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首页> 外文期刊>International Journal of Molecular Sciences >EGF Receptor Stalls upon Activation as Evidenced by Complementary Fluorescence Correlation Spectroscopy and Fluorescence Recovery after Photobleaching Measurements
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EGF Receptor Stalls upon Activation as Evidenced by Complementary Fluorescence Correlation Spectroscopy and Fluorescence Recovery after Photobleaching Measurements

机译：EGF受体在活化时失速，通过互补荧光相关光谱法和光漂白测量后的荧光回收率证明

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To elucidate the molecular details of the activation-associated clustering of epidermal growth factor receptors (EGFRs), the time course of the mobility and aggregation states of eGFP tagged EGFR in the membranes of Chinese hamster ovary (CHO) cells was assessed by in situ mobility assays. Fluorescence correlation spectroscopy (FCS) was used to probe molecular movements of small ensembles of molecules over short distances and time scales, and to report on the state of aggregation. The diffusion of larger ensembles of molecules over longer distances (and time scales) was investigated by fluorescence recovery after photobleaching (FRAP). Autocorrelation functions could be best fitted by a two-component diffusion model corrected for triplet formation and blinking. The slow, 100–1000 ms component was attributed to membrane localized receptors moving with free Brownian diffusion, whereas the fast, ms component was assigned to cytosolic receptors or their fragments. Upon stimulation with 50 nM EGF, a significant decrease from 0.11 to 0.07 μm 2 /s in the diffusion coefficient of membrane-localized receptors was observed, followed by recovery to the original value in ~20 min. In contrast, the apparent brightness of diffusing species remained the same. Stripe FRAP experiments yielded a decrease in long-range molecular mobility directly after stimulation, evidenced by an increase in the recovery time of the slow component from 13 to 21.9 s. Our observations are best explained by the transient attachment of ligand-bound EGFRs to immobile or slowly moving structures such as the cytoskeleton or large, previously photobleached receptor aggregates.
机译：为了阐明表皮生长因子受体（EGFR）活化相关簇的分子细节，通过原位迁移率评估了eGFP标记EGFR在中国仓鼠卵巢（CHO）细胞膜中的迁移时间和聚集状态的时程分析。荧光相关光谱法（FCS）用于探测小分子团在短距离和时间范围内的分子运动，并报告聚集状态。通过光漂白后的荧光恢复（FRAP）研究了较大分子团在更长距离（和时间尺度）上的扩散。自相关函数可以通过针对三线态形成和眨眼校正的两成分扩散模型来最佳拟合。缓慢的100-1000 ms成分归因于膜局部受体随自由布朗扩散而移动，而快速ms成分归因于胞质受体或其片段。用50 nM EGF刺激后，观察到膜定位受体的扩散系数从0.11显着降低到0.07μm2 / s，然后在约20分钟内恢复到原始值。相反，扩散物质的表观亮度保持不变。条纹FRAP实验在刺激后直接导致远距离分子迁移率降低，这可以通过慢速组分的恢复时间从13 s增加到21.9 s来证明。我们的观察结果可以通过将配体结合的EGFR瞬时附着到固定的或缓慢移动的结构（例如细胞骨架或大型的，以前被光漂白的受体聚集体）上来最好地解释。

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