We thank Brian G. Coon and Claudia B. Hanna for helpful discussions and support.
This project was supported by start-up funds from the Dep. of Biological Sciences, Purdue University to R. Claudio Aguilar and an American Cancer Society Institutional Research Grant to R. Claudio Aguilar through the Purdue Cancer Center.

A. Cell Culture and Protein Induction
This section describes cell culture conditions, cell-cycle synchronization with Nocodazole, followed by induction of protein expression from a regulatable promoter in yeast cells.
<ol>
 <li>The wild type yeast strain W303 (leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15) is transformed with high copy (2&mu;) plasmid DNA that contains our gene of interest (ENTH2) under the control of a methionine-repressible promoter (MET25). 2mM methionine is sufficient to suppress promoter activity, while media lacking methionine allows maximal expression. Hence, a range of methionine concentrations can be used to express the protein at the desired level. 1 Transformation of plasmid DNA in W303 cells is done using the LiAc-TE method according to standard procedures.2
 Note: This experiment should be performed by growing the cells overnight in the presence of 2mM methionine to ensure protein expression only at background levels. Next morning, protein expression should be induced by resuspending the cells in media lacking methionine immediately following cell-cycle synchronization (approximately 4h). However, we have found that the ENTH2-dependent cell division phenotype is induced only after a considerable concentration of ENTH2 has built up in the cells (usually after 4-6h of protein induction3), making the time course for analysis of all manifestations of the phenotype very lengthy (at least 16h). We have found that growing the cells overnight in the presence of 0.2mM methionine represses the morphological phenotype but allows subcritical protein concentration that can be rapidly increased to phenotype-inducing levels when transferred to media lacking methionine. This permits detailed analysis of severe manifestations of the cell division defect in a relatively shorter period of time. We hence recommend that the experimenter determine the methionine concentration that is sub-threshold for phenotype induction on a case-by-case basis.</li>
 <li>Pick six colonies from a plate containing recently transformed cells and inoculate in 50 ml of selective media with 0.2mM methionine in a sterilized conical flask using standard sterile techniques. Incubate overnight at 30&deg;C in with shaking at 200 rpm. (For more information on standard yeast culture conditions, see ref. 4) </li>
 <li>Next morning, estimate cell density by measuring the optical density of the culture at 600nm. Transfer the equivalent to 20 OD600nm of cells into a sterile centrifuge tube and harvest by spinning at 2200 rpm for 5min at room temperature.</li>
 <li>Resuspend cells in YPD at a concentration of 0.4 OD600nm/ml and add Nocodazole to a final concentration of 15&mu;g/ml of from a stock of 1mg/ml in DMSO. Incubate for 4h at 30&deg;C with shaking at 200rpm. Note: Alternative arrest methods including arrest at the G0 phase using alpha-factor or at G1-S phase using hydroxyurea can be used. We have found that arrest at mitosis using Nocodazole is simple and effective enough for our purposes.</li>
 <li>After 4h, check percentage of cells arrested in mitosis by counting the number of cells with equal sized buds by viewing under the microscope. If the arrest is greater than 90%, proceed to the following step. Otherwise, maintain cells in Nocodazole until the desired percentage of mitotic arrest is achieved.</li>
 <li>Harvest cells by centrifugation at 2200 rpm for 5min and wash with ice cold water three times. After the final wash, resuspend the pellet in selective media lacking methionine at a cell density of ~ 0.5 OD600nm/ml. Transfer half the volume of cells to a new sterile tube and add methionine to a final 0.2mM concentration. This will serve as a control for effects due to basal levels of protein expression. This marks the first time-point of the experiment (time = 0h). Analyze cells every hour for 6h.</li>
