Overview: Revolutionizing Biology with Nanocrystal Molecules As Probes
Cells orchestrate dynamically cooperative operations of a variety of biomolecular machineries to tightly regulate key biological functions such as signaling, transport, replication, and transcription. In order to investigate such highly complicated dynamic events of biomolecular machineries in living objects; our approach is to use an advanced nanoprobing system, termed nanocrystal Molecules As Probes (nanoMAPs.) The nanoMAPs are constructed by assembling of multiple nanocrystal components in which individual nanocrystal components (i.e. nanocrystal atoms) are either electronically, magnetically, optically, or mechanically coupled to each other. The chemical and physical properties of the nanoMAPs are a function of interparticle couplings, and hence a function of the interparticle distance, the bonding mode, and the nanocrystal molecule geometry (e.g. symmetry). Unlike conventional single molecule- or single nanocrystal-based bioprobes that only report the spatial distribution of target bio-molecules, such unique coupling phenomena of the nanoMAPs provide optical and magnetic transduction of dynamic structural and environmental changes across single biomolecule. We expect that the development of new nanoMAPs will enable deeper understanding of the fundamental dynamics of bio-molecular machineries in living objects. Specifically, we develop a set of new nanoMAP systems that include magnetic, plasmonic, and fluorescent nanoMAPs to investigate structures, forces, interaction, and reaction of key biomolecules related to cell-to-cell communication (Notch), angiogenesis, gene regulation, and neuron signaling.
Single molecule study of membrane receptor tyrosine kinase dynamics
Membrane receptor tyrosine kinases (RTKs) are key regulators of cellular processes and also known to have a critical role in the cancer development and progression. Most RTKs exist as a free monomeric sub-unit, but target ligand binding such as polypeptides and hormones to their extra cellular domain promotes RTK dimerization and even oligomerization. The membrane association of the RTKs then activates intracellular signal cascades related to key cell developments such as proliferation and differentiation. Interestingly, even in the case of genetically identical cells, each cell expresses different frequency and spatial distribution of the RTK assembly and hence behaves differently and sometimes such inhomogeneity of RTK assemblies directs distinctly different fates of each cell. To understand how the dynamic assembly of RTKs determines the phenotypic responses of individual cells, we develop a new nanoMAP based single molecule imaging technique and, by using this new technique, we study real-time RTK dynamics in live cell membranes.
Individually in apoptotic cells: In situ single molecule study using plasmonic nanoMAPs
Apoptosis is a well-choreographed mode of cell death where caspases (cysteine-aspartic acid proteases) take center stage in orchestrating cellular demise. Upon commencement of the caspase activation cascades, hundreds of protein substrates are cleaved and the hallmarks of apoptosis (nuclear condensation, cell shrinkage, membrane blebbing, and DNA fragmentation) begin to take their form. Despite remarkable advances in biochemical characterization of apoptotic lysates, the individualistic behavior of cells in a dying population is lost by synchronization of ensemble analyses. It has been postulated that the trajectory of cell death occurs on the order of hours or days, where once a certain internal signaling threshold has been met the cell follows an irreversible death sentence where it is doomed to die within 10 minutes. This paradigm could only have been brought to light with extensive single molecule and cell analysis. By using plasmonic nanoMAPs that show characteristic optical signals against the apoptotic signal progression, we perform single molecule monitoring of cell to provide signal progression, we perform single molecule monitoring of cell to provide key quantitative hallmarks to help describe apoptosis in diverse cell types, cancerous or otherwise, that are prominently mentioned throughout the cell death literature. Such questions call for the construction of sensitive imaging tools to continuously monitor processes at single molecule resolution that transpire along time courses unanticipated by the observer.
Single molecule kinetic study of motor proteins using nanoMAP sensors
Despite recent progress in understanding the mechanism of biomolecular motor, development of high-resolution single molecule imaging technique is expected to provide deeper insights of undiscovered molecular mechanisms and kinetic processes of the proteins. To investigate molecular mechanisms of the motor protein activity, we develop new nanoMAP plasmon nanorulers and protractors having nano-meter resolution. Unlike conventional single molecule techniques that only provide distance-displacement of target nucleic acids, nanorulers and protractors allows us to directly translate both tranlational and rotational motions of the target biomolecules. This new technique has the potential to provide extremely high sensitivity and spatial resolution.