Work in the Duennwald laboratory is supported by grants from the American Federation for Aging Research (AFAR), the Hereditary Disease Foundation (HDF) and the William Wood Foundation.

1. Expression of Toxic PolyQ-expansion Proteins in Yeast
A systematic analysis has established the precise amino acid sequence of a polyQ-expansion protein that is required to produce toxicity in yeast 7. This toxic polyQ-expansion protein contains an amino-terminal FLAG-tag followed by 17 amino acids from the original sequence of the huntingtin protein, a polyQ region, and a carboxy-terminal fusion to a fluorescent protein (either GFP or CFP, see Figure 1 a). The expression of proteins with polyQ expansions of 46 glutamines or more (e.g. 72 and 103 glutamines) produces toxicity in yeast when expressed under the control of the inducible and relative strong GAL1 promoter 7, 9.
As described previously, polyQ toxicity in yeast is only apparent in cells that carry the protein Rnq1p in its prion conformation, [RNQ+], e.g. the yeast strain W303 9. The toxic polyQ-expansion protein recapitulates central aspects of polyQ-biology in yeast, such as polyQ length-dependent toxicity (see below) and aggregation (Figure 1 b). Notably, toxic polyQ-expansion proteins do not kill yeast cells immediately; they rather impair or arrest the cell cycle and cell devision, thereby slowing down or inhibiting the growth of yeast colonies on plates or liquid cultures (our unpublished data).
2. Potential Problems with Yeast Cells Expressing Toxic PolyQ-expansion Proteins
Yeast cells expressing the otherwise toxic polyQ expansion proteins may not show any growth defect 9. The genetic nature of these suppressors of polyQ-toxicity does not appear to be based on simple Mendelian mutations and thus can occur with relatively high frequency (our unpublished results). We speculate that these spontaneous suppressors are caused by the curing of unidentified prions that function similarly to [RNQ+] in determining polyQ toxicity.These spontaneous suppressors of polyQ toxicity can jeopardize the successful outcome of any experiment that aims to characterize polyQ toxicity or to identify and characterize modifiers of polyQ toxicity.
In order to avoid the frequent occurrence of these spontaneous suppressors, follow the precautionary measures outlined below, which have proven to be very effective:
<ol>
	<li>Use fresh yeast cells for toxicity experiments. Do not store yeast for extended periods of time. Frequently retrieve fresh yeast colonies from frozen stocks.</li>
	<li>Frequently monitor the expression and aggregation of toxic polyQ-expansion proteins by fluorescence microscopy.</li>
	<li>Keep the yeast cells in media that repress the expression of the toxic polyQ toxicity at all times (i.e. in selective media containing glucose as sole carbon source) before starting any toxicity measurements.</li>
	<li>Use at least three independent transformants for each polyQ-toxicity experiment.</li>
</ol>
3. Growth Assays
<ol>
	<li>For each experimental condition, inoculate one colony each from three independent transformants of yeast cells harboring the toxic polyQ-expansion protein in 3 ml of selective yeast media with glucose as sole carbon source.</li>
	<li>Incubate these cultures overnight at 30 &deg;C. Under these conditions, the cells are not expressing the polyQ-expansion protein because the glucose in the medium represses their expression. Do not let these cultures overgrow; keep the OD600 (absorbance of light of 600 nm wavelength) of the overnight cultures below 1.</li>
	<li>We routinely use three different assays to monitor the growth defects of yeast cells expressing the toxic polyQ-expansion protein:</li>
</ol>
3.1 Growth on plates
<ol>
	<li>Dilute the overnight yeast cultures (grown with 220 rpm shaking) to an OD600 of 0.0005 (i.e. a 1:1000 dilution of a OD600 = 0.5 culture) in selective media 7 containing glucose.</li>
	<li>Evenly spread 50 &mu;l (resulting in ca. 700 colonies per plate) of each diluted culture on one plate (10 cm diameter) with selective medium containing glucose as sole carbon source and one plate with selective medium containing galactose as sole carbon source.</li>
	<li>Incubate the plates for three to four days at 30 &deg;C. After the incubation, take photographs of each plate and count the number of colonies on the glucose and the number of colonies on the galactose plates. Under ideal conditions, there should be no or only very few colonies on the galactose plate when using yeast cells expressing a highly toxic polyQ-expansion protein (103Q, Figure 2a).</li>
</ol>
3.2 Spotting assays
This assay is more quantitative than the plating assay described above and can thus reveal even subtle differences in polyQ toxicity with the same experiment on the same plate.
<ol>
	<li>Dilute the overnight cultures grown in medium with glucose to an OD600 of 0.1.</li>
	<li>Pipette 200 &mu;l of these diluted cultures into sterile 96-well plates and prepare five fivefold serial dilutions in sterile water using a multi-channel pipette.</li>
	<li>Using a frogger,(also called a spotter, with 8 x 6 pins for transfer of cell suspensions) transfer the cell suspensions onto plates containing selective media with glucose as sole carbon source and plates containing selective media with galactose as sole carbon source.</li>
	<li>Allow the plates to dry before incubating them at 30 &deg;C for three to four days.</li>
	<li>After the incubation, take photographs of each plate (Figure 2b).</li>
</ol>
3.3. Monitoring polyQ toxicity in yeast by growth of liquid cultures
This protocol is the most quantitative (OD600 numbers) of the three assays described here and can even detect very subtle differences in polyQ toxicity. The aforementioned occurrence of spontaneous suppressors, however, can potentially produce misleading results. I therefore recommend combining this assay with at least one of the two plating assays described above. We propose to use the BioscreenC instrument for these experiments. The BioscreenC is an instrument that automatically measures the optical density of yeast cultures in 100-well plates while incubated at defined temperatures with defined agitation. Other methods for growing yeast cells and measuring their optical density may also apply.
<ol>
	<li>Wash the yeast cells from minimal media containing glucose as sole carbon source three times in 3 ml sterile water.</li>
	<li>Dilute the overnight cultures grown in medium with galactose to an OD600 of 0.1.</li>
	<li>Fill each well of the 100-well BioscreenC plate with 300 &mu;l of the yeast cultures.</li>
	<li>Open the Easy Bioscreen Experiment program. Determine the number of samples you wish to monitor (including blanks and medium-only controls), set the temperature to 30 &deg;C, set the length of the experiments to 3 days, set the measurement intervals to 15 minutes, set the filter to 600 nm /Brown, and set the shaking mode to 15 seconds before each measurement at medium strength.</li>
	<li>The BioscreenC instrument and the attached software will produce Excel spreadsheets of each data point taken during the experiment.</li>
	<li>Prepare growth curves with Excel for each sample and compare growth of different samples (Figure 2c). A detailed description of the analysis of data produced by BioscreenC experiments has been provided before 10.</li>
</ol>
4. Representative Results
<img alt="Figure 1" src="/files/ftp_upload/3461/3461fig1.jpg" /> 
Figure 1. The yeast polyQ model. a) Schematic representation of the toxic polyQ-expansion protein. b) Fluorescence microscopy showing yeast cells expressing a short, non-toxic polyQ expansion protein (25Q, left panel) and yeast cells expressing a long, toxic polyQ-expansion protein (103Q, right panel).
<img alt="Figure 2" src="/files/ftp_upload/3461/3461fig2.jpg" /> 
Figure 2. Representative results of growth assays of yeast cells expressing polyQ-expansion proteins. a) Plating assay. Approximately 700 yeast cells were spread on plates and incubated for three days at 30 &deg;C. The upper panel shows a plate containing glucose medium, i.e. the expression of the toxic polyQ-expansion protein (103Q) is not induced. The lower panel shows a yeast plate containing galactose medium, i.e. the expression of the toxic polyQ-expansion protein is induced. Note that in the experiment shown here, no spontaneous suppressor occurred. b) Spotting assay. Five serial five-fold dilutions of yeast cells harboring either a non-toxic polyQ protein (25Q) or a toxic polyQ-expansion protein (103Q) were spotted on glucose plates that repress the expression of the proteins (left panel) or on galactose plates that induce their expression. The plates were then incubated for three days at 30 &deg;C. c) BioscreenC experiment. The growth of cultures of yeast cells expressing either a non-toxic polyQ protein (25Q) or a toxic polyQ-expansion protein (103Q) were monitored by the BioscreenC instrument. The experimental conditions and the analysis of the BioscreenC experiments are described in the main text.

