DNA Nanotechnology to Map and Manipulate Adhesion Forces at Fluid Interfaces

Cells transmit piconewton (pN) receptor forces to ligands in the extracellular matrix (ECM) and on the surface of adjacent cells. These forces regulate functions ranging from adhesion to clotting and the immune response. Whereas adhesion mechanics on rigid substrates are well characterized, understanding mechanotransduction at cell-cell junctions remains challenging due to a lack of tools. We develop and apply new classes of DNA-based force probes to map and manipulate receptor forces on supported lipid bilayers (SLBs), planar membranes that mimic an adjacent cell. We use these probes to elucidate force balance in podosomes, which are multipurpose protrusive structures that form at cell-cell and cell-ECM interfaces. Podosomes have a core-ring architecture, and previous works demonstrated that the podosome’s actin core generates nanonewton protrusive forces. However, the podosome’s contractile landscape remained poorly understood. In Aim 1 (Chapter 3), we develop and apply Molecular Tension- Fluorescence Lifetime Imaging Microscopy to map integrin receptor forces and clustering on SLBs. We demonstrate that integrin receptors apply pN tension in podosome rings. We then introduce photocleavable probes to site-specifically perturb adhesion forces and apply rupturable DNA-based force probes to test the role of receptor tension in podosome formation and maintenance. These studies confirm a local mechanical feedback between podosome core protrusion and integrin receptor tension. In Aim 2 (Chapter 4), we evaluate structure and energy transfer across a library of DNA-based tension probes using spectroscopy and microscopy. We then demonstrate the functional implications of probe design on cellular imaging. This work expands our understanding of receptor forces in podosome mechanobiology and contributes new insight and tools for studying juxtacrine receptor interactions.Ph.D

DNA mechanotechnology reveals that integrin receptors apply pN forces in podosomes on fluid substrates

International audiencePodosomes are ubiquitous cellular structures important to diverse processes including cell invasion, migration, bone resorption, and immune surveillance. Structurally, podosomes consist of a protrusive actin core surrounded by adhesion proteins. Although podosome protrusion forces have been quantified, the magnitude, spatial distribution, and orientation of the opposing tensile forces remain poorly characterized. Here we use DNA nanotechnology to create probes that measure and manipulate podosome tensile forces with molecular piconewton (pN) resolution. Specifically, Molecular Tension-Fluorescence Lifetime Imaging Microscopy (MT-FLIM) produces maps of the cellular adhesive landscape, revealing ring-like tensile forces surrounding podosome cores. Photocleavable adhesion ligands, breakable DNA force probes, and pharmacological inhibition demonstrate local mechanical coupling between integrin tension and actin protrusion. Thus, podosomes use pN integrin forces to sense and respond to substrate mechanics. This work deepens our understanding of podosome mechanotransduction and contributes tools that are widely applicable for studying receptor mechanics at dynamic interfaces

A Folate-Targeted Nanoplatform for Combined Magnetic Resonance Imaging and Radiation Therapy

From the Washington University Senior Honors Thesis Abstracts (WUSHTA), Volume 6, Spring 2014. Published by the Office of Undergraduate Research. Joy Zalis Kiefer, Director of Undergraduate Research and Associate Dean in the College of Arts & Sciences; Jane Green and Stacy Ross, Editors; Kristin G. Sobotka, Undergraduate Research Coordinator. Mentor: Yan Mei Wan

Site-Selective RNA Splicing Nanozyme: DNAzyme and RtcB Conjugates on a Gold Nanoparticle

Modifying RNA through either splicing or editing is a fundamental biological process for creating protein diversity from the same genetic code. Developing novel chemical biology tools for RNA editing has potential to transiently edit genes and to provide a better understanding of RNA biochemistry. Current techniques used to modify RNA include the use of ribozymes, adenosine deaminase, and tRNA endonucleases. Herein, we report a nanozyme that is capable of splicing virtually any RNA stem–loop. This nanozyme is comprised of a gold nanoparticle functionalized with three enzymes: two catalytic DNA strands with ribonuclease function and an RNA ligase. The nanozyme cleaves and then ligates RNA targets, performing a splicing reaction that is akin to the function of the spliceosome. Our results show that the three-enzyme reaction can remove a 19 nt segment from a 67 nt RNA loop with up to 66% efficiency. The complete nanozyme can perform the same splice reaction at 10% efficiency. These splicing nanozymes represent a new promising approach for gene manipulation that has potential for applications in living cells

Comparing nearshore benthic and pelagic prey as mercury sources to lake fish: the importance of prey quality and mercury content

Mercury (Hg) bioaccumulation in fish poses well-known health risks to wildlife and humans through fish consumption. Yet fish Hg concentrations are highly variable, and key factors driving this variability remain unclear. One little studied source of variation is the influence of habitat-specific feeding on Hg accumulation in lake fish. However, this is likely important because most lake fish feed in multiple habitats during their lives, and the Hg and caloric content of prey from different habitats can differ. This study used a three-pronged approach to investigate the extent to which habitat-specific prey determine differences in Hg bioaccumulation in fish. This study first compared Hg concentrations in common nearshore benthic invertebrates and pelagic zooplankton across five lakes and over the summer season in one lake, and found that pelagic zooplankton generally had higher Hg concentrations than most benthic taxa across lakes, and over a season in one lake. Second, using a bioenergetics model, the effects of prey caloric content from habitat-specific diets on fish growth and Hg accumulation were calculated. This model predicted that the consumption of benthic prey results in lower fish Hg concentrations due to higher prey caloric content and growth dilution (high weight gain relative to Hg from food), in addition to lower prey Hg levels. Third, using data from the literature, links between fish Hg content and the degree of benthivory, were examined, and showed that benthivory was associated with reduced Hg concentrations in lake fish. Taken together, these findings support the hypothesis that higher Hg content and lower caloric content make pelagic zooplankton prey greater sources of Hg for fish than nearshore benthic prey in lakes. Hence, habitat-specific foraging is likely to be a strong driver of variation in Hg levels within and between fish species