Name: assay development for high content quantification of sod1 mutant protein aggregate formation in living cells
Uploaded: 2017-10-04T12:15:00
Description: Watch this Scientific Journal Video about Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells at JoVE.com

Este trabajo fue apoyado por una beca financiada por el gobierno coreano (MSIP) (NRF-2014K1A4A7A01074642) y el programa de apoyo nacional investigación Fundación de Corea (NRF) científico individual (NRF/NRF-2013M3A9B5076486-2015R1D1A1A09057239).

1. producción de lentivirus Nota: la producción y manipulación de vectores lentivirales se llevó a cabo según las pautas de los institutos nacionales de salud (NIH) para investigación con recombinante DNA. La codificación de plásmido el tipo salvaje y mutante SOD1 de A4V etiquetados YFP mejorada (SOD1WT-YFP y SOD1A4V-YFP) se describen en Kim et al. 11 ambos productos de fusión genética fueron amplificados mediante el par de la cartilla PCR 5 &#8242; ...

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I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disulfide+bond+mediates+aggregation,+toxicity,+and+ubiquitylation+of+familial+amyotrophic+lateral+sclerosis-linked+mutant+SOD1.">Disulfide bond mediates aggregation, toxicity, and ubiquitylation of familial amyotrophic lateral sclerosis-linked mutant SOD1.</a> J Biol Chem. 282 (38), 28087-28095 (2007).</li><li>Juneja, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognosis+in+familial+amyotrophic+lateral+sclerosis:+progression+and+survival+in+patients+with+glu100gly+and+ala4val+mutations+in+Cu,Zn+superoxide+dismutase.">Prognosis in familial amyotrophic lateral sclerosis: progression and survival in patients with glu100gly and ala4val mutations in Cu,Zn superoxide dismutase.</a> Neurology. 48 (1), 55-57 (1997).</li><li>Kaur, S. 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R., Rashid, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutant+SOD1+mediated+pathogenesis+of+Amyotrophic+Lateral+Sclerosis.">Mutant SOD1 mediated pathogenesis of Amyotrophic Lateral Sclerosis.</a> Gene. 577 (2), 109-118 (2016).</li><li>de Bruyns, A., Geiling, B., Dankort, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+Modular+Lentiviral+Vectors+for+Effective+Gene+Expression+and+Knockdown.">Construction of Modular Lentiviral Vectors for Effective Gene Expression and Knockdown.</a> Methods Mol Biol. 1448, 3-21 (2016).</li><li>Sirven, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+Transgene+Expression+in+Cord+Blood+CD34+-Derived+Hematopoietic+Cells,+Including+Developing+T+Cells+and+NOD/SCID+Mouse+Repopulating+Cells,+Following+Transduction+with+Modified+TRIP+Lentiviral+Vectors.">Enhanced Transgene Expression in Cord Blood CD34+-Derived Hematopoietic Cells, Including Developing T Cells and NOD/SCID Mouse Repopulating Cells, Following Transduction with Modified TRIP Lentiviral Vectors.</a> Mol Ther. 3 (4), 438-448 (2001).</li><li>Wu, C., Lu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-titre+retroviral+vector+system+for+efficient+gene+delivery+into+human+and+mouse+cells+of+haematopoietic+and+lymphocytic+lineages.">High-titre retroviral vector system for efficient gene delivery into human and mouse cells of haematopoietic and lymphocytic lineages.</a> J gen virol. 91 (Pt 8), 1909-1918 (2010).</li><li>Irshad, H., Veillard, A., Roux, L., Racoceanu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+nuclei+detection,+segmentation,+and+classification+in+digital+histopathology:+A+review-current+status+and+future+potential.">Methods for nuclei detection, segmentation, and classification in digital histopathology: A review-current status and future potential.</a> IEEE Rev Biomed Eng. 7, 97-114 (2014).</li><li>Purschke, M., Rubio, N., Held, K. 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Hay dos enfoques principales para la generación de líneas celulares estables. La primera toma varias semanas y requiere transitoria selección de transfección y resistencia de los vectores de DNA de plásmido integrada genomic. El segundo lleva pocas horas mediante el uso de lentivirus, haciendo este protocolo favorable a la expresión efectiva de la proteína diana en múltiples líneas celulares con un esfuerzo limitado. El viaje-CMV vector19 era un vector constante a utilizar, con eficacia d...

