Sandia National Laboratories, PO Box 5800, MS-0895, Albuquerque, NM 87185, USA.
Small. 2012 Oct 8;8(19):3041-9. doi: 10.1002/smll.201200106. Epub 2012 Jul 17.
Many membrane receptors are recruited to specific cell surface domains to form nanoscale clusters upon ligand activation. This step appears to be necessary to initiate cell signaling, including pathways in innate immune system activation. However, virulent pathogens such as Yersinia pestis (the causative agent of plague) are known to evade innate immune detection, in contrast to similar microbes (such as Escherichia coli) that elicit a robust response. This disparity has been partly attributed to the structure of lipopolysaccharides (LPS) on the bacterial cell wall, which are recognized by the innate immune receptor TLR4. It is hypothesized that nanoscale differences exist between the spatial clustering of TLR4 upon binding of LPS derived from Y. pestis and E. coli. Although optical imaging can provide exquisite details of the spatial organization of biomolecules, there is a mismatch between the scale at which receptor clustering occurs (<300 nm) and the optical diffraction limit (>400 nm). The last decade has seen the emergence of super-resolution imaging methods that effectively break the optical diffraction barrier to yield truly nanoscale information in intact biological samples. This study reports the first visualizations of TLR4 distributions on intact cells at image resolutions of <30 nm using a novel, dual-color stochastic optical reconstruction microscopy (STORM) technique. This methodology permits distinction between receptors containing bound LPS from those without at the nanoscale. Importantly, it is also shown that LPS derived from immunostimulatory bacteria result in significantly higher LPS-TLR4 cluster sizes and a nearly twofold greater ligand/receptor colocalization as compared to immunoevading LPS.
许多膜受体在配体激活时被募集到特定的细胞表面区域，形成纳米级别的簇。这一步似乎是启动细胞信号转导所必需的，包括先天免疫系统激活的途径。然而，像鼠疫耶尔森氏菌（鼠疫的病原体）这样的毒力病原体已知能够逃避先天免疫检测，而与之相似的微生物（如大肠杆菌）则会引发强烈的反应。这种差异部分归因于细菌细胞壁上脂多糖（LPS）的结构，先天免疫受体 TLR4 可以识别这种结构。人们假设，在结合源自鼠疫耶尔森氏菌和大肠杆菌的 LPS 时，TLR4 的空间聚类存在纳米级差异。尽管光学成像可以提供生物分子空间组织的精细细节，但受体聚类发生的尺度（<300nm）与光学衍射极限（>400nm）之间存在不匹配。在过去的十年中，出现了超分辨率成像方法，可以有效地突破光学衍射障碍，在完整的生物样本中获得真正的纳米级信息。本研究首次使用一种新颖的双色随机光学重建显微镜（STORM）技术，在<30nm 的图像分辨率下，对完整细胞上的 TLR4 分布进行可视化，该技术分辨率<30nm。这种方法可以在纳米尺度上区分含有结合 LPS 的受体和不含 LPS 的受体。重要的是，还表明与免疫逃避 LPS 相比，源自免疫刺激细菌的 LPS 导致 LPS-TLR4 簇的尺寸显著增加，并且配体/受体的共定位增加近两倍。
