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

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 than that on the adherent side. The rmax GLUT1 clusters. value corresponding to the maximum of L(r)-r was defined as the Herein, we applied direct stochastic optical reconstruction average size of the analyzed clusters in the region of 2 × 2 μm2, microscopy (dSTORM) (30, 31), one of the super-resolution which showed that GLUT1 formed clusters with an average di- imaging techniques, to observe the spatial distribution of GLUT1 ameter of 250 ± 20 nm on the medium exposed side and 137 ± on HeLa cell membranes, which are found to overexpress GLUT1 16 nm on the adherent side. We also extracted the information (SI Appendix,Fig.S1), then quantitatively analyze the distribution of the amount (Fig. 1F) and size (Fig. 1G) of the GLUT1 clusters characteristics of GLUT1 clusters. By dual-color dSTORM im- on the medium exposed side and adherent side of HeLa cells. aging and inhibitor treatment, we elucidated the critical roles of The results showed that the transporter formed much more and lipid rafts, actin cytoskeleton, and glycosylation in cluster forma- larger clusters on the medium exposed side than on the adherent tion and stability. Moreover, combining dSTORM data with bio- side. To further clarify the sporadic GLUT1 and the molecular chemistry results, we depicted the detailed changes of GLUT1 organization in clusters, we applied semiquantitative analysis clustering and distribution under activation by different stimuli, (Experimental Section) to calculate the amount of distribution of which revealed a probable relationship between the activation and sporadic GLUT1 (one molecule) and clustered GLUT1 containing spatial distribution. more than two molecules (Fig. 1H). The statistical result showed that GLUT1 formed clusters with different numbers of molecules, Results and Discussion ranging from 2 to more than 10, but a few were more than 25. Mapping GLUT1 on Cell Membranes Using STORM. Considering the Small clusters containing two to four molecules were in the ma- fluidity of the cell membrane, we used fixed HeLa cells to ob- jority. Moreover, more large clusters that consisted of more than serve the distribution of GLUT1. When the fixation was not four molecules were found to generate on the medium exposed adequate, proteins on cell membranes would not be completely side, while sporadic GLUT1 were more on the adherent side. immobilized, causing the redistribution of proteins (32, 33). So, By comparison, we found that GLUT1 formed clusters with we first optimized the fixation time and found that the mor- different numbers and sizes on HeLa medium exposed side and phology of GLUT1 has no significant difference on cell mem- adherent side. High-density and large-diameter clusters were branes with different fixation time (SI Appendix, Fig. S2). Thus, seen on the medium exposed side, while sparse and small clusters 20 min for fixation was used in all subsequent experiments. were observed on the adherent side. These discrepancies may be

Fig. 1. GLUT1 proteins form clusters of different sizes and amounts on HeLa medium exposed side and adherent side. (A–D) Reconstructed dSTORM images of GLUT1 on medium exposed side (A) and adherent side (C), and the corresponding magnified images (B and D). (Scale bars: A and C,5μm; B and D,2μm.) (E) Representative Ripley’s K function plots of GLUT1 on different membranes. Data are from 30 stochastically selected regions of 2 × 2 μm2 in 10 cells of three independent experiments. (F) The average number of GLUT1 clusters per μm2.(G) The average cluster area. (H) The number of sporadic GLUT1 and clusters containing different amounts of molecules per μm2. Data in F–H are obtained from 10 cells of three independent experiments (mean ± SD). **P < 0.01 (two- tailed unpaired t test).

