The project will be focused on investigation of the role of mitochondrial nucleoids and their redistribution within the mitochondrial network in relation to mtDNA genetics and pathologies.
- Imaging of mt-nucleoids in dormant, transcriptional, and replication form using 4Pi and STED microscopy and development of in vivo mtDNA hybridization.
- Development of mitochondrial DNA silencing methods and studies of mutated/wild type mtDNA distribution during cell division, disintegration of the mitochondrial network in dependence on stress stimulus and under pathological conditions.
Content of the research
Our department deals with mitochondrial DNA biology. The main objective is to elucidate the structure and physiology of mtDNA nucleoids using 3D super-resolution microscopy. The major methodology used is 3D immunocytochemistry by the direct stochastic optical reconstruction microscopy (dSTORM) or the expression of nucleoid protein conjugates with photoconvertible fluorescent proteins. Moreover, dSTORM probes are developed against specific mtDNA sequences that will be visualized. For example, visualization of D-loop sequences will count the number of mtDNA molecules per nucleoid. Since mtDNA is reduced in numerous pathologies, studies of e. g. diabetic pandreatic beta cells with reduced mtDNA down to 25% will help to elucidate type-2 diabetes development. We are trying to develop the mitochondrial mitochondrial import of exogenous RNA for sequence-specific silencing of mitochondrially encoded genes. Alternatively, we want to employ the mitochnodrial RNA import of wild-typed RNA for substitution of mutated mtDNA in cybrid cell lines derived from patient cells with mtDNA mutations. The above mentioned goal of our department is in order to substitute mtDNA loss in pancreatic beta cells during the development of diabetes. We are using super-resolution microscopy for calculating mitochondrial nucleoid volume calculations together with RT-PCR for mtDNA copy number quantification. The last project adressed in our department is focused on mtDNA replication. We are investigating several nucleoid proteins and their impact on repication.
We are working with prof. MD František Saudek DrSc, from IKEM Diabetes Centre on the diabetes project and prof. Daniel Bogenhagen MD, from Stony Brook University NY USA, on the mitochondrial DNA replication project and wih Dr. Joerg Bewersdorf, from the Department of Cell Biology, Yale University, USA, on super-resolution microscopy development.
The department possesses a Bi-PLANE FPALM super-resolution microscope at the FGU ASCR.
Potential for Cooperation
We would like to establish external collaboration within the microscopic field. We have mastered PALM-dSTORM microscopy and we want to continue working on STEO microscopy.