analyzing nek4-ku70 interaction after etoposide-induced dna damage

Assessing Protein Interaction and Localization in DNA Damage

Cellular DNA damage occurs daily because of the spontaneous chemical reactions and is also increased by exogenous factors such as genotoxic agents (radiation and chemicals such as etoposide) and oxidative stress1,2,3. The cells have a complex machinery that corrects a myriad of different types of DNA damage, from removal of bases to replicative fork torsion or interruption to the most deleterious lesion: the DNA double-strand break4,5.
Several proteins that participate in the DDR have already been characterized, and excellent revisions explore the pathways of non-homologous end join (NHEJ) and homologous recombination (HR), the main pathways implied in double-strand breaks (DSB) repair5,6. The phosphorylation of the histone H2A variant, H2AX, at serine 139 (thereafter named y-H2AX) has been used for many years as a sensitive and reliable marker of the double-strand break. The phosphorylation of H2AX is observed in the first minutes following the damage and can persist for hours7.
The advantage of using the immunofluorescence detection of &#947;-H2AX foci is the characterization of the temporal and spatial distribution of the foci, as well as its co-staining with other proteins. Moreover, immunofluorescence does not require lysis, which preserves cellular context information and makes it more informative. Besides, as &#947;-H2AX is formed de novo, it is not abundantly present in the absence of DNA damage, making it a specific marker7.
H2AX is mostly phosphorylated by protein kinase ataxia-telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PK), the sensors of DSBs. Ku70/80 (XRCC6 X-ray repair cross-complementing 6/ XRCC5 X-ray repair cross-complementing 5) and DNA-protein kinase catalytic subunit (DNA-PKcs) are responsible for DSB identification and the protection of DNA ends, whereas Artemis carries out end-processing to facilitate ligation by the XRCC4-Ligase IV complex. The phosphorylation of H2AX at sites flanking DSBs acts as a signal for the recruitment of several repair proteins and signaling for cell cycle arrest, death, or survival8.
The analysis of changes in y-H2AX foci is the most common assay for studying if the protein of interest is implicated in DNA damage response. Whether a direct role in the DNA damage site is expected, fluorescence microscopy is used to verify the co-localization of the protein of interest with y-H2AX foci. However, except for the new super-resolution fluorescence methods, concluding the local interaction with DNA damage sites can be tricky. Furthermore, immunofluorescence detection usually depends on many protein molecules at the DNA damage site, making it difficult to identify scarce proteins9.
Here, we show an assay to evaluate the localization of proteins involved in the DDR pathway using y-H2AX as a marker of the damage site. This assay can be used to characterize temporal localization under different insults that cause DNA damage.
The Proximity Ligand Assay (PLA) emerged as a tool for estimating interaction between proteins as well as spatial proximity among organelles or cellular structures10,11and allows the temporal localization and co-localization analysis under stress conditions, for instance. The method is simple because it is like conventional immunofluorescence and allows the simultaneous staining of organelle or cellular structure-specific markers, such as mitochondria, endoplasmic reticulum, PML bodies, or DNA double-strand marker, yH2AX. The use of PLA allows the identification of protein interaction during DNA damage in a sensitive, specific, and reliable manner that can be used to monitor the interaction during the cell cycle, in response to different stimuli, and at different times after genotoxic stress.
We show here the use of PLA concomitantly to conventional &#947;-H2AX immunofluorescence to localize Nek4-Ku70 interaction after etoposide-induced DNA damage. Nek4, a Nek (NIMA-related kinases) family member, has no clear role in DNA Damage response. In 2012, Nguyen and colleagues showed that Nek4 interacts with Ku70/Ku80 proteins, and in the Nek4 absence, these proteins are not recruited to the DNA damage site, and H2AX phosphorylation is impaired12. The cellular localization of this interaction is unknown so far. We have observed a reduction in Ku70 phosphorylation in cells expressing Nek4 kinase-dead mutant13but whether Nek4 phosphorylates directly Ku70 remains to be elucidated. Considering that Nek4 interaction with Ku70 is increased after etoposide treatment according to immunoprecipitation studies12, we attempt to localize this interaction using PLA and the &#947;-H2AX marker.
Usually, the interaction studies are based on immunoprecipitation assays, but these are in vitro and do not provide information about the location of the interaction. When possible, an immunoprecipitation of fractioned cells (nucleus, mitochondrial, cellular membrane, or cytosol) can be performed, but performing a time course of the interaction is costly and laborious. Immunofluorescence can give information about the cellular localization of the proteins, but proving the interaction can be difficult and depends on the resolution of the microscopes. Here, we show the advantage of using the Proximity Ligand Assay to monitor the interaction of two proteins in a DNA damage context.