Agregación de proteínas es un proceso biológico por el cual mal grupo de proteínas para arriba y puede actuar como agentes causales de enfermedades neurodegenerativas (amiloidosis). Caracterización de la agregación de la proteína es esencial para entender el papel de los agregados en la disfunción celular, así como en facilitar el descubrimiento de nuevos factores que influyen en la aparición de la patología. La visualización de proteínas etiquetadas de fluorescencia en células vivas es un método eficaz que puede ayudar en el desarrollo de análisis aplicables al alto contenido de proyección (HCS)1,2,3,4.
Esclerosis lateral amiotrófica (ELA) es considerada como una enfermedad proteopathic causada por la presencia de proteínas mal plegadas con la propensión a agregado y se acumulan en las neuronas motoras en ALS familiar (fALS) y esporádicos ALS (sALS)5,6 . Un subconjunto de ~ 20% de los casos de fALS se asocian con mutaciones dominantes en el gen que codifica la enzima citosólica antioxidante cobre-zinc superóxido dismutasa tipo 1 (SOD1)7,8. Se han propuesto varias causas posibles para este trastorno genético derivado, incluyendo los cambios en la estructura y función de las variantes de la SOD1, como aberrante estabilidad, mayor tasa de despliegue y propensión a agregado9, 10. En particular, la única propiedad comprobada y potencialmente tóxica compartidos por ambas variantes vinculada ALS SOD1 y wild-type (WT) SOD1 es una propensión creciente para formar agregados proteicos compartimentado o inclusiones proteínico11,12 . Mal plegada mutante SOD1, es persistentemente polyubiquitinated y degradada por el sistema ubiquitina-proteasoma. Como resultado, un bajo nivel de inhibición del proteasoma actividad conduce a la acumulación del mutante que SOD1 agregados13,14, que las estructuras de forma amorfa compuesta de componentes solubles que pueden intercambiar con soluble mutante SOD1 en el citosol15. En particular, SOD1 mutante A4V (alanina en el codón 4 cambiada a valina) es la mutación más común que causa la ALS y lleva a la neurodegeneración rápida con un tiempo promedio de supervivencia de menos de 2 años después del inicio de la enfermedad16. Bioquímico, SOD1 A4V tiene una tendencia creciente a monomerize, se agregan y forman poros de amiloides; sus poro-como los agregados son similares a los poros de amiloides de otras formas mutantes ligado a la enfermedad, como la α-sinucleína y β-amiloide proteína17. Para estudiar la dinámica de la acumulación de agregados de SOD1, métodos de control solubles e insolubles SOD1 agregadas formas permanecen para ser desarrollado.
Hemos demostrado anteriormente, usando proyección de imagen de células vivas y las células HEK-293 transfectadas transitoriamente con SOD1 de etiquetado proteína fluorescente, que mutaciones asociadas a ALS deterioran SOD1 dimerización y agregación11. Aunque la expresión transitoria de sistemas pueden proporcionar información útil sobre el resultado biológico de sobreexpresión génica a corto plazo, métodos proporciona la integración estable de los genes deseados pueden ser preferidos para el desarrollo del ensayo. Como tal, vectores lentivirales ofrecen la posibilidad de conferir la expresión del gen regulado y a largo plazo en las células mamíferas 18. En este estudio, nos hemos centrado en la generación de líneas celulares estables de transduced con lentivirus recombinante teniendo peso y mutante SOD1 etiquetadas con proteína fluorescente amarilla (YFP). Usando microscopía de imágenes de células vivas y cuantificación automatizada de la agregación de SOD1, desencadenaron y cuantificar eventos de agregación de SOD1 en la inhibición del proteasoma.

