Magnetic quantum dots for active sensing of the nuclear envelope

说明(申请人提供)：发光半导体纳米晶(量子点，量子点)是一种具有优异材料和光学性能的无机荧光团纳米颗粒，包括高量子产率、窄发射和宽激发光谱、极高的光漂白阈值以及高抗光和化学降解。尽管量子点在生物学上的应用引起了广泛的兴趣，但量子点在很大程度上局限于体外传感器的应用，固定组织和细胞的染色，以及作为体内的简单标记。量子点领域面临的关键挑战包括主动操纵、活组织和细胞中的高空间和时间分辨率成像、可控的跨生物膜的量子点转移以及针对细胞器的特异性靶向。我们提出的研究的总体目标是通过开发一类新的磁性量子点(MQD)和新型MQD-蛋白质结合物来直接应对这些挑战。多量子点结合了半导体纳米晶体的独特性质和磁性粒子的主动操纵能力。我们建议使用所得到的多量子点并开发多量子点-蛋白质结合物，以获得在生物膜科学和核包膜微观力学方面的新的和重要的见解。该提案的具体目标是：具体目标1.开发和测试多量子点。我们将开发和制造用于活细胞应用的表面工程多功能多量子点。具体目标2.用已知生化组成和物理性质的模型膜系统测试多量子点。用核膜(NE)脂类和核膜等NE蛋白重建核膜模型。利用多量子点探测这些模拟体系的渗透性。具体目的3.将多量子点注入细胞核，探索活细胞去甲肾上腺素的微观机制。我们将开发一种新的纳米粒子弹道轰击系统，以确保多量子点同时高速率转移到培养中的多个细胞。我们将使用外加磁场来探测气囊注入的多量子点在细胞质和核质中的比例，以及局部去甲肾上腺素的形变随着施加在多量子点上的力的函数。3.我们将研究层蛋白A/C的缺失或致病突变是否会影响NE的微力学。3D。我们将研究肌动蛋白和微管网络是否以及如何影响NE的机械特性。

DESCRIPTION (provided by applicant): Luminescent semiconductor nanocrystals (quantum dots, QDs) are inorganic fluorophore nanoparticles with superior materials and optical properties, including high quantum yield, narrow emission and broad excitation spectra, extremely high photobleaching thresholds, and high resistance to photo- and chemical degradation. Despite widespread interest in QDs for biological applications, QDs have been largely confined to sensor applications in vitro, staining of fixed tissues and cells, and as simple markers in vivo. The key challenges facing the QD field include active manipulation, high spatial and temporal resolution imaging in living tissues and cells, controlled translocation of QDs across biological membranes, and specific targeting to organelles. The overall goal of our proposed research is to address these challenges directly by developing a new class of magnetic quantum dots (MQDs) and novel MQD-protein conjugates. MQDs combine the unique properties of semiconductor nanocrystals with the active manipulation capabilities of magnetic particles. We propose to use the resulting MQDs and develop MQD-protein conjugates to gain new and important insight in biomembrane science and nuclear envelope micromechanics. The specific aims of the proposal are: Specific Aim 1. Develop and test MQDs. We will develop and fabricate surface-engineered multifunctional MQDs for live-cell applications. Specific Aim 2. . Test the MQDs with model membrane systems of known biochemical compositions and physical propertie.. Reconstitute the nuclear envelope (NE) model using NE lipids and NE proteins such as nuclear lamins. Probe the permeability of these mimetic systems using MQDs. Specific Aim 3. Drive MQDs into the nucleus to probe NE micromechanics in live cells. 3a.We will develop a novel nanoparticle ballistic bombardment system to ensure a high rate of transfer of MQDs to many cells in culture simultaneously. Sb.We will use external magnetic fields to probe the fractions of ballistically injected MQDs in the cytoplasm and nucleoplasm as well as local NE deformation as a function of the applied force on the MQDs. 3c.We will investigate whether the depletion of lamin A/C or disease-causing mutations in lamin A/C affects NE micromechanics. 3d. We will investigate whether and how the actin and microtubule networks affect the mechanical properties of the NE.