</ol>
B. Analysis of Phenotype Development
Note: All the analysis methods described below do not need to be performed simultaneously.
Determination of protein expression levels by western Blotting
<ol>
 <li>At every time-point, measure the OD600nm of the culture and harvest 2 OD600nm equivalents of cells by centrifugation at 2200 rpm for 5 mins.</li>
 <li>Resuspend the pellet in 1ml sterile water and transfer the suspension to a 1.5ml eppendorf tube. Repeat the centrifugation to obtain a pellet. Aspirate the supernatant. </li>
 <li>Resuspend the pellet in 50&mu;l SDS-PAGE laemmli sample buffer (50mM Tris pH 6.8, 2% SDS, 10% glycerol, 2% &beta;-mercaptoethanol and 0.006% bromophenol blue). Add 50&mu;l of acid-washed glass beads (0.2-0.4 microns diameter) and vortex for 1 min.</li>
 <li>Boil tubes on a 95&deg;C heat block for 2 mins, vortex again for 30 secs and resume boiling for 5 mins. Freeze the protein sample at -20&deg;C.</li>
 <li>Run protein samples on a 12% SDS-PAGE gel, transfer onto a nitrocellulose membrane and perform Western blotting with appropriate antibodies to detect the protein of interest. In this example, we probe the presence of haemagglutinin (HA) tagged ENTH domains by incubating with mouse anti-HA antibody (Covance, Princeton, NJ) at 1:2000 dilution in Blotto-Tween (1XPBS, 5% non-fat dry milk, 0.1% Tween). HRP tagged goat anti-mouse secondary antibody (Pierce) at a dilution of 1:2500, also in Blotto-Tween, is allowed to bind the primary antibody on the membrane for 1h at room temperature. Supersignal West Pico Chemiluminescent Substrate is used to detect HRP tagged anti mouse antibody. </li>
 <li>Quantify protein expression levels by densitometry. </li>
</ol>
This section describes two methods of tracking phenotype development as a function of increasing protein concentration. The first method is cell-population based, while the second studies it at the level of a single cell.
Quantification of cell-division phenotype and cell death
<ol>
 <li>At each time-point, transfer 1ml cell suspension to a sterile eppendorf tube using sterile techniques and harvest cells by centrifugation at 2200 rpm for 5 mins. </li>
 <li>Aspirate the supernatant and resuspend the pellet in 70&mu;l media, followed by 30&mu;l Methylene blue from a stock of 1mg/ml in sterile water. Methylene blue is a blue vital dye that becomes colorless in the reducing environment of the cytosol hence dead cells can be identified by their blue color. </li>
 <li>Pipette ~6&mu;l of this suspension on a clean glass slide and place a coverslip over the drop. Press the coverslip gently to form a thin, uniform layer of cells between the glass interfaces.</li>
 <li>Divide the coverslip into 9 quadrants and take 3 images per quadrant as indicated in Fig. 2. The number of cells/field should be no larger than 30 cells to allow accurate quantification. Each coverslip can be imaged for approximately 15 mins before air bubbles damage the preparation.</li>
 <li>Count the number of cells showing the phenotype under study. The cell division defect induced upon ENTH2 overexpression leads to cell death and hence, we also count the number of dead cells identified by their blue color.</li>
 <li>Calculate the percentage of phenotypic cells.</li>
 <li>Plot the data as shown in Fig. 3.</li>
</ol>
Visualization of phenotype-specific defects using fluorescence microscopy
Cell-division defective cells, as seen upon ENTH2 overexpression, are often characterized by cell wall defects and septin mislocalization3 (visualized as GFP-fusion proteins). The cell wall of budding yeast can easily be visualized by staining with Calcofluor White (CfW) which binds irreversibly to chitin in yeast cell walls.
<ol>
 <li>At every time-point, aliquot 1ml of culture in a sterile eppendorf tube and harvest cells by centrifugation at 2200 rpm for 5 mins at room temperature. </li>
 <li>Resuspend the cell pellet in 100&mu;l of 1mg/ml CfW solution in sterile water and incubate for 5 mins at room temperature in the dark.</li>
 <li>Harvest cells by centrifugation and wash 3 times with 1X PBS. </li>
