Perforin-2 is a pore-forming effector of endocytic escape in cross-presenting dendritic cells.

During initiation of antiviral and antitumor T cell-mediated immune responses, dendritic cells (DCs) cross-present exogenous antigens on major histocompatibility complex (MHC) class I molecules. Cross-presentation relies on the unusual "leakiness" of endocytic compartments in DCs, whereby internalized proteins escape into the cytosol for proteasome-mediated generation of MHC I-binding peptides. Given that type 1 conventional DCs excel at cross-presentation, we searched for cell type-specific effectors of endocytic escape. We devised an assay suitable for genetic screening and identified a pore-forming protein, perforin-2 (Mpeg1), as a dedicated effector exclusive to cross-presenting cells. Perforin-2 was recruited to antigen-containing compartments, where it underwent maturation, releasing its pore-forming domain. Mpeg1-/- mice failed to efficiently prime CD8+ T cells to cell-associated antigens, revealing an important role for perforin-2 in cytosolic entry of antigens during cross-presentation.

To Kill But Not Be Killed: Controlling the Activity of Mammalian Pore-Forming Proteins.

Pore-forming proteins (PFPs) are present in all domains of life, and play an important role in host-pathogen warfare and in the elimination of cancers. They can be employed to deliver specific effectors across membranes, to disrupt membrane integrity interfering with cell homeostasis, and to lyse membranes either destroying intracellular organelles or entire cells. Considering the destructive potential of PFPs, it is perhaps not surprising that mechanisms controlling their activity are remarkably complex, especially in multicellular organisms. Mammalian PFPs discovered to date include the complement membrane attack complex (MAC), perforins, as well as gasdermins. While the primary function of perforin-1 and gasdermins is to eliminate infected or cancerous host cells, perforin-2 and MAC can target pathogens directly. Yet, all mammalian PFPs are in principle capable of generating pores in membranes of healthy host cells which-if uncontrolled-could have dire, and potentially lethal consequences. In this review, we will highlight the strategies employed to protect the host from destruction by endogenous PFPs, while enabling timely and efficient elimination of target cells.

Characterisation of GLUT4 trafficking in HeLa cells: comparable kinetics and orthologous trafficking mechanisms to 3T3-L1 adipocytes.

Insulin-stimulated glucose transport is a characteristic property of adipocytes and muscle cells and involves the regulated delivery of glucose transporter (GLUT4)-containing vesicles from intracellular stores to the cell surface. Fusion of these vesicles results in increased numbers of GLUT4 molecules at the cell surface. In an attempt to overcome some of the limitations associated with both primary and cultured adipocytes, we expressed an epitope- and GFP-tagged version of GLUT4 (HA-GLUT4-GFP) in HeLa cells. Here we report the characterisation of this system compared to 3T3-L1 adipocytes. We show that insulin promotes translocation of HA-GLUT4-GFP to the surface of both cell types with similar kinetics using orthologous trafficking machinery. While the magnitude of the insulin-stimulated translocation of GLUT4 is smaller than mouse 3T3-L1 adipocytes, HeLa cells offer a useful, experimentally tractable, human model system. Here, we exemplify their utility through a small-scale siRNA screen to identify GOSR1 and YKT6 as potential novel regulators of GLUT4 trafficking in human cells.