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Visualization of GLUT1 Trafficking in Live Cancer Cells by the Use of a Dual-Fluorescence Reporter.
ACS Omega ( IF 3.7 ) Pub Date : 2020-06-23 , DOI: 10.1021/acsomega.0c01054 Zhen-Yan Li 1 , Yu-Ling Shi 2 , Guo-Xiong Liang 3 , Jie Yang 1 , Song-Kuan Zhuang 4 , Jie-Bin Lin 2 , Abdelmoumin Ghodbane 1 , Man-Seng Tam 5 , Zu-Jian Liang 2 , Zhen-Gang Zha 1 , Huan-Tian Zhang 1
ACS Omega ( IF 3.7 ) Pub Date : 2020-06-23 , DOI: 10.1021/acsomega.0c01054 Zhen-Yan Li 1 , Yu-Ling Shi 2 , Guo-Xiong Liang 3 , Jie Yang 1 , Song-Kuan Zhuang 4 , Jie-Bin Lin 2 , Abdelmoumin Ghodbane 1 , Man-Seng Tam 5 , Zu-Jian Liang 2 , Zhen-Gang Zha 1 , Huan-Tian Zhang 1
Affiliation
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Glucose metabolism is an essential process for energy production and cell survival for both normal and abnormal cellular metabolism. Several glucose transporter/solute carrier 2A (GLUT/SLC2A) superfamily members, including glucose transporter 1 (GLUT1), have been shown to mediate the cellular uptake of glucose in diverse cell types. GLUT1-mediated glucose uptake is a transient and rapid process; thus, the real-time monitoring of GLUT1 trafficking is pivotal for a better understanding of GLUT1 expression and GLUT1-dependent glucose uptake. In the present study, we established a rapid and effective method to visualize the trafficking of GLUT1 between the plasma membrane (PM) and endolysosomal system in live cells using an mCherry-EGFP-GLUT1 tandem fluorescence tracing system. We found that GLUT1 localized at the PM exhibited both red (mCherry) and green (EGFP) fluorescence (yellow when overlapping). However, a significant increase in red punctate fluorescence (mCherry is resistant to acidic pH), but not green fluorescence (EGFP is quenched by acidic pH), was observed upon glucose deprivation, indicating that the mCherry-EGFP-GLUT1 functional protein was trafficked to the acidic endolysosomal system. Besides, we were able to calculate the relative ratio of mCherry to EGFP by quantification of the translocation coefficient, which can be used as a readout for GLUT1 internalization and subsequent lysosomal degradation. Two mutants, mCherry-EGFP-GLUT1-S226D and mCherry-EGFP-GLUT1-ΔC4, were also constructed, which indirectly confirmed the specificity of mCherry-EGFP-GLUT1 for monitoring GLUT1 trafficking. By using a series of endosomal (Rab5, Rab7, and Rab11) and lysosomal markers, we were able to define a model of GLUT1 trafficking in live cells in which upon glucose deprivation, GLUT1 dissociates from the PM and experiences a pH gradient from 6.8–6.1 in the early endosomes to 6.0–4.8 in the late endosomes and finally pH 4.5 in lysosomes, which is appropriate for degradation. In addition, our proof-of-concept study indicated that the pmCherry-EGFP-GLUT1 tracing system can accurately reflect endogenous changes in GLUT1 in response to treatment with the small molecule, andrographolide. Since targeting GLUT1 expression and GLUT1-dependent glucose metabolism is a promising therapeutic strategy for diverse types of cancers and certain other glucose addiction diseases, our study herein indicates that pmCherry-EGFP-GLUT1 can be utilized as a biosensor for GLUT1-dependent functional studies and potential small molecule screening.