<ol>
	<li>Soto, C., Estrada, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+misfolding+and+neurodegeneration.">Protein misfolding and neurodegeneration.</a> Arch. Neurol. 65, 184-189 (2008).</li><li>Ross, C. A., Tabrizi, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Huntington's+disease:+from+molecular+pathogenesis+to+clinical+treatment.">Huntington's disease: from molecular pathogenesis to clinical treatment.</a> Lancet Neurol. 10, 83-98 (2011).</li><li>Zoghbi, H. Y., Orr, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamine+repeats+and+neurodegeneration.">Glutamine repeats and neurodegeneration.</a> Annu. Rev. Neurosci. 23, 217-247 (2000).</li><li>Meriin, A. 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L., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+ERAD+and+ER+stress+are+early+and+specific+events+in+polyglutamine+toxicity.">Impaired ERAD and ER stress are early and specific events in polyglutamine toxicity.</a> Genes Dev. 22, 3308-3319 (2008).</li><li>Duennwald, M. L., Jagadish, S., Muchowski, P. J., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flanking+sequences+profoundly+alter+polyglutamine+toxicity+in+yeast.">Flanking sequences profoundly alter polyglutamine toxicity in yeast.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 11045-1150 (2006).</li><li>Duennwald, M. L., Jagadish, S., Giorgini, F., Muchowski, P. 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No conflicts of interest declared.

