Fluorescent Labeling of Yeast

J.J. Baggett1, J.D. Shaw1, C.J. Sciambi1, H.A. Watson1, B. Wendland1

1 Johns Hopkins University, Baltimore, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 4.13
DOI:  10.1002/0471143030.cb0413s20
Online Posting Date:  November, 2003
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Abstract

This unit describes the use of several different fluorescence methods for labeling yeast cells. It includes methods to label the vacuole, the actin cytoskeleton, and chitin deposits on cell walls (bud scars), as well as methods for visualizing specific proteins in live cells with GFP chimeras and in fixed cells by immunofluorescence.

Keywords: yeast; fluorescent labeling; vacuole; endocytic pathway; chitin

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Labeling the Vacuole in Live Cells with CDCFDA
  • Alternate Protocol 1: Labeling Acidic Organelles with Quinacrine in Live Cells
  • Basic Protocol 2: Labeling the Endocytic Pathway in Live Cells with FM4‐64
  • Alternate Protocol 2: Internalization Time‐Course Assay Using FM4‐64
  • Basic Protocol 3: Endocytic Rate Measurement by Lucifer Yellow Uptake in Live Cells
  • Basic Protocol 4: Localization of Proteins in Live Yeast Cells with GFP‐Fusion Proteins
  • Basic Protocol 5: Labeling the Actin Cytoskeleton with Rhodamine‐Phalloidin in Fixed Cells
  • Alternate Protocol 3: Labeling of Live Cells with Rhodamine‐Phalloidin for Colocalization with GFP Fusion Protein
  • Basic Protocol 6: Localization of a Protein in Fixed Yeast Cells with Immunofluorescence
  • Alternate Protocol 4: Localization in Spheroplasted then Fixed Cells with Immunofluorescence
  • Basic Protocol 7: Labeling Cell Wall Chitin Deposition with Calcofluor White
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Labeling the Vacuole in Live Cells with CDCFDA

  Materials
  • Yeast cells (see Strategic Planning)
  • 0.5 M sodium citrate, pH 4.0 (see recipe)
  • 10 mM 5(6)‐carboxy‐2′‐7′‐dichlorofluorescein diacetate (CDCFDA; Molecular Probes) in anhydrous DMSO (store in the dark for 3 months to 1 year at −20°C)
  • PBS (pH 7.4)/2% (w/v) glucose (see recipe)
  • Shaking incubator set to appropriate growth temperature
  • Con A–coated coverslips (see recipe)
  • FITC filter
  • Additional reagents and equipment for yeast cell culture (unit 1.6) and fluorescence microscopy (unit 4.2)

Alternate Protocol 1: Labeling Acidic Organelles with Quinacrine in Live Cells

  • Rich medium (unit 1.6)
  • 1 M HEPES, pH 7.6: adjust to pH 7.6 with KOH (store up to 1 year at room temperature)
  • 20 µM quinacrine (Sigma) in 1 M HEPES, pH 7.6 (store in the dark for 3 months to 1 year at −20°C)
  • 100 mM HEPES (pH 7.6)/2% (w/v) glucose (see recipe), ice cold

Basic Protocol 2: Labeling the Endocytic Pathway in Live Cells with FM4‐64

  Materials
  • Yeast cells (see Strategic Planning)
  • Rich medium (unit 1.6) at growth temperature
  • FM4‐64 (see recipe)
  • Heat block at assay temperature
  • Water bath with shaker set at assay temperature
  • Con A–coated slides (see recipe)
  • Rhodamine/TRITC filter
  • Additional reagents and equipment for yeast cell culture (unit 1.6) and fluorescence microscopy (unit 4.2)

Alternate Protocol 2: Internalization Time‐Course Assay Using FM4‐64

  • FM4‐64 (see recipe), ice cold
  • Rich medium with and without a carbon source (unit 1.6), ice cold

Basic Protocol 3: Endocytic Rate Measurement by Lucifer Yellow Uptake in Live Cells

  Materials
  • Yeast cells (see Strategic Planning)
  • Culture medium, preferably rich (unit 1.6)
  • Rich medium (unit 1.6)
  • 40 mg/ml lucifer yellow (see recipe)
  • Phosphate/azide/fluoride buffer (see recipe), ice cold
  • 16‐G needle
  • Incubator with orbital shaker set at assay temperature
  • Con A–coated coverslips (see recipe)
  • FITC filter
  • Additional reagents and equipment for yeast cell culture (unit 1.6) and fluorescence microscopy (unit 4.2)

Basic Protocol 4: Localization of Proteins in Live Yeast Cells with GFP‐Fusion Proteins

