A rapid, reversible switch for controlling intracellular trafficking

描述（由申请人提供）：很少有技术可以快速和可逆地操纵活细胞内的成分；此外，能够这样做的工具的开发将大大提高科学家理解生物学和治疗疾病的能力。这项研究的长期目标是设计新的分子工具，可以在生物环境（如细胞）内组装和操作。为了实现这一目标，本提案描述了对环境响应多肽（ERPs）相变的研究。假设是erp可以隔离细胞内的关键因子，并且它们可以可逆地打开和关闭活细胞内的分子途径。为了证明这种方法的可行性，erp将被设计用于控制一种普遍存在的细胞过程，称为网格蛋白介导的内吞作用。这一过程在包括癌症和感染在内的许多疾病的调节中都很重要。提出了以下具体目标：1)ERP开关的生物合成和生物物理化学：将使用生物物理方法来表征ERP和溶液中ERP融合的行为，并开发数学模型，使这些系统能够设计为响应任何所需的温度。erp的细胞内行为将在产生erp的人类细胞内观察到，这些细胞融合到荧光蛋白中，可以在显微镜下观察。使用这个结构，我们将评估erp在细胞内自我关联的速度。描述影响胞内ERP相变的参数的相图将与自由溶液中的相图进行比较。2)网格蛋白介导内吞作用的ERP开关：ERP将融合到参与细胞环境中因子摄取的关键蛋白上。这个研究项目的目的是在有选择性地停止细胞通路的一般策略中达到高潮。这一建议在三个主要方面是创新的：(i)这里提出的特定ERP行为从未在活的人类细胞内观察到；（ii）该方法有望成为一种快速、可逆的技术，可以潜在地开启或关闭特定的细胞通路；（iii）这种方法可以推广到任何已知蛋白质相互作用的细胞途径。这种方法的成功演示旨在改变如何进行细胞生物学研究的范式，使对疾病治疗至关重要的生物过程的精确操纵成为可能。

DESCRIPTION (provided by applicant): There are few techniques that can both rapidly and reversibly manipulate components inside living cells; furthermore, the development of tools capable of doing so will significantly enhance the ability of scientists to understand biology and treat disease. The long-term goal of this research is to engineer new molecular tools that can be assembled and operated inside biological environments such as the cell. Towards this goal, this proposal describes an investigation of the phase transition of environmentally-responsive polypeptides (ERPs). The hypothesis is that ERPs can sequester key factors inside cells and that they can reversibly switch on and off molecular pathways inside live cells. To demonstrate the feasibility of this approach, ERPs will be designed to control a ubiquitous cellular process called clathrin-mediated endocytosis. This process is important in the regulation of many diseases, including cancer and infection. The following specific aims are proposed: 1) Biosynthesis and biophysical chemistry of ERP switches: A biophysical approach will be used to characterize the behavior of ERPs and ERP-fusion in solution and mathematical modeling will be developed that permits these systems to be designed to response to any desired temperature. The intracellular behavior of ERPs will be observed inside human cells that produce ERPs fused to a fluorescent protein that can be viewed under a microscope. Using this construct, we will evaluate how quickly the ERPs self-associate inside the cell. A phase diagram describing the parameters that influence the intracellular ERP phase transition will be compared to that in free solution. 2) ERP switching of clathrin-mediated endocytosis: ERPs will be fused to a key protein involved with the uptake of factors in the cellular environment. This research project is intended to culminate in a general strategy for selectively halting cellular pathways. This proposal is innovative in three main ways: (i) the specific ERP behavior proposed here has never been observed inside of live human cells; (ii) this approach is expected to be a rapid, reversible technique that can potentially switch on or off specific cell pathways; and (iii) this approach can be generalized to target any cellular pathway for which a known protein interacts. The successful demonstration of this approach is intended to shift the paradigm for how cellular biology studies are performed, enabling precise manipulation of biological processes that are fundamentally important to the treatment of disease. PUBLIC HEALTH RELEVANCE: Understanding the process by which diseases, such as cancer or infection, proceed at a cellular level is critical to the development of new treatments. This proposal describes the exploration of a novel, enabling technology that is intended to rapidly turn on and off critical cellular process involved with disease. Success of this project will catalyze numerous future studies of many disease processes and culminate with improved treatments for illnesses.