This manuscript outlines three complementary experimental approaches to measure polyQ-toxicity in the model organism yeast based on reduced growth of yeast cells expressing toxic polyQ-expansion proteins. Work in yeast has offered profound insights into the basic cellular and molecular mechanisms of protein misfolding and its ensuing toxicity, including the misfolding and toxicity of polyQ-expansion proteins 9,11,12. Experiments based on the protocols presented here have already helped to identify and characte...

<table border="1">
	<tbody>
		<tr>
			<td>Name of instrument</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Frogger (6x8 pins)</td>
			<td>V&amp;P Scientific, San Diego</td>
			<td>VP 407 AH</td>
		</tr>
		<tr>
			<td>BioscreenC</td>
			<td>Growthcurves USA</td>
			<td>5101370</td>
		</tr>
		<tr>
			<td>100-well Honeycomb plates</td>
			<td>Growthcurves USA</td>
			<td>9602550</td>
		</tr>
	</tbody>
</table>

growth assays to assess polyglutamine toxicity in yeast

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's disease, and Huntington's disease 1. Huntington's disease (HD) is caused by the abnormal expansion of a polyglutamine (polyQ) region within the protein huntingtin. The polyQ-expanded huntingtin protein attains an aberrant conformation (i.e. it misfolds) and causes cellular toxicity 2. At least eight further neurodegenerative diseases are caused by polyQ-expansions, including the Spinocerebellar Ataxias and Kennedy&#x2019;s disease 3.
The model organism yeast has facilitated significant insights into the cellular and molecular basis of polyQ-toxicity, including the impact of intra- and inter-molecular factors of polyQ-toxicity, and the identification of cellular pathways that are impaired in cells expressing polyQ-expansion proteins 3-8. Importantly, many aspects of polyQ-toxicity that were found in yeast were reproduced in other experimental systems and to some extent in samples from HD patients, thus demonstrating the significance of the yeast model for the discovery of basic mechanisms underpinning polyQ-toxicity. 
A direct and relatively simple way to determine polyQ-toxicity in yeast is to measure growth defects of yeast cells expressing polyQ-expansion proteins. This manuscript describes three complementary experimental approaches to determine polyQ-toxicity in yeast by measuring the growth of yeast cells expressing polyQ-expansion proteins. The first two experimental approaches monitor yeast growth on plates, the third approach monitors the growth of liquid yeast cultures using the BioscreenC instrument. 
Furthermore, this manuscript describes experimental difficulties that can occur when handling yeast polyQ models and outlines strategies that will help to avoid or minimize these difficulties. The protocols described here can be used to identify and to characterize genetic pathways and small molecules that modulate polyQ-toxicity. Moreover, the described assays may serve as templates for accurate analyses of the toxicity caused by other disease-associated misfolded proteins in yeast models.

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Growth Assays to Assess Polyglutamine Toxicity in Yeast

This manuscript describes three complementary protocols for assessing the toxicity of polyglutamine (polyQ)-expansion proteins in the yeast Saccharomyces cerevisiae. These protocols can easily be modified to monitor the toxicity of other misfolded proteins in yeast.

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's ...

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13.5K Views.  Boston Biomedical Research Institute. This manuscript describes three complementary protocols for assessing the toxicity of polyglutamine (polyQ)-expansion proteins in the yeast Saccharomyces cerevisiae. These protocols can easily be modified to monitor the toxicity of other misfolded proteins in yeast.

Video: Growth Assays to Assess Polyglutamine Toxicity in Yeast

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 and minimum levels of 0.03 ng ml&#8722;1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, log-phase, and stationary-phase. Each growth phase can reveal different aspects of fitness that are related to various environmental and genetic conditions. High-resolution and quantitative measurements of all 3 phases of growth are generally difficult to obtain. Here we present a detailed method to characterize all 3 growth phases on solid media using an assay called One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). ODELAY quantifies growth phenotypes of individual cells growing into colonies on solid media using time-lapse microscopy. This method can directly observe population heterogeneity with each growth parameter in genetically identical cells growing into colonies. This population heterogeneity offers a unique perspective for understanding genetic and epigenetic regulation, and responses to genetic and environmental perturbations. While the ODELAY method is demonstrated using yeast, it can be utilized on any colony forming microorganism that is visible by bright field microscopy.

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and quantified.

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...