| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2002: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2001: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
Identification of CRMP-2 as a molecule involved in neuronal polarity and axon formation
Formation of an axon and dendrites is a fundamental step in neuronal development. In this research, we found that CRMP-2 was enriched in growing axons of cultured hippocampal neurons. Overexpression of CRMP-2 in the cells led to the formation of supernumerary axons without remarkable increase in the total number of neurites. CRMP-2 not only enhanced the formation of multiple axons during the initial stages of axon specification but also induced axon sprouting from already formed dendrites. Furthermore, expression of truncated CRMP-2 mutants suppressed the formation of primary axon in a dominant negative manner. Thus, local concentrations of CRMP-2 in neurites appear to play a critical role in axon induction of hippocampal neurons, thereby establishing and maintaining neuronal polarity.
In order to examine the molecular mechanisms for CRMP-2 induced axonal formation, we also investigated the CRMP-2 bin
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ding proteins. Here, we identified two activities of CRMP-2: tublin-heterodimer binding and the promotion of microtuble assembly. CRMP-2 bound tublin dimers with higher affinity than it bound microtubules. Association of CRMP-2 with microtubules was enhanced by tublin polymerization in the presence of CRMP-2. The binding property of CRMP-2 with tubulin was apparently distinct from that of Tau, which preferentially bound microtubules. In neurons, overexpression of CRMP-2 promoted axonal growth and branching. A mutant of CRMP-2, lacking the region responsible for microtuble assembly, inhibited axonal growth and branching in a dominant-negative manner. Taken together, our results suggest that CRMP-2 regulate axonal growth and branching as a partner of the tublin hetereodimer, in a different fashion from traditional MAPs.
Search for molecules involved in neuronal polarity and axon formation by protemic strategy
We further started to investigated the molecules involved in axon and polarity formation in addition to CRMP-2 by proteomic strategy. In order to searched molecules involved in axon and polarity formation, we established a 2-DE two-dimensional gel electrophoresis (2-DE) system of highresolution. We utilized fourteen large 2-DE gels (twelve 24 cm x 70 cm gels and two 18 cm x 70 cm gels) which were assembled into a 93 cm x 103 cm cybergel. Our data suggested that the cyber gel can display more than 11,000 protein spots expressed in a 1-10^5 dynamic range in cells. The utilization of present strategy led to about 500% increase in the number of detected spots in comparison to a standard procedure. With this system, we are currently screening molecules which are expressed during the processes of neuronal polarity formation. Less
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