 <li>Resuspend the pellet in 100&mu;l 1X PBS and image under the microscope using UV optics (cell wall) and FITC filter (GFP-septin) at 100X magnification.</li>
 <li>Quantify percentage of cells with cell wall defect and septin mislocalization by following steps 4-6 in section B.2.1</li>
</ol>
B.3. Determination of phenotype development at the single cell level: Time-lapse video microscopy.
<ol>
 <li>Clean a glass slide with a concave depression with 70% alcohol and allow to air dry.</li>
 <li>Boil 0.06g agarose in 5ml selective media lacking methionine in a sterile glass tube in a microwave until the agarose dissolves completely.</li>
 <li>Quickly pipette 200&mu;l of the agarose containing media in the slide depression and invert another clean glass slide over it, making sure that no air bubbles are trapped underneath.</li>
 <li>When the gel solidifies, move the slide on top smoothly away from the depression slide, keeping their surfaces parallel at all times. This ensures that the surface of the agarose bed is flush with the surface of the depression slide. Clean the slide region surrounding the agarose bed with a delicate tissue. The slide is now ready for use.</li>
 <li>At time = 4 hours, transfer 1ml of cells from the culture to a sterile eppendorf tube and harvest by centrifugation at 2200 rpm for 5 mins at room temperature. 
Note: It is recommended a later time-point be chosen to assure high levels of protein expression so that phenotype development is guaranteed. Pilot experiments are required to determine induction times on a case-by-case basis. 
 </li>
 <li>Aspirate the supernatant and resuspend cells in 100&mu;l of fresh selective media lacking methionine.</li>
 <li>Apply petroleum jelly to the edges of the coverslip. Then place a drop of cell suspension at the center of the agarose bed and spread uniformly by gently pressing a coverslip over them.</li>
 <li>Seal the edges of the coverslip with petroleum jelly and fix its position by lining the edges with nail polish. Wait until the nail polish dries.</li>
 <li>Place the slide on a heated stage of a microscope and choose a field that contains no more than 10 cells spaced adequately. As cells divide, the field will get crowded over time.</li>
 <li>Take an image of the field every 5 minutes for ~5 hours. Phenotype progression can be studied by assembling the images into a movie using ImageJ software (<a name="cite">Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/, 1997-2005</a> ) </li>
</ol>
Note: We use Zeiss Axiovert 200M microscope, Zeiss Axiocam MRm monochrome digital camera and Carl Zeiss Axiovision image acquisition software (version 4.4) for live cell imaging. Temperature control is achieved by placing slides on a heated stage maintained at 30&deg;C by the PeCon Tempcontrol 37 device. 
In order to avoid re-adjusting the focus repeatedly, it is convenient to program the image acquisition software to automatically acquire a few z-stack images at every time-point and choose the image with the best focus later when assembling the movie. Some image acquisition softwares have the option of autofocus for time course experiments. It is convenient to use that option in when available. 
Wild type yeast cells grow best at 30&deg;C hence temperature control is critical. In order to ensure adequate heat, it is helpful to provide an external source of heat in addition to the heated stage. For example, we leave the transmitted white light of the microscope on for the entire duration of imaging. 
It is also critical to avoid air-bubbles when preparing the agarose bed and cell sandwich. The air entrapped within the bubbles expands when heated over time and tend to push cells away from the field being imaged. 
It is also important to ensure that individual cell positions within the field do not change appreciably over the duration of the imaging process, especially during re-focusing attempts. Image stabilization plugins are available for ImageJ to rectify small shifts in cell position after the movie has been assembled. (<a href="http://www.cs.cmu.edu/~kangli/code/Image_Stabilizer.html">http://www.cs.cmu.edu/~kangli/code/Image_Stabilizer.html</a>).

