Optogenetic stimulation of TMEM16F to control phospholipid flip-flop

总结 细胞膜上的磷脂翻转可以对细胞信号传导产生深远的影响， 功能，包括细胞凋亡，吞噬作用，血液凝固，膜囊泡脱落， 骨矿化、细胞-细胞融合、受精、病毒感染（包括HIV和SARS-CoV 2） 感染.尽管如此，磷脂的翻转如何导致观察到的细胞反应， 很难捉摸最近发现的磷脂乱序酶和翻转酶， 能够对这些关键的磷脂转运蛋白进行基因操作， 我们对磷脂翻转生物学的理解。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， 阻碍了对各种生理过程中磷脂翻转机制的理解 在真实的时间里。在这个应用中，我们的目标是测试开发一种基因编码的， 光遗传学工具箱，以精确控制磷脂触发器与光在高时空 分辨率和真实的时间。我们的建议是根据我们最近在 在分子水平上发现了钙激活磷脂乱序酶（CaPLSase）TMEM 16 F， 细胞水平。响应于细胞内钙的增加，TMEM 16 F可以快速催化 磷脂触发器，有效地破坏膜环境，并触发广谱 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 控制磷脂翻转的光遗传学工具。首先，我们将共表达各种钙- 调动光遗传学工具以利用其钙敏感特性间接激活TMEM 16 F。 其次，我们将在TMEM 16 F中设计光传感图案，以实现其直接光控制。 活动如果成功，光遗传学工具箱在这个高风险，高回报的 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.