Oligomeric organization of membrane proteins from native membranes at nanoscale spatial and single-molecule resolution
Source abstract
Abstract
The oligomeric organization of membrane proteins in native cell membranes is a critical regulator of their function. High-resolution quantitative measurements of oligomeric assemblies and how they change under different conditions are indispensable to understanding membrane protein biology. We report Native-nanoBleach, a total internal reflection fluorescence microscopy-based single-molecule photobleaching step analysis technique to determine the oligomeric distribution of membrane proteins directly from native membranes at an effective spatial resolution of ~10 nm. We achieved this by capturing target membrane proteins in native nanodiscs with their proximal native membrane environment using amphipathic copolymers. We applied Native-nanoBleach to quantify the oligomerization status of structurally and functionally diverse membrane proteins, including a receptor tyrosine kinase (TrkA) and a small GTPase (KRas) under growth-factor binding and oncogenic mutations, respectively. Our data suggest that Native-nanoBleach provides a sensitive, single-molecule platform to quantify membrane protein oligomeric distributions in native membranes under physiologically and clinically relevant conditions.
Source: europepmc · Quality: candidate unverified · Source record ↗
Research context
OpenAlex topics: RNA Interference and Gene Delivery · Advanced Fluorescence Microscopy Techniques · Cell Adhesion Molecules Research
SciVX discovery tags are terminology-based. OpenAlex topics are source-provided classifications.
References (68)
Show source-provided references
1. Levental, I. & Lyman, E. Regulation of membrane protein structure and function by their lipid nano-environment. Nat. Rev. Mol. Cell Biol. 24, 107–122 (2023).10.1038/s41580-022-00524-4 ↗
2. Sezgin, E., Levental, I., Mayor, S. & Eggeling, C. The mystery of membrane organization: composition, regulation and roles of lipid rafts. Nat. Rev. Mol. Cell Biol. 18, 361–374 (2017).10.1038/nrm.2017.16 ↗
3. Liu, S., Hoess, P. & Ries, J. Super-resolution microscopy for structural cell biology. Annu. Rev. Biophys. 51, 301–326 (2022).10.1146/annurev-biophys-102521-112912 ↗
4. Baddeley, D. & Bewersdorf, J. Biological insight from super-resolution microscopy: what we can learn from localization-based images. Annu. Rev. Biochem. 87, 965–989 (2018).10.1146/annurev-biochem-060815-014801 ↗
5. Jain, A. et al. Probing cellular protein complexes using single-molecule pull-down. Nature 473, 484–488 (2011).10.1038/nature10016 ↗
6. Chung, J. K. et al. K-Ras4B remains monomeric on membranes over a wide range of surface densities and lipid compositions. Biophys. J. 114, 137–145 (2018).10.1016/j.bpj.2017.10.042 ↗
7. Kaliszewski, M. J. et al. Quantifying membrane protein oligomerization with fluorescence cross-correlation spectroscopy. Methods 140–141, 40–51 (2018).10.1016/j.ymeth.2018.02.002 ↗
8. Huang, Y. et al. Molecular basis for multimerization in the activation of the epidermal growth factor receptor. eLife 5, e14107 (2016).10.7554/eLife.14107 ↗
9. Ulbrich, M. H. & Isacoff, E. Y. Subunit counting in membrane-bound proteins. Nat. Methods 4, 319–321 (2007).10.1038/nmeth1024 ↗
10. Low-Nam, S. T. et al. ErbB1 dimerization is promoted by domain co-confinement and stabilized by ligand binding. Nat. Struct. Mol. Biol. 18, 1244–1249 (2011).10.1038/nsmb.2135 ↗
