Acidification of morphologically distinct endosomes in mutant and wild-type Chinese hamster ovary cells.

Abstract

we have shown that there is rapid acidification of endosomal compartments to pH 6.3 by 3 min in wildtype Chinese hamster ovary (CHO) cells. In contrast, early acidification of endosomes is markedly reduced in the CHO mutants, DTF 1-5-4 and DTF 1-5-1. Since these CHO mutants are pleiotropically defective in endocytosis (Robbins, A. R., S. S. Peng, and J. L. Marshall. 1983. J. Cell Biol. 96:1064-1071; Robbins, A. R., C. Oliver, J. L. Bateman, S. S. Krag, C. J. Galloway, and I. Mellman. 1984. J. Cell Biol. 99" 1296-1308, our results are consistent with a requirement for proper acidification of early endocytic compartments in many pH-regulated endocytic processes. In this paper, by measuring the pH of morphologically distinct endosomes using fluorescence microscopy and digital image analysis, we have determined in which of the endocytic compartments the defective acidification occurs. We found that the acidification of both the para-Golgi recycling endosomes and lysosomes was normal in the CHO mutants DTG 1-5-4 and DTF 1-5-1. The mean pH of large endosomes containing either fluorescein-labeled r or fluorescein-isothiocyanate dextran was only slightly less acidic in the mutant cells than in wild-type cells. However, when we examined the pH of individual large (150-250 nm) endosomes, we found that there was an increased number of endosomes with a pH >6.5 in the CHO mutants when compared with wildtype cells. Heterogeneity in the acidification of large endosomes was also seen in DTF 1-5-1 by a combined null point pH method and digital image analysis technique. In addition, both CHO mutants showed a marked decrease in the acidification of the earliest endosomal compartment, a diffusely fluorescent compartment comprised of small vesicles and tubules. We suggest that the defect in endosome acidification is most pronounced in the early, small vesicular, and tubular endosomes and that this defect partially carries over to the large endosomes that are involved in the sorting and processing of ligands. The proper step-wise acidification of the different endosomes along the endocytic pathway may have an important role in the regulation of endocytic processes.I N the preceding paper (40) we examined the kinetics of endosome acidification in wild-type and mutant Chinese hamster ovary (CHO) cells. We found in wild-type cells that there was a rapid (3-5 min) acidification of endosomes to pH 6.2-6.3. With time, ct~-macroglobulin (a2M) ~ and fluorescein-isothiocyanate dextran (F-Dex), molecules that are routed to lysosomes, are found in progressively more acidic endocytic compartments (pH < 6.0), while transferrin Dr. Maxfieid's present address is Departments of Pathology and Physiology, College of Physicians and Surgeons, Columbia University, New York, NY 10032.1. Abbreviations used in this paper: a2M, u2-macroglobulin; a2M-gold, a2-macroglobulin adsorbed to colloidal gold; AA/MA, ammonium acetate/methylamine; F-a2M, fluorescein-labeled ct2-macroglobulin; F-Dex, fluorescein isothioeyanate dextran; F-Tf, fluorescein-labeled transferr...

Kinetics of endosome acidification in mutant and wild-type Chinese hamster ovary cells.

“…One of the possible sites where the defect could occur is in the large, prelysosomal endosomes. However, when we measured the pH of large endosomes, as distinguished by light microscopy, we found that the mean pH was nearly the same in the mutants DTG 1-5-4 and DTF 1-5-1 as in the wild-type cells (32). This suggested that the defect might be at an earlier step in the endocytic pathway.…”

Sorting of membrane components from endosomes and subsequent recycling to the cell surface occurs by a bulk flow process.

“…This sorting is a rapid step (tu2 < 3 min), and as depicted in Fig. 1, takes place in acidic organelles called sorting endosomes (Yamashiro and Maxfield, 1987), having 1. Abbreviations used in this paper: ce2M, ce2-macroglobulin; C6-NBD-PC, C6-NBD-phosphatidylcholine; C6-NBD-SM, N-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-~-aminohexanoyl]-sphingosylphosphorylcholine; CCD, chargedcouped device; Cy3-ct2M, Cy3-1abeled ce2M; DiO-LDL, 3,3tdioctadecyl -oxacarbocyanine-labeled LDL; DiI-LDL, 3,3'-dioctadecylindocarbocyanine-labeled LDL; HF-12, Hepes-buffered Hams F-12 medium; LDL, low density lipoprotein; LDL-R, LDL-receptor; Rh-Tf, rhodamine-labeled Tf; "If, transferrin; Tf-R, "IT receptor; Tx, Texas-red; Tx-Tf, Texas-redlabeled Tf.…”

“…This sorting is a rapid step (tu2 < 3 min), and as depicted in Fig. 1, takes place in acidic organelles called sorting endosomes (Yamashiro and Maxfield, 1987), having…”

Aβ(1–40) Prevents Heparanase-catalyzed Degradation of Heparan Sulfate Glycosaminoglycans and Proteoglycans in Vitro

“…Long HS glycosaminoglycans interact with the peptide better than the short chains at the pH values found in endosomal compartments (pH 5.0 -6.5, Ref. 20), which again may reflect that the size of the glycosaminoglycan influences the 35 S counts retained on the membrane. Alternatively, because of their length, the nascent HS chains may be able to form more stable complexes with A␤(1-40) at these pH values than the heparanase-degraded glycosaminoglycans.…”

“…Experiments by Snow et al (1) suggest that both interactions are necessary for the formation of fibrillar A␤-amyloid, since coinfusion of A␤ and HSPGs into rat brain produced deposits, whereas coinfusion of A␤ and HS glycosaminoglycans did not. This is somewhat surprising, since in vitro, HS chains alone can cause both A␤ and A␤(1-40) to aggregate (3), and heparin converts A␤ (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28) into macrofibers (2). It may be that in vivo, stabilization of the aggregates and decreased amyloid susceptibility to proteolysis require A␤ to interact with both the core protein and HS chains.…”