Several peripheral membrane proteins are known to form membrane pores through multimerization. In many cases, in biochemical reconstitution experiments, a complex distribution of oligomeric states has been observed that may, in part, be irrelevant to their physiological functions. This phenomenon makes it difficult to identify the functional oligomeric states of membrane lipid interacting proteins, for example, during the formation of transient membrane pores. Using fibroblast growth factor 2 (FGF2) as an example, we present a methodology applicable to giant lipid vesicles by which functional oligomers can be distinguished from nonspecifically aggregated proteins without functionality. Two distinct populations of fibroblast growth factor 2 were identified with (i) dimers to hexamers and (ii) a broad population of higher oligomeric states of membrane-associated FGF2 oligomers significantly distorting the original unfiltered histogram of all detectable oligomeric species of FGF2. The presented statistical approach is relevant for various techniques for characterizing membrane-dependent protein oligomerization.

Department of Physics University of Helsinki P O Box 64 FI 00014 Helsinki Finland

Faculty of Mathematics and Physics Charles University Ke Karlovu 2027 3 121 16 Prague Czech Republic

Heidelberg University Biochemistry Center Im Neuenheimer Feld 328 69120 Heidelberg Germany

J Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences Dolejškova 3 182 23 Prague Czech Republic

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Moertelmaier M.; Brameshuber M.; Linimeier M.; Schütz G. J.; Stockinger H. Thinning out Clusters While Conserving Stoichiometry of Labeling. Appl. Phys. Lett. 2005, 87, 26390310.1063/1.2158031. DOI

Gwosch K. C.; Pape J. K.; Balzarotti F.; Hoess P.; Ellenberg J.; Ries J.; Hell S. W. MINFLUX Nanoscopy Delivers 3D Multicolor Nanometer Resolution in Cells. Nat. Methods 2020, 17, 217–224. 10.1038/s41592-019-0688-0. PubMed DOI

Hell S. W.; Wichmann J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 1994, 19, 78010.1364/ol.19.000780. PubMed DOI

Rust M. J.; Bates M.; Zhuang X. Sub-Diffraction-Limit Imaging by Stochastic Optical Reconstruction Microscopy (STORM). Nat. Methods 2006, 3, 793–795. 10.1038/nmeth929. PubMed DOI PMC

Boehr D. D.; McElheny D.; Dyson H. J.; Wrightt P. E. The Dynamic Energy Landscape of Dihydrofolate Reductase Catalysis. Science 2006, 313, 1638–1642. 10.1126/science.1130258. PubMed DOI

Gupta R.; Ghosh S. JNK3 Phosphorylates Bax Protein and Induces Ability to Form Pore on Bilayer Lipid Membrane. Biochim. Open 2017, 4, 41–46. 10.1016/j.biopen.2017.02.001. PubMed DOI PMC

Simonyan L.; Légiot A.; Lascu I.; Durand G.; Giraud M. F.; Gonzalez C.; Manon S. The Substitution of Proline 168 Favors Bax Oligomerization and Stimulates Its Interaction with LUVs and Mitochondria. Biochim. Biophys. Acta, Biomembr. 2017, 1859, 1144–1155. 10.1016/j.bbamem.2017.03.010. PubMed DOI

Aluvila S.; Mandal T.; Hustedt E.; Fajer P.; Choe J. Y.; Oh K. J. Organization of the Mitochondrial Apoptotic BAK Pore: Oligomerization of the BAK Homodimers. J. Biol. Chem. 2014, 289, 2537–2551. 10.1074/jbc.M113.526806. PubMed DOI PMC

Pieta P.; Mirza J.; Lipkowski J. Direct Visualization of the Alamethicin Pore Formed in a Planar Phospholipid Matrix. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 21223–21227. 10.1073/pnas.1201559110. PubMed DOI PMC

Rahaman A.; Lazaridis T. A Thermodynamic Approach to Alamethicin Pore Formation. Biochim. Biophys. Acta, Biomembr. 2014, 1838, 98–105. 10.1016/j.bbamem.2013.09.012. PubMed DOI PMC

Mulvihill E.; Sborgi L.; Mari S. A.; Pfreundschuh M.; Hiller S.; Müller D. J. Mechanism of Membrane Pore Formation by Human Gasdermin-D. EMBO J. 2018, 37, e9832110.15252/embj.201798321. PubMed DOI PMC

James H. P.; Jadhav S. Kinetics of Pore Formation in Stearoyl-Oleoyl-Phosphatidylcholine Vesicles by PH Sensitive Cell Penetrating Peptide GALA. Chem. Phys. Lipids 2021, 241, 10513910.1016/j.chemphyslip.2021.105139. PubMed DOI

