Supplement figure 9.1: PicoGreen dissociates from mtDNA
Figure 9.1 PicoGreen dissociates from the mtDNA: U-2 OS cells were incubated with PicoGreen according to the protocol shown in the material and method section. A) A confocal record directly after the sample preparation. Single nucleoid signals can be identified and the signal reveals a good contrast. B) 3 h after the sample preparation single nucleoid signals can still be identified. However, the background signal is increased, especially in the mitochondrial network as tubular structures can be identified. C) 8 h after the sample preparation single nucleoid signals are very dim. The sample reveals a high level of background and a poor contrast. Scale bar: 3 µm
Supplementary information
142 Supplement figure 9.2: Antibodies against proteins involved in mitochondrial
replication do not label active nucleoids
Figure 9.2 Antibodies against proteins involved in mitochondrial replication do not label active nucleoids: U-2 OS cells and HDFa cells were incubated with antisera against dsDNA to label nucleoids and different proteins involved in mitochondrial transcription. A-B’’) Un U-2 OS cells TWINKLE signal is located in the cytoplasm and the mitochondria (A-A’). The signal reveals an enrichment in dots that partially colocalize with nucleoids (green circles). Upon EtBr treatment (B-B’), this colocalization can still be observed (green circles). C-D’’) The used antibodies against POLγ showed a weak enrichment in some dots in U-2 OS and HDFa cells (blue circles) that do not colocalize with nucleoids. E-F’) In HDFa cells antibodies against mtSSBP show a signal that is enriched at nucleoids (E-E’; green circles). This colocalization of mtSSBP and nucleoids is not impaired by EtBr treatment (F-F’). G-G’) In HDFa cells, TWINKLE antibodies show no signal. Scale bar: 1 µm
Supplementary information
143 Supplement figure 9.3: Antibodies against proteins involved in mitochondrial
transcription do not label active nucleoids
Figure 9.3 Antibodies against proteins involved in mitochondrial transcription do not label active nucleoids: U-2 OS cells and HDFa cells were incubated with antisera against dsDNA to label nucleoids and different proteins involved in mitochondrial transcription. A-B’) In U-2 OS cells, TEFM signal is enriched in defined dots within mitochondria that partially colocalize with nucleoids (A-A’; green circles). This colocalization is not disturbed by EtBr treatment (B-B’). C-D’) Neither in U-2 OS cells nor in HDFa cells, the used antibodies against TFB2M revealed a specific signal. E-F’) In HDFa cells, the used antibodies against POLRMT and TEFM show no specific signal. Scale bar 1 µm
Supplementary information
144 Supplement figure 9.4: Optimized concentration and incubation time for EdU and BrU
Figure 9.4 Optimized concentration and incubation time for EdU and BrU: HDFa cells were incubated with EdU and BrU for different incubation periods and with different concentration. EdU und BrU (magenta) were labeled afterwards. Furthermore antibodies against the β-subunit of the ATP synthase (green) were used to label the mitochondria. A-E) EdU staining: The best results could be obtained with an incubation time of 70 min and a concentration of 20 µM (C). EdU signal is located within the mitochondria. If a lower concentration is used (A) the specific EdU signal disappears and only bright unspecific spots in the cytoplasm are detected (arrowheads). If the concentration of EdU it too high (C), specific signal within the mitochondria is detectable, but additionally bright signal outside the mitochondria appear (arrowheads). When lower incubation times than 70 minutes and a concentration of 20 µM were used (D), no signal was detected. If incubation times were above 70 minutes (E) the signal within the mitochondria appears brighter and more dots were detected. F-J) BrU staining: The best were achieved with an incubation time of 25 min and a concentration of 20 µM (G). Upon a reduced concentration of BrU (F) or a lower incubation time (I) no BrU signal was detected. When either the concentration of BrU (H) or the incubation time (J) were increased, dots within the mitochondria were detectable but an increased labeling of the complete mitochondrial network could be observed. Scale bar:
3 µm
Supplementary information
145 Supplement figure 9.5: STED nanoscopy of BrdU labeled nucleoids
Figure 9.5 STED nanoscopy of BrdU labeled nucleoids: HDFa cells were incubated with 20 µM BrdU for 2 h. Sample was incubated with HCl and nucleases to enable a BrdU staining. Afterwards, BrdU (magenta) and dsDNA (green) were labeled with antisera. A-A’’) A single confocal nucleoid signal that is positive for BrdU. B-B’’) Record of the same section like in A-A’’ applying STED nanoscopy. Two single nucleoids are apparent and both of them are positive for BrdU. The nucleoids in the DNA cannel reveal an irregular shape. C-C’’) multiple confocal nucleoid signals. Only some of the nucleoids are labeled with BrdU. D-D’’) The section shown in C-C’’ was recorded with the STED mode. The nucleoids appear fragmented and single structures can barely be identified. The BrdU channel could be recorded with a good contrast. Scale bar:
200 nm.
