Fibroblast growth factor 2 (FGF2) exits cells by direct translocation across the plasma membrane, a type I pathway of unconventional protein secretion. This process is initiated by phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent formation of highly dynamic FGF2 oligomers at the inner plasma membrane leaflet, inducing the formation of lipidic membrane pores. Cell surface heparan sulfate chains linked to glypican-1 (GPC1) capture FGF2 at the outer plasma membrane leaflet, completing FGF2 membrane translocation into the extracellular space. While the basic steps of this pathway are well understood, the molecular mechanism by which FGF2 oligomerizes on membrane surfaces remains unclear. In the current study, we demonstrate the initial step of this process to depend on C95-C95 disulfide-bridge-mediated FGF2 dimerization on membrane surfaces, producing the building blocks for higher FGF2 oligomers that drive the formation of membrane pores. We find FGF2 with a C95A substitution to be defective in oligomerization, pore formation, and membrane translocation. Consistently, we demonstrate a C95A variant of FGF2 to be characterized by a severe secretion phenotype. By contrast, while also important for efficient FGF2 secretion from cells, a second cysteine residue on the molecular surface of FGF2 (C77) is not involved in FGF2 oligomerization. Rather, we find C77 to be part of the interaction interface through which FGF2 binds to the α1 subunit of the Na,K-ATPase, the landing platform for FGF2 at the inner plasma membrane leaflet. Using cross-linking mass spectrometry, atomistic molecular dynamics simulations combined with a machine learning analysis and cryo-electron tomography, we propose a mechanism by which disulfide-bridged FGF2 dimers bind with high avidity to PI(4,5)P2 on membrane surfaces. We further propose a tight coupling between FGF2 secretion and the formation of ternary signaling complexes on cell surfaces, hypothesizing that C95-C95-bridged FGF2 dimers are functioning as the molecular units triggering autocrine and paracrine FGF2 signaling.

Department of Physics University of Helsinki Helsinki Finland

Heidelberg University Biochemistry Center Heidelberg Germany

Institute for Chemistry and Biochemistry Freie Universität Berlin Berlin Germany

J Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences Prague Czech Republic

Schaller Research Group Department of Infectious Diseases Virology Heidelberg University Hospital Heidelberg Germany

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doi: 10.7554/eLife.88579.1 PubMed

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Abraham MJ, Murtola T, Schulz R, Páll S, Smith JC, Hess B, Lindahl E. GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX. 2015;1–2:19–25. doi: 10.1016/j.softx.2015.06.001. DOI

Akl MR, Nagpal P, Ayoub NM, Tai B, Prabhu SA, Capac CM, Gliksman M, Goy A, Suh KS. Molecular and clinical significance of fibroblast growth factor 2 (FGF2 /bFGF) in malignancies of solid and hematological cancers for personalized therapies. Oncotarget. 2016;7:44735–44762. doi: 10.18632/oncotarget.8203. PubMed DOI PMC

Albritton LM, Tseng L, Scadden D, Cunningham JM. A putative murine ecotropic retrovirus receptor gene encodes A multiple membrane-spanning protein and confers susceptibility to virus infection. Cell. 1989;57:659–666. doi: 10.1016/0092-8674(89)90134-7. PubMed DOI

Amblard I, Dupont E, Alves I, Miralvès J, Queguiner I, Joliot A. Bidirectional transfer of homeoprotein EN2 across the plasma membrane requires PIP2. Journal of Cell Science. 2020;133:jcs244327. doi: 10.1242/jcs.244327. PubMed DOI

Backhaus R, Zehe C, Wegehingel S, Kehlenbach A, Schwappach B, Nickel W. Unconventional protein secretion: membrane translocation of FGF-2 does not require protein unfolding. Journal of Cell Science. 2004;117:1727–1736. doi: 10.1242/jcs.01027. PubMed DOI

Benda A, Beneš M, Mareček V, Lhotský A, Hermens WTh, Hof M. How to determine diffusion coefficients in planar phospholipid systems by confocal fluorescence correlation spectroscopy. Langmuir. 2003;19:4120–4126. doi: 10.1021/la0270136. DOI

Brown DI, Griendling KK. Nox proteins in signal transduction. Free Radical Biology & Medicine. 2009;47:1239–1253. doi: 10.1016/j.freeradbiomed.2009.07.023. PubMed DOI PMC

