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The overall goal of this project is to develop a novel AFM-based scanning technique with nanometer-scale topography, molecular recognition imaging capabilities and pico-Newton force resolution functionality to investigate the lateral distribution of cell surface proteins and glycolipids. The new method will make it possible to determine ligand-induced redistribution of cell surface proteins during cell signaling and other biological events. It will significantly enhance the resolution of imaging microdomains from the current 100 nm level to the nanometer level, and provide biomechanical information about the bindings and anchoring of these molecules to a cell membrane. <P>Objective 1: To develop a new AFM-based scanning technology with capabilities to provide a new multifunctional tool with nano-scale resolution for simultaneous imaging and structural recognition. <P>Objective 2: To use this new AFM-based technology to image the lateral distribution of membrane-bound proteins and glycolipids and characterize their redistribution induced by ligand-binding events.<P> Objective 3: To measure the strengths of molecular bindings and the biomechanical interactions between these molecules.<P> If successful, this research will provide the capacity to simultaneously map the structure and chemomechanical interactions of whole cells, membranes, and individual molecular receptors at or below the nanoscale. This enables systematic, in situ characterization of the mechanisms governing drug-cell interactions at the in vitro level. <P>We will extend the technology to explore dynamic properties of biological systems, ligand-receptor (antibody-antigen, drug-receptor, DNA-protein, DNA-DNA, etc.) by imaging patterns of molecular binding and adhesion on surfaces. It is expected that this dynamic nanomechanical mapping of ligand-receptor interactions at the single-cell level will be key to scientific advances in understanding, identifying, and developing therapies that promote human health. We will also focus in detecting pathogens in food to improve the security and sustainability of US food production systems. <P>The new technology does not require fluorescence, radioactive, or enzyme labeling. It features real-time detection of molecular recognition events with single-molecule sensitivity in combination with conventional AFM-SPM capabilities.
Non-Technical Summary: When studying biological systems in which molecular individuality matters, single-molecule experiments offer several important advantages over ensemble measurements. For example, detailed knowledge about the organization of cell surface microdomains in living cells is extremely important for the understanding of many biological processes. To date, information about the lateral distribution of membrane-bound proteins and glycolipids has come mainly from fluorescence and electron microscopy, which perturb the delicate interactions that are present in the membrane and the spatial resolution of these optical imaging techniques is generally restricted to a few hundred nanometers. The purpose of this study is to develop and use a novel AFM-based approach with multifunctional capabilities that simultaneously provides topography and recognition images as well measurements of the molecular binding strength with pico-Newton force resolution. The new technology does not require fluorescence, radioactive, or enzyme labeling. It features real-time detection of molecular recognition events with single-molecule sensitivity in combination with conventional AFM/SPM capabilities. Using the technology, we will study the lateral organization of proteins and glycolipids in a cell membrane with nanometer resolution, reorganize different proteins involved in the biological binding events, and measure the ligends-receptor interactions. Further, we will detect pathogens in food the same way. <P> Approach: 1. DEVELOP A NEW AFM-BASED SCANNING TECHNOLOGY WITH CAPABILITIES TO PROVIDE A NEW MULTIFUNCTIONAL TOOL WITH NANO-SCALE RESOLUTION FOR SIMULTANEOUS IMAGING AND STRUCUTRAL RECOGNITION. In our new multifunctional AFM system, the funtionalized (e.g. using Ab, adhesion, ligand) tip will be oscillated near-resonance frequency during the scanning operation over an area of interest. The recorded wave signal is then fed into an electronic circuit which splits the wave into maxima (Umax) and minima (Umin). The Umin is used to drive a feedback loop with the AFM controller and provide data for the topological measurements, and the Umax to provide data for the biochemical recognition. This method allows simultaneous attainment of topographic and recognition images. To measure the binding force, a Signal Access Module (SAM) board will be used to separate and measure the force signal using a digital oscilloscope. As a result, this new setup of the AFM-based technology will allow simultaneous topographical imaging, molecular recognition, and intermolecular biomechanical characterization to be performed over an area of interest in a single experiment. During the scanning operation for topographical and recognition imaging, once a ligand/receptor, e.g. protein/lipid binding event occurs we will measure directly the unbinding force needed to break the molecular bond. The obtained information will be used to study the mechanical and structural properties of various molecular bindings. 2. USE THIS NEW AFM-BASED TECHNOLOGY TO IMAGE THE LATERAL DISTRIBUTION OF MEMBRANE-BOUND PROTEINS AND GLYCOLIPIDS AND CHARACTERIZE THEIR REDISTRIBUTION INDUCED BY LIGAND-BINDING EVENTS. This new AFM-based technology will be used to map the lateral organization of proteins and glycolipids in a cell membrane with nanometer resolution, reorganize different proteins involved in the biological binding events, and measure the biomechanical interactions between membrane-bound proteins and the underlying membrane. We will begin with the AFM tip functionalizations and then performing measurements on artificial bilayers and finally move on to the natural cell membranes. AFM tip functionalizations: (1) A number of antibodies, which recognize receptors involved in Toll-like receptor mediated cellular activation, will be modified by N-succinimidyl 3-(acetylthio)proprionate for attachment to amino modified AFM tips. (2) Several lipid-As, which are known to interact with TLR4/MD2, will be prepared by chemical synthesis. The C-6' will be selectively functionalized with an amino group for attachment to an AFM tip. AFM imaging of artificial bilayers: As a first step in assessing our new technology, we will study the specific binding between a glycolipid in an artificial membrane (for its much simpler bilayer structure as compared with a natural cell membrane) with an AFM tip modified with Cholera toxin B subunit. AFM imaging of natural cell membrane: Finally, we will use the knowledge obtained for artificial bilayers to image the natural cell membranes.