Activity of nucleoids does not depend on their distance to the nucleus . 83

The previous experiments revealed the existence of at least two subpopulations of nucleoids: active and inactive ones. It was tested if nucleoids activity depends on their distance to the nucleus as some properties of mitochondria are correlated with distance to the nucleus. One example for that dependence is the abundance of MICOS proteins of the inner mitochondrial membrane (Jans and Wurm et al., 2013). Furthermore, less nucleoids can be identified in the periphery of the cell (Kukat and Wurm et al., 2011).

To analyze the distribution of the engagement of nucleoids into transcription and replication the same dataset like in 3.4.1 was used. HDFa cells were incubated with BrU and EdU for only a short period to label ongoing activity

Fig 3.13 shows how current transcription and replication are distributed within the cell.

Fig 3.13 A displays an HDFa cell after short incubation times with BrU and EdU. The shortest distance of each nucleoids center to the outer rim of the nucleus was estimated. Fig. 3.13 B displays the normalized amount of nucleoids with increasing distance to the nucleus. As mentioned above, it becomes apparent that the amount of nucleoids decreases in the periphery. For Fig 3.1 C-E the amount of nucleoids for each distance to the nucleus was always determined in relation to the number of total nucleoids. Fig. 3.13 C reveals that the fraction of active and inactive nucleoids remains constantly at about 20% from perinuclear areas into the periphery. Fig 3.13 D shows the distribution of replication and transcription within single cells. Both processes appear to be uninfluenced by the distance of the nucleoid to the nucleus. Fluctuations of the values at greater distances from the nucleus are the result of a smaller sample size as less nucleoids can be identified in the periphery of cells. Fig 3.13 E displays the distribution of nucleoids only engaged in transcription or replication, or involved in both processes simultaneously. Again, the position of the nucleoid within the cell shows no effect on nucleoids current activity.

The presented data in Fig 3.11 and 3.12 present evidence that nucleoids within a single cell form two different subpopulations, namely an active and an inactive population.

Whether a nucleoid belongs to the active or inactive subpopulation does not depend on its distance to the nucleus (Fig 3.13). Parameters which are involved in the activation of nucleoids have to be identified in the future.

Results

84

Figure 3.13 Intracellular distribution of mitochondrial transcription and replication: HDFa cells were incubated with 20 µM EdU for 70 min and 20 µM BrU for 25 min. Cells were afterwards processed to label the DNA, incorporated BrU and EdU. More than 50 cells with a total amount of over 18,000 nucleoids were measured with STED nanoscopy. A) Exemplary image to demonstrate the measurement of the distance between nucleus and nucleoid. The shortest distance between the center of each nucleoid signal and the outer rim of the nucleus was quantified. B) Within the cell, more nucleoids are located perinuclear than peripheral. When single cells are analyzed, the amount of nucleoids continuously decreases with an increasing distance to the nucleus. C) The distribution of active nucleoids engaged in replication and/or transcription was analyzed. The fractions of active and inactive nucleoids remains constant over the increasing distance to the nucleus. D) Distribution of nucleoids engaged in transcription and nucleoids engaged in replication was analyzed. No relationship between the distance of a nucleoid to the nucleus and an involvement in one of these processes could be observed. E) It was separated between nucleoids

Results

85

engaged only in transcription or only in replication and nucleoids engaged in both processes simultaneously. Again, the distance to the nucleus does not affect the behavior of nucleoids. Scale bar:

5 µm

Whereas in this section, it was focused on the general activity of the general activity of nucleoids, the next section addresses the switch between transcription and replication of single nucleoids.

3.5 Analysis of single nucleoids in knockdowns of POLRMT and TEFM

Only a few aspects of the regulation of mitochondrial replication and transcription have been identified so far. As both of these processes are coupled in mitochondria, especially the factors that trigger nucleoids towards replication or transcription represent a field of intense research. Recently, the transcription elongation factor TEFM was identified as the molecular switch between transcription and replication of nucleoids (Minczuk et al., 2011; Agaronyan et al., 2015; Posse et al., 2015). Transcription of the light strand can be terminated just 100 bp after the promotor at a sequence termed CSBII. This termination results in the formation of a primer for mitochondrial replication instead of functional transcription. Processivity of mitochondrial transcription throughout CSBII or termination depends on the presence of TEFM (Agaronyan et al., 2015). During transcription the nascent RNA forms a G-quadruplex structure at CSBII (Hillen et al., 2017(2)). In the absence of TEFM, the G-quadruplex structure clashes with the mitochondrial RNA polymerase POLRMT leading to the dissociation of the transcription machinery and a primer that works as a substrate for the mitochondrial DNA polymerase POLγ. If TEFM is present, it forms a RNA exit channel leading to a suppression of an early formation of a G-quadruplex structure of the nascent RNA (Hillen et al., 2017(2)). Hence, the presence of TEFM promotes complete transcription.

Studies of a heterozygous knock out of POLRMT in mice revealed that POLRMT level influence the level of TEFM (Kühl et al., 2015). Reduced POLRMT level has no influence on the level of total transcripts in mice although transcripts from LSP occur with a higher frequency than transcripts from HSP. As LSP-transcription is important for the formation of a replication primer, it was proposed that replication is favored at low POLRMT level (Kühl et al., 2016). The influence of TEFM and POLRMT on nucleoid behavior and activity were analyzed with different biochemical methods like Western and Northern Blots so far (Kühl et al., 2015, Agaronyan et al., 2015; Posse et al., 2015).

In the present study an approach to visualize the activity of single nucleoids with STED nanoscopy was developed. Hence, it can be determined whether single nucleoids are

Results

86 engaged in replication or transcription. To extend the understanding of how POLRMT and TEFM influence the switch between mitochondrial replication and transcription, single nucleoids upon reduction of these factors were analyzed. To ensure a controlled decrease of POLRMT and TEFM level within the cell, gene expression was reduced by RNA interference.