Name: Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast (Video) | JoVE Journal
Uploaded: 2016-10-23T15:00:00.000+00:00
Duration: 10 min 2 s
Description: Learn to quantify endocytic trafficking events in living yeast cells using superecliptic pHluorin and fluorescence microscopy. This method enables direct measurement of vesicular dynamics and mutation effects.

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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 of Mup1 expression, additionally omit methionine from the amino acid/nutrient mixture.</li>
		<li>Prepare YNB plates using 1x YNB (from 10x stock), 2% dextrose (from a sterile 50% w/v stock), 0.7 g/L amino acid/nutrient mixture (see step 1.1.5) and 2.5% agar. Prepare plate medium by autoclaving 25 g of agar and 0.7 g of amino acid/nutrient mixture per 860 ml of water. Allow the mixture to cool to 55 &#176;C, then add 100 ml of 10x YNB and 40 ml of 50% glucose. Pour plates (approximately 30 ml of medium per 10 cm dish), allow the medium to solidify at room temperature, and store plates at 4 &#176;C.</li>
		<li>Prepare 100x amino acid/nutrient stock solution (for liquid medium) by dissolving the following in 100 ml of deionized water: 0.1 g L-Methionine, 0.3 g each of L-leucine and L-lysine, and 0.2 g each of L-histidine, L-tryptophan, adenine and uracil (other amino acids and nutrients may be included as needed for specific strains). Use gentle heat if needed to dissolve, and sterilize through a 0.45 &#181;m nitrocellulose filter. Store at room temperature with protection from light. For plasmid selection, prepare stock solutions lacking specific components as needed.</li>
		<li>Prepare amino acid/nutrient mixture (for plates) by combining the following: 0.5 g of adenine, 4 g of L-leucine, and 2 g each of L-histidine, L-lysine, L-methionine, L-tryptophan, L-tyrosine and uracil. Grind with a mortar and pestle, and store with protection from light. For plasmid selection, prepare mixtures lacking specific components as needed, and use 0.7 g of amino acid mixture per liter of plate medium (see step 1.1.3).</li>
	</ol>
	</li>
	<li>Prepare 8-well chamber slides for microscopy by coating the surface of the slide with concanavalin A (ConA). To each well of the chamber slide, add 25 &#181;l of 2 mg/ml ConA dissolved in water. Using a pipette tip, spread the ConA across the bottom surface of the well, then allow the slide to air dry at room temperature for at least 4-8 hr. Ideally, use chamber slides within 24 hr of ConA coating.</li>
	<li>Generate yeast strains with in-frame fusions of GFP or pHluorin to the endocytic cargo of interest using the polymerase chain reaction (PCR)-based integration method described in Longtine et al. and Goldstein and McCusker25,26. 
	NOTE: Plasmids containing pHluorin tagging cassettes with resistance genes for kanamycin (KANMX6) and nourseothricin (NATMX4) were described previously20.
	<ol>
		<li>Amplify GFP or pHluorin cassettes containing selectable markers with primers containing additional sequences specific to the site of genomic integration20,25.</li>
		<li>Transform the resulting PCR product into the desired yeast strain using the lithium acetate method27.</li>
		<li>Select for integration of the cassette by growing cells on YPD plates containing the appropriate drug. To prepare plates, autoclave 10 g yeast extract, 20 g peptone, 0.1 g tryptophan and 25 g agar in 960 ml water. Before pouring plates, allow the mixture to cool to 55 &#176;C, then add 40 ml of 50% glucose and either G418 to a final concentration of 200 &#181;g/ml for KANMX6 selection, or nourseothricin to a final concentration of 100 &#181;g/ml for NATMX4 selection.</li>
		<li>Confirm integration of the GFP or pHluorin tag in individual colonies by PCR and/or Western blotting using anti-GFP antibodies, as well as by detection of the tagged protein by fluorescence microscopy20. 
		NOTE: Specific yeast strains and plasmids used in this study are listed in Tables 1 and 2, respectively.</li>
	</ol>
	</li>
</ol>
2. Endpoint Assay for Steady-state Fluorescence of Constitutively Internalized Endocytic Cargo
<ol>
	<li>Inoculate cells (~3 small colonies) in 5 ml of YNB medium lacking amino acids or nutrients as required for plasmid maintenance (if applicable). Grow cells for 16-24 hr in an orbital shaking incubator at 30 &#176;C, with 250 rpm shaking. Alternatively, streak a ~1-2 mm colony of cells onto a YNB plate lacking amino acids or nutrients for plasmid selection, and grow cells overnight at 30 &#176;C.</li>
	<li>Measure the density (OD600) of each overnight culture using a spectrophotometer. Prepare a 5 ml dilution at 0.35-0.4 OD600/ml in YNB medium lacking amino acids or nutrients for plasmid maintenance, and grow for approximately 3 hr at 30 &#176;C with shaking. If using cells streaked on a plate, skip this step and proceed with step 2.4 (see below).</li>
	<li>Measure the density (OD600) of cells, which should now be between 0.6-1.0 OD600/ml. Transfer 1.5 ml of culture to a microfuge tube, and pellet cells at 8,000 rpm (6,800 x g) for 2 min. Remove 1,475 &#181;l of supernatant.</li>
	<li>Bring cells to a fluorescence microscope. Before imaging each sample, prepare a slide by resuspending the cells in the remaining medium, and mount 3 &#181;l of cell suspension under a cover slip. Alternatively, prepare 8-well chamber slides as described below in protocol 3, using YNB medium as appropriate for plasmid selection. Ideally, image cells with a 100X, 1.4 or higher numerical aperture (NA) oil immersion objective lens, a 12- or 16-bit camera, and excitation/emission filters optimized for GFP fluorescence. 
	NOTE: If using cells grown by streaking on a plate, prepare the slide by placing 3 &#181;l of fresh YNB medium on a cover slip. Using a pipette tip, collect a small colony of cells from the plate, disperse the cells into the YNB medium, and mount the resulting suspension on a glass slide immediately before imaging.</li>
