Single-molecule analysis of the entire perfringolysin O pore formation pathway
Curation statements for this article:-
Curated by eLife
Evaluation Summary:
This paper presents a single-molecule, multi-color microscopy study of the real-time assembly of perfringolysin O, a member of the membrane attack complex perforin cholesterol-dependent cytolysin superfamily. With the ability to resolve different states of the species in the reaction, simultaneously with membrane leakage, this work informs on key aspects of the mechanism including identifying potential assemblies involved in membrane lysis, and how membrane binding, oligomerization, and pore transitioning depends on concentration and pH. While some additional controls are needed to clarify the interpretation of the results, this study will be of interest to many, including those studying cytolysin mechanisms, but also the broader field of single-molecule studies of membrane binding proteins.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Structural Biology and Molecular Biophysics (eLife)
Abstract
The cholesterol-dependent cytolysin perfringolysin O (PFO) is secreted by Clostridium perfringens as a bacterial virulence factor able to form giant ring-shaped pores that perforate and ultimately lyse mammalian cell membranes. To resolve the kinetics of all steps in the assembly pathway, we have used single-molecule fluorescence imaging to follow the dynamics of PFO on dye-loaded liposomes that lead to opening of a pore and release of the encapsulated dye. Formation of a long-lived membrane-bound PFO dimer nucleates the growth of an irreversible oligomer. The growing oligomer can insert into the membrane and open a pore at stoichiometries ranging from tetramers to full rings (~35 mers), whereby the rate of insertion increases linearly with the number of subunits. Oligomers that insert before the ring is complete continue to grow by monomer addition post insertion. Overall, our observations suggest that PFO membrane insertion is kinetically controlled.
Article activity feed
-
-
-
Evaluation Summary:
This paper presents a single-molecule, multi-color microscopy study of the real-time assembly of perfringolysin O, a member of the membrane attack complex perforin cholesterol-dependent cytolysin superfamily. With the ability to resolve different states of the species in the reaction, simultaneously with membrane leakage, this work informs on key aspects of the mechanism including identifying potential assemblies involved in membrane lysis, and how membrane binding, oligomerization, and pore transitioning depends on concentration and pH. While some additional controls are needed to clarify the interpretation of the results, this study will be of interest to many, including those studying cytolysin mechanisms, but also the broader field of single-molecule studies of membrane binding proteins.
(This preprint has been …
Evaluation Summary:
This paper presents a single-molecule, multi-color microscopy study of the real-time assembly of perfringolysin O, a member of the membrane attack complex perforin cholesterol-dependent cytolysin superfamily. With the ability to resolve different states of the species in the reaction, simultaneously with membrane leakage, this work informs on key aspects of the mechanism including identifying potential assemblies involved in membrane lysis, and how membrane binding, oligomerization, and pore transitioning depends on concentration and pH. While some additional controls are needed to clarify the interpretation of the results, this study will be of interest to many, including those studying cytolysin mechanisms, but also the broader field of single-molecule studies of membrane binding proteins.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)
-
Reviewer #1 (Public Review):
In this paper, the authors present a set of studies that examine the assembly reaction and function of Perfringolysin O (PFO) with POPC/cholesterol membranes. Using single-molecule TIRF microscopy, they are able to visualize PFO labelled with dye AF647 binding to vesicles that are loaded with AF488. With this co-localization experiment, they are able to examine the molecular steps in PFO assembly that couple to functional membrane leakage. This approach offers an increase in resolution that allows a direct observation of the reaction. With this, the behavior of monomers and dimers in the membrane can measured, as well as the higher-order oligomeric reaction and transition to a functional lysis pore. One interesting observation is that lysis occurs at lower oligomer numbers than would be expected for a full …
Reviewer #1 (Public Review):
In this paper, the authors present a set of studies that examine the assembly reaction and function of Perfringolysin O (PFO) with POPC/cholesterol membranes. Using single-molecule TIRF microscopy, they are able to visualize PFO labelled with dye AF647 binding to vesicles that are loaded with AF488. With this co-localization experiment, they are able to examine the molecular steps in PFO assembly that couple to functional membrane leakage. This approach offers an increase in resolution that allows a direct observation of the reaction. With this, the behavior of monomers and dimers in the membrane can measured, as well as the higher-order oligomeric reaction and transition to a functional lysis pore. One interesting observation is that lysis occurs at lower oligomer numbers than would be expected for a full ring pore, and that oligomerization continues to occur after membrane leakage, supporting a model that an incomplete arc pore is functional for membrane lysis. In addition, this approach allows for a full investigation of the kinetics of each step of the reaction, as well as the dependency on concentration and pH. While further technical details would be useful in this paper, the experiments appear to have been carried out carefully and rigorously, providing quantitative access to this complex reaction. Thus, this study will be of interest to many working on the mechanisms of cytolysin, as well as those studying protein binding to membranes by single-molecule microscopy approaches.
