Development of in vitro reconstituted system for investigating intracellular signal trasduction and cellular function in myocardial cells

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The aim of this research is to develop an in vitro real-time analyzing system for investigating intracellular signal transduction with CCD camera. In 1995, we succeeded to develop an in vitro reconstituted system for analyzing of intracellular signal transduction, especially nuclear signal transduction. It was found that nuclear protein was transported to the nucleus within 30 minutes, although non-nuclear proteins were not by using homogenates from heart which means that this system reproduces in vivo nuclear protein transport. In 1996, we examined nuclear transport efficiency with extracts from diseased hearts by this in vitro system. The rate of the nuclear transport was more decreased in spontaneous hypertensive rat than of the control. Furthermore, we found the existence of cellular factors that are activated by nuclear localization signal (NLS) and partially purified these cellular factors (FEBS Letters). In the next step, we should improve this system in order to analyze the real-time behavior of molecules quantitatively with CCD camera. The real-time analytical system will provide the precise evaluation of efficiency of nuclear transport that may modulate the cellular function in heart cells.