| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2000: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1999: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
The plasma membrane contains various forms of invaginations with apparently different physical building principles. To specify the mechanisms of domain formation, particularly those for clathrin-coated pits and caveolae, the dynamical characterization of the lattice assembly should be an important approach. We addressed the effects of poly (ethylene glycol) cholesteryl ether (PEG-Chol) on the structure/function of clathrin-coated pit and caveolae. Addition of the compound to cultured cells induced progressive smoothening of the surface. In these cells were accumulated opened clathrin-coated pits, which ultimately became flat on occasion of the cell rupture by slightly more PEG-Chol. These results strongly suggested that coated-pit elastically differentiates from the bulk membrane phase and also from caveolae. Next, we analyzed the effect of accumulated membrane stress. Whereas simple incubation of PEG-Chol gave no morphological effect, we found numerous plasma membrane tubules decorate
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d with biotinylated PEG-Chol (bPEG-Chol) and SA were introduced. The tubules were homogeneous in diameter (d【approximately equal】100 nm) and contained actin filaments. Biochemically, bPEG-Chol induced tyrosinephosphorylation of proteins. The analysis suggested that local accumulation of lipid-packing defects evoked signaling reaction (s) that mobilize actin to reduce the stress from the main cell body. Next, we describe the responses of K562 human leukemia cells that were treated with okadaic acid, an inhibitor of type 1 and 2A protein dephosphatases, to physical stress on the plasma membrane. While OKA alone induced patchy accumulation of cortical F-actin, application of mechanical stresses induced budding of membrane-bound body containing the whole cell F-actin. The formation of this actin-rich body was prevented by modification of F-actin and also by protein kinase-inhibitors. Moreover, when a non-receptor tyrosine kinase Fyn was knocked-out, such change did not occur. In this project, we suggest the presence of the specific mechanical linkage between actin microfilaments and a defined set of membrane organelles or surfaces proteins in OKA-treated cells. Less
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