Acidification of phagosomes is initiated before lysosomal enzyme activity is detected

P. L. McNeil, L. Tanasugarn, J. B. Meigs, D. L. Taylor

Research output: Contribution to journalArticle

46 Citations (Scopus)

Abstract

We have measured changes of pH in a protein's microenvironment consequent on its binding to the cell surface and incorporation into pinosomes. Changes of pH were measured from single, living cells and selected regions of cells by the fluorescence ratio technique using a photon-counting microspectrofluorimeter. The chemotactic agent and pinocytosis inducer, ribonuclease, labeled with fluorescein (FTC-RNase), adsorbed to the surface of Amoeba proteus, and was pinocytosed by cells in culture media at pH 7.0. The FTC-RNase entered an apparently acidic microenvironment, pH ~6.1, upon binding to the surface of amoebae. Once enclosed within pinosomes, this protein's microenvironment became steadily more acidic, reaching a minimum of pH ~5.6 in <10 min. FTC-RNase pinocytosed by the giant amoeba, Chaos carolinensis, entered pinosomes whose pH was correlated with their cytoplasmic location during the initial 30-40 min after pinocytosis. The majority of pinosomes containing FTC-RNase clustered in the tail ectoplasm of C. carolinensis during this interval and had a pH of ~6.5; those released into endoplasm and carried into the tip of cells had a pH below 5.0. As pinosomes became distributed at random in C. carolinensis (1-2 h after initial pinocytosis), differences in pH between tip and tail pinosomes vanished. We have also measured the pH within single phagosomes of A. proteus. Phagosomal pH dropped steadily to ~5.4 within 5 min after particle ingestion in 70% of the cells measured, and reached this level of acidity within 10 min in 90% of the cells measured. By contrast, stain for the lysosomal enzyme, acid phosphatase, was evident within only 20% of 5-min-old phagosomes visualized by light microscopy, and within only 40% of 10-min-old phagosomes. A microfluorimetric assay was used to simultaneously record changes in pH, and the initial deposition of lysosomal esterases, within phagosomes of single, living Amoeba proteus. Near complete acidification of the phagosome was recorded from some cells before phagosomal fusion was evident by this microfluorimetric assay. From other cells, however, continued acidification of phagosomes was recorded after lysosomal fusion was initiated. We conclude that acidification of phagosomes by A. proteus is initiated but not necessarily completed prior to phagosome-lysosome formation, and that the two events are closely linked in time. Initial acidification of endosomes is a property intrinsic to the plasma membrane which envelops particles at the cell surface, rather than the result of lysosomal fusion with phagosomes.

Original languageEnglish (US)
Pages (from-to)692-702
Number of pages11
JournalJournal of Cell Biology
Volume97
Issue number3
DOIs
StatePublished - Jan 1 1983

Fingerprint

Phagosomes
Pinocytosis
Enzymes
Ribonucleases
Proteus
Amoeba
Endosomes
Esterases
Acid Phosphatase
Lysosomes
Fluorescein
Photons
Culture Media
Microscopy
Proteins
Coloring Agents
Cell Culture Techniques
Eating
Fluorescence

ASJC Scopus subject areas

  • Cell Biology

Cite this

Acidification of phagosomes is initiated before lysosomal enzyme activity is detected. / McNeil, P. L.; Tanasugarn, L.; Meigs, J. B.; Taylor, D. L.

In: Journal of Cell Biology, Vol. 97, No. 3, 01.01.1983, p. 692-702.

