Abstract
The response of cells to forces is critical for their function and occurs via rearrangement of the actin cytoskeleton1. Cytoskeletal remodelling is energetically costly2,3, yet how cells signal for nutrient uptake remains undefined. Here we present evidence that force transmission increases glucose uptake by stimulating glucose transporter 1 (GLUT1). GLUT1 recruitment to and retention at sites of force transmission requires non-muscle myosin IIA-mediated contractility and ankyrin G. Ankyrin G forms a bridge between the force-transducing receptors and GLUT1. This bridge is critical for enabling cells under tension to tune glucose uptake to support remodelling of the actin cytoskeleton and formation of an epithelial barrier. Collectively, these data reveal an unexpected mechanism for how cells under tension take up nutrients and provide insight into how defects in glucose transport and mechanics might be linked.
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Data availability
Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author upon reasonable request.
References
Fletcher, D. A. & Mullins, R. D. Cell mechanics and the cytoskeleton. Nature 463, 485–492 (2010).
Bernstein, B. W. & Bamburg, J. R. Actin-ATP hydrolysis is a major energy drain for neurons. J. Neurosci. 23, 1–6 (2003).
Daniel, J. L., Molish, I. R., Robkin, L. & Holmsen, H. Nucleotide exchange between cytosolic ATP and F-actin-bound ADP may be a major energy-utilizing process in unstimulated platelets. Eur. J. Biochem. 156, 677–684 (1986).
Ferrell, N. et al. Application of physiological shear stress to renal tubular epithelial cells. Methods Cell Biol. 153, 43–67 (2019).
Neville, M. C. Classic studies of mammary development and milk secretion: 1945–1980. J. Mammary Gland Biol. Neoplasia 14, 193–197 (2009).
Guilluy, C. et al. The Rho GEFs LARG and GEF-H1 regulate the mechanical response to force on integrins. Nat. Cell Biol. 13, 722–727 (2011).
Marjoram, R. J., Guilluy, C. & Burridge, K. Using magnets and magnetic beads to dissect signaling pathways activated by mechanical tension applied to cells. Methods 94, 19–26 (2016).
Barry, A. K. et al. α-Catenin cytomechanics—role in cadherin-dependent adhesion and mechanotransduction. J. Cell Sci. 127, 1779–1791 (2014).
Collins, C. et al. Localized tensional forces on PECAM-1 elicit a global mechanotransduction response via the integrin-RhoA pathway. Curr. Biol. 22, 2087–2094 (2012).
Kim, T. J. et al. Dynamic visualization of α-catenin reveals rapid, reversible conformation switching between tension states. Curr. Biol. 25, 218–224 (2015).
Bays, J. L., Campbell, H. K., Heidema, C., Sebbagh, M. & DeMali, K. A. Linking E-cadherin mechanotransduction to cell metabolism through force-mediated activation of AMPK. Nat. Cell Biol. 19, 724–731 (2017).
Bays, J. L. et al. Vinculin phosphorylation differentially regulates mechanotransduction at cell–cell and cell–matrix adhesions. J. Cell Biol. 205, 251–263 (2014).
Tzima, E. et al. A mechanosensory complex that mediates the endothelial cell response to fluid shear stress. Nature 437, 426–431 (2005).
Park, J. S. et al. Mechanical regulation of glycolysis via cytoskeleton architecture. Nature 578, 621–626 (2020).
Camps, M., Vilaro, S., Testar, X., Palacin, M. & Zorzano, A. High and polarized expression of GLUT1 glucose transporters in epithelial cells from mammary gland: acute down-regulation of GLUT1 carriers by weaning. Endocrinology 134, 924–934 (1994).
Yang, Q. et al. PRKAA1/AMPKα1-driven glycolysis in endothelial cells exposed to disturbed flow protects against atherosclerosis. Nat. Commun. 9, 4667 (2018).
