Supramolecular organization of the antigen processing machinery in the ER membrane

• Applicant: Professor Dr. Robert Tampé
• Subject Area: Biochemistry ; Biophysics ; Cell Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 546606888

The endoplasmic reticulum (ER) plays a pivotal role in various cell biological processes, including protein synthesis, quality control, sorting, signal transduction, antigen processing, and protein trafficking to diverse organelles. In recent years, many of these functions have been associated with distinct structures within the ER. However, many questions remain unanswered, in particular how ER morphology determines the function of ER-associated macromolecular machineries and vice versa. Dysfunction of ER membrane-associated and secreted proteins has been linked to various diseases such as diabetes, cancer, cystic fibrosis, and infections. The emergence of new super-resolution microscopy techniques, including DNA-PAINT, 4Pi-SMS, STED and pan-Expansion Microscopy, now offers an unprecedented opportunity to investigate this structure-function relationship at the nanoscale. Sharing a common interest in this question, the Tampé and Bewersdorf labs seek to combine their complementary expertise to pursue this research aim on the example of the peptide loading complex (PLC) and its central constituent, the transporter associated with antigen processing (TAP). PLC/TAP, along with several ER chaperones, assembles the heterodimeric major histocompatibility complex class I (MHC I) and is therefore of key importance for understanding the adaptive immune response. The Tampé lab has long-standing experience in the structural and mechanistic aspects of the PLC and related chaperone machineries and has made seminal contributions to their understanding. The Bewersdorf lab is one of the few groups worldwide with the multimodal imaging capabilities to generate 3D super-resolution data with the spatial resolution required to elucidate the organization of the ER as well as to study its dynamics, and has already pioneered their application to characterize protein clusters and the morphological dynamics of the ER. Using new 3D super-resolution techniques and the TAP/PLC machinery within the ER, we will gain a holistic understanding of how different ER structures and allocations of ER proteins interact and are regulated. More specifically, we will systematically optimize and validate new labeling and super-resolution imaging methods to elucidate the TAP/PLC machinery in the context of ER nanomorphology (Aim 1), quantify the cluster size and stoichiometry of the PLC in the ER (Aim 2), determine its localization with respect to the ER morphology (Aim 3), and modulate the supramolecular organization of the PLC to unravel its role in health and disease (Aim 4). These studies will ultimately shed light on how the ER can fulfill multiple functions in human cells, contributing to a more holistic view of this highly relevant system for the immune response.

• DFG Programme: Research Grants
• International Connection: USA
• Partner Organisation: National Science Foundation (NSF)
• Cooperation Partner: Dr. Jörg Bewersdorf