	<li>Image 4-6 random fields of cells for each condition, using the same acquisition parameters (i.e., filter sets and exposure time) for all images within an experiment. 
	NOTE: Collecting a brightfield or DIC image for each field can be helpful for cells where the pHluorin signal is quenched in MVB and vacuolar compartments.</li>
	<li>Quantify fluorescence intensity of at least 30-50 cells for each condition (see protocol 5).</li>
</ol>
3. Kinetic Assay for Quantification of Endocytosis of Cargos that Undergo Regulated Endocytosis
<ol>
	<li>Inoculate 2-3 colonies of yeast cells (approximately 2 mm in diameter per colony) in 5 ml of YNB medium lacking methionine and additional amino acids or nutrients for maintaining plasmid selection. Grow cultures for 16-24 hr at 30 &#176;C with shaking. 
	NOTE: In this paper, protocols for studying regulated endocytic cargo proteins will make use of the methionine permease, Mup1. Other cargos that undergo regulated endocytosis are also suitable for tagging with pHluorin (for example, the uracil permease Fur4, which accumulates at the plasma membrane in the absence of uracil, and internalizes in response to externally applied uracil); for these cargos, media conditions should be modified to reflect the specific expression, induction and internalization conditions for that cargo.</li>
	<li>Measure the density (OD600) of each overnight culture approximately 3 hr before imaging. Prepare a 5 ml dilution at 0.35-0.4 OD600/ml in YNB medium lacking methionine and additional amino acids or nutrients for plasmid maintenance, and grow at 30 &#176;C with shaking.</li>
	<li>Pre-equilibrate the microscope&#39;s environmental chamber or heated stage (if available) to 30 &#176;C during the 3 hr incubation of step 3.2.</li>
	<li>Transfer 1 ml of cell culture to a microfuge tube, and pellet cells at 8,000 rpm (6,800 x g) for 2 min. Remove 975 &#181;l of supernatant.</li>
	<li>Prepare a ConA-treated, 8-well glass-bottomed chamber slide for imaging (protocol 1.2).</li>
	<li>Add 200 &#181;l of YNB medium lacking methionine and additional amino acids or nutrients for plasmid selection to each well. Resuspend the cell pellet from step 3.4 in the remaining supernatant, and drop 2.5 &#181;l of cell suspension into the center of each well. Disperse cells across the surface of the well by pipetting up and down, and allow cells to settle for 5-10 min.</li>
	<li>Place the chamber slide on an inverted fluorescence microscope equilibrated to 30 &#176;C (see step 3.3). Locate a field of cells using brightfield illumination.</li>
	<li>Add 50 &#181;l of YNB medium containing 100 &#181;g/ml methionine (pre-warmed to 30 &#176;C) to the 200 &#181;l of medium in the well to obtain a final methionine concentration of 20 &#181;g/ml. Avoid disrupting the medium in the well in order to prevent the cells from floating from the bottom.</li>
	<li>Allow the cells to equilibrate for 2 min before beginning imaging. Immediately before capturing the first image, adjust the microscope to the desired focal plane (typically, an equatorial focal plane works best).</li>
	<li>Acquire GFP and brightfield (or DIC) images at 5 min intervals for 45-60 min, using identical acquisition parameters for all images within a time series (for example, 100X 1.4 NA oil immersion objective and 500 msec acquisition time using GFP filter, 100 msec acquisition time using DIC filter). 
	NOTE: If the microscope&#39;s stage and imaging software do not allow for automatic correction of focal plane drift, it may be necessary to manually readjust the focus prior to each imaging time point. Moreover, acquisition times will vary between microscopes due to differences in illumination and filters, and optimal conditions should be determined manually prior to beginning the experiment. If an endocytic cargo other than Mup1 is used, it may be necessary to modify the acquisition time and imaging intervals, as well as the duration of the experiment, to reflect the internalization kinetics of that cargo.</li>
	<li>Quantify fluorescence intensity of individual cells at each time point (see protocol 5), and express values as a percentage of the initial intensity of the first image.</li>
</ol>
4. Endpoint Assay for Quantification of Endocytic Cargos that Undergo Regulated Endocytosis
<ol>
	<li>Prepare cells for imaging as described in protocol 3 (steps 3.1 through 3.6).</li>
	<li>Image 4-5 random fields of cells for each condition immediately before adding methionine, using brightfield and GFP filter sets.</li>
	<li>Add 50 &#181;l of YNB medium containing 100 &#181;g/ml methionine (pre-warmed to 30 &#176;C) to the 200 &#181;l of medium in the well to obtain a final methionine concentration of 20 &#181;g/ml. Avoid disrupting the medium in the well in order to prevent the cells from floating from the bottom.</li>
	<li>Image 4-5 random fields of cells 30 min after addition of methionine, using the same acquisition parameters as for the 0 min time point (step 4.2).</li>
	<li>Quantify fluorescence intensity of at least 30-50 cells for each time point (see protocol 5). Express values as the percentage of Mup1-pHluorin internalized after 30 min using the formula: [Mean intensity at 0 min - Mean intensity at 30 min]/[Mean intensity at 0 min] x 100%. 
	NOTE: It is possible to simultaneously perform up to eight endpoints assays by staggering the start time of each assay. Table 3 provides an example workflow for eight simultaneous assays.</li>
</ol>
5. Post-acquisition Analysis
<ol>
	<li>Export images from acquisition software as a 16-bit tagged-image file format (.tif or .tiff format). 
	NOTE: Other file formats may also be suitable, and should ideally use lossless compression algorithms. 8-bit images are generally not suitable for quantification purposes.</li>
	<li>Open files using ImageJ software (freely available at &#39;Open&#39; option in the &#39;File&#39; menu. Use the fluorescence image for quantification, and the brightfield/DIC image as a reference to identify the location of cells and to exclude dead cells (if present), which appear darker than live cells when viewed by DIC and are autofluorescent when viewed with GFP excitation/emission filters.</li>