11. Kusumi, A., Tsunoyama, T. A., Hirosawa, K. M., Kasai, R. S. & Fujiwara, T. K. Tracking single molecules at work in living cells. Nat. Chem. Biol. 10, 524–532 (2014).10.1038/nchembio.1558 ↗
12. Lelek, M. et al. Single-molecule localization microscopy. Nat. Rev. Methods Primers 1, 39 (2021).10.1038/s43586-021-00038-x ↗
13. Huang, B., Bates, M. & Zhuang, X. Super-resolution fluorescence microscopy. Annu. Rev. Biochem. 78, 993–1016 (2009).10.1146/annurev.biochem.77.061906.092014 ↗
14. Balzarotti, F. et al. Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science 355, 606–612 (2017).10.1126/science.aak9913 ↗
15. Deguchi, T. et al. Direct observation of motor protein stepping in living cells using MINFLUX. Science 379, 1010–1015 (2023).10.1126/science.ade2676 ↗
16. Panda, A. et al. Direct determination of oligomeric organization of integral membrane proteins and lipids from intact customizable bilayer. Nat. Methods 20, 891–897 (2023).10.1038/s41592-023-01864-5 ↗
17. Sydor, A. M., Czymmek, K. J., Puchner, E. M. & Mennella, V. Super-resolution microscopy: from single molecules to supramolecular assemblies. Trends Cell Biol. 25, 730–748 (2015).10.1016/j.tcb.2015.10.004 ↗
18. Duncan, A. L. et al. Protein crowding and lipid complexity influence the nanoscale dynamic organization of ion channels in cell membranes. Sci. Rep. 7, 16647 (2017).10.1038/s41598-017-16865-6 ↗
19. Kiessling, V., Yang, S.-T. & Tamm, L. K. Supported lipid bilayers as models for studying membrane domains. Curr. Top. Membr. 75, 1–23 (2015).10.1016/bs.ctm.2015.03.001 ↗
20. Sako, Y., Minoghchi, S. & Yanagida, T. Single-molecule imaging of EGFR signalling on the surface of living cells. Nat. Cell Biol. 2, 168–172 (2000).10.1038/35004044 ↗
21. Coffman, V. C. & Wu, J.-Q. Counting protein molecules using quantitative fluorescence microscopy. Trends Biochem. Sci. 37, 499–506 (2012).10.1016/j.tibs.2012.08.002 ↗
22. Huang, E. J. & Reichardt, L. F. Trk receptors: roles in neuronal signal transduction. Annu. Rev. Biochem. 72, 609–642 (2003).10.1146/annurev.biochem.72.121801.161629 ↗
23. Lemmon, M. A. & Schlessinger, J. Cell signaling by receptor tyrosine kinases. Cell 141, 1117–1134 (2010).10.1016/j.cell.2010.06.011 ↗
24. Waters, A. M. & Der, C. J. KRAS: the critical driver and therapeutic target for pancreatic cancer. Cold Spring Harb. Perspect. Med. 8, a031435 (2018).10.1101/cshperspect.a031435 ↗
25. Hobbs, G. A., Der, C. J. & Rossman, K. L. RAS isoforms and mutations in cancer at a glance. J. Cell Sci. 129, 1287–1292 (2016).10.1242/jcs.182873 ↗
26. Simanshu, D. K., Nissley, D. V. & McCormick, F. RAS proteins and their regulators in human disease. Cell 170, 17–33 (2017).10.1016/j.cell.2017.06.009 ↗
27. Wang, J. Y. & Doudna, J. A. CRISPR technology: a decade of genome editing is only the beginning. Science 379, eadd8643 (2023).10.1126/science.add8643 ↗
28. Cho, N. H. et al. OpenCell: endogenous tagging for the cartography of human cellular organization. Science 375, eabi6983 (2022).10.1126/science.abi6983 ↗
29. Smith, A. A. A. et al. Lipid nanodiscs via ordered copolymers. Chem 6, 2782–2795 (2020).10.1016/j.chempr.2020.08.004 ↗
30. Esmaili, M. & Overduin, M. Membrane biology visualized in nanometer-sized discs formed by styrene maleic acid polymers. Biochim. Biophys. Acta Biomembr. 1860, 257–263 (2018).10.1016/j.bbamem.2017.10.019 ↗
