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Mechanistic Insights Into GLUT1 Activation and Clustering Revealed by Super-Resolution Imaging

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Mechanistic Insights Into GLUT1 Activation and Clustering Revealed by Super-Resolution Imaging Mechanistic insights into GLUT1 activation and clustering revealed by super-resolution imaging Qiuyan Yana,b, Yanting Lub,c, Lulu Zhoua,b, Junling Chena, Haijiao Xua, Mingjun Caia, Yan Shia, Junguang Jianga, Wenyong Xiongc,1, Jing Gaoa,1, and Hongda Wanga,d,1 aState Key Laboratory of Electroanalytical Chemistry, Research Center of Biomembranomics, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022 Jilin, P. R. China; bSchool of Chemistry and Chemical Engineering, University of Chinese Academy of Sciences, 100049 Beijing, P. R. China; cState Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201 Yunnan, P. R. China; and dLaboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Aoshanwei, Jimo, Qingdao, 266237 Shandong, P. R. China Edited by Nieng Yan, Princeton University, Princeton, NJ, and accepted by Editorial Board Member Alan R. Fersht May 24, 2018 (received for review March 9, 2018) The glucose transporter GLUT1, a plasma membrane protein that super-resolution fluorescence microscopy, which breaks the dif- mediates glucose homeostasis in mammalian cells, is responsible fraction barrier and achieves a lateral resolution in the tens of for constitutive uptake of glucose into many tissues and organs. nanometers (17), has provided a particularly suitable tool to solve Many studies have focused on its vital physiological functions and these problems. Meanwhile, it has been proven that multiprotein close relationship with diseases. However, the molecular mecha- assemblies are dependent on cholesterol environment, and their nisms of its activation and transport are not clear, and its detailed separation and anchoring are related to the actin cytoskeleton (18, distribution pattern on cell membranes also remains unknown. To 19). Nonetheless, it is still unknown whether these factors have address these, we first investigated the distribution and assembly contributions to the spatial distribution of GLUT1. of GLUT1 at a nanometer resolution by super-resolution imaging. Lipid rafts, also known as the detergent-resistant membranes On HeLa cell membranes, the transporter formed clusters with an (DRMs), are membrane domains containing high levels of cho- ∼ average diameter of 250 nm, the majority of which were regu- lesterol, sphingolipids, and specific proteins, which play a sig- lated by lipid rafts, as well as being restricted in size by both the nificant role in cell signaling and protein assembling (20, 21). cytoskeleton and glycosylation. More importantly, we found that Abundant evidence has proved that spatial recruitment and β the activation of GLUT1 by azide or M CD did not increase its clustering of proteins and lipids into lipid rafts is a remarkable membrane expression but induced the decrease of the large clus- feature in a variety of signaling and transferring processes (22, ters. The results suggested that sporadic distribution of GLUT1 23), for instance insulin receptors, integrin, and T cell antigen may facilitate the transport of glucose, implying a potential asso- receptors (22, 24). Even the members of GLUT family (GLUT4 ciation between the distribution and activation. Collectively, our and GLUT1) have been found to associate with DRMs (4, 25). work characterized the clustering distribution of GLUT1 and linked However, due to the use of detergents for extracting lipid rafts its spatial structural organization to the functions, which would BIOCHEMISTRY provide insights into the activation mechanism of the transporter. in these experiments that broke the natural condition of cell membranes, the validity and accuracy of the colocalization between GLUT1 | direct stochastic optical reconstruction microscopy | single molecule | cluster | activation Significance lucose is the primary source of energy and substrate for Many membrane proteins are functioning in aggregations and Gcells, and its transport process is important for both normal associating with microdomains, which range from nanometers and diseased cellular metabolisms (1, 2). Previous studies have shown to micrometers in size. Therefore, it is indispensable to directly that the uptake of glucose and other carbohydrates through the cell analyze these proteins and microdomains in native cell mem- plasma membrane is largely dependent on members of the branes at a single-molecule level. GLUT1 is a ubiquitously glucose transport (GLUT) family (3). Humans have 14 such expressed protein, contributing to basal and growth factor- members, all of which are encoded by SLC2A genes (4). The stimulated glucose uptake in many tissues. It