A Measure of Endosomal PH by Flow Cytometry in Dictyostelium

Overview

Journal BMC Res Notes

Publisher Biomed Central

Specialties Biology
General Medicine

Date 2009 Jan 14

PMID 19138423

Citations 25

Authors

Anna Marchetti

Emmanuelle Lelong

Pierre Cosson

Affiliations

Soon will be listed here.

Abstract

Background: Dictyostelium amoebae are frequently used to study the organization and function of the endocytic pathway, and specific protocols are essential to measure the dynamics of endocytic compartments and their internal pH.

Findings: We have revisited these classical protocols to measure more accurately endosomal pH, making use of a fluorescent probe (Oregon green) more adequate for very acidic pH values. This pH-sensitive probe was combined with a pH-insensitive marker, in order to visualize simultaneously endosome dynamics and pH changes. Finally, a flow cytometer was used to measure endosomal pH in individual cells.

Conclusion: Using these simple protocols the endosomal pH of endocytic compartments can be assessed accurately, revealing the extreme acidity of Dictyostelium lysosomes (pH <3.5).

Citing Articles

Molecular Characterization of Ancient Prosaposin-like Proteins from the Protist .

Ortjohann M, Leippe M Biochemistry. 2024; 63(21):2768-2777.

PMID: 39421968 PMC: 11542183. DOI: 10.1021/acs.biochem.4c00479.

Antibacterial effectors in : specific activity against different bacterial species.

Munoz-Ruiz R, Lamrabet O, Jauslin T, Guilhen C, Bourbon A, Cosson P mSphere. 2024; 9(10):e0047124.

PMID: 39377588 PMC: 11520349. DOI: 10.1128/msphere.00471-24.

Exploring host-pathogen interactions in the Dictyostelium discoideum-Mycobacterium marinum infection model of tuberculosis.

Guallar-Garrido S, Soldati T Dis Model Mech. 2024; 17(7).

PMID: 39037280 PMC: 11552500. DOI: 10.1242/dmm.050698.

Quantitative Analysis of the Inactivation Process of Internalized Bacteria in Dictyostelium Cells.

Kim L, Osorio-Castillo V Methods Mol Biol. 2024; 2814:89-96.

PMID: 38954199 DOI: 10.1007/978-1-0716-3894-1_6.

Chitosan or Cyclodextrin Grafted with Oleic Acid Self-Assemble into Stabilized Polymeric Micelles with Potential of Drug Carriers.

Zlotnikov I, Streltsov D, Belogurova N, Kudryashova E Life (Basel). 2023; 13(2).

PMID: 36836803 PMC: 9964696. DOI: 10.3390/life13020446.

References

Brenot F, Aubry L, Martin J, Satre M, Klein G . Kinetics of endosomal acidification in Dictyostelium discoideum amoebae. 31P-NMR evidence for a very acidic early endosomal compartment. Biochimie. 1992; 74(9-10):883-95. DOI: 10.1016/0300-9084(92)90072-m. View

Eichinger L, Pachebat J, Glockner G, Rajandream M, Sucgang R, Berriman M . The genome of the social amoeba Dictyostelium discoideum. Nature. 2005; 435(7038):43-57. PMC: 1352341. DOI: 10.1038/nature03481. View

Padh H, Ha J, Lavasa M, Steck T . A post-lysosomal compartment in Dictyostelium discoideum. J Biol Chem. 1993; 268(9):6742-7. View

Murphy R, Powers S, Cantor C . Endosome pH measured in single cells by dual fluorescence flow cytometry: rapid acidification of insulin to pH 6. J Cell Biol. 1984; 98(5):1757-62. PMC: 2113196. DOI: 10.1083/jcb.98.5.1757. View

Johnson K . Basics of flow cytometry. Clin Lab Sci. 1991; 5(1):22-4. View

Vergne I, Constant P, LANEELLE G . Phagosomal pH determination by dual fluorescence flow cytometry. Anal Biochem. 1998; 255(1):127-32. DOI: 10.1006/abio.1997.2466. View

Liu T, Mirschberger C, Chooback L, Arana Q, Sacco Z, MacWilliams H . Altered expression of the 100 kDa subunit of the Dictyostelium vacuolar proton pump impairs enzyme assembly, endocytic function and cytosolic pH regulation. J Cell Sci. 2002; 115(Pt 9):1907-18. DOI: 10.1242/jcs.115.9.1907. View

Padh H, Lavasa M, Steck T . Endosomes are acidified by association with discrete proton-pumping vacuoles in Dictyostelium. J Biol Chem. 1991; 266(9):5514-20. View

Aubry L, Klein G, Martiel J, Satre M . Kinetics of endosomal pH evolution in Dictyostelium discoideum amoebae. Study by fluorescence spectroscopy. J Cell Sci. 1993; 105 ( Pt 3):861-6. DOI: 10.1242/jcs.105.3.861. View

10.

Caterina M, Milne J, Devreotes P . Mutation of the third intracellular loop of the cAMP receptor, cAR1, of Dictyostelium yields mutants impaired in multiple signaling pathways. J Biol Chem. 1994; 269(2):1523-32. View

11.

Charette S, Cosson P . Altered composition and secretion of lysosome-derived compartments in Dictyostelium AP-3 mutant cells. Traffic. 2008; 9(4):588-96. DOI: 10.1111/j.1600-0854.2008.00706.x. View

12.

Rivero F, Maniak M . Quantitative and microscopic methods for studying the endocytic pathway. Methods Mol Biol. 2006; 346:423-38. DOI: 10.1385/1-59745-144-4:423. View

13.

Cornillon S, Pech E, Benghezal M, Ravanel K, Gaynor E, Letourneur F . Phg1p is a nine-transmembrane protein superfamily member involved in dictyostelium adhesion and phagocytosis. J Biol Chem. 2000; 275(44):34287-92. DOI: 10.1074/jbc.M006725200. View

14.

Charette S, Cosson P . A LYST/beige homolog is involved in biogenesis of Dictyostelium secretory lysosomes. J Cell Sci. 2007; 120(Pt 14):2338-43. DOI: 10.1242/jcs.009001. View