Study on the buildup principle of the plasma membrane signaling domains.

质膜包含多种形式的内陷，其物理构造原理明显不同。为了明确结构域形成的机制，特别是网格蛋白包覆的凹坑和小泡，晶格组装的动力学表征应该是一个重要的方法。我们研究了聚乙二醇胆固醇醚（PEG-Chol）对网格蛋白包被的坑和小泡结构/功能的影响。将这种化合物添加到培养的细胞中，诱导细胞表面逐渐光滑。在这些细胞中积累了开放的网格蛋白包被的凹坑，这些凹坑最终在细胞破裂的时候变得平坦，稍微多一些PEG-Chol。这些结果有力地表明，包覆坑与体膜相和小泡相具有弹性区别。其次，我们分析了累积膜应力的影响。而PEG-Chol的简单孵育没有形态学上的影响，我们发现许多质膜小管装饰。更多的是生物素化PEG-Chol （bPEG-Chol）和SA。微管直径均匀（d【约等于】100 nm），含有肌动蛋白丝。生物化学方面，bPEG-Chol诱导蛋白质酪氨酸磷酸化。分析表明，脂质堆积缺陷的局部积累引起了信号反应，调动肌动蛋白来减少来自主细胞体的应激。接下来，我们描述了用冈田酸（一种1型和2A蛋白去磷酸酶抑制剂）处理的K562人白血病细胞对质膜物理应激的反应。单独的OKA诱导皮层f -肌动蛋白的斑片状积累，机械应力的应用诱导包含整个细胞f -肌动蛋白的膜结合体的出芽。这种富含肌动蛋白的小体的形成可以通过f -肌动蛋白的修饰和蛋白激酶抑制剂来阻止。此外，当非受体酪氨酸激酶Fyn被敲除时，这种变化不会发生。在这个项目中，我们提出了肌动蛋白微丝与oka处理细胞中一组定义的膜细胞器或表面蛋白之间存在特定的机械联系。少

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 … More 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