Optogenetic stimulation of TMEM16F to control phospholipid flip-flop

摘要 细胞膜上的磷脂触发器可以对细胞信号和 功能，包括细胞凋亡，吞噬，凝血，膜泡脱落， 骨矿化、细胞-细胞融合、受精、包括HIV和SARS-CoV2在内的病毒感染 感染。然而，磷脂是如何导致观察到的细胞反应的 在很大程度上是难以捉摸的。最近对磷脂扰乱酶和翻转酶的鉴定 能够对这些关键的磷脂转运蛋白进行基因操作，这大大促进了 我们对磷脂触发器生物学的理解。然而，磷脂触发器是一种 动态过程和遗传操作只允许我们观察最终结果，这 阻碍理解磷脂触发器在各种生理过程中的作用机制 实时的。在这项应用中，我们的目标是测试开发一种基因编码的 高空时光精密控制磷脂触发器的光遗传工具箱 分辨率和实时。我们的建议是基于我们最近的丰富经验 发现钙激活磷脂加扰酶(CaPLSase)TMEM16F在分子和 细胞水平。TMEM16F能迅速催化细胞内钙离子的升高 磷脂触发器，有效地扰乱膜环境，触发广泛的 细胞变化。在这里，我们将使用两种互补但独立的方法来开发 控制磷脂触发器的光遗传工具。首先，我们将共同表达各种钙- 利用TMEM16F的钙敏感特性，动员光遗传工具间接激活TMEM16F。 其次，我们将在TMEM16F中设计光敏图案，以实现对其光的直接控制 活动。如果成功，光遗传工具箱开发出来的这种高风险、高回报 应用将在膜生物学、细胞生物学、 生理学、血液学、免疫学、病毒学和医学。

SUMMARY Phospholipid flip-flop on cell membranes can exert profound impacts on cellular signaling and functions, including apoptosis, phagocytosis, blood coagulation, membrane vesicle shedding, bone mineralization, cell-cell fusion, fertilization, viral infection including HIV and SARS-CoV2 infections. Nevertheless, how phospholipid flip-flop leads to the observed cellular responses is largely elusive. The recent identifications of phospholipid scramblases and flippases have enabled genetic manipulations of these critical phospholipid transporters, which greatly advanced our understanding on the biology of phospholipid flip-flop. However, phospholipid flip-flop is a dynamic process and the genetic manipulations only allow us to observe the end results, which hinders gaining mechanistic understanding phospholipid flip-flop in various physiological process in real time. In this application, we aim to test the feasibility of developing a genetically encoded, optogenetic toolbox to precisely control phospholipid flip-flop with light at high temporal and spatial resolution and in real time. Our proposal is based on our extensive experience on the recently discovered calcium-activated phospholipid scramblase (CaPLSase) TMEM16F at molecular and cellular levels. In response to intracellular calcium increase, TMEM16F can rapidly catalyze phospholipid flip-flop, efficiently disrupt membrane environment and trigger wide spectrum of cellular changes. Here, we will use two complementary but independent approaches to develop the optogenetic tools to control phospholipid flip-flop. First, we will coexpress various calcium- mobilizing optogenetic tools to indirectly activate TMEM16F utilizing its calcium sensing property. Second, we will engineer light sensing motifs into TMEM16F to enable direct light control of its activities. If successful, the optogenetic toolbox developed in this high-risk, high-reward application will have profound impacts and broad applications in membrane biology, cell biology, physiology, hematology, immunology, virology and medicine.