Murata Laboratory is an exciting, yet demanding laboratory... a leader in the world’s research commitment to discovering the best solutions for present and future human health problems through basic biomedical sciences. Novel and sophisticated cell-based assay techniques coupled with vanguard visualization techniques enable Murata Laboratory to continually deliver breakthrough ideas and solutions for the myriad of health problems that plague humankind.
Traditional research has involved the study of individual genes or proteins. However, the comprehensive collection, integration, and analysis of biological data obtained from microarray studies, proteomics, epigenomics, and a host of other new “omics” fields haveare driving biomedical research in the direction of dealing with and deciphering the biological nature of living organisms as a whole. Organelles and their pathways can now be studied entirely and simultaneously.
In vitro reconstitution assays and cell-based assays have taken on greater significance in validating the interrelationships of pathological process factors in a network. In vitro reconstitution assays require, however, biochemical purification of isolated organelles or cytoskeletal components from disrupted cells or tissues. This inevitably damages the integrity/topology of these components; and what is worse, can also disrupt the relative spatio-temporal relationship itself between organelles and the cytoskeleton. Their integrity and proper relative spatial localization are crucial for normal protein function. As such, the inclusion of spatio-temporal information in vitro reconstitution assays, complemented by the advantages of systems biology, enables more precise identification (determination) of molecular networks.
Cell-based assays provide yet another means for evaluating spatio-temporal information, and are increasing in their relative importance to traditional drug tests and for investigating key mechanisms that cause diseases and their interactions. Most current cell-based assays utilize ‘‘normal’’ cells, however, which do not reflect intracellular disease conditions. The culture and use of primary cells from a patient’s diseased tissues for cell-based assays continues to prove perplexing due to their lack of uniformity.
Murata Lab has developed a powerful cell-based assay system of “disease model cells”.
Using semi-intact cells and a resealing cell technique pioneered by Murata Lab (details follow), this system enables the investigation of perturbed protein networks under pathogenic conditions while retaining the integrity of intracellular structures. What this robust assay system does is enable the validation and elucidation of protein networks (deduced by systems biology) involved in a variety of pathological processes, especially in diabetes, hyperglycemia, and cancers and other chronic diseases.
Information about the morphological changes of organelles, changes to the relative spatio-temporal positioning between organelles and the cytoskeleton, or perturbation of protein localization under pathogenic conditions are observable by fluorescence microscopic observations comparing resealed cells containing either healthy or pathogenic cytosol.
Therefore, using morphological or spatio-temporal information regarding organelles or proteins under pathogenic conditions, we will be able to develop new criteria for diagnosing disease and screening drug candidates based on the morphological changes of protein localization and organelles.
Current research directions include:
- Developing new platforms for the use of disease model cells to investigate the perturbation of protein/gene networks in diabetes, hyperglycemia and cancers, and other typical chronic diseases.
- Developing new platforms for the use of disease model cells for personalized medicine based on advanced microscopy systems and MicroElectroMechanical Systems (MEMS) for analyzing intracellular events in a single cell.
- Exploringthe novel image analysis system of “localizomics” to elucidate pathogenic mechanisms (collaboration with Nikon Corporation).
- Exploring new methods using semi-intact cell resealing technique to investigate and manipulate epigenetic changes in nuclei.
- Elucidating the role of oxidative/ER stress in pathogenic processes.
RESEALING TECHNIQUE for SEMI-INTACT CELLS
Semi-intact cells are generated when bacterial toxins permeabilize the plasma membrane (Fig. 1). Streptolysin O (SLO), a streptococcal pore-forming toxin, is used to permeabilize cells.