Supplementary information
146 Supplement figure 9.6: Models for the fitting of the data of the increasing EdU
incubation times:
Figure 9.6 Models for the fitting of the data of the increasing EdU incubation times: In Fig 3.12 HDFa cells were treated with EdU for different periods of incubation. Both presented models were fitted to the data points. A) The “one population” model: In this model it is assumed that all nucleoids are equally involved in mitochondrial replication and reveal an equal replication rate. When an EdU negative nucleoid finished replication, two EdU positive nucleoids appear. If a nucleoid is already EdU positive it can still be involved in replication. It is furthermore assumed that all nucleoids reveals the same chance to be degraded. B) The “two subpopulations” model: In this model it is assumed that nucleoids reveal two different subpopulations with two different replication rates termed “active / fast” and “inactive / slow”
nucleoids. Previous incorporation of EdU has no influence on nucleoid replication. It is assumed that the fraction of “active / fast” and “inactive / slow” nucleoids is constant during the experiments. Therefore it was assumed that a transfer from “active / fast” to “inactive / slow” nucleoids occur. In the mitochondria this transfer could be realized by a limitation of the involved factors. Limitation of the responsible factors could ensure that only a constant subpopulation is “active / fast”. It is furthermore assumed that all nucleoids reveals the same chance to be degraded.
Supplementary information
147 Supplement Table 9.1: Single measures of the toxicity tests of EdU, BrU and BrU (see Tab 3.1)
Table 9.1 Single measures of the toxicity tests of EdU, BrU and BrU (see Tab 3.1)
untreated Aphidicholin EdU BrdU BrU
1st Round
Cells in G1-Phase (2 h) 86.2% 86.8% 84.5% 85.7% 86%
Cells in S-Phase (2 h) 5.1% 5.1% 5.9% 5.3% 5.2%
Cells in G2-Phase (2 h) 7.3% 6.1% 7.4% 7.4% 7.3%
Viability (2 h) 95.4 87.5% 89.6% 96.1% 91%
Cells in G1-Phase (48 h) 91.3% 82.9% 83.6% 89% 87.6%
Cells in S-Phase (48 h) 2.1% 7.4% 2.9% 2.3% 3%
Cells in G2-Phase (48 h) 5.7% 8.7% 12.7% 7.4% 8.5%
Viability (48 h) 91.7% 69.2% 88.4% 82.3% 86.8%
2nd Round
Cells in G1-Phase (2 h) 88.2% 91% 91.5% 90.5% 90.6%
Cells in S-Phase (2 h) 3.4% 3.2% 2.5% 3.3% 2.6%
Cells in G2-Phase (2 h) 6.9% 6.3% 6.5% 6.4% 6.1%
Viability (2 h) 76% 81.1% 82.3% 82.2% 79.2%
Cells in G1-Phase (48 h) 90.6% 87.9% 86.4% 93.2% 90%
Cells in S-Phase (48 h) 2.5% 4.9% 2.1% 1.5% 2.4%
Cells in G2-Phase (48 h) 6.6% 6.6% 10.7% 4.6% 6.8%
Viability (48 h) 94.9% 90% 94.2% 97% 94.1%
3rd Round
Cells in G1-Phase (2 h) 84.5% 86.4% 81% 81.5% 84.6%
Cells in S-Phase (2 h) 4.6% 4.6% 6.9% 6% 5.2%
