2.2. 2023, Mitochondriální fyziologie

Laboratory name: Laboratory of Mitochondrial Physiology

Laboratory website

PhD project: 3D superresolution microscopy for accessing mitochondrial ultramorphology

3D nanoscopy has not yet assessed mitochondrial cristae morphology, nor the internal structure of mitochondrial DNA (mtDNA) & protein complexes, termed nucleoids. Hence, we’ll survey 3D-redistribution of cristae and their shaping proteins or nucleois employing our prototype Vutara 3D superresolution microscope for stochastic techniques such a PALM and dSTORM. Currently, such an apparatus is offered by Bruker as the commercial microcope with the highest resolution available on the market. We will conduct studies under physiological situations (insulin secretion, hypoxic adaptation, starvation, metabolic-switches) vs. pathology (type-2 diabetes, cancer) using dSTORM with nanobodies or FRET excited PALM/dSTORM. Thus nm changes will be reflected by novel 3D nanoscopy methods. Also mtDNA nucleoids will be studied at increased (physiological) and diminished (pathological) mitochondrial biogenesis, while applying own mitoFISH nanoscopy for D-loop counting. Artificial manipulations of nucleoid size and mtDNA content will be studied as well as nucleoid division. STED microscopy will confirm the expected word priority results. Results will be translated into specific protocols for 3D nanoscopy, specifically developing novel relevant 3D image analyses based upon the Ripley’s K-function and Delaunay algorithm. Molecular cell biology will thus be combined with up-to-date 3D nanoscopy. Note, the molecular biology techniques will be conducted and be ready for the applicants by the coworkers of the Department No.75.

References:

[1] S.J. Sahl, S.W. Hell, S. Jakobs, Fluorescence nanoscopy in cell biology, Nat. Rev. Mol. Cell Biol. 2018, 18 (11), 685–701.

[2] Dlasková A, Engstová H, et al., Ježek P. 3D super-resolution microscopy reflects mitochondrial cristae alternations and mtDNA nucleoid size and distribution. Biochim Biophys Acta. 2018; 1859 (9), 829-844.

[3] Alán L, Špaček T, Ježek P. Delaunay algorithm and principal component analysis for 3D visualization of mitochondrial DNA nucleoids by Biplane FPALM/dSTORM. Eur Biophys J. 2016, 45 (5), 443-61.

Supervisor (email): Mgr. Hana Engstová, Ph.D.  Hana.Engstova@fgu.cas.cz

Candidate’s profile (requirements): We are seeking candidates with master's degree or equivalent in molecular biology, biochemistry, physics or related fields. Candidates should be fluent in English. Experience with in vivo models (mouse, rat) as well as with in vitro cell cultures and molecular biology techniques are advantage.