Europe PMC

This website requires cookies, and the limited processing of your personal data in order to function. By using the site you are agreeing to this as outlined in our privacy notice and cookie policy.

A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast.

Author information

Affiliations

  • 1. Division of Cellular and Molecular Medicine, Howard Hughes Medical Institute, University of California School of Medicine, La Jolla 92093-0668.
      Authors
    • Vida TA 1
    (1 author)

The Journal of Cell Biology, 01 Mar 1995, 128(5):779-792
https://doi.org/10.1083/jcb.128.5.779 PMID: 7533169 PMCID: PMC2120394

This article is in the Europe PMC Open access subset. Refer to the copyright information in the article for licensing details.
Free full text in Europe PMC

Abstract 

We have used a lipophilic styryl dye, N-(3-triethylammoniumpropyl)-4- (p-diethylaminophenyl-hexatrienyl) pyridinium dibromide (FM 4-64), as a vital stain to follow bulk membrane-internalization and transport to the vacuole in yeast. After treatment for 60 min at 30 degrees C, FM 4-64 stained the vacuole membrane (ring staining pattern). FM 4-64 did not appear to reach the vacuole by passive diffusion because at 0 degree C it exclusively stained the plasma membrane (PM). The PM staining decreased after warming cells to 25 degrees C and small punctate structures became apparent in the cytoplasm within 5-10 min. After an additional 20-40 min, the PM and cytoplasmic punctate staining disappeared concomitant with staining of the vacuolar membrane. Under steady state conditions, FM 4-64 staining was specific for vacuolar membranes; other membrane structures were not stained. The dye served as a sensitive reporter of vacuolar dynamics, detecting such events as segregation structure formation during mitosis, vacuole fission/fusion events, and vacuolar morphology in different classes of vacuolar protein sorting (vps) mutants. A particularly striking pattern was observed in class E mutants (e.g., vps27) where 500-700 nm organelles (presumptive prevacuolar compartments) were intensely stained with FM 4-64 while the vacuole membrane was weakly fluorescent. Internalization of FM 4-64 at 15 degrees C delayed vacuolar labeling and trapped FM 4-64 in cytoplasmic intermediates between the PM and the vacuole. The intermediate structures in the cytoplasm are likely to be endosomes as their staining was temperature, time, and energy dependent. Interestingly, unlike Lucifer yellow uptake, vacuolar labeling by FM 4-64 was not blocked in sec18, sec14, end3, and end4 mutants, but was blocked in sec1 mutant cells. Finally, using permeabilized yeast spheroplasts to reconstitute FM 4-64 transport, we found that delivery of FM 4-64 from the endosome-like intermediate compartment (labeled at 15 degrees C) to the vacuole was ATP and cytosol dependent. Thus, we show that FM 4-64 is a new vital stain for the vacuolar membrane, a marker for endocytic intermediates, and a fluor for detecting endosome to vacuole membrane transport in vitro.

Free full text 

Logo of jcellbiolLink to Publisher's site
J Cell Biol. 1995 Mar 1; 128(5): 779–792.
PMCID: PMC2120394
PMID: 7533169

A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast

Copyright and License information Disclaimer

Abstract

We have used a lipophilic styryl dye, N-(3-triethylammoniumpropyl)-4- (p-diethylaminophenyl-hexatrienyl) pyridinium dibromide (FM 4-64), as a vital stain to follow bulk membrane-internalization and transport to the vacuole in yeast. After treatment for 60 min at 30 degrees C, FM 4- 64 stained the vacuole membrane (ring staining pattern). FM 4-64 did not appear to reach the vacuole by passive diffusion because at 0 degree C it exclusively stained the plasma membrane (PM). The PM staining decreased after warming cells to 25 degrees C and small punctate structures became apparent in the cytoplasm within 5-10 min. After an additional 20-40 min, the PM and cytoplasmic punctate staining disappeared concomitant with staining of the vacuolar membrane. Under steady state conditions, FM 4-64 staining was specific for vacuolar membranes; other membrane structures were not stained. The dye served as a sensitive reporter of vacuolar dynamics, detecting such events as segregation structure formation during mitosis, vacuole fission/fusion events, and vacuolar morphology in different classes of vacuolar protein sorting (vps) mutants. A particularly striking pattern was observed in class E mutants (e.g., vps27) where 500-700 nm organelles (presumptive prevacuolar compartments) were intensely stained with FM 4- 64 while the vacuole membrane was weakly fluorescent. Internalization of FM 4-64 at 15 degrees C delayed vacuolar labeling and trapped FM 4- 64 in cytoplasmic intermediates between the PM and the vacuole. The intermediate structures in the cytoplasm are likely to be endosomes as their staining was temperature, time, and energy dependent. Interestingly, unlike Lucifer yellow uptake, vacuolar labeling by FM 4- 64 was not blocked in sec18, sec14, end3, and end4 mutants, but was blocked in sec1 mutant cells. Finally, using permeabilized yeast spheroplasts to reconstitute FM 4-64 transport, we found that delivery of FM 4-64 from the endosome-like intermediate compartment (labeled at 15 degrees C) to the vacuole was ATP and cytosol dependent. Thus, we show that FM 4-64 is a new vital stain for the vacuolar membrane, a marker for endocytic intermediates, and a fluor for detecting endosome to vacuole membrane transport in vitro.

