Mitochondrial fission dynamics and the cytoskeleton

In recent years, research focused on mitochondrial dynamics of fission and fusion has grown exponentially. Rapid progress has been made in the elucidation of the GTPase-driven membrane dynamics itself. However, additional efforts are needed to understand the mitochondrial dynamics in the context of cellular physiology and cell death. Inevitably, this draws attention towards the cytosolic environment of the mitochondria and the cytoskeleton as its structural scaffold. Since the cytoskeleton is physically linked to mitochondria, a dynamics of its interaction with the mitochondrial membrane can be hypothesized. Elucidation of the putative interaction sites themselves requires the lateral resolution of an electron microscope. However, all preparation techniques for visualization of links with cytofilaments applied so far would be unsuitable to reconstruct the cytoskeletal/mitochondrial dynamics. Besides other technique-specific limitations, they all were founded on slow gradient-driven chemical fixation. What is needed instead is the generation of electron microscopic "snap shots" originating from the rapidly immobilized fine structure of the living state. This proposal aims at the creation of technical solutions for systematic electron microscopic studies of cytoskeletal/ mitochondrial interaction sites by introduction of rapid cell fixation techniques to this field of research. This requires the skillful combination of high-pressure-and microwave fixation with modifications of the basic techniques of freeze substitution and embedding of cell monolayers. The technical efforts will be directed towards the elucidation of a putative role of microtubules in the constriction of mitochondria during fission. For this purpose, mitochondrial fission events related to mitochondrial replication- and death will be induced in tissue culture models. By its increase in number, mitochondrial fission will become available for systematic light and electron microscopic studies. The latter will include the fine-structural characterization of the rapidly immobilized mitochondrial constriction sites, their three-dimensional reconstruction, and the localization of molecular components of the constriction apparatus by immunogold labeling. Solid structural evidence for an involvement of microtubules in mitochondrial constriction, obtained by these measures, will open a new chapter in studying mitochondrial dynamics in cell life and death. Furthermore, it will encourage efforts in studying microtubule dynamics in a new, unexpected context.

By studying the kidney epithelial cell line PtK2 as part of our project, unexpectedly, we found a novel, organelle- like membrane array of high complexity. The finding resulted from efforts introducing state-of-the-art preparation techniques for electron microscopy to our research topic "Mitochondrial fission dynamics and the cytoskeleton". Based on the conviction that our observation has relevance for a jet unknown cellular function, we decided to make this novel structure accessible for studies in life science. Supported by electron tomographic 3D-reconstruction in cooperation with Prof. Neumüller (Medical University of Vienna), we found that our observations represent nonlamellar lipid membrane arrays of well-defined size and architecture. Both their lipid membranes and their proteinaceous inner cores are organized according to principles of nanoperiodicity. Strikingly, the proteinaceous core tubules are confined by helix-like threads. Since they differ in their structure from any of the cellular membrane systems reported previously, we named our findings "tubulohelical membrane arrays" (TUHMAs) (Reipert et al., Cell Biol Int. 2009, 33:217-223). By identification of suitable marker antibodies we opened possibilities for systematic studies of TUHMAs using immunofluorescence microscopy. As a result, we found that TUHMAs represent single organelle-like entities which link to various organelles, such as the Golgi complex, the endoplasmic reticulum and annulate lamellae. These indications for exceptional high dynamics were supported by observations of preferential orientations of the membrane arrays, either perpendicularly or in parallel to the cell nucleus. Recognition of microtubules in association with TUHMAs indicated the coupling of such dynamics with alterations of the cytoskeleton. During the last period of our project work, we found a clue for the elucidation of TUHMAs beyond the initial structural description. In its center stands the possibility of a relation between TUHMAs and primary cilia within the ciliary cycle. Consequently, TUHMAs could get importance for medical research on ciliary diseases (so-called ciliopathies) and for understanding of cellular nanomachinery under consideration of supposed biophysical properties (Reipert et al., PMC Biophysics 2010, 3:13).