Castaño-Díez D, Kudryashev M, Arheit M, Stahlberg H. Dynamo: a flexible, user-friendly development tool for subtomogram averaging of cryo-EM data in high-performance computing environments. Journal of Structural Biology. 2012;178:139–151. doi: 10.1016/j.jsb.2011.12.017. PubMed DOI

Castaño-Díez D. The dynamo package for tomography and subtomogram averaging: components for MATLAB, GPU computing and EC2 amazon web services. Acta Crystallographica. Section D, Structural Biology. 2017;73:478–487. doi: 10.1107/S2059798317003369. PubMed DOI PMC

Castaño-Díez D, Kudryashev M, Stahlberg H. Dynamo catalogue: geometrical tools and data management for particle picking in subtomogram averaging of cryo-electron tomograms. Journal of Structural Biology. 2017;197:135–144. doi: 10.1016/j.jsb.2016.06.005. PubMed DOI

Chaudhury S, Gray JJ. Conformer selection and induced fit in flexible backbone protein-protein docking using computational and NMR ensembles. Journal of Molecular Biology. 2008;381:1068–1087. doi: 10.1016/j.jmb.2008.05.042. PubMed DOI PMC

Chiritoiu M, Brouwers N, Turacchio G, Pirozzi M, Malhotra V. GRASP55 and UPR control interleukin-1β aggregation and secretion. Developmental Cell. 2019;49:145–155. doi: 10.1016/j.devcel.2019.02.011. PubMed DOI

Dahl JP, Binda A, Canfield VA, Levenson R. Participation of Na,K-ATPase in FGF-2 secretion: rescue of ouabain-inhibitable FGF-2 secretion by ouabain-resistant Na,K-ATPase alpha subunits. Biochemistry. 2000;39:14877–14883. doi: 10.1021/bi001073y. PubMed DOI

Darden T, York D, Pedersen L. Particle mesh Ewald: an N-log(N) method for Ewald sums in large systems. The Journal of Chemical Physics. 1993;98:10089–10092. doi: 10.1063/1.464397. DOI

Daura X, Gademann K, Jaun B, Seebach D, van Gunsteren WF, Mark AE. Peptide folding: when simulation meets experiment. Angewandte Chemie International Edition. 1999;38:236–240. doi: 10.1002/(SICI)1521-3773(19990115)38:1/2<236::AID-ANIE236>3.0.CO;2-M. DOI

Debaisieux S, Rayne F, Yezid H, Beaumelle B. The ins and outs of HIV-1 Tat. Traffic. 2012;13:355–363. doi: 10.1111/j.1600-0854.2011.01286.x. PubMed DOI

Decker CG, Wang Y, Paluck SJ, Shen L, Loo JA, Levine AJ, Miller LS, Maynard HD. Fibroblast growth factor 2 dimer with superagonist in vitro activity improves granulation tissue formation during wound healing. Biomaterials. 2016;81:157–168. doi: 10.1016/j.biomaterials.2015.12.003. PubMed DOI PMC

Dimou E, Nickel W. Unconventional mechanisms of eukaryotic protein secretion. Current Biology. 2018;28:R406–R410. doi: 10.1016/j.cub.2017.11.074. PubMed DOI

Dimou E, Cosentino K, Platonova E, Ros U, Sadeghi M, Kashyap P, Katsinelos T, Wegehingel S, Noé F, García-Sáez AJ, Ewers H, Nickel W. Single event visualization of unconventional secretion of FGF2. The Journal of Cell Biology. 2019;218:683–699. doi: 10.1083/jcb.201802008. PubMed DOI PMC

Dupont N, Jiang S, Pilli M, Ornatowski W, Bhattacharya D, Deretic V. Autophagy-based unconventional secretory pathway for extracellular delivery of IL-1β. The EMBO Journal. 2011;30:4701–4711. doi: 10.1038/emboj.2011.398. PubMed DOI PMC

Ebert AD, Laussmann M, Wegehingel S, Kaderali L, Erfle H, Reichert J, Lechner J, Beer HD, Pepperkok R, Nickel W. Tec-kinase-mediated phosphorylation of fibroblast growth factor 2 is essential for unconventional secretion. Traffic. 2010;11:813–826. doi: 10.1111/j.1600-0854.2010.01059.x. PubMed DOI