Full Text

The Full Text of this article is available as a PDF (6.4M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Banta LM, Robinson JS, Klionsky DJ, Emr SD. Organelle assembly in yeast: characterization of yeast mutants defective in vacuolar biogenesis and protein sorting. J Cell Biol. 1988 Oct;107(4):1369–1383. [Europe PMC free article] [Abstract] [Google Scholar]
  • Bereiter-Hahn J. Dimethylaminostyrylmethylpyridiniumiodine (daspmi) as a fluorescent probe for mitochondria in situ. Biochim Biophys Acta. 1976 Jan 15;423(1):1–14. [Abstract] [Google Scholar]
  • Betz WJ, Mao F, Bewick GS. Activity-dependent fluorescent staining and destaining of living vertebrate motor nerve terminals. J Neurosci. 1992 Feb;12(2):363–375. [Europe PMC free article] [Abstract] [Google Scholar]
  • Conradt B, Shaw J, Vida T, Emr S, Wickner W. In vitro reactions of vacuole inheritance in Saccharomyces cerevisiae. J Cell Biol. 1992 Dec;119(6):1469–1479. [Europe PMC free article] [Abstract] [Google Scholar]
  • Davis NG, Horecka JL, Sprague GF., Jr Cis- and trans-acting functions required for endocytosis of the yeast pheromone receptors. J Cell Biol. 1993 Jul;122(1):53–65. [Europe PMC free article] [Abstract] [Google Scholar]
  • Franzusoff A, Redding K, Crosby J, Fuller RS, Schekman R. Localization of components involved in protein transport and processing through the yeast Golgi apparatus. J Cell Biol. 1991 Jan;112(1):27–37. [Europe PMC free article] [Abstract] [Google Scholar]
  • Gottlieb TA, Ivanov IE, Adesnik M, Sabatini DD. Actin microfilaments play a critical role in endocytosis at the apical but not the basolateral surface of polarized epithelial cells. J Cell Biol. 1993 Feb;120(3):695–710. [Europe PMC free article] [Abstract] [Google Scholar]
  • Gruenberg J, Howell KE. Membrane traffic in endocytosis: insights from cell-free assays. Annu Rev Cell Biol. 1989;5:453–481. [Abstract] [Google Scholar]
  • Herman PK, Stack JH, DeModena JA, Emr SD. A novel protein kinase homolog essential for protein sorting to the yeast lysosome-like vacuole. Cell. 1991 Jan 25;64(2):425–437. [Abstract] [Google Scholar]
  • Heuser J, Zhu Q, Clarke M. Proton pumps populate the contractile vacuoles of Dictyostelium amoebae. J Cell Biol. 1993 Jun;121(6):1311–1327. [Europe PMC free article] [Abstract] [Google Scholar]
  • Jackson CL, Konopka JB, Hartwell LH. S. cerevisiae alpha pheromone receptors activate a novel signal transduction pathway for mating partner discrimination. Cell. 1991 Oct 18;67(2):389–402. [Abstract] [Google Scholar]
  • Kean LS, Fuller RS, Nichols JW. Retrograde lipid traffic in yeast: identification of two distinct pathways for internalization of fluorescent-labeled phosphatidylcholine from the plasma membrane. J Cell Biol. 1993 Dec;123(6 Pt 1):1403–1419. [Europe PMC free article] [Abstract] [Google Scholar]
  • Köhrer K, Emr SD. The yeast VPS17 gene encodes a membrane-associated protein required for the sorting of soluble vacuolar hydrolases. J Biol Chem. 1993 Jan 5;268(1):559–569. [Abstract] [Google Scholar]
  • Kübler E, Riezman H. Actin and fimbrin are required for the internalization step of endocytosis in yeast. EMBO J. 1993 Jul;12(7):2855–2862. [Europe PMC free article] [Abstract] [Google Scholar]
  • Makarow M. Endocytosis in Saccharomyces cerevisiae: internalization of enveloped viruses into spheroplasts. EMBO J. 1985 Jul;4(7):1855–1860. [Europe PMC free article] [Abstract] [Google Scholar]
  • Makarow M. Endocytosis in Saccharomyces cerevisiae: internalization of alpha-amylase and fluorescent dextran into cells. EMBO J. 1985 Jul;4(7):1861–1866. [Europe PMC free article] [Abstract] [Google Scholar]
  • McConnell SJ, Stewart LC, Talin A, Yaffe MP. Temperature-sensitive yeast mutants defective in mitochondrial inheritance. J Cell Biol. 1990 Sep;111(3):967–976. [Europe PMC free article] [Abstract] [Google Scholar]
  • Pagano RE. Lipid traffic in eukaryotic cells: mechanisms for intracellular transport and organelle-specific enrichment of lipids. Curr Opin Cell Biol. 1990 Aug;2(4):652–663. [Abstract] [Google Scholar]
