4. Discussion
4.2 Functional heterogeneity of nucleoids
Mitochondrial heterogeneity is striking not only in different tissues, but also in different regions of a single cell mitochondria can reveal different (sub-)populations (Kuznetsov et al., 2009). So, mitochondria within single cells can show different functional and structural properties. This functional heterogeneity of mitochondria is not only apparent for different mitochondria in different parts of a cell, but even different subdomains of a single mitochondrion can reveal different functions (Kuznetsov et al., 2004; Kuznetsov et al., 2006). The organelles can vary for instance in their redox state and in their different level of ROS (Romashko et al., 1998; Collins et al., 2002; Bruce et al., 2004;
Kuznetsov et al., 2004). Especially different membrane potentials within mitochondria
Discussion
103 in a single cell and within subdomains of single mitochondria are well documented (Bernard et al., 2008). Numerous studies suggest that the functional heterogeneity of mitochondria is connected to the energy demand in different regions of the cell (reviewed: Kuznetsov et al., 2009). Although many reports discuss the functional heterogeneity of mitochondria, no study existed which show evidence of different functional subpopulations of nucleoids.
In the present study, mitochondrial transcription and replication were visualized simultaneously. It could be demonstrated that single nucleoids can be engaged in transcription and replication at the same time (Fig 3.9). Interestingly, an analysis of over 18,000 nucleoids revealed that the majority of nucleoids within a cell are inactive (Fig.
3.10). Quantification of the data showed that nucleoids are organized in at least two functional subpopulations within cells. Nucleoids can appear active with the tendency to be involved in transcription and replication as well or remain inactive. Furthermore, an analysis of nucleoid replication over a longer time period revealed that not all nucleoids are equally engaged in replication. This further supports the existence of at least two functionally different subpopulation of nucleoids.
Nucleoid gene expression is necessary for the activity of OXPHOS (Larsson et al., 1998, Gustafsson et al., 2016). Different activity level of mtDNA would as a result cause a different functionality of the respiratory chain which would result in a different membrane potential, a different production of ROS and a different redox state of the mitochondria (Appleby et al., 1999; Gustafsson et al., 2016)
4.2.1 Active nucleoids are evenly distributed within the cell
Some reports indicate that the previously mentioned functional heterogeneity of mitochondria is linked to their distance to the nucleus (Park et al 2001; Bruce et al., 2004; Kuznetsov et al., 2009). Also it was suggested that perinuclear mitochondria are triggered towards ATP generation (Dzeja et al., 2002). This could play an important role in the mechanisms that drive nuclear import as well as further functions of the nucleus that need ATP (Dzeja et al., 2002).
In contrast, in the present study active and inactive nucleoids are evenly distributed within the cell if the distance to the nucleus is taken as a reference (Fig 3.13). Neither single nucleoids that are only engaged in transcription, nor nucleoids involved in replication, nor nucleoids engaged in both processes simultaneously show an enrichment in perinuclear regions (Fig 3.13). Hence, the findings of the current study do not explain the increased ATP production of perinuclear mitochondria. This effect could
Discussion
104 be a result of a general increased density of nucleoids in perinuclear areas (Kukat and Wurm et al., 2011)
4.2.2 Activity and inactivity of nucleoids
So far, only a few reports show hints for inactivity of nucleoids in cells (Davis and Clayton, 1996; Piko and Taylor, 1987). Previous studies that used the nucleoside analogue BrdU to label mitochondrial replication achieved contradictory results. On the one hand it was published that nucleoids are equally engaged in replication (Iborra et al., 2004), on the other hand it was reported that a subset of nucleoids within cells show an increased replication rate compared to the remaining nucleoids (Davis and Clayton, 1996).
Nevertheless, in both studies, diffraction limited microscopy was used, thereby lacking the resolution to observe single nucleoids. Furthermore, studies performed in early mouse embryos suggest that a large population of the mtDNA molecules remains inactive (Piko and Taylor, 1987). However, until now, there has been no reliable evidence that nucleoids reveal different activity levels.
The analysis of mitochondrial transcription and replication in this study finally shows that not all nucleoids are active within a cell at a given time point. That gives rise to the question why mitochondria contain such a high amount of nucleoids while not all of them are active. An explanation could be that the high copy number works as a pool to react to environmental stress. In situations with low glucose, respiration becomes more important since the energy need cannot be fulfilled with glycolysis (Auger et al., 2011).
In the future, it has to be tested if this fraction of inactive nucleoids can be stimulated by a reduction of glucose and thereby, the triggering of OXPHOS activity. Experiments with cells growing in media with high glucose concentration or with low glucose but high galactose concentration could reveal if inactive nucleoids work as a pool for potential OXPHOS activity when needed. That could show if cells can regulate the activity of respiration by activation or inactivation of mtDNAs.
4.2.3 Outlook: TFAM as a regulator for nucleoid activity?
A question that remains unanswered is the mechanism that controls the activity and inactivity of nucleoids. This is an important issue for further research.
A molecular switch that triggers nucleoids towards activity or inactivity could be the packaging level of mtDNA by TFAM (Farge et al., 2014; Gustafsson et al. 2016). TFAM is not only essential to initiate mitochondrial transcription but also represents the most abundant nucleoid protein important for the nucleoid maintenance (Bogenhagen,
Discussion
105 2012). At the physiological level of TFAM, an inhomogeneous compaction level was observed in vitro (Farge et al., 2014). It has been proposed in previous reports that the compaction level of mtDNA with TFAM could regulate if a nucleoid is engaged in replication, transcription or both processes together (Gustafsson et al., 2016). A lower compaction level could open the regulatory non coding region (NCR) of the mtDNA and by this grant access to both promotors for the mitochondrial RNA polymerase POLRMT and the second mitochondrial transcription factor TFB2M (Gustafsson et al., 2016). An analysis of nucleoids’ activity upon varying TFAM level should enhance the understanding of TFAM’s influence on nucleoids’ activity.