Farrell, C. L., Yang, J. & Pardridge, W. M. GLUT-1 glucose transporter is present within apical and basolateral membranes of brain epithelial interfaces and in microvascular endothelia with and without tight junctions. J. Histochem. Cytochem. 40, 193–199 (1992).
Capaldo, C. T. & Macara, I. G. Depletion of E-cadherin disrupts establishment but not maintenance of cell junctions in Madin–Darby canine kidney epithelial cells. Mol. Biol. Cell 18, 189–200 (2007).
O’Brien, L. E., Zegers, M. M. & Mostov, K. E. Building epithelial architecture: insights from three-dimensional culture models. Nat. Rev. Mol. Cell Biol. 3, 531–537 (2002).
Yu, W. et al. Formation of cysts by alveolar type II cells in three-dimensional culture reveals a novel mechanism for epithelial morphogenesis. Mol. Biol. Cell 18, 1693–1700 (2007).
Zegers, M. M., O’Brien, L. E., Yu, W., Datta, A. & Mostov, K. E. Epithelial polarity and tubulogenesis in vitro. Trends Cell Biol. 13, 169–176 (2003).
Heuze, M. L. et al. Myosin II isoforms play distinct roles in adherens junction biogenesis. eLife 8, e46599 (2019).
Bennett, V. & Healy, J. Membrane domains based on ankyrin and spectrin associated with cell–cell interactions. Cold Spring Harb. Perspect. Biol. 1, a003012 (2009).
Nelson, W. J., Shore, E. M., Wang, A. Z. & Hammerton, R. W. Identification of a membrane-cytoskeletal complex containing the cell adhesion molecule uvomorulin (E-cadherin), ankyrin, and fodrin in Madin–Darby canine kidney epithelial cells. J. Cell Biol. 110, 349–357 (1990).
Kizhatil, K. et al. Ankyrin-G is a molecular partner of E-cadherin in epithelial cells and early embryos. J. Biol. Chem. 282, 26552–26561 (2007).
Jenkins, P. M. et al. E-cadherin polarity is determined by a multifunction motif mediating lateral membrane retention through ankyrin-G and apical–lateral transcytosis through clathrin. J. Biol. Chem. 288, 14018–14031 (2013).
Cadwell, C. M., Jenkins, P. M., Bennett, V. & Kowalczyk, A. P. Ankyrin-G inhibits endocytosis of cadherin dimers. J. Biol. Chem. 291, 691–704 (2016).
Yang, H. Q. et al. Ankyrin-G mediates targeting of both Na+ and KATP channels to the rat cardiac intercalated disc. eLife 9, e52373 (2020).
Zhou, D. et al. AnkyrinG is required for clustering of voltage-gated Na channels at axon initial segments and for normal action potential firing. J. Cell Biol. 143, 1295–1304 (1998).
Lowe, J. S. et al. Voltage-gated Nav channel targeting in the heart requires an ankyrin-G dependent cellular pathway. J. Cell Biol. 180, 173–186 (2008).
Baines, A. J. The spectrin–ankyrin-4.1–adducin membrane skeleton: adapting eukaryotic cells to the demands of animal life. Protoplasma 244, 99–131 (2010).
Bennett, V. The molecular basis for membrane–cytoskeleton association in human erythrocytes. J. Cell Biochem. 18, 49–65 (1982).
Drenckhahn, D., Schluter, K., Allen, D. P. & Bennett, V. Colocalization of band 3 with ankyrin and spectrin at the basal membrane of intercalated cells in the rat kidney. Science 230, 1287–1289 (1985).
Drenckhahn, D. & Bennett, V. Polarized distribution of Mr 210,000 and 190,000 analogs of erythrocyte ankyrin along the plasma membrane of transporting epithelia, neurons and photoreceptors. Eur. J. Cell Biol. 43, 479–486 (1987).
Khan, A. A. et al. Dematin and adducin provide a novel link between the spectrin cytoskeleton and human erythrocyte membrane by directly interacting with glucose transporter-1. J. Biol. Chem. 283, 14600–14609 (2008).