Research output: Contribution to journalArticle

McNeil, P. L. ; Tanasugarn, L. ; Meigs, J. B. ; Taylor, D. L. / Acidification of phagosomes is initiated before lysosomal enzyme activity is detected. In: Journal of Cell Biology. 1983 ; Vol. 97, No. 3. pp. 692-702.
@article{cf639bdaf31f421c8c4aa3da4e7e1c76,
title = "Acidification of phagosomes is initiated before lysosomal enzyme activity is detected",
abstract = "We have measured changes of pH in a protein's microenvironment consequent on its binding to the cell surface and incorporation into pinosomes. Changes of pH were measured from single, living cells and selected regions of cells by the fluorescence ratio technique using a photon-counting microspectrofluorimeter. The chemotactic agent and pinocytosis inducer, ribonuclease, labeled with fluorescein (FTC-RNase), adsorbed to the surface of Amoeba proteus, and was pinocytosed by cells in culture media at pH 7.0. The FTC-RNase entered an apparently acidic microenvironment, pH ~6.1, upon binding to the surface of amoebae. Once enclosed within pinosomes, this protein's microenvironment became steadily more acidic, reaching a minimum of pH ~5.6 in <10 min. FTC-RNase pinocytosed by the giant amoeba, Chaos carolinensis, entered pinosomes whose pH was correlated with their cytoplasmic location during the initial 30-40 min after pinocytosis. The majority of pinosomes containing FTC-RNase clustered in the tail ectoplasm of C. carolinensis during this interval and had a pH of ~6.5; those released into endoplasm and carried into the tip of cells had a pH below 5.0. As pinosomes became distributed at random in C. carolinensis (1-2 h after initial pinocytosis), differences in pH between tip and tail pinosomes vanished. We have also measured the pH within single phagosomes of A. proteus. Phagosomal pH dropped steadily to ~5.4 within 5 min after particle ingestion in 70{\%} of the cells measured, and reached this level of acidity within 10 min in 90{\%} of the cells measured. By contrast, stain for the lysosomal enzyme, acid phosphatase, was evident within only 20{\%} of 5-min-old phagosomes visualized by light microscopy, and within only 40{\%} of 10-min-old phagosomes. A microfluorimetric assay was used to simultaneously record changes in pH, and the initial deposition of lysosomal esterases, within phagosomes of single, living Amoeba proteus. Near complete acidification of the phagosome was recorded from some cells before phagosomal fusion was evident by this microfluorimetric assay. From other cells, however, continued acidification of phagosomes was recorded after lysosomal fusion was initiated. We conclude that acidification of phagosomes by A. proteus is initiated but not necessarily completed prior to phagosome-lysosome formation, and that the two events are closely linked in time. Initial acidification of endosomes is a property intrinsic to the plasma membrane which envelops particles at the cell surface, rather than the result of lysosomal fusion with phagosomes.",
author = "McNeil, {P. L.} and L. Tanasugarn and Meigs, {J. B.} and Taylor, {D. L.}",
year = "1983",
month = "1",
day = "1",
doi = "10.1083/jcb.97.3.692",
language = "English (US)",
volume = "97",
pages = "692--702",
journal = "Journal of Cell Biology",
issn = "0021-9525",
publisher = "Rockefeller University Press",
number = "3",

}

TY - JOUR

T1 - Acidification of phagosomes is initiated before lysosomal enzyme activity is detected

AU - McNeil, P. L.

AU - Tanasugarn, L.

AU - Meigs, J. B.

AU - Taylor, D. L.