	<li>Perform a background subtraction on the fluorescence image. For images with an uneven background signal, use the &#39;Background Subtraction&#39; algorithm found in the &#39;Process menu&#39;. Use the default setting (rolling ball radius of 50 pixels), which is generally sufficient for quantification purposes. 
	NOTE: An alternative background subtraction method for images with uniform background is described in step 5.3.1.-5.3.2; however, the above method also works for uniform background.
	<ol>
		<li>For images with uniform background (i.e., background intensity is approximately constant at all edges and in the center of the image), perform a manual background subtraction. Click on the &#39;freehand selections&#39; button found in the main ImageJ window, and select 3-5 random regions within the image that do not contain cells.</li>
		<li>Open the &#39;Set Measurements&#39; function located in the &#39;Analyze&#39; menu, select the &#39;Mean Gray Value&#39; parameter, and measure intensity values using the &#39;Measure&#39; function (also found in the &#39;Analyze&#39; menu). Calculate the average value from the 3-5 measured regions, and subtract from all subsequent measurements in that image.</li>
	</ol>
	</li>
	<li>For quantification of kinetic assays (protocol 3), generate a single, stacked image for the time series. Open the fluorescence images from each time point in chronological order, and generate a stack using the &#39;Images to Stack&#39; function (found in the &#39;Image&#39; menu, under the Stacks sub-menu).</li>
	<li>Outline a cell using the freehand selection tool, making sure to include the entire cell but as little area outside of the cell as possible.</li>
	<li>Measure the following parameters: &#39;Area, Integrated Density&#39; and &#39;Mean Gray Value&#39;. Select parameters using the &#39;Set Measurements&#39; function, located in the &#39;Analyze&#39; menu. 
	NOTE: Integrated Density corresponds to the sum of all pixel intensities within the selected region, and Mean Gray Value corresponds to [Integrated Density/Area], which corrects intensity values for cell size.</li>
	<li>Open the &#39;ROI Manager&#39; located in the &#39;Analyze&#39; menu, under the &#39;Tools&#39; sub-menu. In the ROI Manager window, click the &#39;Add&#39; button to enter the highlighted region as a new region of interest (ROI).</li>
	<li>Repeat steps 5.4 through 5.6 for the remaining cells in the image. Exclude any dead cells as assessed by brightfield/DIC and fluorescence (dead cells appear darker in DIC images, and have autofluorescent cytoplasmic signal when viewed with GFP excitation/emission; see Figure 2F), and any cells that touch the edge of the image. Save the selected ROIs as a .zip file by clicking the &#39;More&#39; button in the &#39;ROI Manager&#39; window, and export all measurements to a spreadsheet or statistical analysis software.</li>
	<li>For quantification of kinetic assays (protocol 3), outline and measure a cell from the initial time point as described in steps 5.4 and 5.5. Use the same ROI for measurement of all subsequent time points in the experiment.</li>
	<li>For endpoint assays (protocols 2 and 4), measure a minimum of 30-50 cells for each condition. Perform quantification for at least 2-3 images, and include all cells in each image that meet the criteria outlined in step 5.8 in the analysis.</li>
	<li>Assess statistical significance between samples using one-way ANOVA, followed by Tukey&#39;s multiple comparison test for post hoc analysis.</li>
</ol>
6. Population Analysis of Mup1-pHluorin Endocytosis by Flow Cytometry
<ol>
	<li>Inoculate 2-3 colonies of yeast cells (approximately 2 mm in diameter per colony) in 0.5 ml of YNB medium lacking methionine and additional amino acids or nutrients for maintaining plasmid selection. Grow cells in 5 ml round-bottom tubes for 16-24 hr at 30 &#176;C on a roller drum.</li>
	<li>Add 1.5 ml of YNB medium lacking methionine and additional amino acids or nutrients for plasmid maintenance, and grow at 30 &#176;C for an additional 3 hr on a roller drum.</li>
	<li>Transfer 1 ml of cells into each of two 5 ml round-bottom tubes. Add 0.25 ml of YNB -Met medium to the first tube (-Met condition), and 0.25 ml of YNB medium containing 100 &#181;g/ml methionine to the second tube to give a final methionine concentration of 20 &#181;g/ml (+Met condition). Incubate cells at 30 &#176;C for 45 min on a roller drum.</li>
	<li>Analyze cells by flow cytometry, selecting forward scatter (FS) as a measure of cell size, and Mup1-pHluorin fluorescence intensity from a fluorescein isothiocyanate (FITC) filter as a measure of cargo internalization.</li>
	<li>Use the slider buttons to adjust the voltage of the FS and FITC channels to optimize detection for the size range and fluorescence intensity of the yeast cells being used (for these experiments, settings used were 50 V for FS and 400 V for FITC). 
	NOTE: Voltage for both channels will vary depending on the flow cytometer being used. This step should be done before or during the 45 min treatment with methionine (step 6.3).</li>
	<li>Measure FS and fluorescence intensity for 10,000 cells from each condition, using the high flow rate setting. Generate a scatter plot of FS against fluorescence intensity: click &#39;add graph&#39;, select FITC (x-axis) and FS (y-axis), and graph 1,000 points (the software will display values for 1,000 cells, even though 10,000 cells were measured). 
	NOTE: For maximal consistency, analyze cells 45-50 min after addition of methionine; for experiments involving large numbers of samples, stagger addition of methionine to accommodate the time required for flow cytometry analysis.</li>
	<li>Include -Met and +Met conditions for wild-type (WT) cells as a control. Using the gate function, assign a vertical gate using the WT +Met condition, such that approximately 5% of the brightest cells fall to the right of the gate. Use this gating for all other conditions in the experiment.</li>
</ol>

