Nanoscopy is essential to visualize single nucleoids

To visualize mitochondria and mitochondrial DNA (mtDNA), HDFa cells were incubated with antisera against double stranded (ds)DNA as well as the mitochondrial protein Mic60 and subsequently imaged with a confocal microscope. The cells show numerous dots of extranuclear DNA (magenta) exclusively located within the mitochondria (green, Fig 3.1 A). In confocal images, the mtDNA is found in clusters (Fig 3.1 B-G). STED nanoscopy of this single confocal DNA signals uncovers that these confocal signals can originate from a varying amount of single nucleoids (Fig 3.1 B’-G’). Hence, diffraction limited microscopy can only investigate nucleoid clusters containing an unknown amount of nucleoids, whereas the increased resolution of STED nanoscopy facilitates the investigation of single mitochondrial nucleoids in such clusters.

Figure 3.1 Nanoscopy is essential to visualize single nucleoids: A) HDFa cell incubated with antisera against dsDNA (magenta) and the inner mitochondrial membrane protein Mic60 (green). Mitochondrial DNA (mtDNA) appears as extranuclear DNA signal within the mitochondrial network. B-G’) Comparison between mtDNA signals recorded with conventional confocal microscopy and STED nanoscopy. Identical sections were recorded with the same excitation laser power and the same pixel size. Whereas confocal images show a single DNA signal, STED images reveal different amounts of nucleoids. Raw data with 5%

subtraction of the background. Scale bar in A: 5µm and in 1 and B-G’: 200 nm

Results

58 The visualization of cellular structures in the nanoscale by super-resolution microscopy requires dyes with specific properties, especially high brightness and contrast.

Furthermore, the labeling has to be very specific because small artefacts that are undetectable in confocal microscopy can affect the nanostructure. Moreover, visualization of nucleoids should not influence mtDNA activities as this study focusses on their regulation. Therefore, it was crucial to select a labeling method which is not affecting the replication and transcription.

3.1.1 Antibodies against DNA provide the best properties to label nucleoids

The labeling of nucleoids can be done by labeling of the nucleoid proteins or the nucleic acids. Detecting nucleoids using reporter gene fusion proteins was dismissed as the additional expression of nucleoid proteins can alter mitochondrial replication and transcription (Maniura-Weber et al., 2004, Pohjoismäki et al., 2006; Ikeda et al., 2015;

Kühl et al., 2016). For example, the mitochondrial transcription activator TFAM is a very abundant core structural protein of mitochondrial nucleoids and is a commonly used protein to label nucleoids by its fusion to reporter proteins. However, since it is strongly involved in transcription, additional expression of TFAM is sufficient to stimulate RNA synthesis (Maniura-Weber et al, 2004). Endogenous tagging to circumvent the problem of overexpression was suspended as primary cells are used in some experiments.

In this work, three different methods were tested to label the mitochondrial nucleoids (Fig. 3.2). Cells were incubated either with antibodies against dsDNA (A), antibodies against the nucleoid core protein TFAM (B) or with the DNA intercalator dye PicoGreen (C) and imaged in the confocal- (A-C) or STED-mode (D-F). All three methods give rise to bright signals with very good contrast. Furthermore, the three labeling methods can be used in STED nanoscopy to resolve single nucleoids within a cluster.

The signal of antibodies against dsDNAs reveal nucleoids with a very uniform size and shape like their appearance is described in literature when STED is applied (Fig 3.2 D).

This is in agreement with previous findings (Kukat and Wurm et al., 2011). In contrast, antisera against TFAM result in uneven labeling with inconsistent size and irregular shape (Fig 3.2). This could be a result of inhomogeneous TFAM-binding to mtDNA (Gustafson et al., 2016). Furthermore, TFAM is one of the candidates involved in the regulation of mtDNA activity as its presence primes mitochondrial transcription.

Therefore the labeling of nucleoids should be independent of TFAM to allow a future research of the analysis of nucleoid activity upon varying protein level of this mitochondrial transcription factor. Hence, TFAM is not a suitable nucleoid marker for

Results

59 this study as it does not provide a uniform labeling of nucleoids throughout all experiments.

PicoGreen requires no detection with antibodies as it is fluorescent on its own and shows an increase of fluorescence intensity upon intercalation into the DNA, resolving single nucleoids and displaying good contrast and brightness (Fig 3.2 F). However, PicoGreen has drawbacks making it unsuitable for this study. First, the stain is not specific for dsDNA, but also recognizes structured RNA. Therefore, a crucial step to provide good contrast is the complete digestion of RNA within the cell, but that eliminates the possibility to mark transcription by labeling RNA directly in following steps. Second, labeling of the DNA by PicoGreen has a rather short life time. After preparation of the sample, the dye starts to dissociate from the DNA. About 4 hours after sample preparation, single nucleoids can hardly be identified by the decreased contrast (Supplement Fig. 9.1). The irregular shape of nucleoids labeled with PicoGreen might be a result of the dissociation of the stain (Fig 3.2 F).

Figure 3.2 Different methods to visualize mtDNA: A-C) HDFa cells were incubated with antibodies against dsDNA (A), the nucleoid core protein TFAM (B) and the DNA intercalator PicoGreen. All three methods were suitable to detect mtDNA and images were recorded with conventional confocal microscopy. D-F) same sections like in A-C are recorded with STED nanoscopy using identical excitation power and pixel size. The enhanced resolution reveals that some single confocal signals originate from cluster of several mtDNA molecules. Nucleoids decorated with αdsDNA (D) show a uniform size and shape in contrast to αTFAM (E) and PicoGreen (F) stainings in which nucleoids appear in a higher variety. Scale bar: 500 nm

Results

60 Summarizing, antisera against dsDNA provide the best properties to image nucleoids in this study as they do not influence mtDNA activity at any stage and label nucleoids with high contrast. In the following, structures engaged in the process of replication and transcription respectively will be analyzed based on this visualization of single nucleoids.