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

像通道视紫红质-2这样的遗传编码工具的开发使光的刺激能够控制神经元的膜去极化，引发了我们对大脑功能理解的一连串突破。虽然光作为一种刺激具有优势(例如，精确的空间和时间激活)，但它也有弱点(例如，组织渗透率低和侵入性动物手术)。此外，除了作为一种细胞功能的膜去极化外，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，以确定特定基因在调控下的功能。 我的最终研究目标是设计出基因编码的工具，使之能够在任何想要的刺激下控制任何哺乳动物细胞的功能。我们的团队认为，这是可能的，通过钙离子重新连接结合在所需刺激时产生钙信号的蛋白质与被钙信号激活时控制细胞功能的蛋白质结合在一起。 首先，在提案中，我们将开发基因编码工具，允许钙离子内流和膜去极化由磁场控制。磁场可以渗透到组织中，与基于光或电极的方法相比，无需手术即可刺激大脑深处的神经元。第二，我们将开发钙激活的半胱氨酸天冬氨酸酶。半胱氨酸蛋白酶是细胞凋亡过程中的中心半胱氨酸蛋白酶，其功能是在底物中有天冬氨酸残基后，通过裂解来分解细胞机械。通过将嵌合的caspase与一个嵌合的受体共表达，该嵌合受体会在结合其靶配体时产生钙信号，我们的目标是通过钙重新连接来诱导细胞在任何刺激下死亡。最后，我们将开发可诱导的基因表达系统。通过普遍存在的NFAT途径，钙离子振荡激活基因表达。通过与天然或合成的增强钙振荡的基因共表达嵌合受体，我们的目标是通过钙重新连接来诱导基因表达，以响应任何刺激。 用任何想要的刺激(如磁场)控制任何哺乳动物细胞功能(如钙离子内流、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提出的生物学问题。例如，磁场刺激将允许激活大脑深处的区域，如海马体，以探测关于记忆形成的问题。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，形态原的模式)进行定制，以探索它们在组织发育中的重要性。任何刺激下的基因表达都将扩大目前生物医学研究中可靠的可诱导启动子的匮乏。如果重新连接到磁场，我们可以进行有针对性的深层组织消融，并在大脑和脊髓中进行基因表达。

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.