Name: applications of phluorin for quantitative, kinetic and high-throughput analysis of endocytosis in budding yeast
Uploaded: 2016-10-23T15:00:00
Description: Watch this Scientific Journal Video about Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast at JoVE.com

We would like to thank Gero Miesenb&#246;ck and Tim Ryan for sharing pHluorin cDNA used to generate reagents in this study, Nathan Wright, Joanna Poprawski and Lydia Nyasae for excellent technical assistance, members of the Wendland lab for helpful discussions, and Michael McCaffery and Erin Pryce at the Integrated Imaging Center (Johns Hopkins) for advice and assistance with microscopy and flow cytometry. This work was supported by grants from the National Institutes of Health (to B.W., GM60979) and the National Science Foundation (to B.W., MCB 1024818). K.W. was supported in part by a training grant from the National Institutes of Health (T32-GM007231). A.F.O. was supported by developmental funds from the Department of Biological Sciences at Duquesne University and National Science Foundation CAREER grant 553143.

1. Solutions and Media
<ol>
	<li>Prepare the Following Stocks, Media and Plates:
	<ol>
		<li>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 &#181;m nitrocellulose filter.</li>
		<li>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 ...

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...

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 to allow turnover of damaged proteins.
Studies in yeast and mammalian cells have identified a large number of proteins involved in endocytosis, as well as multiple endocytic pathways that promote internalization of specific cargos or that act in response to a variety of environmental conditions. The best-studied pathway is clathrin-mediated endocytosis (CME), in which clathrin and cytosolic accessory proteins assemble into a coat structure to stabilize the nascent endocytic vesicle. Experiments in the budding yeast Saccharomyces cerevisiae have yielded key insights into the dynamics and order of recruitment for many endocytic proteins1-3. Notably, the CME machinery is highly conserved through evolution such that the majority of CME-related proteins in yeast have human orthologs; thus, budding yeast has been an important tool for understanding mechanisms of endocytosis that are conserved in higher eukaryotes. For example, studies using budding yeast determined that formation and maturation of CME structures (referred to as cortical actin patches in yeast) involves the sequential recruitment of many proteins, beginning with clathrin and cargo-binding adaptor proteins, followed by recruitment of additional endocytic accessory proteins, activation of Arp2/3-mediated actin polymerization, and recruitment of proteins involved in vesicle scission1,2. Recruitment of CME machinery proteins to cortical actin patches is a highly ordered and stereotypical process, and the precise order of recruitment for many proteins has been established with respect to other components of the CME machinery. Importantly, recent studies have confirmed a similar order of recruitment for CME proteins at clathrin-coated pits in mammalian cells4.
In addition to CME, many cell types possess one or more clathrin-independent endocytic (CIE) pathways that rely on alternative mechanisms to promote vesicle formation and internalization from the plasma membrane5-7. In yeast, we recently identified a CIE pathway that utilizes the small GTPase Rho1 and its activating guanine nucleotide exchange factor (GEF), Rom18,9. Rho1 activates the formin Bni1 to promote actin polymerization10,11, which is required for this form of clathrin-independent endocytosis in yeast12. Moreover, the &#945;-arrestin family of proteins recruit the ubiquitin ligase Rsp5 to promote cargo ubiquitination and subsequent internalization through CME13-17, and also promote cargo internalization via the CIE pathway, possibly through direct interaction with proteins involved in CIE18. A variety of CIE pathways also exist in mammalian cells, including clathrin-independent and phagocytic pathways that rely on the Rho1 ortholog, RhoA9,19. The role of RhoA in mammalian CIE is poorly understood; thus, studies in budding yeast may provide additional mechanistic insights that are applicable to mammalian CIE.
Accurate quantification of endocytic events can provide important information about the roles of specific proteins in regulating endocytosis, and can reveal the effects of mutations on cargo internalization or progression through the endocytic pathway. For this purpose, biochemical methods can be utilized to monitor ligand uptake or to measure rates of degradation of endocytic cargo proteins. In living cells, fusion of green fluorescent protein (GFP) and its variants to cargos of interest allows direct visualization of cargo transport. However, GFP-tagged cargos are of limited use for quantification of endocytosis because GFP is resistant to degradation in the lysosome (or vacuole in yeast). Moreover, GFP fluorescence is only partially sensitive to changes in pH, and remains detectable within the vacuole lumen20,21. Consequently, fluorescence of the GFP tag persists in the vacuole long after the remainder of the cargo has been degraded, and whole cell-based quantification of cargo intensity may be inaccurate due to long-term GFP fluorescence in the vacuole.
