Magnetic quantum dots for active sensing of the nuclear envelope

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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.