7034 | www.pnas.org/cgi/doi/10.1073/pnas.1803859115 Yan et al. Downloaded by guest on September 28, 2021 and cholesterol environment (12, 34). Besides, several studies have depicted that lipid rafts exist as nanodomains or microdomains on the cell membrane (14, 35), and our previous studies also observed lipid rafts with the size ranging from 10 to 200 nm (14, 36, 37), which was consistent with the average diameter of GLUT1 (∼200 nm). Accordingly, we hypothesized that GLUT1 clustering might be associated with lipid rafts. To verify this as- sumption, dual-color dSTORM imaging was performed to locate the lipid rafts and GLUT1 on the medium exposed side of HeLa cells (Fig. 2). The merged image of lipid rafts and GLUT1 (Fig. 2C) showed a degree of colocalization between the two. Mander’s coefficients (38), M1 and M2, were used to analyze said colocalization. These coefficients represented the amount of colocalized pixels relative to the total covered by each com- ponent. Based on their values, we sorted the positional rela- tionships into four types: overlap (M1/M2 > 0.66, Fig. 2D), partial overlap (0.33 < M1/M2 <0.66, Fig. 2E), edge connection (0 < M1 and M2 < 0.33, Fig. 2F), and isolation (M1/M2 = 0, Fig. 2G), then we determined the percentage of the four localization states (Fig. 2H). Overlap and partial overlap were collectively referred to as colocalization. The data showed that 35% (over- Fig. 2. Dual-color dSTORM images revealing the relative spatial distribution lap: 11%, partial overlap: 24%) of total GLUT1 was associated of GLUT1 and lipid rafts on the HeLa membrane. (A and B) Reconstructed with lipid rafts, whereas the remaining might be localized in dSTORM images of lipid rafts with Alexa647-conjugated CT-B (A) and GLUT1 other lipid compositions of rafts or nonlipid rafts domains. This labeled with Alexa532-conjugated antibody (B) on the same cell membrane. ’ (C) The merged image of A and B, showing significant colocalization of the hinted us that the clusters formation was not only affected by two. (D–G) Enlarged dual-color dSTORM images of white Inset squares in C, lipid rafts and there might be other factors that contributed to displaying four types of location relationship: overlap (D), partial overlap (E), the aggregation of GLUT1. edge connection (F), and isolation (G). (H) The percentage of the four types of location states. Data are from 10 cells in three independent experiments. Lipid Rafts Disruption Weakens GLUT1 Clustering. The reasons why (Scale bars: A–C,5μm; D–G, 200 nm.) GLUT1 clusters did not totally colocalize with GM1-enriched lipid rafts (CT-B labeled) may be two: (i) other compositions of rafts may colocalize with GLUT1 clusters; and (ii) other factors caused by the varying external environments experienced by may participate in regulating clustering. Hence, we decided to different surfaces. The medium exposed side are entirely ex- further confirm the role of lipid rafts at first. We treated HeLa posed to the external environment and are able to contact with cells with 10 mM methyl-β-cyclodextrin (MβCD) for 20–30 min, more external factors, such as hormones and extracellular growth which can remove the cholesterol from the lipid rafts (39), to BIOCHEMISTRY factors. As previous studies reported that growth factors like IGF investigate whether the alteration of the cholesterol environment and SCF can increase glucose metabolism (34), it is thus possible could influence GLUT1 distribution. We found that a lot of that the changes of glucose levels induced by extracellular factors GLUT1 clusters became smaller or even disappeared after could result in the difference of GLUT1 distribution on the adding MβCD (Fig. 3). The Ripley’s K-function plots indicated medium exposed side and adherent side. that the degree of clustering decreased dramatically and that the average diameter of the clusters dropped from ∼250 nm to Interactions Between GLUT1 and Lipid Rafts. Many studies have ∼140 nm (Fig. 3E). Moreover, the number of clusters per unit been reported that GLUT1 is partly localized to DRM domains area reduced from ∼2.3 ± 0.3 to ∼1.2 ± 0.3 (Fig. 3F), and and that the transporter activity is sensitive to the phospholipid clusters consisting of more than two molecules declined sharply

Fig. 3. The changes of GLUT1 clusters after lipid rafts disruption. (A–D) Reconstructed dSTORM images of GLUT1 on control (A)andMβCD-treated (C)HeLacell membranes and the corresponding magnified images with clusters circled in white (B and D). (Scale bars: A and C,5μm; B and D,2μm.) (E) Ripley’sKfunction plots of GLUT1 on control and MβCD-treated HeLa membranes. (F) The number of clusters per μm2.(G) The number of sporadic GLUT1 and clusters containing a different amount of molecules per μm2. Data are obtained from 10 cells of three independent experiments (mean ± SD). **P < 0.01 (two-tailed unpaired t test).