31. Knowles, T. J. et al. Membrane proteins solubilized intact in lipid containing nanoparticles bounded by styrene maleic acid copolymer. J. Am. Chem. Soc. 131, 7484–7485 (2009).10.1021/ja810046q ↗
32. Zacharias, D. A., Violin, J. D., Newton, A. C. & Tsien, R. Y. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916 (2002).10.1126/science.1068539 ↗
33. Swiecicki, J.-M., Santana, J. T. & Imperiali, B. A strategic approach for fluorescence imaging of membrane proteins in a native-like environment. Cell Chem. Biol. 27, 245–251.e3 (2020).10.1016/j.chembiol.2019.11.008 ↗
34. Goehring, A. et al. Screening and large-scale expression of membrane proteins in mammalian cells for structural studies. Nat. Protoc. 9, 2574–2585 (2014).10.1038/nprot.2014.173 ↗
35. Sniegowski, J. A., Phail, M. E. & Wachter, R. M. Maturation efficiency, trypsin sensitivity, and optical properties of Arg96, Glu222, and Gly67 variants of green fluorescent protein. Biochem. Biophys. Res. Commun. 332, 657–663 (2005).10.1016/j.bbrc.2005.04.166 ↗
36. Xu, Y. et al. Structures of bacterial homologues of SWEET transporters in two distinct conformations. Nature 515, 448–452 (2014).10.1038/nature13670 ↗
37. Khademi, S. et al. Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 A. Science 305, 1587–1594 (2004).10.1126/science.1101952 ↗
38. Kim, D. M. & Nimigean, C. M. Voltage-gated potassium channels: a structural examination of selectivity and gating. Cold Spring Harb. Perspect. Biol. 8, a029231 (2016).10.1101/cshperspect.a029231 ↗
39. Gupta, K. et al. The role of interfacial lipids in stabilizing membrane protein oligomers. Nature 541, 421–424 (2017).10.1038/nature20820 ↗
40. Nemoto, Y. & De Camilli, P. Recruitment of an alternatively spliced form of synaptojanin 2 to mitochondria by the interaction with the PDZ domain of a mitochondrial outer membrane protein. EMBO J. 18, 2991–3006 (1999).10.1093/emboj/18.11.2991 ↗
41. Chen, W. W., Freinkman, E., Wang, T., Birsoy, K. & Sabatini, D. M. Absolute quantification of matrix metabolites reveals the dynamics of mitochondrial metabolism. Cell 166, 1324–1337.e11 (2016).10.1016/j.cell.2016.07.040 ↗
42. Yamashita, A., Singh, S. K., Kawate, T., Jin, Y. & Gouaux, E. Crystal structure of a bacterial homologue of Na+/Cl-dependent neurotransmitter transporters. Nature 437, 215–223 (2005).10.1038/nature03978 ↗
43. Zhang, F. et al. Quantification of epidermal growth factor receptor expression level and binding kinetics on cell surfaces by surface plasmon resonance imaging. Anal. Chem. 87, 9960–9965 (2015).10.1021/acs.analchem.5b02572 ↗
44. Hood, F. E., Sahraoui, Y. M., Jenkins, R. E. & Prior, I. A. Ras protein abundance correlates with Ras isoform mutation patterns in cancer. Oncogene 42, 1224–1232 (2023).10.1038/s41388-023-02638-1 ↗
45. Byrne, P. O., Hristova, K. & Leahy, D. J. EGFR forms ligand-independent oligomers that are distinct from the active state. J. Biol. Chem. 295, 13353–13362 (2020).10.1074/jbc.RA120.012852 ↗
46. Shen, J. & Maruyama, I. N. Nerve growth factor receptor TrkA exists as a preformed, yet inactive, dimer in living cells. FEBS Lett. 585, 295–299 (2011).10.1016/j.febslet.2010.12.031 ↗
47. Ahmed, F. & Hristova, K. Dimerization of the Trk receptors in the plasma membrane: effects of their cognate ligands. Biochem. J. 475, 3669–3685 (2018).10.1042/BCJ20180637 ↗
48. Franco, M. L. et al. Interaction between the transmembrane domains of neurotrophin receptors p75 and TrkA mediates their reciprocal activation. J. Biol. Chem. 297, 100926 (2021).10.1016/j.jbc.2021.100926 ↗
