Analysis of post-lysosomal compartments.
Lysosomes are acidic intracellular compartments and are regarded as degradative and the end point, of the endocytic pathway. Here we provide evidence for the generation of acid hydrolase poor and non-acidic post-lysosomal compartments in NRK cells that have accumulated non-digestible macromolecules, Texas red-dextran (TR-Dex), within lysosomes. When TR-Dex was fed to the cells for 6h, most of the internalized TR-Dex colocalized with a lysosomal enzyme, cathepsin D. With an increase in the chase period, however, the internalized TR-Dex gradually accumulated in cathepsin D-negative vesicles. These vesicles were positive for a lysosomal membrane protein, LGP85, and their formation was inhibited by treatment of the cells with U18666A, which impairs membrane transport out of late endosomal/lysosomal compartments, thereby suggesting that the vesicles are derived from lysosomes. Interestingly, these compartments are non-acidic as judged for the DAMP staining. The results, therefore, suggest that the excess accumulation of non-digestible macromolecules within lysosomes induces the formation of acid hydrolase poor and non-acidic post-lysosomal compartments. The fact that treatment of the cells with lysosomotropic amines or a microtubule-depolymerization agent resulted in extensive colocalization of TR-Dex with cathepsin D further indicates that the formation of the post-lysosomal compartments depends on the lysosomal acidification and microtubule organization. Furthermore, these results suggest bi-directional membrane transport between lysosomes and the post-lysosomal compartments, which implies that the latter are not resting compartments.
Hirota
Yuko
Y
Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, 812-8582 Fukuoka, Japan.
Masuyama
Naoko
N
Kuronita
Toshio
T
Fujita
Hideaki
H
Himeno
Masaru
M
Tanaka
Yoshitaka
Y
D000736:Androstenes / Q000494:pharmacology; D000818:Animals; D018955:CD36 Antigens / Q000096:biosynthesis; D002402:Cathepsin D / Q000096:biosynthesis / Q000737:chemistry; D003911:Dextrans / Q000737:chemistry; D011992:Endosomes / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000378:metabolism; D007668:Kidney; D016464:Lysosomal Storage Diseases / Q000378:metabolism; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000378:metabolism* / Q000502:physiology; D008562:Membrane Glycoproteins; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism; D051381:Rats; D013997:Time Factors; D014966:Xanthenes / Q000494:pharmacology
D000736:Androstenes; D018955:CD36 Antigens; D003911:Dextrans; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D014966:Xanthenes; C070693:Scarb2 protein, rat; C006261:3-beta-(2-(diethylamino)ethoxy)androst-5-en-17-one; C034657:Texas red; D006867:Hydrolases; D002402:Cathepsin D