中文翻译：
通过使用双荧光报告器可视化GLUT1在活癌细胞中的交易。
葡萄糖代谢是正常和异常细胞代谢的能量产生和细胞存活的重要过程。已显示包括葡萄糖转运蛋白1（GLUT1）在内的几个葡萄糖转运蛋白/溶质载体2A（GLUT / SLC2A）超家族成员可介导细胞在多种细胞类型中的摄取。GLUT1介导的葡萄糖摄取是一个短暂而快速的过程。因此，实时监测GLUT1的运输对于更好地了解GLUT1表达和GLUT1依赖性葡萄糖摄取至关重要。在本研究中，我们建立了一种快速有效的方法，使用mCherry-EGFP-GLUT1串联荧光示踪系统可视化GLUT1在质膜（PM）和溶酶体系统之间在活细胞中的运输。我们发现位于PM的GLUT1既显示红色（mCherry）荧光，又显示绿色（EGFP）荧光（重叠时为黄色）。但是，葡萄糖剥夺后，观察到红色点状荧光（mCherry对酸性pH有抵抗力）显着增加，而绿色荧光（EGFP被酸性pH猝灭）却没有观察到，表明mCherry-EGFP-GLUT1功能蛋白被贩运酸性溶酶体系统。此外，我们能够通过对易位系数进行定量来计算mCherry与EGFP的相对比例，可将其用作GLUT1内在化和随后的溶酶体降解的读数。还构建了两个突变体，mCherry-EGFP-GLUT1-S226D和mCherry-EGFP-GLUT1-ΔC4，这间接证实了mCherry-EGFP-GLUT1用于监测GLUT1交易的特异性。通过使用一系列内体标记物（Rab5，Rab7和Rab11）和溶酶体标记物，我们能够定义GLUT1在活细胞中运输的模型，在该模型中，葡萄糖剥夺后GLUT1从PM中解离，pH梯度从6.8内体早期为6.1，晚期内体为6.0-4.8，最后溶酶体的pH为4.5，这适合降解。此外，我们的概念验证研究表明，pmCherry-EGFP-GLUT1追踪系统可以准确反映GLUT1响应小分子穿心莲内酯治疗后的内源性变化。由于针对GLUT1表达和依赖GLUT1的葡萄糖代谢是针对各种类型的癌症和某些其他葡萄糖成瘾性疾病的有前途的治疗策略，
更新日期：2020-07-07
中文翻译：
通过使用双荧光报告器可视化GLUT1在活癌细胞中的交易。
葡萄糖代谢是正常和异常细胞代谢的能量产生和细胞存活的重要过程。已显示包括葡萄糖转运蛋白1（GLUT1）在内的几个葡萄糖转运蛋白/溶质载体2A（GLUT / SLC2A）超家族成员可介导细胞在多种细胞类型中的摄取。GLUT1介导的葡萄糖摄取是一个短暂而快速的过程。因此，实时监测GLUT1的运输对于更好地了解GLUT1表达和GLUT1依赖性葡萄糖摄取至关重要。在本研究中，我们建立了一种快速有效的方法，使用mCherry-EGFP-GLUT1串联荧光示踪系统可视化GLUT1在质膜（PM）和溶酶体系统之间在活细胞中的运输。我们发现位于PM的GLUT1既显示红色（mCherry）荧光，又显示绿色（EGFP）荧光（重叠时为黄色）。但是，葡萄糖剥夺后，观察到红色点状荧光（mCherry对酸性pH有抵抗力）显着增加，而绿色荧光（EGFP被酸性pH猝灭）却没有观察到，表明mCherry-EGFP-GLUT1功能蛋白被贩运酸性溶酶体系统。此外，我们能够通过对易位系数进行定量来计算mCherry与EGFP的相对比例，可将其用作GLUT1内在化和随后的溶酶体降解的读数。还构建了两个突变体，mCherry-EGFP-GLUT1-S226D和mCherry-EGFP-GLUT1-ΔC4，这间接证实了mCherry-EGFP-GLUT1用于监测GLUT1交易的特异性。通过使用一系列内体标记物（Rab5，Rab7和Rab11）和溶酶体标记物，我们能够定义GLUT1在活细胞中运输的模型，在该模型中，葡萄糖剥夺后GLUT1从PM中解离，pH梯度从6.8内体早期为6.1，晚期内体为6.0-4.8，最后溶酶体的pH为4.5，这适合降解。此外，我们的概念验证研究表明，pmCherry-EGFP-GLUT1追踪系统可以准确反映GLUT1响应小分子穿心莲内酯治疗后的内源性变化。由于针对GLUT1表达和依赖GLUT1的葡萄糖代谢是针对各种类型的癌症和某些其他葡萄糖成瘾性疾病的有前途的治疗策略，
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