Phosphoinositide control of antigen cross-presentation by classical dendritic cells

为了收集营养和信息，细胞通过将物质内化到称为核内体的细胞器中来对周围环境进行采样。核内体被一层膜包围，这层膜将内化的物质与细胞的其余部分隔开。通常，内化的大分子，如蛋白质或核酸，不能穿过这层膜，而是被运送到消化细胞器，称为溶酶体，转化成小分子，并纳入细胞的代谢途径或作为废物出口。然而，在某些特殊情况下，未消化的大分子从核内体中释放出来。这在被称为树突状细胞（dc）的免疫细胞中尤为明显。树突状细胞将内吞的大分子，包括核酸和蛋白质，转移到细胞质中，在那里它们可以引发炎症信号，或者在蛋白质的情况下，通过抗原交叉递呈的过程被加工呈递给T细胞。这使得dc在体内平衡和免疫中发挥基本作用，但其发生方式尚不清楚。这是我长期研究计划的重点，将研究细胞如何通过内体膜转移大分子以引发关键的生物学功能。今年，我们在这个问题上取得了进展，表明在树突状细胞的内吞细胞器上形成了大孔。这允许内吞蛋白的逃逸，并且是交叉呈现所必需的。但是这些假定的内体孔是如何形成的呢？我们现在有证据表明，称为磷酸肌苷的脂质驱动内体孔的形成，因此交叉呈现。这一点，再加上磷酸肌苷通过招募蛋白质效应器介导细胞事件的事实，导致了我们的假设，即磷酸肌苷通过招募成孔蛋白效应器来协调大孔的形成，从而调节大分子从核内体的逃逸。在接下来的5年里，我们将确定磷酸肌苷如何协调内体孔形成，以促进交叉呈现的基本DC功能。我们的目标是：i)确定调节内体孔形成和交叉呈递的磷酸肌肽种类ii)确定介导内体孔形成和交叉呈递的蛋白质效应物iii)研究假设的孔形成蛋白APOL7C在内体孔形成中的作用除了dc，内体的大分子逃逸涉及许多细胞类型的关键过程，如炎症信号传导。因此，我们的NSERC DG将对从细胞生理学到有机体稳态等领域产生广泛的影响。我们正处于推动这些发现的理想位置，在这样做的过程中，我们将(i)对交叉呈现的生物学提供前所未有的见解，（ii）为磷酸肌苷及其效应物的研究提供工具箱，并提供（iii） HQP在加拿大学术界和工业界建立职业生涯的培训场所。

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.