Visualization of GLUT1 Trafficking in Live Cancer Cells by the Use of a Dual-Fluorescence Reporter

Zhen-Yan Li; Yu-Ling Shi; Guo-Xiong Liang; Jie Yang; Song-Kuan Zhuang; Jie-Bin Lin; Abdelmoumin Ghodbane; Man-Seng Tam; Zu-Jian Liang; Zhen-Gang Zha; Huan-Tian Zhang

doi:10.1021/acsomega.0c01054

Visualization of GLUT1 Trafficking in Live Cancer Cells by the Use of a Dual-Fluorescence Reporter

ACS Omega. 2020 Jun 23;5(26):15911-15921. doi: 10.1021/acsomega.0c01054. eCollection 2020 Jul 7.

Authors

Affiliations

¹ Institute of Orthopedic Diseases and Department of Bone and Joint Surgery, the First Affiliated Hospital, Jinan University, Guangzhou, Guangdong 510630, China.
² Department of Orthopedics, the Third Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong 510405, China.
³ Department of Pharmacy, Zhongshan Hospital of Traditional Chinese Medicine, Zhongshan, Guangdong 528400, China.
⁴ State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, Fujian 361102, China.
⁵ IAN WO Medical Center, Macao Special Administrative Region, Macao 999078, China.

Abstract

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.