Yan et al. PNAS | July 3, 2018 | vol. 115 | no. 27 | 7035 Downloaded by guest on September 28, 2021 Fig. 4. Comparative analysis of the morphologies of GLUT1 after the disruption of actin cytoskeleton. (A– F) Reconstructed dSTORM images of GLUT1 on control (A), CB-treated (C) and CD-treated (E)cell membranes, and the corresponding magnified images (B, D, and F). (Scale bars: A, C, and E,5μm; B, D, and F,2μm.) (G) The number of localizations per μm2 on these three kinds of membranes. (H) The average cluster area. Data are obtained from 10 cells of three independent experiments (mean ± SD). *P < 0.5, **P < 0.01 (two-tailed unpaired t test).

(Fig. 3G). To rule out the destructive possibility of MβCD on 30 min, which can depolymerize actin cytoskeleton. The average membrane structure or other membrane constituents, we per- cluster area (Fig. 4) and the molecules within clusters (SI Appendix, formed the experiment of cholesterol repletion (MβCD satu- Fig. S7A) decreased significantly compared with that on control rated with cholesterol at a MβCD:cholesterol molar ratio of 8:1). cell membranes. However, CB not only inhibits actin filaments As shown in SI Appendix, Fig. S6A, lipid rafts returned to the from generating networks, but also weakens glucose transport by clustered structure after repletion of cholesterol. The localiza- interacting with the substrate efflux site (40). Our results also tions and size of lipid rafts decreased sharply with MβCD showed that CB-treated cells consumed less glucose than control treatment, while these values increased to the level of control (see Fig. 7). To exclude the effect of glucose transport on GLUT1 cells after cholesterol repletion (SI Appendix, Fig. S6 B and C). distribution, we used another inhibitor cytochalasin D (CD). Thus, these results indicated that MβCD had no effect on other Likewise, it inhibits actin polymerization but hardly affects the constitutes except lipid rafts. In addition, we also validated that cellular permeability to sugars (41), which was supported by our GLUT1 clusters formed again and the average cluster size re- data of glucose consumption as well (see Fig. 7). After CD treat- covered to that on control cell membranes (∼0.12 μm2)(SI Ap- ment, the total localizations of GLUT1 did not change, whereas pendix, Fig. S6 D–F). clusters became scattered and small (Fig. 4 and SI Appendix, Fig. Dual-color imaging, together with the disruption and repletion of S7B). These findings demonstrated that the disruption of actin lipid rafts, demonstrated that the stable existence of most GLUT1 filaments limited the ability of GLUT1 to form clusters, but did not clusters is dependent on the integrality of lipid rafts. The destruc- change its total level, therefore verifying the role of actin cyto- tion of lipid rafts may induce to partial or complete fragmentation skeleton in transporter clustering. of larger GLUT1 clusters. Of note, although the number of large clusters reduced after MβCD treatment, there were a portion of The Impairment of N-Glycosylation Disruption in GLUT1 Clustering. GLUT1 that still formed clusters. This finding was consistent with Glycosylation is a prevalent protein modification with a pro- that only 35% of GLUT1 colocalized with lipid rafts, suggesting found effect on protein stability, folding, and a multitude of bi- other mechanisms in regulation of its distribution. ological processes (42). Since GLUT1 owns an N-glycosylation site, we presupposed that GLUT1 clustering would be related to The Limitation of the Actin Cytoskeleton in GLUT1 Clustering. As glycosylation. To prove this, HeLa cells were pretreated with above mentioned, there are other factors participating in GLUT1 50 μg/mL N-acetyl-β-D-gulcosaminides (NAG) for 30 min, which clustering, and previous studies have reported that various mem- has an effective transglycosylation of N-acetyl-β-D-gulcosaminides, brane channel proteins directly interact with actin (27). Thus, to a common motif of N-linked sugar chains in proteins (36); then clarify whether the actin cytoskeleton plays a role in controlling the nanoscale organizations of GLUT1 on cell membranes were the distribution and clustering of GLUT1, we first imaged GLUT1 investigated. The deglycosylation made GLUT1 clusters disperse, on HeLa membranes treated by 20 μg/mL cytochalasin B (CB) for and almost all of the large clusters disappeared (Fig. 5 A–D).

