2007-2-6 11:40:51
PNAS：史无前例－可以观察生物膜融合的方法
伊利诺伊大学香槟校区的研究人员Taekjip Ha研发出一种新方法，可以将生物膜融合的过程影像化。这张史无前例的详细图片刊载于2006 年12月PNAS的封面故事中。
无容置疑地，了解这种基本的生命现象机制具有很大的生物利益，并且有助于广泛的研究和实验。生物膜是细胞生存和维持细胞基本功能的基础。之前的研究提出了各种生物膜融合的中间体，但是这些方法并未获得实际的影响证据。这项重要的任务是由称为SNAREs 的膜蛋白质所达成的。
研究人员利用荧光共振能量转移（FRET）法，研发出这种观察生物膜融合的方法。在FRET 法中，使用一对绿色和红色染料，只有绿色染料可以被雷射直接激发。如果有些能量从绿色染料转移至红色染料，那么红色染料就会发光，两种染料的距离越近，红色染料发光的效率越高。
研究人员利用荧光显微镜观察SNARE-SNARE间的交互作用，实时地观察到膜融合的影像。这种新方法可以帮助科学家们更进一步了解生物膜产生交互作用的机制，对于基础研究或药物研发都是很有用的工具。
(资料来源 : biocompare)
View larger version (45K):
[in this window]
[in a new window]
|
Fig. 1. Single-liposome fluorescence assay of SNARE-mediated membrane fusion. (a) Schematics of the assay. The v-SNARE liposomes containing membrane fluorescent acceptors are tethered on a PEG-coated quartz slide, and Sso1pHT-reconstituted liposomes doped with membrane fluorescent donors are introduced together with Sec9c to induce fusion. The mixing of donor and acceptor dyes caused by fusion between the cognate liposomes leads to increase in E, which is being monitored by wide-field total internal reflection (TIR) microscopy. (b) Negative staining electron micrograph of the Sso1pHT-reconstituted liposomes (1200 EX; JEOL, Tokyo, Japan). (Scale bar: 100 nm.) (c–f) Final E distribution of the products of SNARE-driven single-liposome fusion (after 30 min of reaction) in the absence of Sec9c (c) and at the Sec9c/Sso1pHT ratio of 2:1 (d and e) and 1:1 (f). The fusion reactions of c, d, and f were induced on surface (as illustrated in a), whereas in e, fusion was induced in bulk solution and the products were subsequently immobilized on the quartz surface for observation. We notice that after 30 min of reaction the fraction of the population that remains at the docked state varies significantly sample by sample.
| |
View larger version (34K):
[in this window]
[in a new window]
|
Fig. 2. Multiple intermediate states of SNARE-induced fusion. (a–f) Single-liposome fusion time traces; full fusion events with no intermediates (classified as  class, a), one intermediate state ( 1 class, b and c), two intermediate states (2 class, d and e), and three intermediate states (3 class, f). (Upper) Shown is the fluorescence intensity time traces of the donor (ID, green) and the acceptor (IA, red) channels. (Lower) Shown is the corresponding FRET efficiency (blue) where the intermediate states are marked with black bars. In b, docking of a t-SNARE liposome is followed by a gradual FRET increase (between the two arrows), which culminates with the first intermediate state. (g–i) FRET histograms of the first intermediate state for the subclasses 1, 2, and 3 (g). The narrow distribution is not disturbed when traces obtained at the different Sec9c/Sso1pHT ratios of 2:1 (h) and 1:1 (i) are separately considered. The histograms are fitted with the Gaussian distributions, and the center (Ec) and the standard deviation ( ) of the Gaussians are shown. (j) Schematic illustration of a typical single-liposome fusion time trace. (k) Pathway of SNARE-driven membrane fusion. For the class , docking of a t-SNARE liposome and close contacting between two liposomes (state D) are followed by an obligatory intermediate state, the hemifusion state (state H). The number of premature closings of the fusion pore (F', F'', and so on) between the hemifusion state and the full fusion state (state F) then determines the subclass such as 1, 2, and 3. Class evolves from the D to F state without discernible intermediate states. In contrast, the FRET increase of class  is too gradual for intermediates to be identified, which could be caused by many pore-flickering phenomena. | |
英文原文链接：
http://www.pnas.org/cgi/content/full/103/52/19731?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=1&author1=Taekjip+Ha&andorexacttitle=and&andorexacttitleabs=and&andorexactfulltext=and&searchid=1&FIRSTINDEX=0&sortspec=relevance&fdate=11/1/2006&tdate=12/31/2006&resourcetype=HWCIT