is overexpressed first characterized glucose transporter, GLUT1, is widely in almost all tumors. Herein, by direct stochastic optical re- expressed and responsible for the constant uptake of glucose (5, construction microscopy, we previously mapped GLUT1 on 6). Many researchers have been attracted to focus on its vital native cell membranes and highlighted key contributions of physiological and pathophysiological sense (7, 8), and its over- the lipid raft, cytoskeleton, and glycosylation to the formation expression has become an important hypoxic marker in malignant of clusters. Moreover, we elucidated that the clustered distri- tumors and a prognostic indicator for tumorigenesis (7, 9). bution of the transporter was associated with its activation, Recently, the structure and distribution pattern of GLUT1 has which is crucial to advance our understanding of the trans- ’ also drawn wide concern. Some studies have found that it is an porter s spatial organization and activation mechanism. inward-open uniporter with a single N-glycosylation site (10, 11), Author contributions: J.G. and H.W. designed research; Q.Y., Y.L., and W.X. performed and some have showed a markedly punctate staining pattern of research; H.X., M.C., Y.S., and J.J. contributed new reagents/analytic tools; Q.Y., L.Z., and GLUT1 on cell membranes under deconvolution fluorescence J.C. analyzed data; and Q.Y., W.X., J.G. and H.W. wrote the paper. microscopy (12). However, the diffraction-limited resolution The authors declare no conflict of interest. made it very difficult to reveal the detailed structure of GLUT1. This article is a PNAS Direct Submission.N.Y.isaguesteditorinvitedbythe For example, issues on whether membrane GLUT1 forms clus- Editorial Board. ters as a working unit in the same way as many other membrane Published under the PNAS license. proteins, such as GPI-anchored proteins, epidermal growth re- 1To whom correspondence may be addressed. Email: [email protected], ceptors (EGFRs), and Toll-like receptors (13–15), and which [email protected], or [email protected]. transmutation causes an acute increase of the maximal velocity This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (Vmax) for glucose uptake following exposure to osmotic or 1073/pnas.1803859115/-/DCSupplemental. metabolic stimuli (12, 16), have not been clarified. Fortunately, Published online June 18, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1803859115 PNAS | July 3, 2018 | vol. 115 | no. 27 | 7033–7038 Downloaded by guest on September 28, 2021 GLUT1 and lipid rafts is still debatable. Besides, actin as a major Cultured HeLa cells present adherent growth and their adherent cytoskeleton protein is also found to be involved in almost all side and medium exposed side face different environments, biological events, contributing to the mechanical properties and which we think may influence the distribution of GLUT1. To test shapes of cells (26). Some ion channel proteins on cell mem- this idea, we used dSTORM to investigate the spatial distribu- branes have been identified binding to actin directly or indirectly tion of GLUT1 on both the medium exposed side and adherent through the actin binding proteins (27). Nevertheless, whether side (see Experimental Section and SI Appendix, Fig. S3 for de- actin filaments have an effect on the distribution of energy tail). The reconstructed dSTORM images and the corresponding channel protein, GLUT1, remains unknown. magnified pictures showed that GLUT1 tended to form elliptic As an important glucose transporter, the activation and the and dense clusters on the medium exposed side (Fig. 1 A and B) transport of GLUT1 has been explored as well. Several studies but sparse clusters with irregular shapes on the adherent side have suggested that the activation of the transporter by meta- (Fig. 1 C and D). The same phenomenon was also observed on bolic stresses is mediated by translocating of GLUT between OS-RC-2 cell (human renal carcinoma cell) membranes (SI intracellular storage pools and the cell surface or involves acti- Appendix, Fig. S4). vation (“unmasking”) of individual transporters preexisting in To quantify the features of the clusters, we used Ripley’sK the plasma membranes (28, 29). Little is known about whether function (13) to analyze the spatial clustering in nanoscale do- the activation changes the distribution pattern of GLUT1 and mains (see SI Appendix, Fig. S5 for detail). The maxima of the how the transporters assemble and organize with or without L(r)-r (medium exposed side: 180 ± 20, adherent side: 101 ± 4) activation. The current uncertainty on these topics calls for new in Fig. 1E indicated that the degree of clustering on the medium methods capable of directly monitoring the size and stability of exposed side was higher
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