Engling A, Backhaus R, Stegmayer C, Zehe C, Seelenmeyer C, Kehlenbach A, Schwappach B, Wegehingel S, Nickel W. Biosynthetic FGF-2 is targeted to non-lipid raft microdomains following translocation to the extracellular surface of CHO cells. Journal of Cell Science. 2002;115:3619–3631. doi: 10.1242/jcs.00036. PubMed DOI

Evans DJ, Holian BL. The nose–hoover thermostat. The Journal of Chemical Physics. 1985;83:4069–4074. doi: 10.1063/1.449071. DOI

Evans R, O’Neill M, Pritzel A, Antropova N, Senior A, Green T, Žídek A, Bates R, Blackwell S, Yim J, Ronneberger O, Bodenstein S, Zielinski M, Bridgland A, Potapenko A, Cowie A, Tunyasuvunakool K, Jain R, Clancy E, Kohli P, Jumper J, Hassabis D. Protein complex prediction with AlphaFold-Multimer. Bioinformatics. 2021;01:463034. doi: 10.1101/2021.10.04.463034. DOI

Evavold CL, Ruan J, Tan Y, Xia S, Wu H, Kagan JC. The pore-forming protein gasdermin D regulates interleukin-1 secretion from living macrophages. Immunity. 2018;48:35–44. doi: 10.1016/j.immuni.2017.11.013. PubMed DOI PMC

Florkiewicz RZ, Anchin J, Baird A. The inhibition of fibroblast growth factor-2 export by cardenolides implies a novel function for the catalytic subunit of Na+,K+-ATPase. The Journal of Biological Chemistry. 1998;273:544–551. doi: 10.1074/jbc.273.1.544. PubMed DOI

García-Sáez AJ, Ries J, Orzáez M, Pérez-Payà E, Schwille P. Membrane promotes tBID interaction with BCL(XL) Nature Structural & Molecular Biology. 2009;16:1178–1185. doi: 10.1038/nsmb.1671. PubMed DOI

Goddard TD, Huang CC, Meng EC, Pettersen EF, Couch GS, Morris JH, Ferrin TE. UCSF ChimeraX: meeting modern challenges in visualization and analysis. Protein Science. 2018;27:14–25. doi: 10.1002/pro.3235. PubMed DOI PMC

Gray JJ, Moughon S, Wang C, Schueler-Furman O, Kuhlman B, Rohl CA, Baker D. Protein-protein docking with simultaneous optimization of rigid-body displacement and side-chain conformations. Journal of Molecular Biology. 2003;331:281–299. doi: 10.1016/s0022-2836(03)00670-3. PubMed DOI

Hagen WJH, Wan W, Briggs JAG. Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution subtomogram averaging. Journal of Structural Biology. 2017;197:191–198. doi: 10.1016/j.jsb.2016.06.007. PubMed DOI PMC

Hakim M, Fass D. Cytosolic disulfide bond formation in cells infected with large nucleocytoplasmic DNA viruses. Antioxidants & Redox Signaling. 2010;13:1261–1271. doi: 10.1089/ars.2010.3128. PubMed DOI

Heilig R, Dick MS, Sborgi L, Meunier E, Hiller S, Broz P. The gasdermin-D pore acts as a conduit for IL-1β secretion in mice. European Journal of Immunology. 2018;48:584–592. doi: 10.1002/eji.201747404. PubMed DOI

Huang J, Rauscher S, Nawrocki G, Ran T, Feig M, de Groot BL, Grubmüller H, MacKerell AD., Jr CHARMM36m: an improved force field for folded and intrinsically disordered proteins. Nature Methods. 2017;14:71–73. doi: 10.1038/nmeth.4067. PubMed DOI PMC