<ol>
	<li>R&#246;nicke, V., Graulich, W., Mumberg, D., M&#252;ller, R., Funk, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+conditional+promoters+for+expression+of+heterologous+proteins+in+Saccharomyces+cerevisiae.">Use of conditional promoters for expression of heterologous proteins in Saccharomyces cerevisiae.</a> Methods Enzymol. 283, 313-322 (1997).</li><li>Gietz, R. D., Woods, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> High efficiency transformation with lithium acetate in Molecular Genetics of Yeast: A Practical Approach. , 121-134 (1994).</li><li>Mukherjee, D., Coon, B. G., Edwards, D. F., Hanna, C. B., Longhi, S. A., McCaffery, J. M., Wendland, B., Retegui, L. A., Bi, E., Aguilar, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yeast+endocytic+protein+Epsin+2+functions+in+a+cell-division+signaling+pathway.">The yeast endocytic protein Epsin 2 functions in a cell-division signaling pathway.</a> J Cell Sci. 122, 2453-2463 (2009).</li><li>Sherman, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Getting started with yeast in Guide to Yeats genetics and Molecular Biology. , 3-21 (1991).</li></ol>

This protocol describes how to monitor the development of a morphological phenotype (yeast cell unable to undergo proper cell division) upon protein overexpression. When doing this procedure it's important to remember to harvest yeast cells by pelleting at the recommended centrifugation speed as faster speeds may damage cells and obscure results. Methylene blue and Calcofluor white should be added to live cells just prior to imaging as they are toxic. This procedure can also be easily adapted for phenotypes observed unde...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Nocodazole</td>
<td>Reagent</td>
<td>Sigma (St. Louis, MO)</td>
<td>M1404</td>
<td>&nbsp;</td>
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<td>Methylene Blue 1% (w/v)</td>
<td>Reagent</td>
<td>VWR International</td>
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<td>Calcofluor White</td>
<td>Reagent</td>
<td>Sigma (St. Louis, MO)</td>
<td>F3543</td>
<td>&nbsp;</td>
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<td>Petroleum Jelly</td>
<td>Reagent</td>
<td>VWR International</td>
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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 phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Watch this Scientific Journal Video about Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast at JoVE.com

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 phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

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

analysis-development-morphological-phenotype-as-function-protein

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10.7K visualizações.  Purdue University. Deleção do gene e superexpressão da proteína são métodos comuns para o estudo funções das proteínas. Neste artigo, descrevemos um protocolo para análise do desenvolvimento do fenótipo como uma função da concentração de proteína na população e de uma única célula em níveis Saccharomyces cerevisiae.

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

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

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 yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

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

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis 

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), a plant cysteine protease, by real-time label-free analysis using Biacore X100. Several cystatin B variants with point mutations in areas of interaction with papain, are produced. For each cystatin B variant we determine its specific binding concentration using calibration-free concentration analysis (CFCA) and compare the values obtained with total protein concentration as determined by A280. After that, the kinetics of each cystatin B variant binding to papain is measured using single-cycle kinetics (SCK). We show that one of the four cystatin B variants we examine is only partially active for binding. This partial activity, revealed by CFCA, translates to a significant difference in the association rate constant (ka) and affinity (KD), compared to the values calculated using total protein concentration. Using CFCA in combination with kinetic analysis in a structure-function study contributes to obtaining reliable results, and helps to make the right interpretation of the interaction mechanism.

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

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 hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, 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 ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular function of many proteins. PTMs of kinetochore proteins that associate with centromeric DNA mediate faithful chromosome segregation to maintain genome stability. Biochemical approaches such as mass spectrometry and western blot analysis are most commonly used for identification of PTMs. Here, a protein purification method is described that allows the detection of both sumoylation and ubiquitination of the kinetochore proteins, Ndc10 and Ndc80, in Saccharomyces cerevisiae. A strain that expresses polyhistidine-Flag-tagged Smt3 (HF-Smt3) and Myc-tagged Ndc10 or Ndc80 was constructed and used for our studies. For detection of sumoylation, we devised a protocol to affinity purify His-tagged sumoylated proteins by using nickel beads and used western blot analysis with anti-Myc antibody to detect sumoylated Ndc10 and Ndc80. For detection of ubiquitination, we devised a protocol for immunoprecipitation of Myc-tagged proteins and used western blot analysis with anti-Ub antibody to show that Ndc10 and Ndc80 are ubiquitinated. Our results show that epitope tagged-protein of interest in the His-Flag tagged Smt3 strain facilitates the detection of multiple PTMs. Future studies should allow exploitation of this technique to identify and characterize protein interactions that are dependent on a specific PTM.

This manuscript describes the detection of sumoylation and ubiquitination of kinetochore proteins, Ndc10 and Ndc80, in the budding yeast Saccharomyces cerevisiae.

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular ...

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.

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 ...

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 regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

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.

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