1. Research Background:

Our group aims at understanding the underlying physical principles and molecular mechanisms that govern membrane organization and dynamics during regulated cell death. A key open question is how membrane behavior and signaling are coordinated by the interplay between multiple molecular components and the membrane mechanical properties. We focus our research on the mitochondrial alterations during apoptosis as well as on membrane permeabilization as a common feature in the execution of regulated cell death.

To these aims, we have established a multi-disciplinary group that develops new microscopy tools and combines biophysics, biochemistry and cell biology in reconstituted minimal systems and in single cells to analyze mitochondrial permeabilization and additional alterations during apoptosis from a quantitative perspective. We also apply these methods to study the molecular mechanisms of membrane permeabilization that execute other forms of regulated cell death.

2. Research Questions Addressed By The Group:

One main research line in the lab aims to unravel the molecular mechanisms involved in the permeabilization of mitochondria during apoptosis. In our group, we study the stoichiometry and interaction preferences of BCL-2 proteins complexes by exploiting the single molecule toolbox implemented in the lab. We also aim to define the composition, dynamics and structure of apoptotic foci and to understand how they are integrated to orchestrate function. In addition,  we are interested in understanding the mechanisms underlying mitochondrial dynamics and the contacts of mitochondria with other cellular membranes.

3. Possible Projects:

MECHANISM AND FUNCTION OF BOK-INDUCED CELL DEATH

BOK is a poorly understood member of the BCL-2 family of proteins that has been proposed to function as a pro-apoptotic, BAX-like effector. However, the molecular mechanism and structural properties of BOK pores remain enigmatic. In preliminary work, we directly visualized BOK pores in liposomes by electron microscopy, which appeared similar to those induced by BAX, in line with comparable oligomerization properties quantified by single molecule imaging. In addition, super-resolution STED imaging revealed that BOK organized into dots and ring-shaped assemblies in apoptotic mitochondria, also reminiscent of those found for BAX and BAK. Yet, unlike BAX and BAK, the apoptotic activity of BOK was independent of and unaffected by other BCL-2 proteins. These results suggest that, while BOK pores are structurally similar to those of BAX and BAK, its activity is kept in check by limited mitochondrial localization instead of interaction with BCL-2 family members.

Important questions about the mechanism and function of BOK remain open, including 1) the mechanisms and interaction partners that regulate its subcellular localization and thereby killing activity, 2) the cellular conditions under which BOK relocalizes to mitochondria to induce cell death and 3) the additional functions of BOK outside mitochondria. This questions will be addressed in the context of the project using a newly generated cell line with endogenously tagged BOK that allows unprecedented visualization with advanced microscopy, a genetic screening for identifying BOK regulators as well as mass spectrometry analysis of interaction partners.

4. Applied methods and model organisms:

Superresolution microscopy (STORM, STED, SIM, DNA-PAINT), CRISPR/Cas9, cell lines and primary cells, mass spectrometry, molecular and cell biology, quantitative biology

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:

1. Cosentino K, Hertlein V, Jenner A, Dellmann T, Gojkovic M, Peña-BlancoA, Dadsena S, Wajngarten N, Danial JSH, Thevathasan JV, Mund M, Ries J & Garcia-Saez AJ. (2022) The interplay between BAX and BAK tunes apoptotic pore growth to control mitochondrial DNA-mediated inflammation. Mol Cell, 82(5):933-949.
2. Jenner A, Peña-Blanco A, Salvador-Gallego R, Ugarte-Uribe B, Zollo C, Ganief T, Bierlmeier J, Mund M, Lee JE, Ries J, Schwarzer D, Macek B & Garcia-Saez AJ. (2022) DRP1 interacts directly with BAX to induce its activation and apoptosis. EMBO J. 41(8):e108587.
3. Flores-Romero H, Hohorst L, John M, Albert MC, King LE, Beckmann L, Szabo T, Hertlein V, Luo X, Villunger A, Frenzel LP, Kashkar H & Garcia-Saez AJ. (2022) BCL-2-family protein tBID can act as a BAX-like effector of apoptosis. EMBO J. 41(2):e108690.
4. Pedrera L, Espiritu RA, Ros U, Weber J, Schmitt A, Stroh J, Hailfinger S, von Karstedt S & García-Sáez AJ. (2021) Ferroptotic pores induce Ca2+ fluxes and ESCRT-III activation to modulate cell death kinetics. Cell Death Differ. 28(5):1644-1657.
5. Danial JSH & Garcia-Saez AJ. (2019) Quantitative analysis of super-resolved structures using ASAP. Nat Methods, 16(8):711-714. 
6. Bleicken S, Hantusch A, Das KK, Frickey T & Garcia-Saez AJ (2017) Quantitative interactome of a membrane Bcl-2 network identifies a hierarchy of complexes for apoptosis regulation. Nat Commun, 8:73.
7. Ros U, Peña-Blanco A, Hänggi K, Kunzendorf U, Krautwald S, Wong WW-L & Garcia-Saez AJ (2017). Necroptosis execution is mediated by plasma membrane nanopores independent of calcium. Cell Rep, 19:175-187.
8. Salvador-Gallego R, Mund M, Cosentino K, Schneider J, Unsay J, Schraermeyer U, Engelhardt J, Ries J* & Garcia-Saez AJ* (2016). Bax assembly into rings and arcs in apoptotic mitochondria is linked to membrane pores. EMBO J, 35:389-401.
9. Subburaj Y, Cosentino K, Axmann M, Pedrueza-Villalmanzo E, Hermann E, Bleicken S, Spatz J & Garcia-Saez AJ (2015). Bax monomers form dimer units in the membrane that further self-assemble into multiple oligomeric species. Nat Commun, 6:8042.
10. Bleicken S, Jeschke G, Stegmueller C, Salvador-Gallego R, Garcia-Saez AJ* & Bordignon E* (2014). Structural model of active Bax at the membrane. Mol Cell, 56:496-505.