PY - 1983/1/1

Y1 - 1983/1/1

N2 - We have measured changes of pH in a protein's microenvironment consequent on its binding to the cell surface and incorporation into pinosomes. Changes of pH were measured from single, living cells and selected regions of cells by the fluorescence ratio technique using a photon-counting microspectrofluorimeter. The chemotactic agent and pinocytosis inducer, ribonuclease, labeled with fluorescein (FTC-RNase), adsorbed to the surface of Amoeba proteus, and was pinocytosed by cells in culture media at pH 7.0. The FTC-RNase entered an apparently acidic microenvironment, pH ~6.1, upon binding to the surface of amoebae. Once enclosed within pinosomes, this protein's microenvironment became steadily more acidic, reaching a minimum of pH ~5.6 in <10 min. FTC-RNase pinocytosed by the giant amoeba, Chaos carolinensis, entered pinosomes whose pH was correlated with their cytoplasmic location during the initial 30-40 min after pinocytosis. The majority of pinosomes containing FTC-RNase clustered in the tail ectoplasm of C. carolinensis during this interval and had a pH of ~6.5; those released into endoplasm and carried into the tip of cells had a pH below 5.0. As pinosomes became distributed at random in C. carolinensis (1-2 h after initial pinocytosis), differences in pH between tip and tail pinosomes vanished. We have also measured the pH within single phagosomes of A. proteus. Phagosomal pH dropped steadily to ~5.4 within 5 min after particle ingestion in 70% of the cells measured, and reached this level of acidity within 10 min in 90% of the cells measured. By contrast, stain for the lysosomal enzyme, acid phosphatase, was evident within only 20% of 5-min-old phagosomes visualized by light microscopy, and within only 40% of 10-min-old phagosomes. A microfluorimetric assay was used to simultaneously record changes in pH, and the initial deposition of lysosomal esterases, within phagosomes of single, living Amoeba proteus. Near complete acidification of the phagosome was recorded from some cells before phagosomal fusion was evident by this microfluorimetric assay. From other cells, however, continued acidification of phagosomes was recorded after lysosomal fusion was initiated. We conclude that acidification of phagosomes by A. proteus is initiated but not necessarily completed prior to phagosome-lysosome formation, and that the two events are closely linked in time. Initial acidification of endosomes is a property intrinsic to the plasma membrane which envelops particles at the cell surface, rather than the result of lysosomal fusion with phagosomes.

AB - We have measured changes of pH in a protein's microenvironment consequent on its binding to the cell surface and incorporation into pinosomes. Changes of pH were measured from single, living cells and selected regions of cells by the fluorescence ratio technique using a photon-counting microspectrofluorimeter. The chemotactic agent and pinocytosis inducer, ribonuclease, labeled with fluorescein (FTC-RNase), adsorbed to the surface of Amoeba proteus, and was pinocytosed by cells in culture media at pH 7.0. The FTC-RNase entered an apparently acidic microenvironment, pH ~6.1, upon binding to the surface of amoebae. Once enclosed within pinosomes, this protein's microenvironment became steadily more acidic, reaching a minimum of pH ~5.6 in <10 min. FTC-RNase pinocytosed by the giant amoeba, Chaos carolinensis, entered pinosomes whose pH was correlated with their cytoplasmic location during the initial 30-40 min after pinocytosis. The majority of pinosomes containing FTC-RNase clustered in the tail ectoplasm of C. carolinensis during this interval and had a pH of ~6.5; those released into endoplasm and carried into the tip of cells had a pH below 5.0. As pinosomes became distributed at random in C. carolinensis (1-2 h after initial pinocytosis), differences in pH between tip and tail pinosomes vanished. We have also measured the pH within single phagosomes of A. proteus. Phagosomal pH dropped steadily to ~5.4 within 5 min after particle ingestion in 70% of the cells measured, and reached this level of acidity within 10 min in 90% of the cells measured. By contrast, stain for the lysosomal enzyme, acid phosphatase, was evident within only 20% of 5-min-old phagosomes visualized by light microscopy, and within only 40% of 10-min-old phagosomes. A microfluorimetric assay was used to simultaneously record changes in pH, and the initial deposition of lysosomal esterases, within phagosomes of single, living Amoeba proteus. Near complete acidification of the phagosome was recorded from some cells before phagosomal fusion was evident by this microfluorimetric assay. From other cells, however, continued acidification of phagosomes was recorded after lysosomal fusion was initiated. We conclude that acidification of phagosomes by A. proteus is initiated but not necessarily completed prior to phagosome-lysosome formation, and that the two events are closely linked in time. Initial acidification of endosomes is a property intrinsic to the plasma membrane which envelops particles at the cell surface, rather than the result of lysosomal fusion with phagosomes.

UR - http://www.scopus.com/inward/record.url?scp=0020512756&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0020512756&partnerID=8YFLogxK

U2 - 10.1083/jcb.97.3.692

DO - 10.1083/jcb.97.3.692

M3 - Article

C2 - 6885916

AN - SCOPUS:0020512756

VL - 97

SP - 692

EP - 702

JO - Journal of Cell Biology

JF - Journal of Cell Biology

SN - 0021-9525

IS - 3

ER -