<table>
	<tbody>
		<tr>
			<td>ALLN (C20H37N3O4)</td>
			<td>Millipore</td>
			<td>208719</td>
			<td></td>
		</tr>
		<tr>
			<td>MG132 (C26H41N3O5)</td>
			<td>Sigma-Aldrich</td>
			<td>C2211</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxomicin (C28H50N4O7)</td>
			<td>Sigma-Aldrich</td>
			<td>E3652</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H-3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Opera</td>
			<td>Perkin Elmer</td>
			<td>OP-QEHS-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Opera EvoShell software</td>
			<td>Perkin Elmer</td>
			<td>Ver 1.8.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Operetta</td>
			<td>Perkin Elmer</td>
			<td>OPRT1288</td>
			<td></td>
		</tr>
		<tr>
			<td>Harmony Imaging software</td>
			<td>Perkin Elmer</td>
			<td>Ver 3.0.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Columbus Image analysis software</td>
			<td>Perkin Elmer</td>
			<td>Ver 2.3.2</td>
			<td></td>
		</tr>
		<tr>
			<td>CyBi Hummingwell liquid handling</td>
			<td>CyBio AG</td>
			<td>OL 3387 3 0110</td>
			<td></td>
		</tr>
	</tbody>
</table>

assay development for high content quantification of sod1 mutant protein aggregate formation in living cells

Esclerosis lateral amiotrófica (ELA) es una enfermedad neurodegenerativa fatal que puede ser causada por mutaciones heredadas en el gene codificación cobre-zinc superóxido dismutasa 1 (SOD1). La inestabilidad estructural del SOD1 y la detección de inclusiones de SOD1-positivo en pacientes de ALS familiar apoyan un posible papel causal de SOD1 mal plegadas o agregados en patología de ALS. En este estudio, describimos el desarrollo de un análisis celular diseñado para cuantificar la dinámica de agregación de SOD1 en células vivas por alto contenido métodos de detección. Uso de vectores lentivirales, generamos líneas estables de la célula expresando salvaje-tipo y del mutante SOD1 A4V etiquetadas con proteína fluorescente amarilla y encontró que ambas proteínas se expresaron en el citosol sin ningún signo de agregación. Curiosamente, sólo SOD1 A4V había expresado estable HEK-293, pero no en líneas celulares U2OS o SH-SY5Y, agregados al tratamiento del inhibidor de proteasoma. Nos muestran que es posible cuantificar la agregación basada en análisis de dosis respuesta de diferentes inhibidores de la proteasoma y seguimiento de la cinética de la formación de agregados por microscopia Time-lapse. Nuestro enfoque presenta la posibilidad de cuantificar el efecto de mutaciones de la ALS en el papel de SOD1 en la formación de agregados, así como la detección de pequeñas moléculas que impiden la agregación de SOD1 A4V.

Línea de celular estable de generación mediante lentivirus: La estrategia general para el control de agregados de proteína SOD1 se ilustra en la figura 1. En un primer paso, hemos generado un vector de expresión lentivirales para la entrega de estable gene SOD1 en líneas celulares (paso 1). Dos vectores lentivirales codificación etiquetado YFP SOD1 WT y SOD1 A4V (SOD1WT-YFP y SOD1A4V-YFP, respectivamente) con embalaje y sobre plásmidos...

Watch this Scientific Journal Video about Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells at JoVE.com

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Se describe un método para cuantificar la agregación de proteínas mal plegadas. Nuestros detalles del protocolo lentivirales inducida por generación de línea celular estable, proyección de imagen confocal automatizado y análisis de imágenes de agregados proteicos. Como una aplicación ilustrativa, se estudió el efecto de pequeñas moléculas en la promoción de la agregación de SOD1 en una forma dependiente de dosis y tiempo.

