Research Area: Cell Death and Biophysics, molecular and cellular Biology, Biochemistry and advanced Microscopy
1. Research Background
Apoptotic cell death is essential for diverse processes such as development, immune function or tissue homeostasis, and it is often deregulated in disease. Mitochondrial outer membrane permeabilization (MOMP) is a central event in the execution of apoptosis and plays a key role in its inflammatory outcome. Knowledge of the architecture of the macromolecular machineries mediating MOMP is crucial for understanding their function and for the clinical use of apoptosis. Our recent work revealed that Bax and Bak dimers form distinct line, arc and ring-like assemblies at specific apoptotic foci to mediate MOMP. However, the molecular structure and the mechanisms controlling the spatiotemporal formation and range of action of apoptotic foci are currently not known.
2. Research questions addressed by the group:
Molecular mechanisms of Bcl-2 proteins:
Bcl-2 family members are essential regulators of apoptosis that control permeabilization of the mitochondrial outer membrane, leading to release of apoptotic factors into the cytosol, activation of caspases and cell death. The Bcl-2 family has antiapoptotic and proapoptotic members as well as protein sensors for apoptotic stimuli and initiation of apoptosis. The reasons why Bcl-2 family members have opposing functions despite their high degree of homology remain obscure, and the basis for interaction between different members of the family to decide whether or not apoptosis is induced remains poorly understood. Translocation of Bcl-2 members to the mitochondrial membrane is of high interest, since the effect of the membrane environment on their function is unknown. Our research focuses on the role of the membrane and specific lipids, and on the interaction network between the Bcl-2 members that decides whether or not apoptosis is induced.
Model membranes and optical microscopy:
To investigate dynamic processes in biological membranes we apply model systems of different complexity, ranging from pure lipid bilayers to cultured cells. A common feature of these systems is that they can be visualized with optical microscopy and can be used for experiments involving time-lapse microscopy, FRAP, FRET, FCS, AFM and other advanced microscopy techniques. In recent years, we have focused on implementing new methods to quantify the stoichiometry and molecular architecture of macromolecular complexes based on single molecule imaging and super resolution microscopy.
3. Possible projects:
We offer a multi-disciplinary project that combines multiple approaches aimed at determining the molecular architecture and dynamics of assembly of apoptotic foci. These include:
- Live cell imaging to validate and image the distribution dynamics of protein candidates colocalizing and/or interacting with Bax/Bak during apoptosis, followed by interaction tests using using live-cell Dimerization Dependent Fluorescent Protein (ddFP) imaging.
- Functional analysis of novel apoptotic foci components by knockdown or knockout, effect on mitochondrial morphology and cristae structure (EM), electrochemical potential, calcium flux, mitophagy and contacts with the ER by advanced microscopy
- Quantitative analysis of novel apoptotic foci components: mobility of GFP-tagged apoptotic foci constituents in healthy cells and upon clustering into apoptotic foci; molecular exchange by cytosol/MOM shuttling combining fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), and single particle tracking (SPT); changes in the spatial distribution will be correlated with hallmarks of apoptosis progression.
4. Applied Methods and model organisms:
Our work focuses on the study of membrane processes from a quantitative point of view. To address our questions in the fields of protein-membrane interactions, in pore formation and in membrane dynamics during regulated cell death, we use quantitative methods based on advanced fluorescence microscopy applied to a combination of synthetically reconstituted systems and living cells.
We pioneered the use of single molecule techniques to investigate the mechanism of pore formation by the apoptotic executors Bax and Bak and the interaction of the Bcl-2 proteins.
5. Desirable skills and qualifications:
We are looking for a highly motivated and enthusiastic person with a Masters’ degree in biophysics, cell biology or biochemistry. Experience in cell death signaling, mitochondrial biology or advanced light microscopy techniques would be a bonus, but is not a prerequisite.
6. References and key publications:
- Danial JSH, Garcia-Saez AJ. Quantitative analysis of super-resolved structures using ASAP. Nat Methods. 2019; 16(8):711-714
- Flores-Romero H, García-Sáez AJ. The Incomplete Puzzle of the BCL2 Proteins. Cells. 2019 Sep. 8(10): pii: E1176
- Bleicken S, Hantusch A, Das KK, Frickey T, Garcia-Saez AJ. Quantitative interactome of a membrane Bcl-2 network identifies a hierarchy of complexes for apoptosis regulation. Nat Commun. 2017 July; 8:73
- Danial JS, Garcia-Saez AJ. Improving certainty in single molecule imaging. Curr Opin Struct Biol. 2017 May; 46:24-30
- Ros U*, Peña-Blanco A, Hänggi K, Kunzendorf U, Krautwald S, Wong WW-L, Garcia-Saez AJ*. Necroptosis execution is mediated by plasma membrane nanopores independent of calcium. Cell Rep. 2017 Apr; 19(1):175-187
- Cosentino K, Garcia-Saez AJ. Bax and Bak pores: are we closing the circle? Trends in Cell Biol. 2017 Apr; 27(4):266-275
- Salvador-Gallego R, Mund M, Cosentino K, Schneider J, Unsay J, Schraermeyer U, Engelhardt J, Ries J*, García-Sáez AJ*. Bax assembly into rings and arcs in apoptotic mitochondria is linked to membrane pores. EMBO J. 2016 Feb 15; 35(4):389-401
- Subburaj Y, Cosentino K, Axmann M, Pedrueza-Villalmanzo E, Hermann E, Bleicken S, Spatz J, García-Sáez AJ. Bax monomers form dimer units in the membrane that further self-assemble into multiple oligomeric species. Nature Commun. 2015 Aug 14; 6:8042
- Bleicken S, Jeschke G, Stegmueller C, Salvador-Gallego R, García-Sáez AJ*, Bordignon E*. Structural model of active Bax at the membrane. Molecular Cell. 2014;56(4):496-505
- Bleicken S, Landeta O, Landajuela A, Basañez G*, García-Sáez AJ*. Proapoptotic Bax and Bak Proteins Form Stable Protein-permeable Pores of Tunable Size. J Biol Chem. 2013; 288(46):33241-52