  • Paravicini G, Horazdovsky BF, Emr SD. Alternative pathways for the sorting of soluble vacuolar proteins in yeast: a vps35 null mutant missorts and secretes only a subset of vacuolar hydrolases. Mol Biol Cell. 1992 Apr;3(4):415–427. [Europe PMC free article] [Abstract] [Google Scholar]
  • Pringle JR, Preston RA, Adams AE, Stearns T, Drubin DG, Haarer BK, Jones EW. Fluorescence microscopy methods for yeast. Methods Cell Biol. 1989;31:357–435. [Abstract] [Google Scholar]
  • Pryer NK, Wuestehube LJ, Schekman R. Vesicle-mediated protein sorting. Annu Rev Biochem. 1992;61:471–516. [Abstract] [Google Scholar]
  • Raths S, Rohrer J, Crausaz F, Riezman H. end3 and end4: two mutants defective in receptor-mediated and fluid-phase endocytosis in Saccharomyces cerevisiae. J Cell Biol. 1993 Jan;120(1):55–65. [Europe PMC free article] [Abstract] [Google Scholar]
  • Raymond CK, Howald-Stevenson I, Vater CA, Stevens TH. Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants. Mol Biol Cell. 1992 Dec;3(12):1389–1402. [Europe PMC free article] [Abstract] [Google Scholar]
  • Riezman H. Endocytosis in yeast: several of the yeast secretory mutants are defective in endocytosis. Cell. 1985 Apr;40(4):1001–1009. [Abstract] [Google Scholar]
  • Robinson JS, Klionsky DJ, Banta LM, Emr SD. Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. Mol Cell Biol. 1988 Nov;8(11):4936–4948. [Europe PMC free article] [Abstract] [Google Scholar]
  • Robinson JS, Graham TR, Emr SD. A putative zinc finger protein, Saccharomyces cerevisiae Vps18p, affects late Golgi functions required for vacuolar protein sorting and efficient alpha-factor prohormone maturation. Mol Cell Biol. 1991 Dec;11(12):5813–5824. [Europe PMC free article] [Abstract] [Google Scholar]
  • Rothman JE, Orci L. Molecular dissection of the secretory pathway. Nature. 1992 Jan 30;355(6359):409–415. [Abstract] [Google Scholar]
  • Salzman NH, Maxfield FR. Quantitative fluorescence techniques for the characterization of endocytosis in intact cells. Subcell Biochem. 1993;19:95–123. [Abstract] [Google Scholar]
  • Singer B, Riezman H. Detection of an intermediate compartment involved in transport of alpha-factor from the plasma membrane to the vacuole in yeast. J Cell Biol. 1990 Jun;110(6):1911–1922. [Europe PMC free article] [Abstract] [Google Scholar]
  • Singer-Krüger B, Frank R, Crausaz F, Riezman H. Partial purification and characterization of early and late endosomes from yeast. Identification of four novel proteins. J Biol Chem. 1993 Jul 5;268(19):14376–14386. [Abstract] [Google Scholar]
  • Vida TA, Huyer G, Emr SD. Yeast vacuolar proenzymes are sorted in the late Golgi complex and transported to the vacuole via a prevacuolar endosome-like compartment. J Cell Biol. 1993 Jun;121(6):1245–1256. [Europe PMC free article] [Abstract] [Google Scholar]
  • Vida TA, Graham TR, Emr SD. In vitro reconstitution of intercompartmental protein transport to the yeast vacuole. J Cell Biol. 1990 Dec;111(6 Pt 2):2871–2884. [Europe PMC free article] [Abstract] [Google Scholar]
  • Weisman LS, Bacallao R, Wickner W. Multiple methods of visualizing the yeast vacuole permit evaluation of its morphology and inheritance during the cell cycle. J Cell Biol. 1987 Oct;105(4):1539–1547. [Europe PMC free article] [Abstract] [Google Scholar]
Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

Full text links 

Read article at publisher's site: https://doi.org/10.1083/jcb.128.5.779

Read article for free, from open access legal sources, via Unpaywall: https://rupress.org/jcb/article-pdf/128/5/779/1258690/779.pdfUnpaywall

Citations & impact 

Impact metrics

915
Citations
Jump to Citations
1
Data citation
Jump to Data

Citations of article over time

Alternative metrics

Altmetric item for https://www.altmetric.com/details/42350362
Altmetric
Discover the attention surrounding your research
https://www.altmetric.com/details/42350362

Article citations

Go to all (915) article citations

Data 

Similar Articles 

To arrive at the top five similar articles we use a word-weighted algorithm to compare words from the Title and Abstract of each citation.

Funding 

Funders who supported this work.

NIGMS NIH HHS (1)