Author Spotlight: Combining Proximity Ligand Assay with Gamma-H2AX Staining to Characterize Protein Interactions in DNA Damage Response

Proximity Ligand Assay to Localize Proteins in DNA Damage Sites

The DNA damage response is a cellular system that maintains genome stability and integrity. Dysfunctions in this process cause several disease such as cancer, age-related disease, and chronic inflammation. Our goal is to identify proteins that cooperate in this process, which allows the identification of alternative targets for therapeutic integration.To characterize a protein's participation in the DNA damage response, immunofluorescence studies with the phosphorylated form of the H2AX histone, a marker of DNA damage, are usually applied. Moreover, protein collection assays such as co-immunoprecipitation can be performed after DNA damage to identify the protein's role in signaling. Interaction studies are usually based on immunoprecipitation assays, but these are in vitro and do not provide information about the location of the interaction.Immunofluorescence can give information about the cell localization of the proteins, but proving the interaction can be difficult and depends on the resolution of the microscope. We show here that a simple protocol combining proximity ligand assay with Gamma-H2AX staining allows the spatial and temporal characterizations of proteins'interaction in DNA damage context. Moreover, we have provided a user-friendly macro for data analysis.

Analyzing Nek4-Ku70 Interaction After Etoposide-Induced DNA Damage

After performing the PLA dot acquisition to localize protein interaction, open images from different conditions To adjust the parameters for analysis in ImageJ. Determine and specify these parameters along with the data in the macro program. To remove the background, click Process and Subtract Background.Use the preview function to test different rolling ball radii. For noise removal, click Process, Noise, and Despeckle. Then click Process and Smooth in the nucleus menu to apply the smooth function and improve filling.Now, click Image, then Type, and select 8-bit to convert to an 8-bit image. After that, adjust the threshold by going to Image, Adjust, and finally, Threshold. Adjust the threshold to visualize all the nuclei or dots in the image, while avoiding oversaturation and structures collapsing.Also, note the values and test in different images. To convert to a mask, click Process, Binary, and Convert to Mask. For the nucleus, click Process, Binary, and Fill Holes, followed by Process, Binary, and Watershed.For the PLA dots, click Process, Binary, and Watershed. For counting nuclei, measure the area or length of the different nuclei to estimate the average size. Use an approximate value to analyze particles by clicking Analyze and Analyze Particles.Set the size to 15 infinity and check the options. Show mask, display results, clear results, add to manager and exclude on edges. To count PLA dots, measure their area or radius to estimate their average size.For each ROI on the ROI manager, click in Analyze and Analyze Particles. Set the size to 0.02 to 3. Check the options, show mask, display results, clear results, summarize, and exclude on edges.Save the results showing the number of dots per nucleus in each nucleus present in the image. Nek4 Ku70 interaction increased in the nucleus following DNA damage after 20 minutes, one hour and three hours of etoposide treatment compared to control and DMSO treated cells. Etoposide treatment significantly increased Nek5 topoisomerase 2-beta interaction compared to the DMSO control.Gamma-H2AX staining increased after 20 minutes of etoposide treatment and remained elevated for up to three hours, indicating sustained DNA damage response.

analyzing-nek4-ku70-interaction-after-etoposide-induced-dna-damage

Research

JoVE Journal

Biology

22 vistas. Después de realizar la adquisición de puntos PLA para localizar la interacción de proteínas, abra imágenes de diferentes condiciones para ajustar los parámetros para el análisis en ImageJ. Determine y especifique estos parámetros junto con los datos en el programa de macros. Para eliminar el fondo, haga clic en Procesar y restar fondo.Utilice la función de vista previa para probar diferentes radios de bola rodante. Para eliminar ruido, haga cl...