In order to overcome the drawbacks of vacuolar GFP fluorescence, we previously made use of superecliptic pHluorin, a pH-sensitive variant of GFP that fluoresces brightly at neutral pH, but loses fluorescence in acidic environments such as the lumen of the vacuole/lysosome20,22,23. Placing a pHluorin tag on the cytoplasmic tail of endocytic cargos permits visualization of the cargos at the plasma membrane and on early endosomes, where the pHluorin tag remains exposed to the cytoplasm (Figure 1A). As early endosomes mature, the endosomal sorting complex required for transport (ESCRT) machinery packages surface-localized cargos into vesicles that bud into the lumen of the endosome, generating multivesicular bodies (MVBs)24. For cargos that have been incorporated into internal MVB vesicles, the pHluorin tag faces toward the vesicle lumen. Internal MVB vesicles are acidified; thus, pHluorin-tagged cargos lose fluorescence on early endosomes as they mature into MVBs20. Subsequent fusion of the MVB with the vacuole delivers the MVB luminal contents for degradation, and pHluorin tags remain quenched in the acidic environment of the vacuole lumen.
This paper provides detailed descriptions of quantitative endocytic assays using cargos with cytoplasmic pHluorin tags in yeast. Strains expressing pHluorin-tagged cargos can be used in kinetic and/or endpoint assays, depending on the properties and trafficking behavior of the specific cargo. In addition, some pHluorin-tagged cargos are amenable to high-throughput analysis methods, including flow cytometry. Importantly, the pHluorin tag is a versatile tool for studying endocytic events in living cells, allowing quantification of endocytosis and comparison of endocytic function in wild-type and mutant strains.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adenine</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">A8626-25G</td>
			<td style="width:167px;">Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto-agar</td>
			<td>Fisher</td>
			<td>BP1423-2</td>
			<td>Use for preparation of plate media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Difco Yeast Nitrogen Base (without amino acids)</td>
			<td>BD</td>
			<td>291920</td>
			<td>Use for preparation of liquid and plate media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Concanavalin A</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">C5275-5MG</td>
			<td>Use for coating of chamber slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextrose</td>
			<td>Fisher</td>
			<td>BP350-1</td>
			<td>Use for preparation of liquid and plate media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Histidine</td>
			<td>Fisher</td>
			<td>BP382-100</td>
			<td>Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Leucine</td>
			<td>Acros</td>
			<td>125121000</td>
			<td>Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Lysine</td>
			<td>Fisher</td>
			<td>BP386-100</td>
			<td>Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Methionine</td>
			<td>Fisher</td>
			<td>BP388-100</td>
			<td>Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Tryptophan</td>
			<td>Fisher</td>
			<td>BP395-100</td>
			<td>Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Tyrosine</td>
			<td>Acros</td>
			<td>140641000</td>
			<td>Use for preparation of amino acid mixture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nunc Lab-Tek Chambered Coverglass (8-well)</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">155411</td>
			<td>Use for kinetic and endpoint assays of Mup1-pHluorin internalization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Uracil</td>
			<td>Sigma</td>
			<td>U0750-100G</td>
			<td>Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Axiovert 200 inverted microscope</td>
			<td style="width:71px;">Carl Zeiss</td>
			<td style="width:119px;">Custom Build</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100X/1.4 Plan-Apochromat Oil Immersion Objective Lens</td>
			<td style="width:71px;">Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td>Objective should be 100X, 1.4NA or higher</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Sensicam</td>
			<td style="width:71px;">Cooke Corporation</td>
			<td style="width:119px;"></td>
			<td>Camera should have 12-bit or higher dynamic range</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">X-Cite 120PC Q Illumination Source</td>
			<td style="width:71px;">Excelitas Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Slidebook 5 software</td>
			<td style="width:71px;">Intelligent Imaging Innovations</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">ImageJ software</td>
			<td style="width:71px;">National Institutes of Health</td>
			<td style="width:119px;"></td>
			<td>http://imagej.nih.gov/ij/</td>
		</tr>
	</tbody>
</table>

applications of phluorin for quantitative, kinetic and high-throughput analysis of endocytosis in budding yeast

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.

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)...

Watch this Scientific Journal Video about Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast at JoVE.com

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

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.

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

Research

JoVE Journal

Biology

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...