The video outlines a protocol for imaging and analyzing transfected HeLa cells. It details steps for fluorescence imaging, background correction, and segmentation to measure cell components. The impact of treatments on mitochondrial DNA levels and gene expression is quantified, revealing varying effects on mitochondrial DNA copy number linked to specific siRNA transfections.

quantitative image analysis to measure the mtdna and gene expression levels in the transfected hela cells

Hochdurchsatz-Quantifizierung der Synthese und Verteilung von mtDNA

For most eukaryotic cells mitochondria are essential organelles, as they play a crucial role in numerous cellular processes. First and foremost, mitochondria are the key energy suppliers of cells1. Mitochondria are also involved in regulating cellular homeostasis (for instance, intracellular redox2 and the calcium balance3), cell signaling4,5, apoptosis6, the synthesis of different biochemical compounds7,8, and the innate immune response9. Mitochondrial dysfunction is associated with various pathological states and human diseases10. 
The functioning of mitochondria depends on the genetic information located in two separate genomes: the nuclear and mitochondrial genomes. The mitochondrial genome encodes a small number of genes compared to the nuclear genome, but all the mtDNA-encoded genes are essential for human life. The mitochondrial protein machinery necessary to maintain the mtDNA is encoded by nDNA. The basic components of the mitochondrial replisome, as well as some mitochondrial biogenesis factors, have already been identified (reviewed in previous research11,12). However, mitochondrial DNA replication and maintenance mechanisms are still far from being understood. In contrast to nDNA, the mitochondrial genome exists in multiple copies, which provides an additional layer for regulating mitochondrial gene expression. Much less is currently known about the distribution and segregation of mtDNA within organelles, to what extent these processes are regulated, and if they are, which proteins are involved13. The segregation pattern is crucial when cells contain a mixed population of wild-type and mutated mtDNA. Their unequal distribution may lead to the generation of cells with a detrimental amount of mutated mtDNA.
So far, the protein factors necessary for mtDNA maintenance have been identified mainly by biochemical methods, bioinformatic analyses, or through disease-associated studies. In this work, in order to ensure a high chance of identifying factors that have previously escaped identification, a different strategy is described. The method is based on the labeling of mtDNA during replication or repair with 5-bromo-2'-deoxyuridine (BrdU), a nucleoside analog of thymidine. BrdU is readily incorporated into nascent DNA strands during DNA synthesis and, in general, is used for monitoring the replication of nuclear DNA14. However, the procedure developed here has been optimized for detecting BrdU incorporated into mtDNA using the immunofluorescence of anti-BrdU antibodies.
The approach allows for the high-throughput quantification of mtDNA synthesis and distribution in human cells cultured in vitro. A high-throughput strategy is necessary to conduct tests under different experimental conditions in a relatively short time; therefore, it is proposed in the protocol to utilize a multi-well format for cell culturing and automated fluorescence microscopy for imaging. The protocol includes the transfection of human HeLa cells with an siRNA library and the subsequent monitoring of mtDNA replication or repair using the metabolic labeling of newly synthesized DNA with BrdU. This approach is combined with immunostaining of the DNA with the help of anti-DNA antibodies. Both parameters are analyzed using quantitative fluorescence microscopy. Additionally, mitochondria are visualized with a specific dye. To demonstrate the specificity of the protocol, BrdU staining was tested on cells devoid of mtDNA (rho0 cells), on HeLa cells upon the silencing of well-known mtDNA maintenance factors, and on HeLa cells after treatment with an mtDNA replication inhibitor. The mtDNA levels were also measured by an independent method, namely qPCR.

Autor im Rampenlicht: Bildbasierte Hochdurchsatz-Quantifizierung der mitochondrialen DNA-Synthese und -Verteilung  

Bildbasierte Hochdurchsatz-Quantifizierung der mitochondrialen DNA-Synthese und -Verteilung

The protocol aims to identify proteins with dysfunction leads to changes in the number of mitochondrial DNA molecules. This research may also reveal the factors involving the distribution of mitochondrial DNA within the mitochondria. Mitochondrial replication and maintenance mechanisms are still not fully understood.Much less is known about regulating the distribution of mitochondrial genomes within organoids and the proteins involved in it. As a result, identifying and characterizing some new players will be of great importance. The main advantage of the technique is its high-throughput and high content protocol.We can simultaneously test many experimental conditions and measure various parameters during a single experiment. In genome-wide studies, the proposed protocol provides an opportunity to outline a global picture of how nuclear encoded genetic information regulates its mitochondrial counterpart. Additionally, it has the potential to identify proteins whose dysfunction causes mitochondrial DNA stress and activates the interferon response pathway.

