Cells release extracellular vesicles (ECVs) that can act as signaling platforms to modulate the strength of an immune response, influence differentiation during development, promote the coagulation of platelets, or induce the metastasis of tumor cells. Many ECVs form by budding outwards from the plasma membrane, but the molecular mechanisms that regulate external vesicle budding are unknown. It has been observed that the outer leaflet of the membrane bilayer of ECVs contains lipids that are normally sequestered to the inner leaflet of the plasma membrane, leading to the hypothesis that disruption of lipid asymmetry might have a role in ECV formation. However, a molecular link between lipid asymmetry and ECV budding has not been found. In preliminary data, we identify a potential molecular link between lipid asymmetry and ECV budding by showing that loss of the conserved lipid flippase TAT-5 causes the large-scale budding of ECVs in C. elegans embryos. Flippases maintain lipid asymmetry by transferring specific lipids from the outer leaflet of the membrane bilayer to the inner leaflet. Loss of TAT-5 results in excess exposure of the lipid phosphatidylethanolamine (PE) on cell surfaces, thereby correlating loss of PE asymmetry with an increase in vesicle budding. We also show that known regulators of viral budding are required for ECV budding induced by loss of TAT-5, suggesting that budding occurs via conserved mechanisms. The goals of this proposal are to determine what factors work in opposition to TAT-5 to induce vesicle budding, and to test the hypothesis that PE externalization causes ECV budding. In preliminary data, we show that proteins known to regulate viral budding (RAB-11 and the ESCRT complex) also regulate ECV budding in C. elegans embryos.
In Aim 1, we test how RAB-11 and the ESCRT complex regulate ECV budding.
This aim will expand our understanding of how RAB-11 and the ESCRT complex are recruited to the plasma membrane to form and release buds.
In Aim 2, we test whether any lipid scramblases that disrupt lipid asymmetry also induce ECV budding. Scramblases act in opposition to flippases and disrupt lipid asymmetry by randomizing lipid localization between the bilayers. The identification of a scramblase that regulates ECV production would provide further support for the hypothesis that loss of lipid asymmetry induces ECV budding. Finally, in Aim 3, we alter PE levels (via disruption of lipid biosynthetic pathways) to test the hypothesis that PE regulates ECV budding. In summary, the proposed research will test whether TAT-5 directly links PE asymmetry to the dynamic budding of vesicles from the plasma membrane. This work provides the groundwork for future studies determining whether mammalian cells use the same molecular mechanisms to regulate ECV budding during blood clotting, the immune response, and metastasis. Because these mechanisms may also be co-opted by viruses as they bud from the host plasma membrane, our studies may also reveal new antiviral targets.
Cells can communicate with each other via released extracellular vesicles. No proteins are currently known to regulate extracellular vesicle budding out from the cell surface. Understanding how extracellular vesicles bud may provide important diagnostic and therapeutic insights into immune responses, blood clotting disorders, metastatic cancer, and viral spread.