Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Summary

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

Abstract

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Introduction

Vesicular trafficking plays an important role in maintaining organelle identity and function in eukaryotic cells, and is a key mechanism for regulating protein and membrane composition of individual cellular compartments. At the plasma membrane, fusion of exocytic vesicles delivers new proteins and membranes to the surface of the cell, whereas vesicles generated through endocytosis remove membrane and proteins from the surface for subsequent recycling or targeting to the lysosome. Thus, endocytosis is important for nutrient uptake and for responses to the extracellular environment. Exocytosis and endocytosis are balanced to regulate plasma membrane surface area, and t....

Protocol

1. Solutions and Media

1. Prepare the Following Stocks, Media and Plates:
   1. To prepare 10x YNB, dissolve 67 g of yeast nitrogen base lacking amino acids in 1 L of water. Sterilize by filtration through a 0.22 µm nitrocellulose filter.
   2. Prepare YNB medium using 1x YNB (from 10x stock) with 2% dextrose (from a sterile 50% w/v stock) and 1x amino acid/nutrient mixture (from 100x stock, see step 1.1.4). For plasmid selection, omit individual amino acids or nutrients as needed. For induction of Mup1 expression, additionally omit methionine from the amino acid/nutrient mixture.
   3. Prepare YNB plates using 1x YNB (from 10x stock), 2%....

Results

Steady-state localization and quantification of the constitutively internalized endocytic cargo protein Ste3

To demonstrate that pHluorin-tagged cargos can be utilized for quantification of endocytosis in living cells, localization of chimeric GFP and pHluorin fusions with the cytoplasmic C-terminal tail of Ste3, the a-factor pheromone receptor in yeast, were compared. Ste3 is a G protein-coupled receptor (GPCR).......

Discussion

Applications of pHluorin for quantification of endocytic events in yeast cells makes use of transmembrane cargos in which the fluorescent tag is fused to the cytoplasmic tail of the protein (Figure 1A). For the assays described here, cargos are brightly fluorescent when the pHluorin tag is exposed to neutral environments, but become quenched when the pHluorin tag encounters acidic conditions. Thus, a pHluorin-tagged endocytic cargo is readily detected at the cytoplasmic face of the plasma membrane and on.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Adenine	Sigma	A8626-25G	Use for preparation of amino acid mixture and stock solution
Bacto-agar	Fisher	BP1423-2	Use for preparation of plate media
BD Difco Yeast Nitrogen Base (without amino acids)	BD	291920	Use for preparation of liquid and plate media
Concanavalin A	Sigma	C5275-5MG	Use for coating of chamber slides
Dextrose	Fisher	BP350-1	Use for preparation of liquid and plate media
L-Histidine	Fisher	BP382-100	Use for preparation of amino acid mixture and stock solution
L-Leucine	Acros	125121000	Use for preparation of amino acid mixture and stock solution
L-Lysine	Fisher	BP386-100	Use for preparation of amino acid mixture and stock solution
L-Methionine	Fisher	BP388-100	Use for preparation of amino acid mixture and stock solution
L-Tryptophan	Fisher	BP395-100	Use for preparation of amino acid mixture and stock solution
L-Tyrosine	Acros	140641000	Use for preparation of amino acid mixture
Nunc Lab-Tek Chambered Coverglass (8-well)	Thermo Scientific	155411	Use for kinetic and endpoint assays of Mup1-pHluorin internalization
Uracil	Sigma	U0750-100G	Use for preparation of amino acid mixture and stock solution
Axiovert 200 inverted microscope	Carl Zeiss	Custom Build
100X/1.4 Plan-Apochromat Oil Immersion Objective Lens	Carl Zeiss		Objective should be 100X, 1.4NA or higher
Sensicam	Cooke Corporation		Camera should have 12-bit or higher dynamic range
X-Cite 120PC Q Illumination Source	Excelitas Technologies
Slidebook 5 software	Intelligent Imaging Innovations
ImageJ software	National Institutes of Health		http://imagej.nih.gov/ij/