Cells in G2-Phase (2 h) 10% 8.4% 11.6% 11.5% 9.7%
Viability (2 h) 95.3% 95.7% 96.5% 95.5% 95.3%
Cells in G1-Phase (48 h) 89.9% 79.2% 77.4% 88.1% 90.9%
Cells in S-Phase (48 h) 3% 10.2% 3.3% 2.2% 3.3%
Cells in G2-Phase (48 h) 5.8% 9% 17.6% 8.1% 4.5%
Viability (48 h) 96.2% 84.1% 91.2% 82.5% 94.9%
Affidavit
148
Affidavit
I declare, that I prepared the PhD thesis entitled
“Analysis of mitochondrial transcription and replication on the single nucleoid level”
submitted on 30th March, 2018 is the result of my own work and prepared with no other sources than quoted.
Göttingen, 30th March, 2018
Christian Brüser
Acknowledgements
149
Acknowledgements
Es ist vollbracht! Alle Experimente sind abgeschlossen, alle Proben gemessen, jede Erkenntnis niedergeschrieben. Nun fehlt es den Leuten zu danken die mir direkt oder indirekt dabei halfen.
Ich danke Herrn Prof. Dr. Stefan Jakobs für die Möglichkeit an diesem interessanten Thema zu arbeiten und für seine fortwährende Unterstützung die einen wichtigen Beitrag zum Gelingen dieser Arbeit darstellte.
Darüber hinaus möchte ich Herrn Prof. Dr. Peter Rehling für die Übernahme des Koreferats danken. Besonders für die konstruktiven Diskussionen und das Interesse an dieser Arbeit möchte ich mich bedanken.
Zudem bedanke ich mich bei Herrn Prof. Dr. Ahmed Mansouri für die hilfreichen Ratschläge, Besprechungen und die freundliche Begleitung dieser Arbeit.
Herrn Prof. Dr. Patrick Cramer, Herrn PD Dr. Wilfried Kramer und Herrn Prof. Dr. Thomas Teichmann danke ich für ihre Teilnahme an meiner Disputation.
Ein großer Dank gebührt Tanja Gilat, Sylvia Löbermann, Rita Schmitz-Salue und Ellen Rothermel für alle Hilfestellungen und die perfekte Organisation des Labors. Auch möchte ich bei Jan Keller-Findeisen bedanken, da er mir eine enorme Hilfe bei der Auswertung der Daten war. Zudem möchte ich mich bei Jaydev Jethwa, Stefan Stoldt, und Nickels Jensen für die Korrektur meiner Arbeit bedanken.
Ein besonderer Dank geht an alle Mitglieder der Abteilung für NanoBiophotonics. Viele Aufgaben lösen sich, wenn man den Kopf frei bekommt und man die Probleme für einen Moment vergisst. Deshalb: Danke für das Film-gucken, das Pokern, das Trinken, das Essen, das Laufen, das Schwimmen, das Ausflüge machen, das Feiern, das Pumpen, das Kaffeepause machen, das Reden, das Lachen, das Zug fahren, das Spielen, das Rätseln und den Fußball. Danke für alle Hilfestellungen, jeden guten Rat und jedes nette Wort.
Danke dafür, dass es für jede Frage eine Antwort gab.
Der folgende Satz steht so bereits in meiner vorigen Arbeit: Die letzten Worte gehören Isabelle, meiner Mutter und meinem Vater. Danke für alles.
Manches ändert sich nie.