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Methods of Analysis for Imaging and Detecting Ions and Molecules
The principal research topics of Dr. Umezawa's work are directed to develop new analytical methods for molecular imaging. His work is to implement “seeing what was unseen” in single live cells and interfacial molecular recognition chemistry.
Methods of non-destructive analysis of cellular signaling processes in live cells have been explored extensively in Dr. Umezawa's laboratory for the past several years. His approach for this has been to develop genetically encoded fluorescent and bioluminescent indicators to pinpoint each cellular process in single living cells. |
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Prof. Yoshio Umezawa
Department of Chemistry, School of Science,
The University of Tokyo
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Chemistry-facilitated intermolecular electron tunneling is another approach to molecular imaging that Umezawa has made. The chemical imaging was obtained from a distinctive chemical affinity between the imaging tip of scanning tunneling microscopy (STM) and the substrate that alters the tunneling current. This was achieved by tailored chemical modification of the STM tips.
1. Illuminating Molecular Processes in Single Living Cells
Umezawa has developed optical probes that the intracellular signaling can be monitored in vivo in living cells by genetically encoded intracellular fluorescent and bioluminescent probes or indicators, which include second messengers such as guanosine 3E5Ecyclic monophosphate (cGMP), inositol 1,4,5-trisphosphate (IP3), phosphatidylinositol-3,4,5-trisphosphate (PIP3), and nitric oxide (NO), protein phosphorylations, protein-protein interactions, and protein localizations. These probes are of general use not only for fundamental biological studies, but also for assay and screening of possible pharmaceutical or toxic chemicals that inhibit or facilitate cellular signaling pathways.
Umezawa describes an amplifier-coupled fluorescent indicator for NO to visualize physiological nanomolar dynamics of NO in living cells with a detection limit of 0.1 nM. This genetically encoded high sensitive indicator revealed that approximately 1 nM of NO, which is enough to relax blood vessels, is generated in vascular endothelial cells even in the absence of shear stress. Nanomolar range of basal endothelial NO thus revealed appears to be fundamental to vascular homeostasis (Proc. Natl. Acad. Sci., USA, 102, 14515, 2005).
PIP3 regulates diverse cellular functions, including cell proliferation and apoptosis, and has roles in the progression of diabetes and cancer. However, little is known about its production. Umezawa et al. has developed fluorescent indicators for PIP3 based on FRET. These novel PIP3 indicators are composed of two distinctly coloured mutants of green fluorescent protetin (GFP) and a PIP3-binding domain. The PIP3 level was observed by dual-emission ratio imaging, thereby allowing stable observation without the problem of artifacts described above. Furthermore, these indicators were fused with localization sequences to direct them to the plasma membrane or endomembranes, allowing localized analysis of PIP3 concentrations. Using these fluorescent indicators, Umezawa and co-workers analyzed the spatio-temporal regulation of the PIP3 production in single living cells (Nature Cell Biology, 5, 1016, 2003).
Umezawa's group developed genetically encoded fluorescent indicators for visualizing protein phosphorylations in living cells. The approach is that a substrate domain for a kinase protein of interest is fused with a phosphorylation recognition domain via a flexible linker sequence. The tandem fusion unit consisting of the substrate domain, linker sequence, and phosphorylation recognition domain is sandwiched with two fluorescent proteins of different colors, cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), which serve as the donor and acceptor fluorophores for FRET. As a result of the phosphorylation of the substrate domain and subsequent binding of the phosphorylated substrate domain with the adjacent phosphorylation recognition domain, FRET is induced between the two fluorescent units, which causes phosphorylation-dependent changes in the fluorescence emission ratios of the donor and acceptor fluorophores. Upon activation of the phosphatases, the phosphorylated substrate domain is dephosphorylated and the FRET signal is decreased (Nature Biotechnology, 20, 287, 2002). To monitor protein-protein interactions (PPIs), a new method with general applicability was developed by Umezawa et al. based on protein splicing. In this process, an intervening protein sequence is excised and the flanking protein fragments are spliced together. In this splicing system, the flanking pieces are the N-and C-terminal fragments of GFP. The intervening sequence is DnaE, splicing protein derived from Synechocystis. An interaction between protein A and B brings the two parts of DnaE close enough to fold together properly and initiate the splicing and linking of the two GFP halves with a peptide bond. Reconstitution of GFP is monitored by its fluorescence.
