Genetically encoded tools to control any mammalian cell function with any desired stimulus

像通道视紫红质-2这样的基因编码工具的发展，使得光的刺激能够控制神经元的膜去极化，在我们对大脑功能的理解上引发了源源不断的突破。虽然光作为一种刺激有优点（例如精确的空间和时间激活），但它也有缺点（例如低组织穿透性和侵入性动物手术）。此外，除了作为细胞功能的膜去极化之外，生物学家还寻求控制细胞死亡以确定特定细胞类型的生物学作用，并控制基因表达以确定受调节的特定基因的功能。******我的最终研究目标是设计基因编码工具，使任何哺乳动物细胞功能的控制与任何期望的刺激。我们的研究小组认为，这是可能的，通过Ca2+重新布线结合蛋白质产生Ca2+信号结合所需的刺激与蛋白质控制细胞功能时，Ca2+信号激活。******首先在提案中，我们将开发遗传编码工具，以允许Ca2+内流和膜去极化由磁场控制。磁场可穿透组织，与基于光或电极的方法相比，无需手术就能刺激大脑深处的神经元。其次，我们将开发Ca2+激活的半胱天冬酶。半胱天冬酶是凋亡细胞死亡中的中心半胱氨酸蛋白酶，其作用是在底物中有天冬氨酸残基后裂解细胞机制。通过与嵌合受体共表达嵌合caspase，嵌合受体在结合其靶配体时产生Ca2+信号，我们的目标是通过Ca2+重新布线诱导细胞死亡以响应任何刺激。最后，我们将开发可诱导的基因表达系统。通过无处不在的NFAT途径，Ca2+振荡激活基因表达。通过与增强Ca2+振荡的天然或合成基因共表达嵌合受体，我们的目标是通过Ca2+重新布线诱导基因表达以响应任何刺激。******用任何期望的刺激（例如磁场）控制任何哺乳动物细胞功能（例如Ca2+内流，细胞死亡和基因表达）的能力将使我们能够提出目前不可能的生物学问题。例如，磁场刺激可以激活大脑深处的区域，如海马体，以探索有关记忆形成的问题。将细胞消融与任何刺激重新连接，将允许细胞死亡根据微环境（例如形态因子的模式）进行定制，以探索它们在组织发育中的重要性。将基因表达与任何刺激重新连接起来，将扩大目前用于生物医学研究的可靠诱导启动子的匮乏。如果重新连接到磁场上，我们就可以在大脑和脊髓中进行有针对性的深层组织消融和基因表达。

The development of genetically encoded tools like channelrhodopsin-2 has allowed the stimuli of light to control membrane depolarization of neurons, sparking a steady stream of breakthroughs in our understanding of brain function. While light as a stimuli has strengths (e.g. precise spatial and temporal activation), it also has weaknesses (e.g. low tissue penetration and invasive animal surgery). Furthermore, other than membrane depolarization as a cell function, biologists have sought control over cell death to determine the biological role of the particular cell types and control over gene expression to determine the function of particular genes under regulation. ******My ultimate research objective is to engineer genetically encoded tools that will allow the control of any mammalian cell function with any desired stimuli. Our group believes this is possible through Ca2+ rewiring combining proteins that generate Ca2+ signals upon binding desired stimuli with proteins that control cell function when activated by Ca2+ signals.******First in the proposal, we will develop genetically-encoded tools to allow Ca2+ influx and membrane depolarization to be controlled by magnetic fields. Magnetic fields are permeable through tissues that will allow neurons to be stimulated deep inside the brain with no surgery in contrast with light-based or electrode-based approaches. Second, we will develop Ca2+-activated caspases. Caspases are central cysteine proteases in the apoptotic cell death that function to dismantle the cell machinery by cleaving after an aspartate residue in their substrate. By co-expressing the chimeric caspase with a chimeric receptor that generates a Ca2+ signal in response to binding its target ligand, we aim to induce cell death in response to any stimuli via Ca2+ rewiring. Lastly, we will develop inducible gene expression systems. Through the ubiquitous NFAT pathway, Ca2+ oscillations activate gene expression. By co-expressing a chimeric receptor with natural or synthetic genes that enhance Ca2+ oscillations, we aim to induce gene expression in response to any stimuli via Ca2+ rewiring.******The ability to control any mammalian cell function (e.g. Ca2+ influx, cell death and gene expression) with any desired stimuli (e.g. magnetic fields) would allow us to ask biological questions that are currently not possible. For example, magnetic field stimuli would allow activation of the areas deep inside the brain such as the hippocampus to probe questions about the formation of memories. Cell ablation rewired to any stimuli would allow cell death to be customized to the microenvironment (e.g. patterns of morphogens) to probe questions about their importance in development of tissues. Gene expression rewired to any stimuli would expand the current paucity of reliable inducible promoters for biomedical research. If rewired to magnetic fields, we could perform targeted deep tissue ablation and gene expression in the brain and spinal cord.