Nanodelivery of functional proteins to phagosomal membranes

项目摘要 摘要细胞外小泡是一种携带跨膜蛋白的细胞膜结构。 以及腔内货物，包括可溶性细胞质蛋白质和核酸。他们是一个 异质颗粒组，通常根据亚细胞来源、尺寸和 Tetraspanin表面表达。循环中的电动汽车可以作为纳米容器，能够识别 靶细胞通过膜受体，通过膜融合传递货物，a 臭名昭著的低概率过程。与受体细胞的相互作用可导致它们获得 EV表面蛋白和腔内容物，尽管控制这种交换的机制仍然存在 不清楚。其复杂的货物和表面标志在以下方面变化很大 功能影响，并依赖于它们被释放的组织上下文。一个中环 拟议工作的重点是分离和鉴定具有功能能力的Small EVS(30-150 nm)携带感兴趣的离子通道，然后该离子通道可以被转移到幼稚的， 非表达细胞。功能显著的EV介导的通道从一个细胞转位到 另一种是在EV生物合成过程中每个小泡需要高密度的通道表达 供体细胞与细胞膜和内吞/吞噬间隔室的可靠融合 受体细胞。为了促进EV膜的融合，我们将共表达病毒蛋白FusoGen 血凝素亚型7(HA)。蓝光激活的基因工程钙离子载体的表达 响应光打开选择性钙离子通道的通道开关(BACCS)将是 用于筛选策略的设计，探索囊泡融合和通道转移。光-- 敏感的探针将作为无反应的功能性钙通道转移的筛选工具 通过从表达Baccs-ORAI的细胞中分离的EV获得受体细胞。一旦我们有了 建立了EV介导的渠道转移的最佳条件，我们将利用我们在 阴离子通道囊性纤维化跨膜电导调节剂的研究 小鼠肺泡巨噬细胞和上皮细胞及EV介导的CFTR转移的研究 在囊性纤维化(CF)疾病的背景下，使用过继转移技术将CFTR-/-细胞。

Project Summary Extracellular vesicles (EVs) are cell-derived membranous structures carrying transmembrane proteins and luminal cargo including soluble cytoplasmic proteins and nucleic acids. They are a heterogeneous group of particles generally classified according to subcellular origin, dimension, and tetraspanin surface expression. Circulating EVs may act as nanocontainers capable of recognizing target cells through membrane receptors and communicating cargo through membrane fusion, a notoriously low probability process. Interaction with recipient cells can lead to their acquisition of EV surface proteins and luminal contents although the mechanisms controlling this exchange remain unclear. Their complex cargo and well as surface markers are highly variable with respect to functional impact and dependent upon the tissue context from which they are released. A central focus of the proposed work is the isolation and identification of functionally competent small EVs (30-150 nm) carrying an ion channel of interest which can then be transferred to a naïve, non-expressing cell. Functionally significant EV-mediated channel translocation from one cell to another would require a high density of channel expression per vesicle during EV biosynthesis in the donor cell and reliable fusion with the plasma membrane and endocytic/phagocytic compartments of recipient cells. In order to facilitate EV membrane fusion we will co-express the viral protein fusogen hemagglutinin subtype 7 (HA). Expression of a genetically engineered blue light-activated Ca2+ channel switch (BACCS) which opens the Ca2+ selective ORAI ion channel in response to light will be used in the design of screening strategies exploring vesicle fusion and channel transfer. The light- sensitive probe will be used as screening tool for functional Ca2+ channel transfer in non-responsive recipient cells via isolated EVs derived from BACCS-ORAI expressing cells. Once we have established optimal conditions for EV-mediated channel transfer, we will leverage our experience in the study of the anion channel Cystic Fibrosis Transmembrane conductance Regulator (CFTR) in murine alveolar macrophages (AMs) and epithelial cells and study the EV-mediated transfer of CFTR to cftr-/- cells in the context of the disease of cystic fibrosis (CF) using adoptive transfer techniques.