Force Sensing with Nanotubes

描述(申请人提供)：大多数人类恶性肿瘤起源于上皮源性。转化的上皮细胞从原发肿瘤部位扩散，并通过产生移行结构(如丝足、内足和板足)侵入周围组织。移行结构的相对丰度与肿瘤细胞的转移潜能有关。移行结构是通过重塑转化的上皮细胞前沿的肌动蛋白而形成的。生长因子(如表皮生长因子，EGF)通过靶向转化细胞质膜上的受体(如表皮生长因子受体)启动肌动蛋白重塑。虽然EGF途径有很好的特征，但我们缺乏实验证据来量化有助于迁移的力，包括粘附性牵引、阻性粘性阻力和突出力。这项工作将专注于发生在活细胞前沿的突出力。当肌动蛋白聚合释放的化学能对质膜产生推动力时，它们就会产生，这是由于相对于生物聚合物，溶液中单体肌动蛋白的浓度更高。当肌动蛋白束附着在质膜上时，F-肌动蛋白的解聚会对质膜产生拉力。这种反向化学反应是由主体中相对于生物聚合物的较低浓度的单体推动的。鲜有实验测量活细胞内推力的大小和时间过程，也没有测量F-肌动蛋白解聚产生的拉力。这项工作的目标是确定F-肌动蛋白束快速增长末端的推力和拉力的时间进程和大小。我们将使用膜作为传感器来确定癌细胞运动水平上的力。我们将通过EGF或EGF途径中的活性效应分子刺激转化的上皮细胞形成迁移结构。我们将使用光学镊子来测量力量，荧光显微镜来成像F-肌动蛋白，并开发方法学来同时测量两者。这项工作将提供转化的上皮细胞前沿的肌动蛋白马达的功能模型，并通过测量一个前沿的突出力来推动该领域对癌症侵袭阶段细胞迁移的理解。这将为研究这种化学马达的转导机制提供定量的实验方法，并从根本上深入了解细胞膜和肌动蛋白马达的整合操作。这项工作将为设计用于纳米技术的力传感器提供基础。

DESCRIPTION (provided by applicant): Most human malignancies are of epithelial origin. Transformed epithelial cells spread from the primary tumor site and invade surrounding tissues through the production of migratory structures (e.g., filopodia, invadopodia, and lamellapodia). The relative abundance of migratory structures is correlated with the metastatic potential of tumor cells. Migratory structures form by remodeling actin at the leading edge of transformed epithelial cells. Growth factors (e.g., epidermal growth factor, EGF) initiate actin remodeling by targeting their receptors (e.g., epidermal growth factor receptor) on the plasma membrane of transformed cells. Although the EGF pathway is well characterized, we lack experimental evidence to quantify forces that contribute to migration, including adhesive traction, resistive viscous drag, and protrusive forces. This work will focus on protrusive forces that occur at the leading edge of a living cell. They arise when the chemical energy released upon actin polymerization produces a pushing force against the plasma membrane, driven by the greater concentration of monomeric actin in the solution relative to the biopolymer. When an actin bundle is attached to the plasma membrane, depolymerization of F-actin produces a pulling force on the membrane. This reverse chemical reaction is driven by a lower concentration of monomer in the bulk relative to the biopolymer. Experimental measurements of the magnitude and time course of the pushing force in living cells are lacking and there are no measurements of the pulling force arising from the depolymerization of F-actin. The goal of this work is to determine the time course and magnitude of the pushing and pulling forces at the fast-growing end of an F-actin bundle. We will use the membrane as a sensor to determine the force at the motor-level in cancer cells. We will stimulate transformed epithelial cells to form migratory structures by EGF or with active effectors within the EGF pathway. We will use optical tweezers to measure the force, fluorescence microscopy to image F- actin, and develop methodology to measure both simultaneously. This work will provide a functional model of the actin motor at a leading edge of transformed epithelial cells, and advance the field in understanding cell migration during the invasive stage of cancer by providing measurements of the protrusive force at one leading edge. It will provide a quantitative experimental method to investigate the transduction machinery of this chemical motor, and fundamental insight into the integrated operations of the cell membrane and actin motor. This work will provide a basis to design force sensors for applications in nanotechnology.