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<li>Maldonado-Báez, L., Dores, M. R., Perkins, E. M., Drivas, T. G., Hicke, L., Wendland, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interaction+between+Epsin%2FYap180+adaptors+and+the+scaffolds+Ede1%2FPan1+is+required+for+endocytosis%5BTitle%5D)+AND+%22Mol+Biol+Cell%22%5BJournal%5D)">Interaction between Epsin/Yap180 adaptors and the scaffolds Ede1/Pan1 is required for endocytosis.</a> Mol Biol Cell. 19 (7), 2936-2948 (2008).</li>
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<li>Wendland, B., McCaffery, J. M., Xiao, Q., Emr, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+fluorescence-activated+cell+sorter-based+screen+for+yeast+endocytosis+mutants+identifies+a+yeast+homologue+of+mammalian+eps15%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">A novel fluorescence-activated cell sorter-based screen for yeast endocytosis mutants identifies a yeast homologue of mammalian eps15.</a> J Cell Biol. 135 (6), 1485-1500 (1996).</li>
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<li>Ghaddar, K., Merhi, A., Saliba, E., Krammer, E. M., Prevost, M., Andre, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Substrate-Induced+Ubiquitylation+and+Endocytosis+of+Yeast+Amino+Acid+Permeases%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">Substrate-Induced Ubiquitylation and Endocytosis of Yeast Amino Acid Permeases.</a> Mol Cell Biol. 34 (24), 4447-4463 (2014).</li>
<li>Becuwe, M., Léon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Integrated+control+of+transporter+endocytosis+and+recycling+by+the+arrestin-related+protein+Rod1+and+the+ubiquitin+ligase+Rsp5%5BTitle%5D)+AND+%22eLife%22%5BJournal%5D)">Integrated control of transporter endocytosis and recycling by the arrestin-related protein Rod1 and the ubiquitin ligase Rsp5.</a> eLife. 3, (2014).</li>
<li>Henne, W. M., Buchkovich, N. J., Zhao, Y., Emr, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Endosomal+Sorting+Complex+ESCRT-II+Mediates+the+Assembly+and+Architecture+of+ESCRT-III+Helices%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">The Endosomal Sorting Complex ESCRT-II Mediates the Assembly and Architecture of ESCRT-III Helices.</a> Cell. 151 (2), 356-371 (2012).</li>
<li>Nickerson, D. P., Russell, M. R. G., Lo, S. -Y., Chapin, H. C., Milnes, J. M., Merz, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Termination+of+Isoform-Selective+Vps21%2FRab5+Signaling+at+Endolysosomal+Organelles+by+Msb3%2FGyp3%5BTitle%5D)+AND+%22Traffic%22%5BJournal%5D)">Termination of Isoform-Selective Vps21/Rab5 Signaling at Endolysosomal Organelles by Msb3/Gyp3.</a> Traffic. 13 (10), 1411-1428 (2012).</li>
<li>Nickerson, D. P., Merz, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((LUCID%3A+A+Quantitative+Assay+of+ESCRT-Mediated+Cargo+Sorting+into+Multivesicular+Bodies%5BTitle%5D)+AND+%22Traffic%22%5BJournal%5D)">LUCID: A Quantitative Assay of ESCRT-Mediated Cargo Sorting into Multivesicular Bodies.</a> Traffic. 16 (12), 1318-1329 (2015).</li>
<li>Dulic, V., Egerton, M., Elguindi, I., Raths, S., Singer, B., Riezman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Yeast+endocytosis+assays%5BTitle%5D)+AND+%22Methods+Enzymol%22%5BJournal%5D)">Yeast endocytosis assays.</a> Methods Enzymol. 194, 697-710 (1991).</li>
<li>Silve, S., Volland, C., Garnier, C., Jund, R., Chevallier, M. R., Haguenauer-Tsapis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Membrane+insertion+of+uracil+permease%2C+a+polytopic+yeast+plasma+membrane+protein%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">Membrane insertion of uracil permease, a polytopic yeast plasma membrane protein.</a> Mol Cell Biol. 11 (2), 1114-1124 (1991).</li>
<li>Miliaras, N. B., Park, J. -H., Wendland, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Function+of+the+Endocytic+Scaffold+Protein+Pan1p+Depends+on+Multiple+Domains%5BTitle%5D)+AND+%22Traffic%22%5BJournal%5D)">The Function of the Endocytic Scaffold Protein Pan1p Depends on Multiple Domains.</a> Traffic. 5 (12), 963-978 (2004).</li>
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The authors declare no competing interests.

