large sized endosomes. At this time, endosomes in cells from dor alleles and dor1car1 cells do not show any detectable pro–cathepsin L staining (unpublished data). When a 15-min pulse of F-Dex is chased for longer times in cells from wild-type and car1 animals an increasing fraction of F-Dex–containing endosomes shows staining for pro–cathepsin L as seen qualitatively in Fig. 7, B and C, and quantitatively in Fig. 7 F. However, in none of the dor alleles, is significant pro–cathepsin L staining detected even at late times (Fig. 7, D–F; dor4, unpublished data). This defect is completely rescued in cells overexpressing Dor (dor4/Ydor+; Fig. 7 F and Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.20010166/DC1).

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