Desarrollo de ensayos para cuantificación de contenido alto de Sod1 mutante de la proteína total formación en células vivas

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

The overall goal of this experiment is to develop an assay for studying the effect of small molecules in modulating the aggregation of misfolded proteins in a time and dose dependent manner. This method can help answer the key questions for development of cellular assay, and it's use is for high-content screening techniques. The main advantage of this technique is to provide a simple method to detect and quantify protein aggregation occurring in several neurodegenerative disorders.The implication of this technique extends towards potential therapy of neurodegenerative diseases, such as ALS, because it proposes a simple method that allow to develop cellular assay and perform screening for small molecule moderators. To start virus production, prior to plasmid transfection split HEK-293 T-cells at 30 to 40%confluence in a 10 centimeter tissue culture dish and 10 milliliters of cell culture medium. Incubate the culture plate in a 5%carbon dioxide incubator at 37 degrees Celsius overnight.On the following day, prepare DNA transfection solution with packaging plasmids lentiviral vector, 125 microliters of one molar calcium chloride and add distilled water to adjust the volume to 500 microliters. Gently add 500 microliters of 0.05 molar HEPS buffer and incubate for 10 minutes at room temperature. After that, replace HEK-293 T-cell medium with 10 milliliters pre-warmed antibiotic-free fresh culture medium mixed with one milliliter of DNA transfection solution.Incubate at 37 degrees Celsius and 5%carbon dioxide incubator over night. Replace cell supernatent with fresh culture medium on the following day and incubate again overnight. Finally, on the following day harvest the supernatent and transfer to a 15 milliliter tube.To remove the dead cells and debris, centrifuge the tube at 500 times g for five minutes. To further purify the supernatent, pass it through a 0.45 micrometer filter using a 60-milliliter syringe. Then, immediately dispense the filtered supernatent into 300-microliter single use aliquots.To prepare the cells for transduction, split them to be infected at 60%confluence in a six-well tissue culture plate in two milliliters of DMAM supplemented with 10%FBS. Incubate the cells in a 37-degree Celsius incubator at 5%carbon dioxide overnight. On the following day, thaw one aliquot of the frozen lentivirus on ice for each use.Meanwhile, warm the cell culture medium containing the serum compatible with the cell line of interest. Once the virus is fully thawed, dilute it in DMEM in new 1.5 milliliter micro-centrifuge tubes. Then adjust the volume to one milliliter with reduced serum medium.Next, add two microliters of polybrene to one milliliter of virus per medium and mix well by pipetting. Add one milliliter of this mixture to the cells and incubate at 37 degrees Celsius for 24 hours. After incubation, remove the virus containing medium and replace it with normal DMEM supplemented with 10%FBS and return the cells to the incubator.Monitor the cell growth and change the medium every two days When the cells reach confluence, expand each well of the six-well dish into a 10-centimeter tissue culture plate. Move the plates to the incubator. Finally, save 1.5 times 10 to the 4th cells per well and 50 microliters of DMEM on 384 well assay plates.Then check the expression level of the YFP-tagged protein under the microscope. If the expression shows signal-to-noise ratio equal or greater than three, prepare cell stock for the corresponding cell line. For the compound transfer, on a 384-well storage polypropylene plate, add serial dilutions of compounds by the standard one to three dilution series.Then, using a liquid handler equipped with a 384-capillary head, distribute 0.25 microliters of proteasome inhibitors on empty, flat-bottomed black 384-well assay plates. Then seed 1.5 times 10 to the 4th cells on these plates. Incubate at 37 degrees Celsius and 5%carbon dioxide for 24 hours.Prior to imaging the cell line treated with proteasome inhibitors, add 10 microliters of pre-warmed DMEM with staining solution and incubate at room temperature for 10 minutes. After staining, start the automated microscope operating software by selecting the microscope tab. First, select the configuration tab and then select 20 times objective in the correct plate type.To allow proper focus with different plate types, make sure that the caller is set to the correct value on the objective. Next, select exposure one. Set focus height to zero micrometers, then select focus.Once it is focused, expose camera one. To optimize the exposure plane, adjust the focus height and click on take height. Change exposure times and laser power for a maximum pixel intensity of approximately 3000 and save exposure parameters.Select experiment definition tab, create a layout and sublayout. Next, drag and drop the relevant layout exposure, reference image, skew crop file, and sublayout. Then, save the experiment.Finally, select automatic experiment tab and acquire images. For two-channel images, first select the software algorithm to segment the primary objects, such as nuclei, cytosol, and aggregates. First, distribute 0.25 microliters of proteasome inhibitor dissolved in 100%DMSO or DMSO on 384-well flat-bottomed plates.On the same plate, manually seed 1.5 times 10 to the 4th cells per well in 50-microliters of DMEM. Then set up the microscope system environmental control unit to 37 degrees Celsius and 5%carbon dioxide. Operate the microscope in wide-field florescence mode and as software settings, choose LWD 20 times objective non-confocal mode, four fields per well, and capture interval of one hour.Then, select the appropriate algorithm to segment the primary object such as cytosol and aggregates. Adjust background threshold and contrast parameters if required. The tested cell lines transduced with SOD-1 wild type and SOD-1 A4V mutant remained healthy, showed high expression levels, and longterm wide phi-labeling irrespective of the SOD form.Treatment with proteasome inhibitor ALLN for 24 hours promoted the accumulation of SOD-1 A4V YFP aggregates in HEK-293 cells but not in SOD-1 Wild Type YFP transduced cells. Furthermore, there were very low levels of aggregation in U2 OS and SH-SY5Y cell lines SOD-1 A4V YFP transduced with the same vectors. After seeding the cells in the presence or absence of ALLN, they were monitored for aggregate formation for a period of 50 hours.ALLN aggregate formation reached a plateau at 24 hours and remained stable over the next 12 hours. Furthermore, cell density significantly decreased after 28 hours as a result of the cytotoxic effect of ALLN, while cell number increased in the DMSO-treated negative control. SOD-1 A4V YFP cells cultured in the presence of proteasome inhibitors for 24 hours were stained with Hoechst solution.The relative toxicity for three different proteasome inhibitors was quatified by measuring the number of adherent cell lines remaining in the well labeled with Hoechst dye. The total cell number decreased in response to increased dose of protease inhibitors. Inversely, the percentage of cells expressing SOD-1 A4V aggregates increased with the concentration of protease inhibitor.When doing this process it is important to remember that the cell quality will drastically influence your result. So it's important to determine that the cells are microplasma free and are healthy all along the steps. Following this process, another imaging method can be used in order to answer additional questions regarding the degradation of protein or the interaction of the protein with others using resonance energy transfer.After watching this video, you should have a good understanding of how to generate the tools to test the fate of protein in multiple living cells using high-content image analysis. So this method can provide insight into protein aggregation as it occurs in simple cellular models. It can also be applied to other systems such as neuron differentiated from patient specific induced pluripotent stem cells.