Iacobucci C, Piotrowski C, Aebersold R, Amaral BC, Andrews P, Bernfur K, Borchers C, Brodie NI, Bruce JE, Cao Y, Chaignepain S, Chavez JD, Claverol S, Cox J, Davis T, Degliesposti G, Dong M-Q, Edinger N, Emanuelsson C, Gay M, Götze M, Gomes-Neto F, Gozzo FC, Gutierrez C, Haupt C, Heck AJR, Herzog F, Huang L, Hoopmann MR, Kalisman N, Klykov O, Kukačka Z, Liu F, MacCoss MJ, Mechtler K, Mesika R, Moritz RL, Nagaraj N, Nesati V, Neves-Ferreira AGC, Ninnis R, Novák P, O’Reilly FJ, Pelzing M, Petrotchenko E, Piersimoni L, Plasencia M, Pukala T, Rand KD, Rappsilber J, Reichmann D, Sailer C, Sarnowski CP, Scheltema RA, Schmidt C, Schriemer DC, Shi Y, Skehel JM, Slavin M, Sobott F, Solis-Mezarino V, Stephanowitz H, Stengel F, Stieger CE, Trabjerg E, Trnka M, Vilaseca M, Viner R, Xiang Y, Yilmaz S, Zelter A, Ziemianowicz D, Leitner A, Sinz A. First community-wide, comparative cross-linking mass spectrometry study. Analytical Chemistry. 2019;91:6953–6961. doi: 10.1021/acs.analchem.9b00658. PubMed DOI PMC

Joliot A, Prochiantz A. Unconventional secretion, gate to homeoprotein intercellular transfer. Frontiers in Cell and Developmental Biology. 2022;10:926421. doi: 10.3389/fcell.2022.926421. PubMed DOI PMC

Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K, Bates R, Žídek A, Potapenko A, Bridgland A, Meyer C, Kohl SAA, Ballard AJ, Cowie A, Romera-Paredes B, Nikolov S, Jain R, Adler J, Back T, Petersen S, Reiman D, Clancy E, Zielinski M, Steinegger M, Pacholska M, Berghammer T, Bodenstein S, Silver D, Vinyals O, Senior AW, Kavukcuoglu K, Kohli P, Hassabis D. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596:583–589. doi: 10.1038/s41586-021-03819-2. PubMed DOI PMC

Kaptan S, Vattulainen I. Autoencoder based clustering for Molecular Dynamics Simulations. swh:1:rev:100d4d85a50fd6667d1427d8edea19aed98854b5Software Heritage. 2023 https://archive.softwareheritage.org/swh:1:dir:cccd3788289c9ffb11c777b3d8136a2dd09b52f0;origin=https://gitlab.com/molintel/autoencoder-based-clustering-for-md;visit=swh:1:snp:c7452f995d2929f94e0d1b26630cf015170b7330;anchor=swh:1:rev:100d4d85a50fd6667d1427d8edea19aed98854b5

Kastrup JS, Eriksson ES, Dalbøge H, Flodgaard H. X-ray structure of the 154-amino-acid form of recombinant human basic fibroblast growth factor: comparison with the truncated 146-amino-acid form. Acta Crystallographica Section D Biological Crystallography. 1997;53:160–168. doi: 10.1107/S0907444996012711. PubMed DOI

Katsinelos T, Zeitler M, Dimou E, Karakatsani A, Müller H-M, Nachman E, Steringer JP, Ruiz de Almodovar C, Nickel W, Jahn TR. Unconventional secretion mediates the trans-cellular spreading of tau. Cell Reports. 2018;23:2039–2055. doi: 10.1016/j.celrep.2018.04.056. PubMed DOI

Katsinelos T, McEwan WA, Jahn TR, Nickel W. Identification of cis-acting determinants mediating the unconventional secretion of tau. Scientific Reports. 2021;11:12946. doi: 10.1038/s41598-021-92433-3. PubMed DOI PMC

La Venuta G, Zeitler M, Steringer JP, Müller H-M, Nickel W. The startling properties of fibroblast growth factor 2: how to exit mammalian cells without a signal peptide at hand. The Journal of Biological Chemistry. 2015;290:27015–27020. doi: 10.1074/jbc.R115.689257. PubMed DOI PMC

La Venuta G, Wegehingel S, Sehr P, Müller H-M, Dimou E, Steringer JP, Grotwinkel M, Hentze N, Mayer MP, Will DW, Uhrig U, Lewis JD, Nickel W. Small molecule inhibitors targeting tec kinase block unconventional secretion of fibroblast growth factor 2. The Journal of Biological Chemistry. 2016;291:17787–17803. doi: 10.1074/jbc.M116.729384. PubMed DOI PMC