-
Reviewer #2 (Public Review):
This work investigates the mechanism of pore formation for large nanopore. It is known that water soluble monomers are excreted by cells and then oligomerize on targeted membranes. However, the molecular details of this process, especially for large pores, is still largely debated.
In particular, it is not clear how nucleation and growth happen. What is the minimal amount of protomer that can sustain nucleation? Is the pore-forming protein growing one protomer at the time or by adding dimer, trimer, tetramers? It has also been observed that arc-pores can be formed. However, what is not clear is how such arc pores can close to form full pores.
Here the authors are attempting to answer much of these questions. The experiments are well performed. The authors use microscopy to image the formation of labelled …
Reviewer #2 (Public Review):
This work investigates the mechanism of pore formation for large nanopore. It is known that water soluble monomers are excreted by cells and then oligomerize on targeted membranes. However, the molecular details of this process, especially for large pores, is still largely debated.
In particular, it is not clear how nucleation and growth happen. What is the minimal amount of protomer that can sustain nucleation? Is the pore-forming protein growing one protomer at the time or by adding dimer, trimer, tetramers? It has also been observed that arc-pores can be formed. However, what is not clear is how such arc pores can close to form full pores.
Here the authors are attempting to answer much of these questions. The experiments are well performed. The authors use microscopy to image the formation of labelled promoters. They used a clever use of fluorescent labels to image both the association with the membranes and the punctuation mechanism. Lipids are labeled with a dye that remains transiently bound, which allows the localization of the liposome. Then the interaction of fluorescently tagged protomers is investigate. Finally, the mechanism of pore formation is observed by measuring the efflux quenched dyes.
This work is clear, well written, and the interpretation is at large convincing. The authors conclude that monomeric PFO interact with the membranes only transiently (for less than a second. However, when they encounter a second monomer, they oligomerize and remain (almost) permanently bound to the membrane. It is the dimer that is then the nucleation for the pore and individual protomers are then added sequentially.
-
Reviewer #3 (Public Review):
They demonstrate that PFO binds the membrane as a monomer with a short binding half-life and that its dimerization nucleates the growth of a stable oligomer. They show that PFO oligomerizes by addition of monomers from the solution in an irreversible process and that small oligomers can insert into the membrane leading to perforation. Their thorough analysis quantifies binding rates, oligomerization rates, insertion rates and the relationships between them, thereby providing new understanding how these processes interrelate to define the stochastic process of pore formation.
The study is rigorous and well-presented. The authors' conclusions are largely supported by the results and enforced by mathematical models on the PFO binding and pore-formation kinetics. One of the main strengths of the manuscript lies …
Reviewer #3 (Public Review):
They demonstrate that PFO binds the membrane as a monomer with a short binding half-life and that its dimerization nucleates the growth of a stable oligomer. They show that PFO oligomerizes by addition of monomers from the solution in an irreversible process and that small oligomers can insert into the membrane leading to perforation. Their thorough analysis quantifies binding rates, oligomerization rates, insertion rates and the relationships between them, thereby providing new understanding how these processes interrelate to define the stochastic process of pore formation.
The study is rigorous and well-presented. The authors' conclusions are largely supported by the results and enforced by mathematical models on the PFO binding and pore-formation kinetics. One of the main strengths of the manuscript lies on the nature of the new method, that provides high throughput analysis of thousands of pore events in individual liposomes in parallel with the single molecule analysis of pore forming protein self-assembly. Furthermore, the new insight provided into the mechanism of PFO is of high interest and will impact our understanding of pore forming proteins in general.
-