Mammalian membrane trafficking as seen through the lens of bacterial toxins.

A fundamental question of eukaryotic cell biology is how membrane organelles are organised and interact with each other. Cell biologists address these questions by characterising the structural features of membrane compartments and the mechanisms that coordinate their exchange. To do so, they must rely on variety of cargo molecules and treatments that enable targeted perturbation, localisation, and labelling of specific compartments. In this context, bacterial toxins emerged in cell biology as paradigm shifting molecules that enabled scientists to not only study them from the side of bacterial infection but also from the side of the mammalian host. Their selectivity, potency, and versatility made them exquisite tools for uncovering much of our current understanding of membrane trafficking mechanisms. Here, we will follow the steps that lead toxins until their intracellular targets, highlighting how specific events helped us comprehend membrane trafficking and establish the fundamentals of various cellular organelles and processes. Bacterial toxins will continue to guide us in answering crucial questions in cellular biology while also acting as probes for new technologies and applications.

Endoplasmic reticulum chaperone Gp96 controls actomyosin dynamics and protects against pore-forming toxins.

During infection, plasma membrane (PM) blebs protect host cells against bacterial pore-forming toxins (PFTs), but were also proposed to promote pathogen dissemination. However, the details and impact of blebbing regulation during infection remained unclear. Here, we identify the endoplasmic reticulum chaperone Gp96 as a novel regulator of PFT-induced blebbing. Gp96 interacts with non-muscle myosin heavy chain IIA (NMHCIIA) and controls its activity and remodelling, which is required for appropriate coordination of bleb formation and retraction. This mechanism involves NMHCIIA-Gp96 interaction and their recruitment to PM blebs and strongly resembles retraction of uropod-like structures from polarized migrating cells, a process that also promotes NMHCIIA-Gp96 association. Consistently, Gp96 and NMHCIIA not only protect the PM integrity from listeriolysin O (LLO) during infection by Listeria monocytogenes but also affect cytoskeletal organization and cell migration. Finally, we validate the association between Gp96 and NMHCIIA in vivo and show that Gp96 is required to protect hosts from LLO-dependent killing.

Control of cytoskeletal dynamics during cellular responses to pore forming toxins.

Following damage by pore forming toxins (PFTs) host cells engage repair processes and display profound cytoskeletal remodeling and concomitant plasma membrane (PM) blebbing. We have recently demonstrated that host cells utilize similar mechanisms to control cytoskeletal dynamics in response to PFTs and during cell migration. This involves assembly of cortical actomyosin bundles, reorganisation of the endoplasmic reticulum (ER) network, and the interaction between the ER chaperone Gp96 and the molecular motor Non-muscle Myosin Heavy Chain IIA (NMHCIIA). Consequently, Gp96 regulates actomyosin activity, PM blebbing and cell migration, and protects PM integrity against PFTs. In addition, we observed that PFTs increase association of Gp96 and ER vacuoles with the cell surface or within PM blebs loosely attached to the cell body. Similarly, gut epithelial cells damaged by PFTs in vivo were shown to release microvilli structures or directly purge cytoplasmic content. Cytoplasmic purging involves profound cytoskeletal remodeling and ER vacuolation, suggesting that our observations recapitulate recovery processes in vivo. Here, we discuss our findings in light of the current understanding of PM repair mechanisms and in vivo recovery responses to PFTs.