Watch this Scientific Journal Video about Quantitative Image Analysis to Measure the mtDNA and Gene Expression Levels in the Transfected HeLa Cells at JoVE.com

Quantitative Image Analysis to Measure the mtDNA and Gene Expression Levels in the Transfected HeLa Cells  

Quantitative Bildanalyse zur Messung der mtDNA- und Genexpressionsniveaus in den transfizierten HeLa-Zellen

Um mit der Bildgebung transfizierter HeLa-Zellen zu beginnen, wählen Sie eine ausreichend lange Belichtungszeit für die einzelnen Fluoreszenzkanäle im Live-View-Modus basierend auf den von der Bildgebungssoftware erzeugten Intensitätshistogrammen. Legen Sie die entsprechende Anzahl von Ebenen für die Bildgebung auf der Z-Achse fest. Wählen Sie die entsprechende Anzahl von Sichtfeldern aus, die pro Vertiefung angezeigt werden sollen.Um mit der quantitativen Analyse der aufgenommenen Bilder zu beginnen, führen Sie eine Hintergrundkorrektur für alle Bilder aus allen Fluoreszenzkanälen durch. Wählen Sie je nach Größe der analysierten Objekte die entsprechende Filtergröße aus. Beginnen Sie als Nächstes mit der Bildsegmentierung, indem Sie eine Maske des Hauptobjekts erstellen, die auf dem Verhältnis der Intensität des Fluoreszenzsignals zum Hintergrund für den Kanal basiert, der den Zellkernen entspricht.Erstellen Sie Masken für die Unterobjekte, die BrdU und mitochondriale DNA-Spots darstellen, indem Sie die Fluoreszenzkanäle verwenden, die für die gegebenen Strukturen geeignet sind. Stellen Sie die Parameter ein und messen Sie die Intensität der einzelnen Pixel innerhalb jeder Maske für alle Fluoreszenzkanäle. Um die Gesamtfluoreszenzintensitäten für den BrdU oder den mitochondrialen DNA-Kanal zu erhalten, summieren Sie die Intensitäten aller Unterobjekte, die einem bestimmten Zellkern zugeordnet sind.Um die mittlere Fluoreszenzintensität zu erhalten, dividieren Sie die gesamte Fluoreszenzintensität durch die Summe der Fläche der Unterobjekte, die einem bestimmten Zellkern zugeordnet sind. Die Bildgebungsergebnisse wurden durch die Messung der mitochondrialen DNA und der Genexpression mittels quantitativer real-time PCR verifiziert. Die Behandlung mit Didesoxycytidin oder DDC führte zu einer sehr starken Abnahme der mitochondrialen DNA-Spiegel.Ein schwächerer Effekt wurde beim Stummschalten von TFAM- und TWINKLE-Helikasen beobachtet, während die Transfektion von Zellen mit mitochondrialer DNA-Polymerase gamma oder POLG-siRNA einen bescheidenen Effekt auf die mitochondriale DNA-Kopienzahl hatte. Die Quantifizierung der Helikase-Expression von TFAM und TWINKLE bestätigte eine effiziente Reduktion der mRNA-Expression auf etwa 15 bis 20 % der Kontrollwerte. In POLG war die mRNA-Expression nur auf 30% reduziert, was den unerwartet bescheidenen Effekt auf die mitochondriale DNA-Kopienzahl erklärt.

quantitative-image-analysis-to-measure-mtdna-gene-expression-levels

Research

JoVE Journal

Biochemistry

Quantitative Image Analysis to Measure the mtDNA and Gene Expression Levels in the Transfected HeLa Cells

105 Views.  University of Warsaw. To begin the imaging of transfected HeLa cells select a sufficiently long exposure time for the individual fluorescence channels in live view mode based on the intensity histograms generated by the imaging software. Set the appropriate number of planes for imaging on the Z axis. Select the appropriate number of fields of view to display per well.To start the quantitative analysis of acquired images, perform background correction for all the images from all the fluorescence channels. De...