Lee J, Cheng X, Swails JM, Yeom MS, Eastman PK, Lemkul JA, Wei S, Buckner J, Jeong JC, Qi Y, Jo S, Pande VS, Case DA, Brooks CL, MacKerell AD, Klauda JB, Im W. CHARMM-GUI input generator for NAMD, GROMACS, AMBER, OpenMM, and CHARMM/OpenMM simulations using the CHARMM36 additive force field. Journal of Chemical Theory and Computation. 2016;12:405–413. doi: 10.1021/acs.jctc.5b00935. PubMed DOI PMC

Legrand C, Saleppico R, Sticht J, Lolicato F, Müller H-M, Wegehingel S, Dimou E, Steringer JP, Ewers H, Vattulainen I, Freund C, Nickel W. The Na,K-ATPase acts upstream of phosphoinositide PI(4,5)P2 facilitating unconventional secretion of Fibroblast growth factor 2. Communications Biology. 2020;3:141. doi: 10.1038/s42003-020-0871-y. PubMed DOI PMC

Lennicke C, Cochemé HM. Redox metabolism: ROS as specific molecular regulators of cell signaling and function. Molecular Cell. 2021;81:3691–3707. doi: 10.1016/j.molcel.2021.08.018. PubMed DOI

Liu X, Zhang Z, Ruan J, Pan Y, Magupalli VG, Wu H, Lieberman J. Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores. Nature. 2016;535:153–158. doi: 10.1038/nature18629. PubMed DOI PMC

Liu F, Lössl P, Scheltema R, Viner R, Heck AJR. Optimized fragmentation schemes and data analysis strategies for proteome-wide cross-link identification. Nature Communications. 2017;8:15473. doi: 10.1038/ncomms15473. PubMed DOI PMC

Liu L, Zhang M, Ge L. Protein translocation into the ERGIC: an upstream event of secretory autophagy. Autophagy. 2020;16:1358–1360. doi: 10.1080/15548627.2020.1768668. PubMed DOI PMC

Locker JK, Griffiths G. An unconventional role for cytoplasmic disulfide bonds in vaccinia virus proteins. The Journal of Cell Biology. 1999;144:267–279. doi: 10.1083/jcb.144.2.267. PubMed DOI PMC

Lolicato F, Nickel W. A role for liquid-ordered plasma membrane nanodomains coordinating the unconventional secretory pathway of fibroblast growth factor 2? Frontiers in Cell and Developmental Biology. 2022;10:864257. doi: 10.3389/fcell.2022.864257. PubMed DOI PMC

Lolicato F, Saleppico R, Griffo A, Meyer A, Scollo F, Pokrandt B, Müller H-M, Ewers H, Hähl H, Fleury J-B, Seemann R, Hof M, Brügger B, Jacobs K, Vattulainen I, Nickel W. Cholesterol promotes clustering of PI(4,5)P2 driving unconventional secretion of FGF2. The Journal of Cell Biology. 2022;221:e202106123. doi: 10.1083/jcb.202106123. PubMed DOI PMC

Lutz R, Bujard H. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Research. 1997;25:1203–1210. doi: 10.1093/nar/25.6.1203. PubMed DOI PMC

Malhotra V. Unconventional protein secretion: an evolving mechanism. The EMBO Journal. 2013;32:1660–1664. doi: 10.1038/emboj.2013.104. PubMed DOI PMC

Mastronarde DN. Automated electron microscope tomography using robust prediction of specimen movements. Journal of Structural Biology. 2005;152:36–51. doi: 10.1016/j.jsb.2005.07.007. PubMed DOI

Mastronarde DN, Held SR. Automated tilt series alignment and tomographic reconstruction in IMOD. Journal of Structural Biology. 2017;197:102–113. doi: 10.1016/j.jsb.2016.07.011. PubMed DOI PMC

Meng Y, Roux B. Efficient determination of free energy landscapes in multiple dimensions from biased umbrella sampling simulations using linear regression. Journal of Chemical Theory and Computation. 2015;11:3523–3529. doi: 10.1021/ct501130r. PubMed DOI PMC

