title: Revealing Assembly of a Pore-Forming Complex Using Single-Cell Kinetic Analysis and Modeling.
abstract: Many biological processes depend on the sequential assembly of protein complexes. However, studying the kinetics of such processes by direct methods is often not feasible. As an important class of such protein complexes, pore-forming toxins start their journey as soluble monomeric proteins, and oligomerize into transmembrane complexes to eventually form pores in the target cell membrane. Here, we monitored pore formation kinetics for the well-characterized bacterial pore-forming toxin aerolysin in single cells in real time to determine the lag times leading to the formation of the first functional pores per cell. Probabilistic modeling of these lag times revealed that one slow and seven equally fast rate-limiting reactions best explain the overall pore formation kinetics. The model predicted that monomer activation is the rate-limiting step for the entire pore formation process. We hypothesized that this could be through release of a propeptide and indeed found that peptide removal abolished these steps. This study illustrates how stochasticity in the kinetics of a complex process can be exploited to identify rate-limiting mechanisms underlying multistep biomolecular assembly pathways.
authors.affiliation: Global Health Institute, Faculty of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland; The Institute of Bioengineering, Faculty of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors.forename: Mirko
authors.initials: M
authors.lastname: Bischofberger
authors.affiliation: Global Health Institute, Faculty of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors.forename: Ioan
authors.initials: I
authors.lastname: Iacovache
authors.affiliation: The Brain Mind Institute, Faculty of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors.forename: Daniel
authors.initials: D
authors.lastname: Boss
authors.affiliation: The Institute of Bioengineering, Faculty of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
authors.forename: Felix
authors.initials: F
authors.lastname: Naef
authors.affiliation: Global Health Institute, Faculty of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland. Electronic address: gisou.vandergoot@epfl.ch.
authors.forename: F Gisou
authors.initials: FG
authors.lastname: van der Goot
authors.affiliation: Center of Synthetic and Systems Biology, University of Edinburgh, Edinburgh, United Kingdom. Electronic address: nacho.molina@ed.ac.uk.
authors.forename: Nacho
authors.initials: N
authors.lastname: Molina
mesh_terms: D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism*; D004912:Erythrocytes / Q000166:cytology; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D010446:Peptide Fragments / Q000378:metabolism; D052899:Pore Forming Cytotoxic Proteins / Q000737:chemistry* / Q000378:metabolism*; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D001427:Bacterial Toxins; D010446:Peptide Fragments; D052899:Pore Forming Cytotoxic Proteins; C032676:aerolysin