Fig. 5. The glycosylation regulates the GLUT1 clustering on HeLa cell membranes. (A–D) Reconstructed dSTORM images of GLUT1 on control (A) and NAG- treated (C) cell membranes, and the corresponding magnified images (B and D). (Scale bars: A and C, 5 μm; B and D,2μm.) (E) Ripley’s K function analysis of GLUT1 under the two conditions. (F) The average number of localizations per μm2.(G) The average number of GLUT1 clusters per μm2. Data are obtained from 10 cells of three independent experiments (mean ± SD). *P < 0.05 (two-tailed unpaired t test).

7036 | www.pnas.org/cgi/doi/10.1073/pnas.1803859115 Yan et al. Downloaded by guest on September 28, 2021 Fig. 6. NaN3 activation of GLUT1 changed the molecular organization of it clusters. (A and B) Reconstructed dSTORM images of GLUT1 on control (A) and NaN3-treated (B) cell membranes. (Scale bars: 5 μm.) (C) Ripley’s K function analysis of GLUT1 under the two conditions. (D) The average number of localizations per μm2.(E) The average number of GLUT1 clusters per μm2.(F) The number of sporadic GLUT1 and clusters containing different amount of molecules per μm2. Data are obtained from 10 cells in three independent experiments (mean ± SD). ns, no significance (two-tailed unpaired t test).

From the Ripley’s K function analysis (Fig. 5E), we found that localizations of GLUT1 remained stable (Fig. 6D), and even the the degree of clustering and the diameter of GLUT1 clusters fell cluster number reduced slightly on azide-treated cell membranes down significantly (from ∼250 nm to ∼130 nm). Further statis- (Fig. 6E). However, the degree of clustering and diameter of tical data indicated that there was a distinguishable decline in clusters reduced, which was only ∼170 ± 25 nm (Fig. 6C). both the number of localizations (Fig. 5F) and clusters (Fig. 5G). Moreover, the number of monomer GLUT1 increased following Moreover, sporadic molecules and clusters containing two mol- activation, and small clusters with less than five molecules went up ecules increased, whereas other large clusters with more than significantly (Fig. 6F). These results indicated that the activation two molecules fell down (SI Appendix, Fig. S7C). The reasons for of GLUT1 might change the molecular organization of clusters the decrease of cluster size and number might be two: One is that and inhibit the formation of large clusters on cell membranes. deglycosylation directly inhibits the formation of clusters, and the At this time, we focused on the results of MβCD treatment other is that the lower level of total GLUT1 could not generate again. The cluster number decreased (Fig. 3F) and only mono- larger and more clusters as it could on control cell membranes. mer GLUT1 increased, but small clusters with less than four However, we could not determine which one or both were the molecules were not as many as those on control cells (Fig. 3G). right reasons. Even so, the results clearly supported the hypothesis The results indicated that the inhibition of MβCD on GLUT1 BIOCHEMISTRY that glycosylation was an indispensable element in maintaining of clustering was stronger than that of sodium azide. It is easy to the distribution and clustering of GLUT1 on cell membranes. understand. MβCD has two functions, i.e., disrupting lipid rafts and activating glucose transporter, both of which can weaken the The Effect of GLUT1 Activation on Its Clustering. Exposure to os- clustering. Additionally, we also tested the influence of CB, CD, motic or metabolic stimuli like MβCD or azide can increase the and NAG treatment on the activity of glucose transport (Fig. 7). maximal transfer rate of GLUT1 (12, 34), we wondered what Only CB reduced the glucose uptake compared with control changes would be brought to GLUT1’s membrane distribution by groups. CB has two antagonistic effects on clustering. One is activating. So, we first performed glucose uptake assay to confirm inhibiting actin polymerization that could limit the aggregation of the activation of sodium azide and MβCD on GLUT1. As shown GLUT1, the opposite one is inhibiting GLUT1 activity that may in Fig. 7, the glucose consumption increased, and the cell viability induce the formation of clusters. Therefore, the changes of clus- did not change after treatment with azide or MβCD, validating tering features after adding CB were not as obvious as those with that these two reagents can effectively activate glucose transporter. CD treatment (Fig. 4), because CD only affects the actin cyto- Then we used 5 mM sodium azide to stimulate the adherent skeleton. For NAG groups, we thought the glucose consumption HeLa cells for 30 min before staining. From the reconstructed would decrease due to the disruption of N-glycosylation, while it dSTORM images, most of the molecules on treated membranes did not change as we expected. This might be because there is seemed to form clusters in the same way as those on control another family of glucose transporters, sodium-linked glucose membranes (Fig. 6 A and B). Further Ripley’s K function anal- transporters (SGLTs) (43), which are not affected by the degly- ysis and statistical results showed some differences in the size, cosylation of NAG. Together, the detailed molecular mechanism number, and molecular organization of GLUT1 clusters. The total for the clustering of GLUT1 and glucose uptake is complex; even

