Institute for Genetics, Faculty of Math. Nat. Sciences
Apoptotic cell death is essential for development, immune function or tissue homeostasis, and it is often deregulated in disease. Mitochondrial outer membrane permeabilization (MOMP) is central for apoptosis execution and plays a key role in its inflammatory outcome. The Bcl-2 family of proteins is form by pro- and anti-apoptotic members that interact with each other to control MOMP. During apoptosis, the Bcl-2 proteins BAX and BAK accumulate at discrete sites on the MOM5 in structures called apoptotic foci, where they undergo conformational changes concomitant with oligomerization followed by MOMP. Knowing the architecture of the macromolecular machineries mediating MOMP is crucial for understanding their function and for the clinical use of apoptosis.
Beyond apoptosis, it has been recently recognized that several forms of necrotic cell death are also regulated by genetically encoded signaling pathways that we are only beginning to understand. Common to the execution of necroptosis, pyroptosis and ferroptosis is the permeabilization of the plasma membrane that releases the cellular contents to the microenvironment and initiates immune responses.
Our research: The Garcia-Saez group aims at understanding the underlying physical principles and molecular mechanisms that govern membrane organization and dynamics. A key open question is how membrane behavior and signaling are coordinated by the interplay between multiple molecular components and the membrane mechanical properties. To address these questions, we focus on the mitochondrial alterations during apoptosis as well as on membrane permeabilization as a common feature in the execution of regulated cell death.
Our goals: 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.
A second line of research in the group focuses on other forms of regulated cell death. Membrane pore formation is emerging as a common theme in regulated forms of regulated cell death, including new pathways like necroptosis, pyroptosis or ferroptosis, which we are studying with similar strategies.
Our successes: Our work has contributed to our understanding of mitochondrial permeabilization. We have built the first 3D model for the structure of active, full-length Bax in the membrane that reveals a key conformational change critical for pore formation. We have shown that Bax does not adopt a unique oligomeric state, but rather exists as a mixture of oligomeric species based on dimer units. We also discovered that active Bax clusters into a broad distribution of distinct architectures, including full rings, as well as linear and arc-shaped oligomeric assemblies with both rings and arcs being able to perforate the membrane. Altogether, our data shed new light on the supramolecular organization of Bax during apoptosis and support a novel molecular mechanism in which Bax fully or partially delineates pores of different sizes to permeabilize the mitochondrial outer membrane.
We have also pioneered the use of single molecule techniques to understand complex formation between Bcl-2 proteins both in solution and in the membrane environment. We have addressed the question how the intricate, fine-tuned interaction network formed by the Bcl-2 family of proteins determines cell fate using novel, quantitative systems approaches.
Our methods/techniques: 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.
Figure 1: A) BAX apoptotic foci (green) in mitochondria (magenta) of a dying cell.
B) BAX/BAK apoptotic foci in cell death signaling and inflammation.
Figure 2: Model for mitochondria permeabilization by BAX. A) Supramolecular assemblies of BAX into lines, arcs and rings in cells undergoing apoptosis visualized by super-resolution and atomic force microscopy. B) Structural model of BAX in the membrane built from electron paramagnetic resonance data.