Research

JoVE Journal

Neuroscience

High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue

Alta sensibilidad de detección de 5 hidroximetilcitosina en el tejido cerebral Balb / C

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research.   Mammalian DNA is enzymatically ...

Investigación

Neurociencias

Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

Preparación de la vida aislada de Vertebrados células visuales para imágenes de fluorescencia

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells1-4.  ...

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Un ensayo de migración celular cuantitativa para murinos entéricas Neuronales Progenitores

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout ...

A High Content Imaging Assay for Identification of Botulinum Neurotoxin Inhibitors

Un alto contenido de imágenes de Ensayo para la Identificación de la neurotoxina botulínica Inhibidores

Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Ensayo sencillo para cuantificación de Prevención Social en<em&gt; Drosophila melanogaster</em

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

De alta resolución de cuantificación del comportamiento guiado Olor en<em&gt; Drosophila melanogaster</em&gt; Uso de la<em&gt; Flywalk</em&gt; Paradigma

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

Novel Assay for Cold Nociception in Drosophila Larvae

Novel Assay for Cold Nociception in Drosophila Larvae

Ensayo novedoso para la nocicepción en frío<em&gt; Drosophila</em&gt; Las larvas

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

High-throughput Analysis of Locomotor Behavior in the Drosophila Island Assay

Análisis de alto rendimiento del comportamiento Locomotor en el ensayo de la isla de Drosophila

Advances in next-generation sequencing technologies contribute to the identification of (candidate) disease genes for movement disorders and other ...

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

Alta resolución imagen Confocal de la barrera hemato - encefálica: proyección de imagen, reconstrucción 3D y cuantificación de transcitosis

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Un análisis de gradiente de temperatura para determinar las preferencias térmicas de larvas de Drosophila

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...