Jiao F.; Ruan Y.; Scheuring S.. High-Speed Atomic Force Microscopy to Study Pore-Forming Proteins. In Methods in Enzymology; Academic Press Inc, 2021; Vol. 649, pp 189–21710.1016/bs.mie.2021.01.033. PubMed DOI

Stahelin R.Monitoring Peripheral Protein Oligomerization on Biological Membranes. In Methods in Cell Biology; Academic Press Inc., 2013; Vol. 117, pp 359–371. PubMed PMC

Subburaj Y.; Cosentino K.; Axman M.; Pedrueya-Villalmanyo E.; Hermann E.; Bleicken S.; Spatz J.; Garcı A. J. Bax Monomers Form Dimer Units in the Membrane That Further Self-Assemble into Multiple Coexisting Species. Nat. Commun. 2015, 6, 804210.1038/ncomms9042. PubMed DOI PMC

Steringer J. P.; Lange S.; Čujová S.; Šachl R.; Poojari C.; Lolicato F.; Beutel O.; Müller H. M.; Unger S.; Coskun Ü.; Honigmann A.; Vattulainen I.; Hof M.; Freund C.; Nickel W. Key Steps in Unconventional Secretion of Fibroblast Growth Factor 2 Reconstituted with Purified Components. eLife 2017, 6, e2898510.7554/eLife.28985. PubMed DOI PMC

Šachl R.; Čujová S.; Singh V.; Riegerová P.; Kapusta P.; Müller H. M.; Steringer J. P.; Hof M.; Nickel W. Functional Assay to Correlate Protein Oligomerization States with Membrane Pore Formation. Anal. Chem. 2020, 92, 14861–14866. 10.1021/acs.analchem.0c03276. PubMed DOI

Scomparin C.; Lecuyer S.; Ferreira M.; Charitat T.; Tinland B. Diffusion in Supported Lipid Bilayers: Influence of Substrate and Preparation Technique on the Internal Dynamics. Eur. Phys. J. E 2009, 28, 211–220. 10.1140/epje/i2008-10407-3. PubMed DOI

Wagner M. L.; Tamm L. K. Tethered Polymer-Supported Planar Lipid Bilayers for Reconstitution of Integral Membrane Proteins: Silane-Polyethyleneglycol-Lipid as a Cushion and Covalent Linker. Biophys. J. 2000, 79, 1400–1414. 10.1016/S0006-3495(00)76392-2. PubMed DOI PMC

Honigmann A.; Mueller V.; Hell S. W.; Eggeling C. STED Microscopy Detects and Quantifies Liquid Phase Separation in Lipid Membranes Using a New Far-Red Emitting Fluorescent Phosphoglycerolipid Analogue. Faraday Discuss. 2013, 161, 77–89. 10.1039/c2fd20107k. PubMed DOI

Dimou E.; Cosentino K.; Platonova E.; Ros U.; Sadeghi M.; Kashyap P.; Katsinelos T.; Wegehingel S.; Noé F.; García-Sáez A. J.; Ewers H.; Nickel W. Single Event Visualization of Unconventional Secretion of FGF2. J. Cell Biol. 2019, 218, 683–699. 10.1083/jcb.201802008. PubMed DOI PMC

Ries J.; Petrášek Z.; García-Sáez A. J.; Schwille P. A Comprehensive Framework for Fluorescence Cross-Correlation Spectroscopy. New J. Phys. 2010, 12, 11300910.1088/1367-2630/12/11/113009. DOI

Benda A.; Beneš M.; Mareček V.; Lhotský A.; Hermens W. T.; Hof M. How to Determine Diffusion Coefficients in Planar Phospholipid Systems by Confocal Fluorescence Correlation Spectroscopy. Langmuir 2003, 19, 4120–4126. 10.1021/la0270136. DOI

Humpolíčková J.; Gielen E.; Benda A.; Fagulova V.; Vercammen J.; VandeVen M.; Hof M.; Ameloot M.; Engelborghs Y. Probing Diffusion Laws within Cellular Membranes by Z-Scan Fluorescence Correlation Spectroscopy. Biophys. J. 2006, 91, L23–L25. 10.1529/biophysj.106.089474. PubMed DOI PMC

Chen Y.; Wei L. N.; Müller J. D. Probing Protein Oligomerization in Living Cells with Fluorescence Fluctuation Spectroscopy. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 15492–15497. 10.1073/pnas.2533045100. PubMed DOI PMC

Ulbrich M. H.; Isacoff E. Y. Subunit Counting in Membrane-Bound Proteins. Nat. Methods 2007, 4, 319–321. 10.1038/nmeth1024. PubMed DOI PMC