Jiang, W. et al. Interaction of glucose transporter 1 with anion exchanger 1 in vitro. Biochem. Biophys. Res. Commun. 339, 1255–1261 (2006).
Zhang, J. et al. Energetic regulation of coordinated leader-follower dynamics during collective invasion of breast cancer cells. Proc. Natl Acad. Sci. USA 116, 7867–7872 (2019).
DeCamp, S. J. et al. Epithelial layer unjamming shifts energy metabolism toward glycolysis. Sci. Rep. 10, 18302 (2020).
Zanotelli, M. R. et al. Energetic costs regulated by cell mechanics and confinement are predictive of migration path during decision-making. Nat. Commun. 10, 4185 (2019).
Kannan, N. & Tang, V. W. Synaptopodin couples epithelial contractility to α-actinin-4-dependent junction maturation. J. Cell Biol. 211, 407–434 (2015).
Maiers, J. L., Peng, X., Fanning, A. S. & DeMali, K. A. ZO-1 recruitment to α-catenin—a novel mechanism for coupling the assembly of tight junctions to adherens junctions. J. Cell Sci. 126, 3904–3915 (2013).
Tang, V. W. & Goodenough, D. A. Paracellular ion channel at the tight junction. Biophys. J. 84, 1660–1673 (2003).
Peng, X., Cuff, L. E., Lawton, C. D. & DeMali, K. A. Vinculin regulates cell-surface E-cadherin expression by binding to β-catenin. J. Cell Sci. 123, 567–577 (2010).
Acknowledgements
We thank P. Rubenstein and G. DeWane for comments. This work is supported by National Institutes of Health grants R35GM136291 to K.A.D. and P30CA086862 to the Holden Comprehensive Cancer Center. R.-M.M. was supported by Agence Nationale de la Recherche ‘POLCAM’ (ANR-17-CE13-0013) and ‘CODECIDE’ (ANR-17-CE13-0022) Ligue Contre le Cancer (Equipe labellisée 2019). National Institutes of Health predoctoral fellowships T32GM067795 and 1F31GM135962-01 supported A.M.S. American Heart Association grant 16PRE26701111 supported J.L.B.
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A.M.S. designed and performed experiments, analysed data and wrote the manuscript. J.L.B. and S.R.M. performed experiments. K.A.D. helped with experimental design, wrote the manuscript and directed the project. R.-M.M. provided reagents. All authors provided detailed comments.
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Extended data
Extended Data Fig. 1 GLUT1 mediates force-induced glucose uptake.
a-b, Inhibition of GLUT3 has no effect on glucose uptake. MCF-10A cells expressing shRNAs targeting GLUT3 (shGLUT3.1, shGLUT3.2) or a scramble sequence (scrGLUT3) were employed. a, GLUT3 expression was measured using immunoblotting. The graph beneath the immunoblot represents the average amount of GLUT3 normalized to the loading control ± SEM, n = 3 biologically independent samples. b, Glucose uptake was monitored. The graphs represent the average glucose taken up into the cells ± SEM, n = 3 biologically independent samples c, Tensile forces stimulate glucose uptake in a GLUT1-dependent manner. Tensile force was applied to MCF-10A cells as described in Fig. 1g. Glucose uptake was monitored. The graphs represent the average glucose taken up into the cells ± SEM, n = 3 biologically independent samples. Where indicated, the cells were pre-incubated with inhibitors against the various GLUTs. d, Plating cells on soft substrates does not change glucose uptake. MDCKII cells were plated on soft elastic collagen substrates and glucose uptake was monitored. The graphs represent the average glucose taken up into the cells ± SEM, n = 3 biologically independent samples e-g, Controls for the studies in Fig. 2a showing that GLUT1 localizes to cell-cell junctions when cells expressing a scramble sequence are employed. MDCKII cells expressing a shRNA encoding a scramble sequence in GLUT1 were processed, visualized, and quantified as described in Fig. 2a. Representative images are shown in (e) and a quantification of the amount of junctional GLUT1 (f) and β-catenin (g) is plotted. Data are represented as a box and whisker plot with median, 10th, 25th, 75th, and 90th percentiles shown. Scale bars=10 μM. n = 50 junctions over 5 fields of view h, Co-localization of GLUT1 and β-catenin occurs in cell-cell junctions. Orthogonal sectional views of cells are shown. Scale bars=10 μM. i, GLUT1 protein levels were unaffected by shear. Cell lysates were probed for GLUT1 expression. The graph beneath the immunoblots represents the average amount of GLUT1 normalized to the loading control ± SEM, n = 3 biologically independent samples. For all experiments, significance was calculated using a two-tailed unpaired Student t-test.