49. Van, Q. N. et al. RAS nanoclusters: dynamic signaling platforms amenable to therapeutic intervention. Biomolecules 11, 377 (2021).10.3390/biom11030377 ↗
50. Abankwa, D., Gorfe, A. A. & Hancock, J. F. Ras nanoclusters: molecular structure and assembly. Semin. Cell Dev. Biol. 18, 599–607 (2007).10.1016/j.semcdb.2007.08.003 ↗
51. Nan, X. et al. Ras-GTP dimers activate the mitogen-activated protein kinase (MAPK) pathway. Proc. Natl Acad. Sci. USA 112, 7996–8001 (2015).10.1073/pnas.1509123112 ↗
52. Ambrogio, C. et al. KRAS dimerization impacts MEK inhibitor sensitivity and oncogenic activity of mutant KRAS. Cell 172, 857–868.e15 (2018).10.1016/j.cell.2017.12.020 ↗
53. Kessler, D. et al. Drugging an undruggable pocket on KRAS. Proc. Natl Acad. Sci. USA 116, 15823–15829 (2019).10.1073/pnas.1904529116 ↗
54. Tran, T. H. et al. The small molecule BI-2852 induces a nonfunctional dimer of KRAS. Proc. Natl Acad. Sci. USA 117, 3363–3364 (2020).10.1073/pnas.1918164117 ↗
55. Sarkar-Banerjee, S. et al. Spatiotemporal analysis of K-Ras plasma membrane Interactions reveals multiple high order homo-oligomeric complexes. J. Am. Chem. Soc. 139, 13466–13475 (2017).10.1021/jacs.7b06292 ↗
56. Buscail, L., Bournet, B. & Cordelier, P. Role of oncogenic KRAS in the diagnosis, prognosis and treatment of pancreatic cancer. Nat. Rev. Gastroenterol. Hepatol. 17, 153–168 (2020).10.1038/s41575-019-0245-4 ↗
57. Muzumdar, M. D. et al. Survival of pancreatic cancer cells lacking KRAS function. Nat. Commun. 8, 1090 (2017).10.1038/s41467-017-00942-5 ↗
58. Sligar, S. G. & Denisov, I. G. Nanodiscs: a toolkit for membrane protein science. Protein Sci. 30, 297–315 (2021).10.1002/pro.3994 ↗
59. Boldog, T., Grimme, S., Li, M., Sligar, S. G. & Hazelbauer, G. L. Nanodiscs separate chemoreceptor oligomeric states and reveal their signaling properties. Proc. Natl Acad. Sci. USA 103, 11509–11514 (2006).10.1073/pnas.0604988103 ↗
60. Du, Z. & Lovly, C. M. Mechanisms of receptor tyrosine kinase activation in cancer. Mol. Cancer 17, 58 (2018).10.1186/s12943-018-0782-4 ↗
61. Lindhoud, S., Carvalho, V., Pronk, J. W. & Aubin-Tam, M.-E. SMA-SH: modified styrene-maleic acid copolymer for functionalization of lipid nanodiscs. Biomacromolecules 17, 1516–1522 (2016).10.1021/acs.biomac.6b00140 ↗
62. Wood, E. R. et al. Discovery and in vitro evaluation of potent TrkA kinase inhibitors: oxindole and aza-oxindoles. Bioorg. Med. Chem. Lett. 14, 953–957 (2004).10.1016/j.bmcl.2003.12.002 ↗
63. Tinevez, J.-Y. et al. TrackMate: an open and extensible platform for single-particle tracking. Methods 115, 80–90 (2017).10.1016/j.ymeth.2016.09.016 ↗
64. Jaqaman, K. et al. Robust single-particle tracking in live-cell time-lapse sequences. Nat. Methods 5, 695–702 (2008).10.1038/nmeth.1237 ↗
65. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).10.1038/nmeth.2019 ↗
66. Karandur, D. et al. Breakage of the oligomeric CaMKII hub by the regulatory segment of the kinase. eLife 9, e57784 (2020).10.7554/eLife.57784 ↗
67. Mi, L.-Z. et al. Simultaneous visualization of the extracellular and cytoplasmic domains of the epidermal growth factor receptor. Nat. Struct. Mol. Biol. 18, 984–989 (2011).10.1038/nsmb.2092 ↗
68. Bhattacharyya, M. et al. MATLAB Codes for Native-nanoBleach (1.0.1) (Zenodo, 2023); https://doi.org/10.5281/zenodo.842932110.5281/zenodo.8429321 ↗