| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2001: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2000: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1999: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
Annexin is a Ca- and phospholipid-binding protein. It is found in a wide variety of organisms from lower eucaryotes to human. However, its cellular function is poorly understood. We have analyzed intracellular functions of annexin, by a technology called MicroCALI which transiently inactivates specific proteins in a live cell, thereby enabling alternative approaches to cellular function analysis of a protein other than genetic approaches.
1. Technical Achievements:
A. Setting-up of MicroCALI. We purchased external laser units, and other parts from Olympus Inc, and Luminex Inc., and finished setting-up the usable MicroCALI system with a conventional versatile microscopy Olympus AX-70, which we already fine-tuned for computerized operation.
B. Purification of recombinant annexin and fluorescence labeling. The annexin gene from Physarum plasmodium was transformed to E. coli and successfully expressed in E. coli, in a soluble form of protein. We biochemically purified it to a 100 % purity by
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FPLC and chemically labeled it with Alexia fluorochrome. To minimize protein denaturation, we tried to keep the fluorescence/protein ratio low.
C. Incorporation of fluorescence-labeled annexin into live cell. We succeed in this by use of glass-bead method.
D. Setting-up of a fast, ultra-sensitive microscope recording system. For recording at video rate of a low-light, low-signal images, we finished setting-up of a very fast, and sensitive fluorescence microscopy recording system which includes Mahatmas Photomaps SIT camera, Photometric PXL- 1400 cooled CCD digital camera, and a control software for full automatic operation.
2. Scientific Achievements:
A. Live recording of fluorescent annexin in Physarum amoebae. Intracellular annexin is localized in reticular organelles, and the annexin signal kept moving repeating fusion and fission. Most movements are directed forward to the pseudopodia. The maximum speed sometimes exceeded 60 urn/min, pretty faster than cell migration rate (10 um/min).
B. Annexin accumulates at the cell periphery in the pseudopodial region. Annexin forms a belt-shaped zone immediately beneath the pseudopodia plasma membrane. The belts disappear and then seedpods protrude, suggesting that the plasma membrane precursor for protrusion is bound with annexin.
C. Involvement of annexin in plasma membrane resealing. Annexin is involved in plasma membrane repair. Annexin is concentrated in regions of plasma membrane damage. Anti-annexin, microinjected, inhibited plasma membrane resealing.
D. Identification of annexin-binding proteins. It is plausible that annexin co-works with other accessory proteins in membrane protrusion and resealing. By immunoprecipitation method, we have identified 116 kD, and 87 kD proteins. Those two proteins directly bind to annexin as revealed by west-western blotting method. Less
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