文獻：Ultrafast Diffusion of a Fluorescent Cholesterol Analog in Compartmentalized Plasma Membranes

作者：Nao Hiramoto-Yamaki, Kenji A. K. Tanaka, Kenichi G. N. Suzuki, Koichiro M. Hirosawa, Manami S. H. Miyahara, Ziya Kalay, Koichiro Tanaka, Rinshi S. Kasai … See all authors 

文獻鏈接：https://onlinelibrary.wiley.com/doi/full/10.1111/tra.12163

The insolubility of a PM molecule upon cold-Triton treatment has often been associated with its partitioning into raft domains in the live-cell PM, although this has never been proved (6 7, 20; reviewed in 32). Therefore, in this research, we examined the cold-Triton solubilities of Bdp-Chol and other phospholipid probes incorporated in the PM, for comparisons with those of other prototypical raft and non-raft molecules in the literature, such as GPI-anchored proteins (e.g. CD59, Thy1 and DAF) and transferrin receptor, respectively. The HASM and COS-7 PMs that incorporated Bdp-Chol, fluorescent phospholipid probes, [Cy3-conjugated phosphatidylethanolamines (Cy3-PEs)], collectively representing Cy3-dioleoyl-PE, Cy3-dimyristoyl-PE and Cy3-dipalmitoyl-PE, abbreviated as Cy3-DOPE, Cy3-DMPE and Cy3-DPPE, respectively) or Bodipy FL (Life Technologies)-conjugated GM1 in its alkyl chain (Bdp?-GM1), were extracted with cold Triton (1%, at 2.8°C for 15?min; Figure S1). Bdp-Chol, as well as the prototypical raft-associated molecular complex of the endogenous ganglioside GM1 and Cy3-conjugated cholera toxin B subunit, mostly remained in the DRMs. In contrast, all three species of Cy3-PEs and Bdp?-GM1 were almost entirely solubilized; i.e. Cy3-DMPE, Cy3-DPPE and Bdp?-GM1 are cold-Triton soluble, like the prototypical non-raft molecule Cy3-DOPE. Cy5-DMPE 36 and Bdp?-GM1 21 were previously assumed to be raft-associated without any experimental data, but the results obtained here indicate that these molecules are not prototypical DRM-associated molecules. The present results are consistent with the data obtained by Sezgin et al. 24. Meanwhile, the partitioning of Bdp-Chol in DRM suggested that Bdp-Chol could be a good analogue for native cholesterol, consistent with previous data (see Introduction).

在這項研究中，我們檢查了Bdp-Chol和PM中摻入的其他磷脂探針的冷Triton溶解度，分別與文獻中其他原型筏和非筏分子的溶解度進行比較，如GPI錨定蛋白（如CD59、Thy1和DAF）和轉鐵蛋白受體。

將含有Bdp-Chol、熒光磷脂探針[Cy3-共軛磷脂酰乙醇胺（Cy3-PE）]的HASM和COS-7 PM（統(tǒng)稱為Cy3-dioleoyl-PE、Cy3-dimyristoy-PE和Cy3-dipalmitoy-PE，分別縮寫為Cy3-DOPE、Cy3-DMPE和Cy3 DPPE）或其烷基鏈中的Bodipy FL（Life Technologies）共軛GM1（Bdp?-GM1）用冷Triton（1%，2.8°C）提取15分鐘?min）。

Bdp-Chol以及內源性神經節(jié)苷脂GM1和Cy3結合的霍亂毒素B亞基的原型筏相關分子復合物大多留在DRMs中。相比之下，所有三種Cy3-PE和Bdp?-GM1幾乎完全溶解；即Cy3-DMPE、Cy3-DPPE和Bdp?-GM1是冷溶于Triton的，就像典型的非筏分子Cy3-DOPE一樣。

Cy5-DMPE 36和Bdp?-GM1 21之前被認為與筏相關，但沒有任何實驗數據，但這里獲得的結果表明，這些分子不是典型的DRM相關分子。目前的結果與Sezgin等人24獲得的數據一致。

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