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><tbody><tr><td>Adenine</td><td>Sigma</td><td>A8626-25G</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>Bacto-agar</td><td>Fisher</td><td>BP1423-2</td><td>Use for preparation of plate media</td></tr><tr><td>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><td>Concanavalin A</td><td>Sigma</td><td>C5275-5MG</td><td>Use for coating of chamber slides</td></tr><tr><td>Dextrose</td><td>Fisher</td><td>BP350-1</td><td>Use for preparation of liquid and plate media</td></tr><tr><td>L-Histidine</td><td>Fisher</td><td>BP382-100</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>L-Leucine</td><td>Acros</td><td>125121000</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>L-Lysine</td><td>Fisher</td><td>BP386-100</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>L-Methionine</td><td>Fisher</td><td>BP388-100</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>L-Tryptophan</td><td>Fisher</td><td>BP395-100</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>L-Tyrosine</td><td>Acros</td><td>140641000</td><td>Use for preparation of amino acid mixture</td></tr><tr><td>Nunc Lab-Tek Chambered Coverglass (8-well)</td><td>Thermo Scientific</td><td>155411</td><td>Use for kinetic and endpoint assays of Mup1-pHluorin internalization</td></tr><tr><td>Uracil</td><td>Sigma</td><td>U0750-100G</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>Axiovert 200 inverted microscope</td><td>Carl Zeiss</td><td>Custom Build</td><td></td></tr><tr><td>100X/1.4 Plan-Apochromat Oil Immersion Objective Lens</td><td>Carl Zeiss</td><td></td><td>Objective should be 100X, 1.4NA or higher</td></tr><tr><td>Sensicam</td><td>Cooke Corporation</td><td></td><td>Camera should have 12-bit or higher dynamic range</td></tr><tr><td>X-Cite 120PC Q Illumination Source</td><td>Excelitas Technologies</td><td></td><td></td></tr><tr><td>Slidebook 5 software</td><td>Intelligent Imaging Innovations</td><td></td><td></td></tr><tr><td>ImageJ software</td><td>National Institutes of Health</td><td></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

