Phosphoinositide control of antigen cross-presentation by classical dendritic cells

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To gather nutrients and information, cells sample their surroundings by internalizing material into organelles called endosomes. Endosomes are surrounded by a membrane which partitions the internalized material from the rest of the cell. Typically, internalized macromolecules, such as proteins or nucleic acids, cannot cross this membrane and are instead delivered to digestive organelles, called lysosomes, for conversion into small molecules and incorporation into the cell's metabolic pathways or for export as waste. In some exceptional instances, however, large undigested macromolecules are released from endosomes. This is especially evident in immune cells called dendritic cells (DCs). DCs transfer endocytosed macromolecules, including nucleic acids and proteins, into the cytosol where they can elicit inflammatory signalling or, in the case of proteins, are processed for presentation to T cells in a process known as antigen cross-presentation. This allows DCs to perform fundamental roles in homeostasis and immunity, yet how it happens is not known. This is the focus of my long-term research program, which will investigate how cells transfer macromolecules across endosomal membranes to elicit critical biological functions. This year, we made progress on this question by showing that large pores form on endocytic organelles in DCs. This allows for the escape of endocytosed proteins and is required for cross-presentation. But how do these putative endosomal pores form? We now have evidence that lipids called phosphoinositides drive endosomal pore formation and therefore cross-presentation. This, along with the fact that phosphoinositides mediate cellular events by recruiting protein effectors, leads to our hypothesis that phosphoinositides regulate macromolecular escape from endosomes by coordinating the formation of large pores through the recruitment of pore-forming protein effectors. Over the next 5 years, we will determine how phosphoinositides coordinate endosomal pore formation to facilitate the fundamental DC function of cross-presentation. Our aims are to: i)Identify the phosphoinositide species that regulate endosomal pore formation and cross-presentation ii)Identify the protein effectors that mediate endosomal pore formation and cross-presentation iii)Investigate the role of the putative pore-forming protein APOL7C in endosomal pore formation Beyond DCs, macromolecular escape from endosomes is implicated in key processes, such as inflammatory signalling, in many cell types. Our NSERC DG will therefore have widespread implications for fields ranging from cellular physiology to organismal homeostasis. We are in the ideal position to drive these discoveries and, in doing so, will (i) deliver unprecedented insight into the biology of cross-presentation, (ii) generate a toolbox for the investigation of phosphoinositides and their effectors and provide a (iii) training venue for HQP to build careers in academia and industry in Canada.