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传导产生深远的影响 功能，包括细胞凋亡、吞噬作用、凝血、膜囊泡脱落、 骨矿化、细胞间融合、受精、病毒感染（包括 HIV 和 SARS-CoV2） 感染。然而，磷脂触发器如何导致观察到的细胞反应尚不清楚 很大程度上难以捉摸。磷脂扰乱酶和翻转酶的最新鉴定 能够对这些关键的磷脂转运蛋白进行基因操作，这极大地推进了 我们对磷脂触发器生物学的理解。然而，磷脂触发器是 动态过程和基因操作只能让我们观察到最终结果， 阻碍了对磷脂触发器在各种生理过程中的机制的理解 实时。在此应用中，我们的目标是测试开发基因编码的可行性， 光遗传学工具箱可在高时空光下精确控制磷脂触发器 分辨率和实时性。我们的建议是基于我们最近的丰富经验 在分子和技术上发现了钙激活磷脂扰乱酶（CaPLSase）TMEM16F 细胞水平。响应细胞内钙增加，TMEM16F 可以快速催化 磷脂触发器，有效破坏膜环境并引发广谱 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 控制磷脂触发器的光遗传学工具。首先，我们将共表达各种钙- 利用光遗传学工具利用其钙传感特性间接激活 TMEM16F。 其次，我们将光感应图案设计到 TMEM16F 中，以实现对其直接光控制 活动。如果成功，光遗传学工具箱将在这个高风险、高回报的领域开发出来。 应用将对膜生物学、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.