Unlike an earlier protein interaction assay, the split-GFP system involves the reconstitution of GFP and does not require that the PPIs occur near the cell nucleus and reporter genes or that an enzyme substrate be present. This will make the method generally more useful and enable screening of the interactions in the cytosol or at the inner membrane level (Anal. Chem., 73, 5866, 2001).
One of the most distinct features of eukaryotic cells, in particular mammalian cells, is different compartmentalization of each protein. A method was developed that allows rapid identification of novel proteins compartmentalized in mitochondria by screening large-scale cDNA libraries. The principle is based on reconstitution of split GFP by protein splicing with DnaE (Nature Biotechnology, 21, 287, 2003). Nucleocytoplasmic trafficking of functional proteins plays a key role in regulating gene expressions in response to extracellular signals. Umezawa's group developed a genetically encoded bioluminescent indicator for monitoring the nuclear trafficking of target proteins in vitro and in vivo. The principle is based on reconstitution of split fragments of Renilla luciferase (Rluc) by protein splicing with a DnaE intein. A target cytosolic protein fused to the amino-terminal half of Rluc is expressed in mammalian cells. If the protein translocates into the nucleus, the Rluc moiety meets the carboxy-terminal half of Rluc, which is localized in the nucleus with a fused nuclear localization signal, and full-length Rluc is reconstituted by protein splicing. The bioluminescence is thereby emitted with coelenterazine as the substrate. The principle of the approach is an extension of the earlier developed method to identifying mitochondrial proteins. The method of cell-based screening with the genetically encoded indicator provided a quantitative measure of the extent of nuclear-translocation of androgen receptor upon stimulating with various chemicals (Proc. Natl. Acad. Sci., USA, 101, 11542, 2004).
2. Chemistry-Facilitated Intermolecular Electron Tunneling
Molecular tips in scanning tunneling microscopy can directly detect intermolecular electron tunneling between sample and tip molecules and reveal the tunneling facilitation through chemical interactions that provide overlap of respective electronic wave functions, that is, hydrogen-bond, metal-coordination-bond, and charge-transfer interactions. Nucleobase molecular tips were prepared by chemical modification of underlying metal tips with thiol derivatives of adenine, guanine, cytosine, and uracil and the outmost single nucleobase adsorbate probes intermolecular electron tunneling to or from a sample nucleobase molecule. Umezawa et al. found that the electron tunneling between a sample nucleobase and its complementary nucleobase molecular tip was much facilitated compared with its noncomplementary counterpart. The complementary nucleobase tip was thereby capable of electrically pinpointing each nucleobase (Proc. Natl. Acad. Sci., USA, 103, 10, 2006).
A fullerene molecular tip was used to detect electron tunneling from a single porphyrin molecule. Electron tunneling was found to occur locally from an electron-donating moiety of the porphyrin to the fullerene through charge-transfer interaction between them. In addition, electron tunneling within the single fullerene-porphyrin pair exhibited rectifying behavior in which electrons can be driven only at the direction from the porphyrin to the fullerene. Umezawa et al. demonstrated that localized electron tunneling enables us to spatially visualize the frontier orbital of the porphyrin involved in electron tunneling. In addition, rectification demonstrates that the fullerene-porphyrin pair constitutes a molecular rectifier. Umezawa believes that molecular tips bring insight into intermolecular electron transmission toward realization of molecular electronics as shown here (Proc. Natl. Acad. Sci., USA, 102, 5659, 2005).
Chemically selective imaging using molecular tips may be coined “intermolecular tunneling microscopy” as its principle goes and is of general significance for novel molecular imaging of chemical identities at the membrane and solid surfaces.
Professor Umezawa has thus developed methods of analysis for visualizing and detecting key signaling molecules and processes in living cells to see what was otherwise unseen. He has also invented molecular tips in STM, to visualize molecular recognition processes at membrane solid interfaces. These remarkable cutting-edge techniques have been promised to become key analytical methods for studying chemistry and biology. In recognition of Dr. Umezawa's important contribution to chemistry, the Chemical Society of Japan has decided to confer upon him this year's Chemical Society of Japan Award.
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