Limits...
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.

Show MeSH

Related in: MedlinePlus

Endosomal membrane association of Dor and morphological progression in car1/Ydor+. (A, C, and D) Hemocytes from car1/Ydor+ (A and D) and +/Ydor+ (C) were incubated with F-Dex (green) for 15 min followed by a chase of 2 h, fixed and immunostained (red) for Deep-orange (α-Dor; A and C) or pro–cathepsin L (α-proCathepsin L; D), and imaged on a confocal microscope. Insets in A, C, and D show magnified views of areas marked by an asterisk (top, antibody; middle, F-Dex; bottom, merge). Bold arrows indicate antibody-stained structures lacking endocytic probes. (B) Cells from car1/Ydor+ were incubated with LR-Dex (red) and A488-mBSA (green) for 5 min, and morphology of endosomes was visualized after a 2-h chase by confocal microscopy in living cells. Insets show magnified views of areas marked by an asterisk (top, Dex; middle, mBSA; bottom, merge). Bars: (shown in B corresponds to A–D) 5 μm; (insets) 1 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172926&req=5

fig10: Endosomal membrane association of Dor and morphological progression in car1/Ydor+. (A, C, and D) Hemocytes from car1/Ydor+ (A and D) and +/Ydor+ (C) were incubated with F-Dex (green) for 15 min followed by a chase of 2 h, fixed and immunostained (red) for Deep-orange (α-Dor; A and C) or pro–cathepsin L (α-proCathepsin L; D), and imaged on a confocal microscope. Insets in A, C, and D show magnified views of areas marked by an asterisk (top, antibody; middle, F-Dex; bottom, merge). Bold arrows indicate antibody-stained structures lacking endocytic probes. (B) Cells from car1/Ydor+ were incubated with LR-Dex (red) and A488-mBSA (green) for 5 min, and morphology of endosomes was visualized after a 2-h chase by confocal microscopy in living cells. Insets show magnified views of areas marked by an asterisk (top, Dex; middle, mBSA; bottom, merge). Bars: (shown in B corresponds to A–D) 5 μm; (insets) 1 μm.

Mentions: Endosomes in car1 cells show a wild-type loss of Car immunostaining (Fig. 9 H, gray bars) but are slowed down in the loss of Dor immunoreactivity (Fig. 9, D and E, gray bars, and G), suggesting a function for Car in the removal of Dor from endosomal membranes. To test this role of Car, we examined the kinetics of removal of Dor in cells from animals (car1/Ydor+) that overexpress Dor. Mere overexpression of Dor (in cells from +/Ydor+ animals carrying a duplication of dor) shows normal endosomal Dor dissociation kinetics (Fig. 10 C) and endosomal morphological progression (unpublished data). However, overexpression of Dor in cells with mutant Car (car1) results in a prolonged association (persistent even at 2 h) of Dor on endosomal compartments (Fig. 10 A compared with Fig. 9 E); endosomal morphological transition to small sized compartments is also blocked (Fig. 10 B compared with Fig. 6 G). Furthermore, these endosomes become fusion inaccessible at 30 min, indicating that they mature with wild-type kinetics (unpublished data). These results provide evidence that Car regulates removal of Dor from endosomal membranes; car1 mutation impairs this ability. Arrest of endosomes at the stage of large sized compartments suggests that Car and Dor are required for the morphological transition from large Rab7-positive to small sized endosomes. Endosomal degradation is unchanged with respect to cells from car1 animals (unpublished data) consistent with normal delivery of pro–cathepsin L (Fig. 10 D). Thus, blockage of morphological progression of the endosomes in cells from car1/Ydor+ animals is uncoupled from an inhibition of Golgi vesicle delivery (Fig. 10 D) and endosomal degradation.


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

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

Endosomal membrane association of Dor and morphological progression in car1/Ydor+. (A, C, and D) Hemocytes from car1/Ydor+ (A and D) and +/Ydor+ (C) were incubated with F-Dex (green) for 15 min followed by a chase of 2 h, fixed and immunostained (red) for Deep-orange (α-Dor; A and C) or pro–cathepsin L (α-proCathepsin L; D), and imaged on a confocal microscope. Insets in A, C, and D show magnified views of areas marked by an asterisk (top, antibody; middle, F-Dex; bottom, merge). Bold arrows indicate antibody-stained structures lacking endocytic probes. (B) Cells from car1/Ydor+ were incubated with LR-Dex (red) and A488-mBSA (green) for 5 min, and morphology of endosomes was visualized after a 2-h chase by confocal microscopy in living cells. Insets show magnified views of areas marked by an asterisk (top, Dex; middle, mBSA; bottom, merge). Bars: (shown in B corresponds to A–D) 5 μm; (insets) 1 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172926&req=5

fig10: Endosomal membrane association of Dor and morphological progression in car1/Ydor+. (A, C, and D) Hemocytes from car1/Ydor+ (A and D) and +/Ydor+ (C) were incubated with F-Dex (green) for 15 min followed by a chase of 2 h, fixed and immunostained (red) for Deep-orange (α-Dor; A and C) or pro–cathepsin L (α-proCathepsin L; D), and imaged on a confocal microscope. Insets in A, C, and D show magnified views of areas marked by an asterisk (top, antibody; middle, F-Dex; bottom, merge). Bold arrows indicate antibody-stained structures lacking endocytic probes. (B) Cells from car1/Ydor+ were incubated with LR-Dex (red) and A488-mBSA (green) for 5 min, and morphology of endosomes was visualized after a 2-h chase by confocal microscopy in living cells. Insets show magnified views of areas marked by an asterisk (top, Dex; middle, mBSA; bottom, merge). Bars: (shown in B corresponds to A–D) 5 μm; (insets) 1 μm.
Mentions: Endosomes in car1 cells show a wild-type loss of Car immunostaining (Fig. 9 H, gray bars) but are slowed down in the loss of Dor immunoreactivity (Fig. 9, D and E, gray bars, and G), suggesting a function for Car in the removal of Dor from endosomal membranes. To test this role of Car, we examined the kinetics of removal of Dor in cells from animals (car1/Ydor+) that overexpress Dor. Mere overexpression of Dor (in cells from +/Ydor+ animals carrying a duplication of dor) shows normal endosomal Dor dissociation kinetics (Fig. 10 C) and endosomal morphological progression (unpublished data). However, overexpression of Dor in cells with mutant Car (car1) results in a prolonged association (persistent even at 2 h) of Dor on endosomal compartments (Fig. 10 A compared with Fig. 9 E); endosomal morphological transition to small sized compartments is also blocked (Fig. 10 B compared with Fig. 6 G). Furthermore, these endosomes become fusion inaccessible at 30 min, indicating that they mature with wild-type kinetics (unpublished data). These results provide evidence that Car regulates removal of Dor from endosomal membranes; car1 mutation impairs this ability. Arrest of endosomes at the stage of large sized compartments suggests that Car and Dor are required for the morphological transition from large Rab7-positive to small sized endosomes. Endosomal degradation is unchanged with respect to cells from car1 animals (unpublished data) consistent with normal delivery of pro–cathepsin L (Fig. 10 D). Thus, blockage of morphological progression of the endosomes in cells from car1/Ydor+ animals is uncoupled from an inhibition of Golgi vesicle delivery (Fig. 10 D) and endosomal degradation.

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