Merezhko M, Brunello CA, Yan X, Vihinen H, Jokitalo E, Uronen RL, Huttunen HJ. Secretion of tau via an unconventional non-vesicular mechanism. Cell Reports. 2018;25:2027–2035. doi: 10.1016/j.celrep.2018.10.078. PubMed DOI

Merezhko M, Uronen RL, Huttunen HJ. The cell biology of tau secretion. Frontiers in Molecular Neuroscience. 2020;13:569818. doi: 10.3389/fnmol.2020.569818. PubMed DOI PMC

Mirdita M, Schütze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M. ColabFold: making protein folding accessible to all. Nature Methods. 2022;19:679–682. doi: 10.1038/s41592-022-01488-1. PubMed DOI PMC

Monteleone M, Stanley AC, Chen KW, Brown DL, Bezbradica JS, von Pein JB, Holley CL, Boucher D, Shakespear MR, Kapetanovic R, Rolfes V, Sweet MJ, Stow JL, Schroder K. Interleukin-1β maturation triggers its relocation to the plasma membrane for gasdermin-D-dependent and -independent secretion. Cell Reports. 2018;24:1425–1433. doi: 10.1016/j.celrep.2018.07.027. PubMed DOI

Müller H-M, Steringer JP, Wegehingel S, Bleicken S, Münster M, Dimou E, Unger S, Weidmann G, Andreas H, García-Sáez AJ, 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. The Journal of Biological Chemistry. 2015;290:8925–8937. doi: 10.1074/jbc.M114.622456. PubMed DOI PMC

Navarro PP, Stahlberg H, Castaño-Díez D. Protocols for subtomogram averaging of membrane proteins in the dynamo software Package. Frontiers in Molecular Biosciences. 2018;5:82. doi: 10.3389/fmolb.2018.00082. PubMed DOI PMC

Nawrocka D, Krzyscik MA, Opaliński Ł, Zakrzewska M, Otlewski J. Stable fibroblast growth factor 2 dimers with high pro-survival and mitogenic potential. International Journal of Molecular Sciences. 2020;21:4108. doi: 10.3390/ijms21114108. PubMed DOI PMC

Netto LES, Machado LESF. Preferential redox regulation of cysteine-based protein tyrosine phosphatases: structural and biochemical diversity. The FEBS Journal. 2022;289:5480–5504. doi: 10.1111/febs.16466. PubMed DOI

Nickel W. Unconventional secretion: an extracellular trap for export of fibroblast growth factor 2. Journal of Cell Science. 2007;120:2295–2299. doi: 10.1242/jcs.011080. PubMed DOI

Nickel W, Seedorf M. Unconventional mechanisms of protein transport to the cell surface of eukaryotic cells. Annual Review of Cell and Developmental Biology. 2008;24:287–308. doi: 10.1146/annurev.cellbio.24.110707.175320. PubMed DOI

Nickel W, Rabouille C. Mechanisms of regulated unconventional protein secretion. Nature Reviews. Molecular Cell Biology. 2009;10:148–155. doi: 10.1038/nrm2617. PubMed DOI

Nickel W. The unconventional secretory machinery of fibroblast growth factor 2. Traffic. 2011;12:799–805. doi: 10.1111/j.1600-0854.2011.01187.x. PubMed DOI

Nordzieke DE, Medraño-Fernandez I. The plasma membrane: a platform for intra- and intercellular redox signaling. Antioxidants. 2018;7:168. doi: 10.3390/antiox7110168. PubMed DOI PMC

Pallotta MT, Nickel W. FGF2 and IL-1β - explorers of unconventional secretory pathways at a glance. Journal of Cell Science. 2020;133:jcs250449. doi: 10.1242/jcs.250449. PubMed DOI

Parrinello M, Rahman A. Polymorphic transitions in single crystals: a new molecular dynamics method. Journal of Applied Physics. 1981;52:7182–7190. doi: 10.1063/1.328693. DOI

Paszke A, Gross S, Massa F, Lerer A, Bradbury J, Chanan G, Killeen T, Lin Z, Gimelshein N, Antiga L, Desmaison A, Kopf A, Yang E, DeVito Z, Raison M, Tejani A, Chilamkurthy S, Steiner B, Fang L, Bai J, Chintala S. Advances in Neural Information Processing Systems 32. Curran Associates, Inc; 2019. Pytorch: an imperative style, high-performance deep learning library; pp. 8024–8035.

