Poster Presentation 50th Lorne Proteins Conference 2025

Investigating the conformation of apoptotic pores using expansion microscopy (#328)

Bochen Zhu 1 2 , Richard Birkinshaw 1 2 , Niall Geoghegan 1 2 , Kelly Rogers 1 2 , Peter Czabotar 1 2
  1. The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
  2. Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia

Apoptosis is a regulated cell death pathway that plays important roles in health and disease. The intrinsic pathway of apoptosis is initiated by excessive intracellular stress that activates the B cell lymphoma-2 (BCL-2) family members BAX and BAK to dimerise on the mitochondrial outer membrane. Further oligomerisation of BAX/BAK dimers form pores that result in mitochondrial outer membrane permeabilisation (MOMP) that is often considered as the point-of-no-return of the intrinsic pathway of apoptosis [1].

Previous studies suggest that BAX/BAK pores are consistent with a toroidal pore model that the pore lumen is lined by both proteins and lipids [2, 3]. Two models were proposed for the conformation of BAX/BAK pores, including the clamp model [4] and the asymmetric model [5]. However, due to the small size and dynamic nature of BAX/BAK pores, it is difficult to validate these two models by structural biology techniques such as X-ray crystallography and cryo-electron microscopy (cryo-EM).

Expansion microscopy (ExM) is a novel method for super-resolution light microscopy [6]. In ExM, samples are embedded in a swellable hydrogel can be isotropically expanded 4-to-20-fold along all axes [6]. Imaging the expanded samples effectively improves the resolution by the expansion factor of the hydrogel and allows visualisation of molecular structures smaller than the diffraction limit of light at ~250 nm. Recently, Shaib et al. (2024) demonstrated the potential of ExM to visualise protein shapes using confocal microscopes [7]. This provides opportunities to visualise protein complexes that are difficult to study with traditional structural biology techniques.

This project aims to use ExM to directly visualise of BAX/BAK pores assembled on liposomes and isolated mitochondria. It is hoped that physical expansion of the sample can provide high enough resolution to distinguish between the clamp and asymmetric models of BAX/BAK pore conformation. This may provide insights into the mechanism of BAX/BAK pore assembly and infer the regulation of BAX/BAK activity in different physiological and pathological conditions.

  1. Czabotar & García-Sáez (2023). Mechanisms of BCL-2 family proteins in mitochondrial apoptosis. Nat. Rev. Mol. Cell Biol., 24(10), 732-748.
  2. Qian et al. (2008). Structure of transmembrane pore induced by Bax-derived peptide: Evidence for lipidic pores. PNAS, 105(45), 17379-17383.
  3. Salvador‐Gallego et al. (2016). Bax assembly into rings and arcs in apoptotic mitochondria is linked to membrane pores. EMBO J., 35(4), 389-401.
  4. Bleicken et al (2014). Structural model of active Bax at the membrane. Mol. Cell, 56(4), 496-505.
  5. Mandal et al. (2016). Assembly of Bak homodimers into higher order homooligomers in the mitochondrial apoptotic pore. Sci. Rep., 6(1), 30763.
  6. Wang et al. (2024). Single-shot 20-fold expansion microscopy. Nat. Methods.
  7. Shaib et al. (2024). One-step nanoscale expansion microscopy reveals individual protein shapes. Nat. Biotechnol.