SLO binds to cholesterol in the plasma membrane at 4 ̊C, and forms a ring-shaped multimer that creates 30-nm diameter pores in the plasma membrane. These pores allow both depletion of the cytosol of the permeabilized cells, and delivery of various molecules such as proteins, nucleotides, and small chemical compounds into the cells. The cytosol of a cell in a particular phase of the cell cycle, in a particular state of differentiation, or in disease condition with that from a cell with a different phenotype can be replaced. In addition, since the integrity of organelles and the cytoskeleton remains largely intact in semi-intact cells, these cells can be considered “cell-type test tubes” that enable examination of molecular functions at the level of intracellular substructures. A unique advantage of this system is that information about spatio-temporal changes in protein localization and in the morphology of intracellular structures under different conditions imparted by the specific intracellular environment can be obtained. In particular, the maintenance of the integrity of the organelles and their configuration in semi-intact cells make this system suitable for analyzing membrane trafficking between organelles in as intrinsic an environment as possible. Murata Lab has been working on a mammalian, semi-intact cell system to reconstitute cell cycle-dependent changes in organelle morphology and membrane trafficking between organelles.
We recently observed that SLO-induced pore formation can be reversed by calcium ions under appropriate conditions, and that such “resealed cells” restored various intracellular functions that were difficult to reconstitute in the original semi-intact cell system due to the permeabilization of the plasma membranes.
Resealing renders semi-intact cells intact again and some of the resealed cells proliferated for several days. Hence, the cell resealing technique is a unique method that enables molecules,or a variety of cytosolic factorsdelivered into living cells, to exert their effects on the cells for what is considered a substantial period of time of at least four hours, if not more. Recently, Murata Lab has established a basic protocol for preparing “disease model cells” from semi-intact cells using pathogenic cytosol or its control “healthy model cells” using healthy cytosol. One example is establishing diabetes model cells (Db cells) by introducing cytosol prepared from the liver of db/db diabetes model mice. The important goal of assays using “disease model cells” is detecting various phenotypic differences between healthy and pathogenic (diseased) cytosol. By focusing on such phenotypic differences, the effects of stress or other artifacts induced during permeabilization and resealing can become controllable.
Given that phenotypic differences can be attributed to functional differences between the two types of cytosol, it seems quite likely that the cytosolic factorsthat perturb the reconstituted reactions in disease model cells by biochemical analyses can be controlled. For example, immunodepletion or addition of function-blocking antibodies to the cytosol should enable key pathological factorsto be identified. Thus, established disease model cells can be used for both drug testing and for identifing and evaluating the etiologic factors contributing to disease progression at the cellular level. To investigate the physiological events in semi-intact or resealed cells, a laser scanning fluorescence microscope equipped with 34-channel parallel imaging across the complete wavelength range is used. The acquired images are processed so that the localization of proteins and the morphology of the organelles under disease condition can be evaluated quantitatively at the subcellular level (collaboration with NIKON Corporation).
Murata Lab is currently involved in developing a support system for drug discovery based on the spatio-temporal information using our proprietary disease model cell system (patent pending) in conjunction with advanced imaging techniques. Such morphological and spatial information is a novel approach for understanding diseases, and will take the lead as a new standard for drug development.
Murata Lab seeks highly motivated, passionate researchers at all stages of their careers (postdocs, graduate students, undergraduates, and technical staff) to join our team. Our research focuses on understanding biological systems through reconstituting and developing model cells using semi-intact cells and our innovative resealing technique. If you are interested in developing new cell technology that takes advantage of your background in cell biology, biochemistry, biophysics, and bioinformatics, or other bioscience fields, please contact us. Our laboratory is well-equipped: numerous tools and equipment for basic molecular biology and biochemical experiments enable our team to design and validate the broadest spectrum of experiments.
Please send your application to firstname.lastname@example.org. In your e-mail, include the following:
- CV including list of publications
- short summary of your present and future research interests (limit to one page)
- names of two references (for postdoctoral candidates)
Department of Life Sciences
Graduate School of Arts and Sciences
The University of Tokyo
202A, Building 3, 3-8-1Komaba, Meguro-ku, Tokyo 153-8902, Japan