DETECT and PROTECT – surviving insults to intracellular compartments

To protect the cell interior from pathogens and other harmful agents, it is essential that our cells respond swiftly and with precision. One key defense mechanism in intracellular compartments like endosomes, lysosomes, and phagosomes is the process called “Conjugation of ATG8 proteins to Single Membranes” (CASM). This involves the attachment of ATG8 proteins to damaged membranes, forming a temporary docking platform for protective cellular responses. Our research has revealed that, alongside ATG16L1, the protein TECPR1 plays a vital role in CASM. TECPR1 is activated when sphingomyelin—normally confined to the inner leaflet of endolysosomal membranes—is exposed to the cytosol due to membrane damage. This exposure enables TECPR1 to recruit ATG8 proteins and initiate protective cellular responses. Remarkably, this mechanism continues to function even when ATG16L1 is inhibited, such as during infections by bacteria like Salmonella, which specifically block ATG16L1 activity. Defects in CASM weaken our defenses against pathogens and increase susceptibility to autoimmune diseases. Understanding how TECPR1 and ATG16L1 contribute to CASM provides new insights into how cells detect and respond to membrane damage. The goal of the "DETECT and PROTECT" project is to understand how damaged membranes are recognized and to map the ATG8-dependent processes the cell initiates in response. With insights into how TECPR1 identifies damage—triggered by the exposure of sphingomyelin—the next step is to uncover the protective processes that are activated. This will provide a greater understanding of the mechanisms involved in infections and the failures that occur in certain autoimmune diseases.

Membrane compartments within the cell are exposed to different kinds of stress (e.g. pathogenic insults). Meeting stress-inducing agents with a swift response is key to minimize harmful effects. One such response is single membrane ATG8 conjugation (SMAC), where ATG8 proteins are attached to the surface of the compromised compartment. This provides a transient docking platform for proteins involved in protective countermeasures. In this project I aim to characterize the machinery that mediates ATG8 conjugation on compromised endolysosomes. In Work Package 1 (WP1) I aim to characterize the targeting mechanism of the ATG8 conjugation machinery. Furthermore, by conducting an unbiased image based siRNA screen (WP2) I aim to identify additional machinery components involved in the SMAC pathway. As ATG8 conjugation on membranes provides a docking platform for other proteins, I also aim to identify such SMAC specific effector proteins using a proteomics approach. The identified proteins will be categorized according to function in order to understand which SMAC dependent countermeasures are initiated on compromised membranes (WP3). In WP4 we set out to mechanistically characterize the candidate proteins identified in WP2 and WP3 in further detail.