Imaging 3' phosphoinositides in vivo

描述（由申请人提供）：目前对离体细胞内信号传导的理解与活动物或患者背景下的细胞内信号传导之间存在巨大差距。在3'磷酸肌醇（3' PI）代谢的研究中，这一差距最为显著。磷脂酰肌醇3 '-激酶（PI 3 K）及其合成的3' PI是控制细胞代谢、细胞周期进程、细胞凋亡和运动的调节网络的重要组成部分。通过用于3 'PI合成或降解的酶的突变，3' PI信号传导的失调有助于癌症和自身免疫性疾病;因此，PI 3 K的几种同种型是治疗药物的靶标。尽管3 'PI对人类健康很重要，但目前还不知道控制它们的多个信号输入如何在多细胞环境中整合。要解决这些重要问题，需要对活组织中细胞内的3 'PI进行成像的方法。虽然通过荧光显微镜观察表达荧光蛋白（FP）嵌合体和信号蛋白的3 'PI结合结构域的细胞，3' PI已经离体定位在活细胞中，但是缺乏原位监测活细胞中3 'PI水平的方法。这些障碍可以通过开发使用2-光子显微镜定位升高浓度的3 'PI的试剂和方法来消除，这上级用于成像组织和器官中的荧光的单光子显微镜方法。由于3 'PI在人类健康中的重要性，并且由于膜中的PI 3 K活化通常在细胞膜中产生瞬时的高浓度的3' PI，因此本工作的目的是开发在体内细胞中定位升高浓度的3 'PI的技术。这些研究将改进和扩展荧光显微镜方法，用于通过Forester共振能量转移（FRET）检测3 'PI结合FP嵌合体。中心假设是，细胞膜中局部增加的3 'PI浓度可以通过表达3' PI结合FP嵌合体的细胞的双光子FRET显微镜在体内成像，使用将局部升高的3 'PI浓度报告为二元信号的探针和方法。中心假设将通过解决三个具体目标进行测试，通过专门研究几个基本技术的调查人员的协调努力。目标1将开发用于双光子显微镜的3 'PI的比率报告基因，适应现有的用于共表达的3' PI结合FP嵌合体和游离FP的比率定位的方法。Aim 2将开发基于FRET的3 'PI报告基因。我们的工作假设是，3 'PI可以通过表达3' PI结合FP嵌合体的细胞的FRET显微镜定位。我们将鉴定在膜上达到足够高的浓度以产生FRET而没有来自直接激发的受体FP的信号交叉的嵌合体，从而检测作为二元（开/关）信号的阈上3 'PI水平。Aim 3将在体内对3 'PI成像。通过双光子显微镜成像细胞将在鼠淋巴结制备物中进行优化，使用表达FP的细胞或由校准的、连接的供体和受体FP组成的FRET标准品。在树突细胞中表达的3 'PI特异性FRET探针将通过双光子显微镜成像以在原位趋化性期间定位PI 3 K信号传导。该项目的预期成果将是获得成像和分析体内细胞内PI 3 K依赖性信号传导所需的对比度的新型试剂和方法。它的主要影响是，它将改变目前的体内研究， 状态，通过跟踪细胞运动来推断机制，到分析细胞如何整合影响其在体内行为的多个信号输入的机制。体内细胞内的成像信号对于理解免疫反应和肿瘤中癌细胞的动力学将是重要的。

DESCRIPTION (provided by applicant): There is a huge gap between current understanding of intracellular signaling ex vivo and intracellular signaling in the context of the living animal r patient. Nowhere is this gap more striking than in the study of 3' phosphoinositide (3'PI) metabolism. Phosphoinositide 3'-kinases (PI3K) and the 3'PIs they synthesize are essential components of the regulatory networks controlling cellular metabolism, cell cycle progression, apoptosis and movement. The dysregulation of 3'PI signaling, through mutations of enzymes for 3'PI synthesis or degradation, contributes to cancer and autoimmune diseases; hence, several isoforms of PI3K are targets of therapeutic drugs. Despite the importance of 3'PIs for human health, it is not known how multiple signaling inputs controlling them are integrated in the context of the multicellular environment. Addressing these important questions will require methods for imaging 3'PIs inside cells in living tissues. Although 3'PIs have been localized in living cells ex vivo by fluorescence microscopy of cells expressing chimeras of fluorescent proteins (FP) and 3'PI-binding domains of signaling proteins, methods to monitor 3'PI levels in living cells in situ are lacking. These obstacles could be removed by the development of reagents and methods which localize elevated concentrations of 3'PIs using 2-photon microscopy, which is superior to single photon microscopic methods for imaging fluorescence in tissues and organs. Because of the importance of 3'PIs in human health, and because PI3K activation in membranes often produces transient, high concentrations of 3'PIs in cell membranes, the objective of the present work is to develop technologies which localize elevated concentrations of 3'PIs in cells in vivo. These studies will improve and extend fluorescence microscopic methods for detecting 3'PI- binding FP chimeras by Forester resonance energy transfer (FRET). The central hypothesis is that locally increased 3'PI concentrations in cellular membranes can be imaged in vivo by 2-photon FRET microscopy of cells expressing 3' PI-binding FP chimeras, using probes and methods which report locally elevated 3'PI concentrations as binary signals. The central hypothesis will be tested by addressing three specific aims, through the coordinated efforts of investigators specializing in several essential technologies. Aim 1 will develop ratiometric reporters of 3'PIs for 2-photon microscopy, adapting existing methods for ratiometric localization of 3'PI-binding FP chimeras co-expressed and free FPs. Aim 2 will develop FRET-based reporters of 3'PIs. Our working hypothesis is that 3'PIs can be localized by FRET microscopy of cells expressing 3'PI-binding FP chimeras. We will identify chimeras which attain sufficiently high concentrations on membranes to produce FRET without signal crossover from directly excited acceptor FPs, thereby detecting suprathreshold 3'PI levels as binary (on/off) signals. Aim 3 will image 3'PIs in vivo. Imaging cells by 2-photon microscopy will be optimized in murine lymph node preparations, using cells expressing FPs or FRET standards consisting of calibrated, linked donor and acceptor FPs. 3'PI-specific FRET probes expressed in dendritic cells will be imaged by 2-photon microscopy to localize PI3K signaling during chemotaxis in situ. The expected outcomes of this project will be novel reagents and methods which obtain the contrast necessary to image and analyze PI3K-dependent signaling inside cells in vivo. Its major impact is that it will transform in vivo studies from their current state, which infers mechanism through the tracking of cell movements, to a mechanism for analyzing how cells integrate multiple signal inputs affecting their behavior in the body. Imaging signals inside cells in vivo will be important for understanding immune responses and the dynamics of cancer cells in tumors.