Microspectrofluorometry by digital image processing: measurement of cytoplasmic pH.

114

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...

Section: Resultsmentioning

confidence: 99%

“…To measure the pH of specific endocytic compartments we have used fluorescence microscopy and digital image analysis (18,35,37). We have previously shown in wild-type CHO cells that the recycling endosomes have a pH of 6.4, while the large endosomes have a pH of 5.2 (41).…”

mentioning

confidence: 99%

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

Yamashiro

Maxfield

1987

114

“…8 for effects of pathlength and accessible volume on fluorescence intensity, we generated a ratio image in which Rh-aldolase fluorescence was divided by FITC-dextran fluorescence; bright areas in this image have a high concentration of aldolase relative to dextran (12,70,77). A relatively increased concentration of Rh-aldolase in zones around stress fibers is visualized as the bright, thin lines in Fig.…”

Section: The Cellular Distribution Of Aldolase Is Relatively Uniformmentioning

confidence: 99%

Aldolase exists in both the fluid and solid phases of cytoplasm [published erratum appears in J Cell Biol 1988 Dec;107(6 Pt 1):following 2463]

Pagliaro

Taylor

1988

115

Abstract. We have prepared a functional fluorescent analogue of the glycolytic enzyme aldolase (rhodamine [Rh]-aldolase), using the succinimidyl ester of carboxytetramethyl-rhodamine. Fluorescence redistribution after photobleaching measurements of the diffusion coefficient of Rh-aldolase in aqueous solutions gave a value of 4.7 × 10 -7 cm2/s, and no immobile fraction. In the presence of filamentous actin, there was a 4.5-fold reduction in diffusion coefficient, as well as a 36% immobile fraction, demonstrating binding of Rh-aldolase to actin. However, in the presence of a 100-fold molar excess of its substrate, fructose 1,6-diphosphate, both the mobile fraction and diffusion coefficient of Rh-aldolase returned to control levels, indicating competition between substrate binding and actin cross-linking. When Rh-aldolase was microinjected into Swiss 3T3 cells, a relatively uniform intracellular distribution of fluorescence was observed. However, there were significant spatial differences in the in vivo diffusion coefficient and mobile fraction of Rh-aldolase measured with fluorescence redistribution after photobleaching. In the perinuclear region, we measured an apparent cytoplasmic diffusion coefficient of 1.1 × 10 -7 cm2/s with a 23% immobile fraction; while measurements in the cell periphery gave a value of 5.7 × 10 -g cm2/s, with no immobile fraction. Ratio imaging of Rh-aldolase and FITCdextran indicated that FITC-dextran was relatively excluded from stress fiber domains. We interpret these data as evidence for the partitioning of aldolase between a soluble fraction in the fluid phase and a fraction associated with the solid phase of cytoplasm. The partitioning of aldolase and other glycolytic enzymes between the fluid and solid phases of cytoplasm could play a fundamental role in the control of glycolysis, the organization of cytoplasm, and cell motility. The concepts and experimental approaches described in this study can be applied to other cellular biochemical processes.TIaOUGH glycolytic metabolism is well-defined at the biochemical level, the dynamic and spatial organization of glycolysis in living cells has not been characterized to date. After the elucidation of mitochondrial structure and function 35 years ago (28) it was generally assumed that a corresponding organelle for glycolysis must exist. Efforts to isolate and to characterize such an organelle were unsuccessful, leading de Duve (26) to conclude that the longsought"glycolytic particle" did not exist. As a result, the current concept of in vivo glycolytic metabolism is based on an assumption of dilute solution chemistry, due largely to the absence of evidence for a discrete glycolytic organelle.There has been evidence for over 20 years that some glycoiytic enzymes bind to structural proteins, particularly actin.

“…The digital image analysis method was used by Yamashiro et al (16) and Tycko et al (18) to measure the pH of individual endocytic compartments within the same cell, and by Tanasugarn et al (26) to measure cytoplasmic and pinosome pH within the same cell. Activated monocytes inhibit L. pneumophila multiplication in two ways (13).…”

Section: Nonactivated Monocytesmentioning

confidence: 99%

Legionella pneumophila inhibits acidification of its phagosome in human monocytes.

Horwitz¹,

Maxfield²

1984

362

229

We used quantitative fluorescence microscopy to measure the pH of phagosomes in human monocytes that contain virulent Legionella pneumophila, a bacterial pathogen that multiplies intracellularly in these phagocytes. The mean pH of phagosomes that contain live L. pneumophila was 6.1 in 14 experiments. In the same experiments, the mean pH of phagosomes containing dead L. pneumophila averaged 0.8 pH units lower than the mean pH of phagosomes containing live L. pneumophila, a difference that was highly significant (P < 0.01 in all 14 experiments). In contrast, the mean pH of phagosomes initially containing live E. coil, which were then killed by monocytes, was the same as for phagosomes initially containing dead E. coli. The mean pH of L. pneumophila phagosomes in activated monocytes, which inhibit L. pneumophila intracellular multiplication, was the same as in nonactivated monocytes.To simultaneously measure the pH of different phagosomes within the same monocyte, we digitized and analyzed fluorescence images of monocytes that contained both live L. pneumophila and sheep erythrocytes. Within the same monocyte, live L. pneumophila phagosomes had a pH of ~6.1 and sheep erythrocyte phagosomes had a pH of ~5.0 or below.This study demonstrates that L. pneumophila is capable of modifying the pH of its phagocytic vacuole. This capability may be critical to the intracellular survival and multiplication of this and other intracellular pathogens.Legionella pneumophila, the agent of Legionnaires' disease, multiplies intracellularly in human mononuclear phagocytes within a membrane-bound cytoplasmic phagosome (1, 2).Live L. pneumophila inhibits fusion of the phagosome with monocyte lysosomes and induces the formation of a novel ribosome-lined phagosome. In contrast, formalin-killed L. pneumophila does not inhibit fusion or induce the formation of the specialized phagosome, and the dead bacteria are degraded within the phagolysosome (3, 4).The intraphagosomal pH has been measured or estimated in several bacteria and other particles that are phagocytized by mammalian phagocytes and later reside in phagolysosomes (5-11). These phagolysosomes become acidified. Since live and formalin-killed L. pneumophila have different intracellular destinies, we were interested in comparing the pH of phagosomes containing these two types of bacteria and exploring the possibility that alteration of intraphagosomal pH may play a role in L. pneumophila intracellular survival.In this paper, we used quantitative fluorescence microscopy to measure the pH of monocyte phagosomes that contain L.