An AFM-based pit-measuring method for indirect measurements of cell-surface membrane vesicles.
An AFM-based pit-measuring method for indirect measurements of cell-surface membrane vesicles.
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DOI：
10.1016/j.bbrc.2014.02.114
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发表时间：
2014-03
影响因子：
3.1
通讯作者：
Xiaojun Zhang;Yuan Chen;Yong Chen
中科院分区：
文献类型：
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作者：
Xiaojun Zhang;Yuan Chen;Yong Chen
许多细胞类型脱落的循环膜囊泡具有多种功能，并与许多疾病相关。尽管循环膜囊泡已被广泛表征，但由于缺乏有效的测量方法，人们对细胞表面膜囊泡在释放之前的状态知之甚少。最近，作为一种强大的微米或纳米级成像工具，原子力显微镜（AFM）已被应用于测量循环膜囊泡。然而，由于膜囊泡和细胞表面之间的表面形态相似，AFM 直接成像/识别和测量细胞结合的膜囊泡似乎非常困难。因此，迄今为止尚未见关于细胞表面膜囊泡的AFM研究报道。在这项研究中，我们发现空气干燥可以诱导大多数细胞表面膜囊泡转变为更容易被 AFM 检测到的凹坑。在此基础上，我们开发了一种基于AFM的凹坑测量方法，并首次使用AFM间接测量培养内皮细胞的细胞表面膜囊泡。使用这种方法，我们观察并定量测量了至少两个细胞表面膜囊泡群体，一个纳米尺度群体（直径<500 nm，峰值在~250 nm）和微米尺度群体（从500 nm到~2 μm，峰值在~0.8 μm），而共焦显微镜仅检测到微米尺度群体。基于 AFM 的坑测量方法对于研究细胞表面膜囊泡和研究膜囊泡形成/释放的机制可能有用。
Circulating membrane vesicles, which are shed from many cell types, have multiple functions and have been correlated with many diseases. Although circulating membrane vesicles have been extensively characterized, the status of cell-surface membrane vesicles prior to their release is less understood due to the lack of effective measurement methods. Recently, as a powerful, micro- or nano-scale imaging tool, atomic force microscopy (AFM) has been applied in measuring circulating membrane vesicles. However, it seems very difficult for AFM to directly image/identify and measure cell-bound membrane vesicles due to the similarity of surface morphology between membrane vesicles and cell surfaces. Therefore, until now no AFM studies on cell-surface membrane vesicles have been reported. In this study, we found that air drying can induce the transformation of most cell-surface membrane vesicles into pits that are more readily detectable by AFM. Based on this, we developed an AFM-based pit-measuring method and, for the first time, used AFM to indirectly measure cell-surface membrane vesicles on cultured endothelial cells. Using this approach, we observed and quantitatively measured at least two populations of cell-surface membrane vesicles, a nanoscale population (<500 nm in diameter peaking at ∼250 nm) and a microscale population (from 500 nm to ∼2 μm peaking at ∼0.8 μm), whereas confocal microscopy only detected the microscale population. The AFM-based pit-measuring method is potentially useful for studying cell-surface membrane vesicles and for investigating the mechanisms of membrane vesicle formation/release.