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)...

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 ...

The overall goal of this procedure is to quantify vesicular trafficking events in the endocytic pathway of living cells. This method can help answer key questions in the vesicular trafficking field such as how mutation or changes in protein expression affect rates and efficiency of trafficking events. The main advantage of this technique is that it allows direct quantification of endocytic trafficking and living cells by measuring the intensity of fluorescently-tagged cargo proteins.After preparing solutions and media according to the text protocol, inoculate two to three colonies of yeast cells in five milliliters of YNB medium, lacking additional nutrients for maintaining plasmid selection. Grow the cultures at 30 degrees Celsius with shaking for 16 to 24 hours. Approximately three hours before imaging, measure the OD 600 of each overnight culture.Then prepare five milliliter dilutions at an OD 600 of 0.35 to 0.4 in YNB medium, lacking additional nutrients for plasmid maintenance and grow at 30 degrees Celsius with shaking for three hours. During the incubation, pre-equilibrate the microscopes environmental chamber or heated stage to 30 degrees Celsius. After preparing a can trea

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Biology

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

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

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

Here we present a protocol to quantify phagocytosis of fluorescent particles by adherent macrophage cell line using a fluorometric method. This method facilitates a high throughput quantification of particle internalization as well as the resulting actin polymerization.

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

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

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Efferocytosis, the phagocytic removal of apoptotic cells, is required to maintain homeostasis and is facilitated by receptors and signaling pathways that allow for the recognition, engulfment, and internalization of apoptotic cells. Herein, we present a fluorescence microscopy protocol for the quantification of efferocytosis and the activity of efferocytic signaling pathways.

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

Fission yeast is used here as a heterologous host to express bacterial cytoskeletal proteins such as FtsZ and MreB as translational fusion proteins with GFP to visualize their polymerization. Also, compounds that affect polymerization are identified by imaging using a fluorescence microscope.

Fission Yeast as a Platform for Antibacterial Drug Screens Targeting Bacterial Cytoskeleton Proteins

This protocol details two methods of yeast cell cycle arrest and optional release, and elaborates on the use of fluorescence microscopy to study cell cycle-dependent processes in S. cerevisiae.

Manipulation and Analysis of Cell Cycle-Dependent Processes in Budding Yeast

Learn to quantify endocytic trafficking events in living yeast cells using superecliptic pHluorin and fluorescence microscopy. This method enables direct measurement of vesicular dynamics and mutation effects.

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

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