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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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Multivesicular late endosomes mature into small dense organelles in hemocytes from wild-type animals. (A and B) Hemocytes derived from wild-type larvae were incubated according to the pulse–chase–pulse protocol outlined in A with F-Dex (green) as first pulse and Cy3-mBSA (red) as second pulse, fixed, and imaged on wide-field microscope. (B) Outline of the predictions of maturation (left) and vesicle shuttle (right) models. This is in terms of kinetics of loss of fusion accessibility (top) and the change in ratio of amount of the first pulse to amount of colocalized second pulse in an endosome as a function of chase time (bottom). (C–F) Comparison of a 5- (C; F-Dex, middle; mBSA, top inset) with a 45-min chase time (D; F-Dex, arrow; mBSA, open arrowhead) between the two probes shows that the percentage of endosomes containing first probe and accessible to second probe reduces with increasing chase times. Insets in C and D show magnified views of areas marked by an asterisk. Histograms show kinetics of loss of fusion accessibility (E) and the relative ratio of amount of first pulse remaining in an endosome to amount of colocalized second pulse in the same endosome as a function of chase time (F). The data in E and F represent the median ± SD derived from two experiments. Bars: (shown in C corresponds to C and D) 5 μm; (insets) 1 μm.
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fig4: Multivesicular late endosomes mature into small dense organelles in hemocytes from wild-type animals. (A and B) Hemocytes derived from wild-type larvae were incubated according to the pulse–chase–pulse protocol outlined in A with F-Dex (green) as first pulse and Cy3-mBSA (red) as second pulse, fixed, and imaged on wide-field microscope. (B) Outline of the predictions of maturation (left) and vesicle shuttle (right) models. This is in terms of kinetics of loss of fusion accessibility (top) and the change in ratio of amount of the first pulse to amount of colocalized second pulse in an endosome as a function of chase time (bottom). (C–F) Comparison of a 5- (C; F-Dex, middle; mBSA, top inset) with a 45-min chase time (D; F-Dex, arrow; mBSA, open arrowhead) between the two probes shows that the percentage of endosomes containing first probe and accessible to second probe reduces with increasing chase times. Insets in C and D show magnified views of areas marked by an asterisk. Histograms show kinetics of loss of fusion accessibility (E) and the relative ratio of amount of first pulse remaining in an endosome to amount of colocalized second pulse in the same endosome as a function of chase time (F). The data in E and F represent the median ± SD derived from two experiments. Bars: (shown in C corresponds to C and D) 5 μm; (insets) 1 μm.

Mentions: We next probed the process by which endosomal cargo is trafficked between large sized endosomes and small dense endosomes formed in wild-type cells. There are mainly two ways this trafficking can happen: (1) via transformation of the large sized endosomes into the small dense endosomes (maturation process) or (2) vesicle budding from large sized endosomes, fusing with small dense endosomes (vesicle shuttle) (Fig. 4 B). These processes have distinct predictions for the mixing of endosomal contents between two temporally separated endocytic probe pulses (Fig. 4 A). Measuring the ratio of the amount of the two probes in colocalized endosomal compartments at different chase times provides a method of distinguishing between the vesicle shuttle and maturation models of endosomal trafficking between two types of organelles (Stoorvogel et al., 1991; Dunn and Maxfield, 1992).


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

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

Multivesicular late endosomes mature into small dense organelles in hemocytes from wild-type animals. (A and B) Hemocytes derived from wild-type larvae were incubated according to the pulse–chase–pulse protocol outlined in A with F-Dex (green) as first pulse and Cy3-mBSA (red) as second pulse, fixed, and imaged on wide-field microscope. (B) Outline of the predictions of maturation (left) and vesicle shuttle (right) models. This is in terms of kinetics of loss of fusion accessibility (top) and the change in ratio of amount of the first pulse to amount of colocalized second pulse in an endosome as a function of chase time (bottom). (C–F) Comparison of a 5- (C; F-Dex, middle; mBSA, top inset) with a 45-min chase time (D; F-Dex, arrow; mBSA, open arrowhead) between the two probes shows that the percentage of endosomes containing first probe and accessible to second probe reduces with increasing chase times. Insets in C and D show magnified views of areas marked by an asterisk. Histograms show kinetics of loss of fusion accessibility (E) and the relative ratio of amount of first pulse remaining in an endosome to amount of colocalized second pulse in the same endosome as a function of chase time (F). The data in E and F represent the median ± SD derived from two experiments. Bars: (shown in C corresponds to C and D) 5 μm; (insets) 1 μm.
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fig4: Multivesicular late endosomes mature into small dense organelles in hemocytes from wild-type animals. (A and B) Hemocytes derived from wild-type larvae were incubated according to the pulse–chase–pulse protocol outlined in A with F-Dex (green) as first pulse and Cy3-mBSA (red) as second pulse, fixed, and imaged on wide-field microscope. (B) Outline of the predictions of maturation (left) and vesicle shuttle (right) models. This is in terms of kinetics of loss of fusion accessibility (top) and the change in ratio of amount of the first pulse to amount of colocalized second pulse in an endosome as a function of chase time (bottom). (C–F) Comparison of a 5- (C; F-Dex, middle; mBSA, top inset) with a 45-min chase time (D; F-Dex, arrow; mBSA, open arrowhead) between the two probes shows that the percentage of endosomes containing first probe and accessible to second probe reduces with increasing chase times. Insets in C and D show magnified views of areas marked by an asterisk. Histograms show kinetics of loss of fusion accessibility (E) and the relative ratio of amount of first pulse remaining in an endosome to amount of colocalized second pulse in the same endosome as a function of chase time (F). The data in E and F represent the median ± SD derived from two experiments. Bars: (shown in C corresponds to C and D) 5 μm; (insets) 1 μm.
Mentions: We next probed the process by which endosomal cargo is trafficked between large sized endosomes and small dense endosomes formed in wild-type cells. There are mainly two ways this trafficking can happen: (1) via transformation of the large sized endosomes into the small dense endosomes (maturation process) or (2) vesicle budding from large sized endosomes, fusing with small dense endosomes (vesicle shuttle) (Fig. 4 B). These processes have distinct predictions for the mixing of endosomal contents between two temporally separated endocytic probe pulses (Fig. 4 A). Measuring the ratio of the amount of the two probes in colocalized endosomal compartments at different chase times provides a method of distinguishing between the vesicle shuttle and maturation models of endosomal trafficking between two types of organelles (Stoorvogel et al., 1991; Dunn and Maxfield, 1992).

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