Fig. 7. Glucose uptake assay with different treat- ments. (A) Normalized cell viability under the different conditions. (B) The glucose consumption of every group, which was normalized by living cell numbers. (C) The glucose consumption relative to control. Data are from three independent experiments (mean ± SD). *P < 0.05; **P < 0.01; ns, no significance (two-tailed unpaired t test).

Yan et al. PNAS | July 3, 2018 | vol. 115 | no. 27 | 7037 Downloaded by guest on September 28, 2021 so, our findings revealed a potential relationship between GLUT1 Experimental Section clustering and activation and suggested that small clusters may be Cell Culture. Cells were cultured in Dulbecco’s modified Eagle medium more beneficial to glucose transport. (DMEM; HyClone) supplemented with 10% FBS (Gibco) and antibiotics and maintained in a humidified environment with 5% CO2 at 37 °C. Before the Conclusions imaging experiment, HeLa cells were divided into a dish where a clean In summary, by the combination of dSTORM imaging and coverslip was placed and cultured at least 24 h. proposed single molecule analysis methods, we observed that most of GLUT1 was aggregated in clusters on the HeLa mem- Glucose Uptake Analysis. The glucose consumption of HeLa cells with different treatment was measured as described in SI Appendix, SI Materials and Methods. brane and found a precise spatial association between GLUT1 and lipid rafts, which resolved the debate surrounding on the Preparation of dSTORM Samples. Cells were fixed, blocked, and labeled with localization of the transporter in membrane domains. Regarding antibodies and sealed on microscopes as described in SI Appendix, SI Ma- the organizational mechanism of GLUT1 clusters, our study terials and Methods. revealed that not only the lipid rafts’ environment can stabilize their existence, but also the actin cytoskeleton and N-glycosylation Superresolution Imaging. The raw data were captured by our home-built play important roles in the clusters’ formation. Furthermore, un- dSTORM and analyzed by Quickpalm as described in SI Appendix, SI Mate- der the activation by MβCD and sodium azide, we found that rials and Methods. many transporters were prone to distribute sporadically or form ’ small clusters, which might facilitate glucose uptake. The ability to Data Analysis. The spatial distribution of molecules was analyzed by Ripley sK ’ function and the molecular organization of clusters was estimated by directly visualize the transporter s distribution at a nanometric semiquantitative analysis method as described in SI Appendix, SI Materials resolution provided crucial information of clustering features, and Methods. clarified many regulatory factors, and captured a probable relationship between GLUT1 distribution and activation. With ACKNOWLEDGMENTS. This work was financially supported by National Key further studies such as mutation and simultaneously observation R&D Program of China Grant 2017YFA0505300 (to H.W.), National Nature of multiple glucose transporters, these initial observations may Science Foundation of China Grants 21727816, 21525314, 21721003 (to H.W.), 21703231 (to J.G.), 21503213 (to M.C.), and 31330082 (to J.J.), and form a significant step forward in our understanding of the mo- Yunnan Provincial Science and Technology Department of China Grants lecular mechanism of GLUT clustering and glucose uptake. 2017FA044 and 2013HA023 (to W.X.).

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