Extended Data Fig. 2 Cell stiffening is a transient process that can be visualized by staining cells with F-actin and E-cadherin.
a-c, Recovery of the actin cytoskeleton after application of shear stress revealed reinforcement is a transient event. Cells were left resting or subjected to shear stress for 2 h and then allowed to recover to the indicated times. Cells were examined using confocal microscopy under identical laser settings, and the average corrected fluorescence intensity of E-cadherin (e) or F-actin (f) in junctions are plotted. n = 45–50 junctions over 5 fields of view. d-f, Actin cytoskeleton reinforcement and E-cadherin enrichment in cell-cell junctions in response to shear occurs when similar focal planes are visualized. MCDKII or MDCKII cells expressing a shRNA against a scrambled GLUT1 sequence (scrGLUT1) or E-cadherin (shEcad) were left resting or exposed to shear stress for 2 hours. The cells were fixed, stained with phalloidin to visualize F-actin, DAPI to visualize the nucleus, or antibodies against E-cadherin to visualize cell-cell junctions. The cells were examined using confocal microscopy using identical laser settings, and the average corrected fluorescence intensity of E-cadherin (b) or F-actin (c) in junctions are plotted. n = 30 junctions over 3 fields of view. All immunofluorescence data are represented as a box and whisker plot with median, 10th, 25th, 75th, and 90th percentiles shown. Scale bars=10 μM. For all experiments, significance was calculated using a two-tailed unpaired Student t-test.
Extended Data Fig. 3 Control studies for the effects of shear on reinforcement of the actin cytoskeleton.
MDCKII or MDCKII cells expressing a shRNA against GLUT1 were left resting (no shear) or subjected to shear stress (shear). a-b, Controls for the data in Fig. 3a. Images were collected and quantifications were obtained as described in Fig. 3. No shear control images for Fig. 3a are shown, and quantification of E-cadherin enrichment for the data in Fig. 3a are shown in b. n = 45–50 junctions over 5 fields of view. c-e, Cells reinforce their actin cytoskeletons on soft and stiff matrices to a similar extent. Prior to application of shear, cells were plated on soft collagen elastic matrices or stiff matrices. The cells were fixed, stained, visualized, and quantified as described in Fig. 3a. Representative images are shown (c), and the junctional enrichment of E-cadherin (d) and F-actin (e) are shown. n = 50 junctions over 5 fields of view. f-g, Inhibition of GLUT1 does not globally affect phosphorylation. MDCKII cells were left resting (-) or subjected to orbital shear stress (+). Cells were immediately lysed and whole cell lysates were probed using immunoblotting with antibodies that recognize either AMPK phosphorylated in its activation loop (pAMPK) and then stripped and re-probed for total AMPK (f), or antibodies that recognized pCRKL at the Abl-specific site and stripped and re-probed with antibodies against total CRKL (g). The immunoblot graphs show the average fold-change ± SEM, n = 4 biologically independent samples (f), n = 3 biologically independent samples (g). h, The graph represents the average corrected fluorescence intensity of E-cadherin in 50 junctions for the data in Fig. 3c. i, The graph represents the average corrected fluorescence intensity of GFP-E-cadherin in junctions for the data in Fig. 3e. n = 50 junctions over 5 fields of view. All immunofluorescence data are represented as a box and whisker plot with median, 10th, 25th, 75th, and 90th percentiles shown. Scale bars=10 μM. For all experiments, significance was calculated using a two-tailed unpaired Student t-test.