Balasubramanian H.; Sankaran J.; Pandey S.; Goh C. J. H.; Wohland T. The Dependence of EGFR Oligomerization on Environment and Structure: A Camera-Based N&B Study. Biophys. J. 2022, 121, 4452–4466. 10.1016/j.bpj.2022.11.003. PubMed DOI PMC

Fukushima R.; Yamamoto J.; Kinjo M. Empirical Bayes Method Using Surrounding Pixel Information for Number and Brightness Analysis. Biophys. J. 2021, 120, 2156–2171. 10.1016/j.bpj.2021.03.033. PubMed DOI PMC

Dunsing V.; Luckner M.; Zühlke B.; Petazzi R. A.; Herrmann A.; Chiantia S. Optimal Fluorescent Protein Tags for Quantifying Protein Oligomerization in Living Cells. Sci. Rep. 2018, 8, 1063410.1038/s41598-018-28858-0. PubMed DOI PMC

Widengren J.; Mets U.; Rigler R. Fluorescence Correlation Spectroscopy of Triplet States in Solution: A Theoretical and Experimental Study. J. Phys. Chem. A 1995, 99, 13368–13379. 10.1021/j100036a009. DOI

Angelova M. I.; Dimitrov D. S. Liposome Electroformation. Faraday Discuss. Chem. Soc. 1986, 81, 303–311. 10.1039/DC9868100303. DOI

Dimou E.; Nickel W. Unconventional Mechanisms of Eukaryotic Protein Secretion. Curr. Biol. 2018, 28, R406–R410. 10.1016/j.cub.2017.11.074. PubMed DOI

Bikfalvi A.; Klein S.; Pintucci G.; Rifkin D. B. Biological Roles of Fibroblast Growth Factor-2. Endocr. Rev. 1997, 18, 26–45. 10.1210/er.18.1.26. PubMed DOI

Temmerman K.; Ebert A. D.; Müller H. M.; Sinning I.; Tews I.; Nickel W. A Direct Role for Phosphatidylinositol-4,5-Bisphosphate in Unconventional Secretion of Fibroblast Growth Factor 2. Traffic 2008, 9, 1204–1217. 10.1111/j.1600-0854.2008.00749.x. PubMed DOI

Temmerman K.; Nickel W. A Novel Flow Cytometric Assay to Quantify Interactions between Proteins and Membrane Lipids. J. Lipid Res. 2009, 50, 1245–1254. 10.1194/jlr.D800043-JLR200. PubMed DOI PMC

Steringer J. P.; Bleicken S.; Andreas H.; Zacherl S.; Laussmann M.; Temmerman K.; Contreras F. X.; Bharat T. A. M.; Lechner J.; Müller H. M.; Briggs J. A. G.; García-Sáez A. J.; Nickel W. Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P 2)-Dependent Oligomerization of Fibroblast Growth Factor 2 (FGF2) Triggers the Formation of a Lipidic Membrane Pore Implicated in Unconventional Secretion. J. Biol. Chem. 2012, 287, 27659–27669. 10.1074/jbc.M112.381939. PubMed DOI PMC

Müller H. M.; Steringer J. P.; Wegehingel S.; Bleicken S.; Münster M.; Dimou E.; Unger S.; Weidmann G.; Andreas H.; García-Sáez A. J.; Wild K.; Sinning I.; Nickel W. Formation of Disulfide Bridges Drives Oligomerization, Membrane Pore Formation, and Translocation of Fibroblast Growth Factor 2 to Cell Surfaces. J. Biol. Chem. 2015, 290, 8925–8937. 10.1074/jbc.M114.622456. PubMed DOI PMC

Sparn C.; Dimou E.; Meyer A.; Saleppico R.; Wegehingel S.; Gerstner M.; Klaus S.; Ewers H.; Nickel W. Glypican-1 Drives Unconventional Secretion of Fibroblast Growth Factor 2. eLife 2022, 11, e7554510.7554/eLife.75545. PubMed DOI PMC

Zehe C.; Engling A.; Wegehingel S.; Schäfer T.; Nickel W. Cell-Surface Heparan Sulfate Proteoglycans Are Essential Components of the Unconventional Export Machinery of FGF-2. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 15479–15484. 10.1073/pnas.0605997103. PubMed DOI PMC

Nickel W. The Unconventional Secretory Machinery of Fibroblast Growth Factor 2. Traffic 2011, 12, 799–805. 10.1111/j.1600-0854.2011.01187.x. PubMed DOI

Nickel W. Unconventional Secretion: An Extracellular Trap for Export of Fibroblast Growth Factor 2. J. Cell Sci. 2007, 120, 2295–2299. 10.1242/jcs.011080. PubMed DOI