ABOUT

The plasma membrane serves as a vital cellular barrier, constantly facing assaults from external factors. Plasma membrane damage frequently occurs under physiological conditions, especially in tissues subjected to mechanical or biochemical stress, such as the gut, muscles, and skin. To cope with damage, cells possess intrinsic self-repair capabilities through mechanisms that remain poorly elucidated. However, it appears that these mechanisms are conserved across various sources of damage.
During infections, pathogens strategically target the integrity of the host plasma membrane to disrupt epithelial barriers, manipulate the immune response, and facilitate their dissemination. Several highly pathogenic human bacteria secrete pore-forming toxins, which serve as major virulence factors by inserting in the host cell’s plasma membrane, creating transmembrane pores and thus enabling the pathogens to manipulate their host environment and establish infection. Our research delves into the molecular intricacies of plasma membrane repair mechanisms activated in response to infectious damage.

RESEARCH

We are deeply immersed in unraveling the intricate intracellular processes governing plasma membrane rearrangements and actomyosin cytoskeleton remodeling, which are pivotal events for membrane resealing and thus constitute a critical aspect of cell survival and defense against infections. By elucidating these mechanisms, we aim to enrich our comprehension of how host cells endure infectious damage, thereby laying the groundwork for innovative therapeutic strategies to combat infections and promote tissue repair.

The disruption of plasma membrane integrity triggers uncontrolled exchanges between the intracellular and extracellular environments, precipitating the loss of cellular homeostasis. Notably, our research has revealed that the massive influx of calcium through the toxin-based pores induces endoplasmic reticulum (ER) disruption and prompts the recruitment of ER-derived vacuoles to the cell cortex. This process occurs concurrently with profound remodeling of the cortical actomyosin cytoskeleton and extensive plasma membrane blebbing. Importantly, we have established a correlation between plasma membrane blebbing, actomyosin cytoskeleton remodeling, and the resolution of damage, demonstrating their regulation by ER proteins.

Based on available data, we postulate that the recruitment of ER-derived vacuoles to the cell cortex serves a dual purpose: firstly, as a source of new membrane for damage repair, and secondly, to polarize essential intracellular machinery to the site of injury. To further investigate our hypothesis, we employ a multifaceted approach encompassing proximity-labeling proteomics, live-cell imaging, and advanced high-resolution and combinatory electron microscopy techniques.

To induce damage in host cell lines and deeply investigate common repair mechanisms, we employ a variety of damaging agents, including:

1. Purified recombinant pore-forming toxins, such as PLY (Pneumolysin), LLO (Listeriolysin O), and PFO (Perfringolysin O);
2. Bacterial pathogens known to produce pore-forming toxins, including Streptococcus pneumoniae, Listeria monocytogenes, and Clostridium perfringens;
3. Influenza A virus, which causes significant damage to epithelial lung cells.

In our laboratory, we employ a diverse range of cellular models for our research. These include commercially available epithelial cell lines, genetically modified cell lines, 3D cell models cultured at the air-liquid interface, and stem cell-derived 3D cell models. To evaluate infection outcomes, we commonly utilize zebrafish or C. elegans infection models.

We are currently addressing several key issues, including:

1. Characterizing novel repair machineries, with a specific emphasis on calcium-responsive proteins;
2. Resolving the cellular ultrastructure in the vicinity of the pore and the plasma membrane blebbing, to uncover new cellular compartments involved in the repair process;
3. Elucidating the molecular intricacies of the crosstalk between the endoplasmic reticulum (ER) and cytoskeletal components;
4. Investigating the spatiotemporal regulation of repair events.

Collaborators

• J. Leong, Tufts University, Boston-USA
• E. Bou Ghanem, University at Buffalo, Buffalo-USA
• A. Lacy-Hulbert, Benaroya Research Institute, Seattle-USA
• S. Mostowy, Imperial College, London-UK
• J. Sellers, NIH-NHLBI, Bethesda-USA
• F. Impens, VIB-UGhent, Gent-Belgium
• D. Cabanes, i3S, Porto-Portugal
• A. X. Carvalho, i3S, Porto-Portugal