Characterizing mitochondrial function and structure – Mitochondrial regulation of apoptosis and visualization of mitochondria - endoplasmic reticulum interactions

Abstract

Mitochondria are the powerhouse of the cell generating most of the energy for cellular mechanisms in form of ATP (adenosine triphosphate). Another important role of mitochondria is the regulation of programmed cell death, apoptosis. Apoptosis, in turn, is regulated by a complex protein network, the Bcl-2 protein family. There is a fine balance within the about 25 Bcl-2 proteins between pro- and antiapoptotic proteins. If control gets lost, cells may gain resistance to apoptosis, they can divide in an uncontrolled manner resulting in tumors and diseases like cancer may develop. The second leading cause of cancer deaths in women is cervical cancer. For proving that human papillomaviruses (HPV) are the causative agents of cervical cancer, Harald zur Hausen, a scientist of the German Cancer Research Center, Heidelberg, was awarded the Nobel Prize in 2008. In this study, one aim is to find out which antiapoptotic proteins are responsible for cervical cancer cells to become resistant to apoptosis. Using a specific method called BH3 profiling, Bcl-xL upregulation in cervical cancer cells is suggested to be responsible for apoptosis resistance. This data is supported by knock-down of Bcl-xL in cervical cancer cells finding them to regain apoptosis sensitivity. Furthermore, Bcl-2 proteins have been shown to control Ca2+ influx from ER (endoplasmic reticulum) into mitochondria and thus regulate apoptosis since high mitochondrial Ca2+ levels lead to the induction of apoptosis. Ca2+ transfer takes place at very tight interaction sites between mitochondria and ER called ER-mitochondria contact sites or mitochondria associated membranes (MAMs). More and more proteins are becoming evident to play a role in keeping those two organelles in close contact. However, the structural organization of those proteins is unknown. Since the distance between mitochondria and ER at their contact sites is only between 10 and 25 nm, it is impossible to resolve those structures with conventional confocal microscopy. This is why in this study the high resolution microscopy technique STED (stimulated emission depletion) is used to visualize MAMs. Thus, the second aim of this thesis is to image ER-mitochondria contact sites via STED microscopy to gain a deeper structural insight of these sites. A labeling strategy was established for the used STED microscopy setup to image ER, mitochondria and, as an example for a MAM protein of interest, Grp78. High resolution images could be obtained for mitochondria and Grp78 and the distribution of Grp78 with respect to the ER could be visualized. Using the here established MAM labeling strategy for STED microscopy and studying further MAM proteins in the future will enable the structural analysis of ER-mitochondria contact sites in more detail. In summary, this study provides insights into mitochondrial function and structure. 1) apoptosis regulation in cancer cells via the Bcl-2 protein network is studied, and 2) the interaction of mitochondria and the ER is visualized via high resolution STED microscopy to gain a deeper insight into the structural organization of the MAMs.