Multiplex Imaging of Signaling Pathways in Cell Motility

细胞运动信号通路的多重成像

• 批准号: 10681274
• 负责人: Louis Hodgson
• 金额: $ 64.07万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10681274
• 关键词: Area; Biology; Biosensor; Cells; Cellular biology; Chemotaxis; Data; Development; Disease; Fibroblasts; Fluorescence Resonance Energy Transfer; Guanosine Triphosphate Phosphohydrolases; Health; Human; Human body; Imaging Device; Immune; Inflammatory; Invaded; Macrophage; Modality; Molecular; Neoplasm Metastasis; Normal Cell; Pathway interactions; Process; Proteins; Role; Signal Pathway; Signal Transduction; Signaling Protein; System; Techniques; Technology; Time; Visualization; cell motility; chemokine; design; imaging approach; malignant breast neoplasm; migration; molecular imaging; monomer; multiplexed imaging; neoplastic cell; novel; optogenetics; response; rho GTP-Binding Proteins; temporal measurement; tool; tumor

Abstract: Fluorescent biosensor techniques in cell biology now allow for the real-time interrogation of molecular processes as they occur inside living cells at spatial and temporal resolutions of microns and seconds, respectively. I have a prior and continued focus on the development of novel Förster resonance energy transfer (FRET)- based biosensor technologies that utilize monomeric fluorescent proteins for exceptional sensitivity and probe reversibility. In addition, a single-chain construction is used to facilitate quantitative analysis. I recently pioneered the near-infrared (NIR)-FRET biosensor modality, which included the first simultaneous, orthogonal visualization of cyan-yellow fluorescent protein-based FRET and NIR-FRET biosensors in single living cells. The resulting data were the first true multiplex analysis of two important molecular switches in living cells, the Rac1 and RhoA GTPases. This analysis revealed the direct coordination of these GTPases during cell migration in real-time. Herein, I propose to study the coordination of Rho GTPases associated with important signaling pathways by designing new biosensors for specific signaling nodes and utilizing the direct multiplex FRET imaging approach. Specifically, I will first target the local-level coordination of Rho GTPase signaling in fibroblast cells during migration, chemotaxis, and directional guidance. The coordination of RhoA versus Rac1 GTPases in fibroblasts will be investigated by determining the role of a downstream target protein, the formin mDia1, which is hypothesized to coordinate RhoA and Rac1 during cell motility. The direct multiplex imaging approach will be used to evaluate pairwise biosensor signals. In addition, the RhoA and Rac1 pathways will be perturbed with optogenetic tools to determine the GTPase coordination that is important for controlling cellular morphodynamics. Next, these approaches will be applied to two systems that have important implications for human health and disease. First, macrophage motility and directional guidance, which requires the coordination of Rho GTPases during the chemotactic response to inflammatory chemokines, will be studied. Then, the multiplex imaging and perturbation approaches will be applied to breast cancer invasion and migration, which are critical to controlling tumor metastasis. Collectively, the coordination between Rho GTPases and the associated molecular signaling that governs cell motility will be identified through the development of new biosensors that enable direct multiplex probing of signaling networks.

抽象的： 细胞生物学中的荧光生物传感器技术现在允许对分子过程进行实时询问 当它们发生在活细胞内部，分别是微米和秒的空间和临时分辨率。 在新型Förster共振能量传递（FRET）的发展上有先验和继续关注 - 利用单体荧光蛋白具有特殊敏感性并证明的基于生物传感器的技术 可逆性。此外，单链结构用于促进定量分析。我最近开创了开创性 近红外（NIR） - fret生物传感器模态，其中包括第一个同时可视化 单个活细胞中的青色黄色荧光蛋白基和NIR-FRET生物传感器的组合。结果 数据是活细胞中两个重要分子开关的第一个真实多重分析，Rac1和RhoA GTPases。该分析揭示了这些GTPase在细胞迁移期间的直接配位。 在此，我建议研究与重要信号通路相关的Rho GTPases的协调 通过为特定信号节点设计新的生物传感器并使用直接的多重FRET成像 方法。具体而言，我将首先针对成纤维细胞中Rho GTPase信号的局部级别配位 在迁移，趋化性和方向指导期间。 Rhoa与Rac1 GTPases的协调 成纤维细胞将通过确定下游靶蛋白的作用，formin mdia1来研究成纤维细胞。 假设在细胞运动过程中协调RhoA和Rac1。直接的多重成像方法将是 用于评估成对生物传感器信号。此外，Rhoa和Rac1途径将受到干扰 确定GTPase配位的光遗传学工具对于控制细胞形态动力学很重要。 接下来，这些方法将应用于两个对人类健康具有重要意义的系统和 疾病。首先，巨噬细胞的运动性和定向指导，需要协调Rho GTPases 在对炎性趋化因子的趋化反应期间，将研究。然后，多重成像和 扰动方法将应用于乳腺癌的侵袭和迁移，这对于控制至关重要 肿瘤转移。总体而言，Rho GTPases与相关的分子信号之间的协调 控制细胞运动的情况将通过开发新的生物传感器来识别，这些生物传感器能够直接多重 探测信号网络。

项目成果

StarD13 negatively regulates invadopodia formation and invasion in high-grade serous (HGS) ovarian adenocarcinoma cells by inhibiting Cdc42.

• DOI: 10.1016/j.ejcb.2021.151197
• 发表时间: 2022-01
• 期刊: European journal of cell biology
• 影响因子: 6.6
• 作者: Abdellatef S;Fakhoury I;Haddad MA;Jaafar L;Maalouf H;Hanna S;Khalil B;El Masri Z;Hodgson L;El-Sibai M
• 通讯作者: El-Sibai M

Metalloprotease ADAMTS-1 decreases cell migration and invasion modulating the spatiotemporal dynamics of Cdc42 activity.

• DOI: 10.1016/j.cellsig.2020.109827
• 发表时间: 2021-01
• 期刊: Cellular signalling
• 影响因子: 4.8
• 作者: de Assis Lima M;da Silva SV;Serrano-Garrido O;Hülsemann M;Santos-Neres L;Rodríguez-Manzaneque JC;Hodgson L;Freitas VM
• 通讯作者: Freitas VM

Spatial and temporal dynamics of RhoA activities of single breast tumor cells in a 3D environment revealed by a machine learning-assisted FRET technique.

• DOI: 10.1016/j.yexcr.2021.112939
• 发表时间: 2022-01-15
• 期刊: Experimental cell research
• 影响因子: 3.7
• 作者: Cheung BCH;Hodgson L;Segall JE;Wu M
• 通讯作者: Wu M

Multiplex Imaging of Rho GTPase Activities in Living Cells.

• DOI: 10.1007/978-1-0716-1593-5_4
• 发表时间: 2021
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Bhalla RM;Hülsemann M;Verkhusha PV;Walker MG;Shcherbakova DM;Hodgson L
• 通讯作者: Hodgson L