Extended Data Fig. 4 Ankyrin G mediates GLUT1 and E-cadherin complex.
a-c, Ankyrin G is required for reinforcement of the actin cytoskeleton. MCF-10A cells with depressed levels of ankyrin G (shANKG) or a control cell line (scrANKG) were left resting (no shear) or exposed to orbital shear stress. The cells were fixed, stained with antibodies against E-cadherin or phalloidin, and examined by confocal microscopy. Representative images are shown in a. The graphs represent the average corrected fluorescence intensity of F-actin (b) or E-cadherin (c) in junctions. n = 50 junctions over 5 fields of view. Data are represented as a box and whisker plot with median, 10th, 25th, 75th, and 90th percentiles shown. d, Shear stress has no effect on ankyrin G expression. Cells were subject to shear or left resting. The soluble fraction was isolated by lysing cells in extraction buffer as described in the Materials and Methods, and the insoluble fraction was obtained by solubilizing the pellet in 2x sample buffer. The graphs beneath the blots indicate the amount of ankyrin G in each fraction normalized to the load control, β-actin ± SEM, n = 3 biologically independent samples. e, Shear induces spectrin recruitment to cell-cell contacts. MDCKII cells were fixed and stained with antibodies recognizing β-catenin and spectrin. n = 50 junctions over 5 fields of view. Representative images are shown and an average Pearson correlation coefficient ± SEM is reported beneath the images. Scale bars=10 μM. For all experiments, significance was calculated using a two-tailed unpaired Student t-test.
Extended Data Fig. 5 Ankyrin G mediates force-induced complex between E-cadherin and GLUT1.
a-c, Wildtype E-cadherin and the PolyA mutant E-cadherin protein are expressed at similar levels. MDCKII cells with depressed E-cadherin levels (shEcad) were rescued with either wild-type GFP-E-cadherin (shEcad/WT) or PolyA GFP-E-cadherin (shEcad/Poly A. a, Whole cell lysates were obtained and blotted with antibodies against E-cadherin or a loading control, β-actin. The graph beneath the immunoblot represents the average E-cadherin normalized to β-actin ± SEM. n = 3 biologically independent samples. b-c, The PolyA mutant E-cadherin does not bind ankyrin G. The indicated cells were either left resting (-) or subjected to orbital shear stress (+), lysed, and co-precipitation of E-cadherin with ankyrin G (b) or co-precipitation of ankyrin G with GFP-E-cadherin (c) was examined. The graphs beneath the immunoblots image show the average ankyrin G or E-cadherin binding as a function of GFP-E-cadherin levels or ankyrin G ± SEM. n = 3 biologically independent samples. (d and e) The PolyA mutant does not recruit spectrin to cell-cell contacts in response to force. The indicated cells were either left resting or subjected to shear stress. Cells were fixed and stained with an antibody recognizing spectrin. Representative images are shown in d, and the graph in e represents the average corrected fluorescence intensity of spectrin in junctions. The data are represented as a box and whisker plot with median, 10th, 25th, 75th, and 90th percentiles shown. n = 50 junctions over 5 fields of view. Scale bars=10 μM. For all experiments, significance was calculated using a two-tailed unpaired Student t-test.
Supplementary information
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Salvi, A.M., Bays, J.L., Mackin, S.R. et al. Ankyrin G organizes membrane components to promote coupling of cell mechanics and glucose uptake. Nat Cell Biol 23, 457–466 (2021). https://doi.org/10.1038/s41556-021-00677-y
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DOI: https://doi.org/10.1038/s41556-021-00677-y
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