Organisation and dynamics of mitochondria and their RNA

The organisation of molecules into dynamic cells, and collaboration of many of those cells over a billion years led to the evolution of human life. During the last century, biologists then began to unravel the marvels of cellular organisation with ever increasing detail. We now know, single cells not only comprise a defining DNA, but also a multitude of organelles. Mitochondria are such organelles, and produce cellular energy, at the heart of cellular metabolism. For this, they transcribe their own genome into mtRNA, and assemble respiratory chains. In recent decades, novel approaches, and technology enabled to shed new light on the spatial organisation of sub-cellular and mitochondrial processes. Thereby mtRNA was discovered to accumulate into small foci inside the mitochondrial matrix by fluorescence microscopy. Certain mitochondrial proteins also have also been found to conglomerate in these mitochondrial RNA granules (MRGs), but merely nothing is known about the structural, dynamic, and biophysical properties of MRGs, and their functional importance remains elusive. The development of superresolution microscopy has led to a revolution and revival of imaging methods for cell biology. This technology now enables investigation of sub-cellular biology at the nanometre scale, and to illuminate MRGs inside mitochondria. In this thesis I investigate the organisational principles governing single cell and mitochondrial biology. First, I explore the application of superresolution microscopy to study the spatial organisation of nuclear DNA domains (TADs). I find high throughput STORM microscopy (htSTORM) allows to monitor effects of drug treatment on spatial rearrangement of a cancer associated TAD, together with established bulk-sample analysis. Next, I study the organisation of mitochondrial dynamics, by live-cell structured illumination microscopy (SIM). How different molecular pathways associated with mitochondrial proliferation or degradation are organised has long been a matter of debate. We discover a pattern for spatial organisation of mitochondrial fission, which distribute along the mitochondrial network in a bimodal manner. With this framework we distinguish different types of fissions, and show that distinct molecular features are associated with mitochondrial proliferation, or degradation through mitophagy. I find actin to be involved in proliferative midzone fission, whereas peripheral fission is associated with elevated reactive oxygen levels, and precedes mitophagy. By htSTORM, SIM and additional microscopy methods I then elucidate the biophysical organisation of intra-mitochondrial processes and MRGs. I show that the MRG ultrastructure consists of compacted RNA embedded within a dynamic protein cloud. Furthermore, MRGs associate with the inner mitochondrial membranes, and their distribution is governed by mitochondrial dynamics. MRGs can now be understood as nanoscopic, robust and fluid compartments, likely to influence intra-mitochon