  Materials
  • Yeast cells expressing GFP‐protein chimeras ( appendix 3A; also see Strategic Planning)
  • 1 M Tris·Cl, pH 7.5 ( appendix 2A)
  • PBS (pH 7.4)/2% (w/v) glucose (see recipe)
  • Incubator set to appropriate growth temperature
  • Incubator set to assay temperature
  • Con A–coated coverslips (see recipe)
  • FITC filter
  • Additional reagents and equipment for yeast cell culture (unit 1.6) and fluorescence microscopy (unit 4.2)

Basic Protocol 5: Labeling the Actin Cytoskeleton with Rhodamine‐Phalloidin in Fixed Cells

  Materials
  • Yeast cells (see Strategic Planning)
  • Rich or selective medium (unit 1.6)
  • In‐medium fixation cocktail (see recipe)
  • Overnight fixation cocktail (see recipe)
  • PBS, pH 7.4 (see recipe)
  • 0.2% (v/v) Triton X‐100 in SHA buffer (see recipe)
  • PBS containing phalloidin and DAPI (see recipe)
  • DABCO antifade (see recipe)
  • Water bath with shaker set at assay temperature
  • End‐over‐end rotator
  • Con A–coated coverslips (see recipe)
  • Rhodamine/TRITC and UV/DAPI filters
  • Additional reagents and equipment for yeast cell culture (unit 1.6) and fluorescence microscopy (unit 4.2)

Alternate Protocol 3: Labeling of Live Cells with Rhodamine‐Phalloidin for Colocalization with GFP Fusion Protein

  • Yeast cells expressing GFP‐fusion constructs ( appendix 3A; also see Strategic Planning)
  • Permeabilization/staining solution (see recipe)
  • FITC and rhodamine/TRITC filters
  • Additional reagents and equipment for yeast cell culture (unit 1.6) and fluorescence microscopy (unit 4.2)

Basic Protocol 6: Localization of a Protein in Fixed Yeast Cells with Immunofluorescence

  Materials
  • Yeast cell cultures (see Strategic Planning)
  • 1× and 5× fixation cocktail (see recipe)
  • SHA buffer (see recipe)
  • Spheroplasting solution (see recipe)
  • 1% (v/v) Triton X‐100 in SHA buffer
  • 2 mg/ml poly‐L‐lysine (Sigma; store up to 3 months to 1 year at −20°C)
  • WT buffer (see recipe)
  • Primary and secondary antibodies
  • Fixed or spheroplasted cells from a strain with the target antigen deleted (optional)
  • 1 mg/ml DABCO antifade (see recipe)
  • Nail polish
  • Incubators set at growth temperature and 30°C
  • Standard slides or slides coated with Teflon to form wells (Polysciences)
  • Pipet tip connected to an aspirator
  • Moisture chamber (see recipe)
  • Weight (e.g., book)
  • Fluorescence filters
  • Additional reagents and equipment for yeast cell culture (unit 1.6) and fluorescence microscopy (unit 4.2)
NOTE: It is recommended that 1× AEBSF or similar protease inhibitors be added to all prepared solutions used in this protocol. This reduces loss of rare antigens due to activity remaining from vacuolar proteases.

Alternate Protocol 4: Localization in Spheroplasted then Fixed Cells with Immunofluorescence

  Materials
  • Yeast cell cultures (see Strategic Planning)
  • Softening medium: 0.1 M Tris·Cl, pH 9.4 ( appendix 2A)/10 mM DTT (freshly prepared)
  • Spheroplasting medium (see recipe)
  • 37% (v/v) formaldehyde
  • SHA buffer (see recipe)
  • SHA buffer/1% LDAO (see recipe)
  • Incubator set to growth temperature
  • 30°C incubator with orbital shaker
  • Additional reagents and equipment for yeast cell culture (unit 1.6) and permeablizing and staining cells for proteins (see protocol 9, steps to )
NOTE: It is recommended that 1× AEBSF or similar protease inhibitors be added to all solutions prepared for this protocol. This reduces loss of rare antigens due to activity remaining from vacuolar proteases.

Basic Protocol 7: Labeling Cell Wall Chitin Deposition with Calcofluor White

  Materials
  • Yeast cells (see Strategic Planning)
  • 1 mg/ml CfW (see recipe)
  • Incubator set to growth temperature
  • Con A–coated coverslips (see recipe)
  • UV/DAPI filter
  • Additional reagents and equipment for yeast cell culture (unit 1.6) and fluorescence microscopy (unit 4.2)
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Figures

Videos

Literature Cited

Literature Cited
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   Adams, A.E. and Pringle, J.R. 1991. Staining of actin with fluorochrome‐conjugated phalloidin. Methods Enzymol. 194:729‐731.
   Amberg, D.C. 1998. Three‐dimensional imaging of the yeast actin cytoskeleton through the budding cell cycle. Mol. Biol. Cell 9:3259‐3262.
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