Pedregosa F, Varoquaux G, Gramfort A, Michel V, Thirion B, Grisel O, Blondel M, Prettenhofer P, Weiss R, Dubourg V, Vanderplas J, Passos A, Cournapeau D, Brucher M, Perrot M, Duchesnay E. Scikit-learn: machine learning in python. Journal of Machine Learning Research. 2011;12:2825–2830.

Pettersen EF, Goddard TD, Huang CC, Meng EC, Couch GS, Croll TI, Morris JH, Ferrin TE. UCSF ChimeraX: structure visualization for researchers, educators, and developers. Protein Science. 2021;30:70–82. doi: 10.1002/pro.3943. PubMed DOI PMC

Piersimoni L, Sinz A. Cross-linking/mass spectrometry at the crossroads. Analytical and Bioanalytical Chemistry. 2020;412:5981–5987. doi: 10.1007/s00216-020-02700-x. PubMed DOI PMC

Plotnikov AN, Schlessinger J, Hubbard SR, Mohammadi M. Structural basis for FGF receptor dimerization and activation. Cell. 1999;98:641–650. doi: 10.1016/s0092-8674(00)80051-3. PubMed DOI

Plotnikov AN, Hubbard SR, Schlessinger J, Mohammadi M. Crystal structures of two FGF-FGFR complexes reveal the determinants of ligand-receptor specificity. Cell. 2000;101:413–424. doi: 10.1016/s0092-8674(00)80851-x. PubMed DOI

Rabouille C. Pathways of unconventional protein secretion. Trends in Cell Biology. 2017;27:230–240. doi: 10.1016/j.tcb.2016.11.007. PubMed DOI

Rappsilber J, Mann M, Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nature Protocols. 2007;2:1896–1906. doi: 10.1038/nprot.2007.261. PubMed DOI

Rayne F, Debaisieux S, Yezid H, Lin Y-L, Mettling C, Konate K, Chazal N, Arold ST, Pugnière M, Sanchez F, Bonhoure A, Briant L, Loret E, Roy C, Beaumelle B. Phosphatidylinositol-(4,5)-bisphosphate enables efficient secretion of HIV-1 Tat by infected T-cells. The EMBO Journal. 2010;29:1348–1362. doi: 10.1038/emboj.2010.32. PubMed DOI PMC

Rubin DB. The Bayesian Bootstrap. The Annals of Statistics. 1981;9:130–134. doi: 10.1214/aos/1176345338. DOI

Šachl R, Čujová S, Singh V, Riegerová P, Kapusta P, Müller HM, Steringer JP, Hof M, Nickel W. Functional assay to correlate protein oligomerization states with membrane pore formation. Analytical Chemistry. 2020;92:14861–14866. doi: 10.1021/acs.analchem.0c03276. PubMed DOI

Schäfer T, Zentgraf H, Zehe C, Brügger B, Bernhagen J, Nickel W. Unconventional secretion of fibroblast growth factor 2 is mediated by direct translocation across the plasma membrane of mammalian cells. The Journal of Biological Chemistry. 2004;279:6244–6251. doi: 10.1074/jbc.M310500200. PubMed DOI

Schatz M, Tong PBV, Beaumelle B. Unconventional secretion of viral proteins. Seminars in Cell & Developmental Biology. 2018;83:8–11. doi: 10.1016/j.semcdb.2018.03.008. PubMed DOI

Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A. Fiji: an open-source platform for biological-image analysis. Nature Methods. 2012;9:676–682. doi: 10.1038/nmeth.2019. PubMed DOI PMC

Schlessinger J, Plotnikov AN, Ibrahimi OA, Eliseenkova AV, Yeh BK, Yayon A, Linhardt RJ, Mohammadi M. Crystal structure of a ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR binding and dimerization. Molecular Cell. 2000;6:743–750. doi: 10.1016/s1097-2765(00)00073-3. PubMed DOI

Seelenmeyer C, Wegehingel S, Tews I, Künzler M, Aebi M, Nickel W. Cell surface counter receptors are essential components of the unconventional export machinery of galectin-1. The Journal of Cell Biology. 2005;171:373–381. doi: 10.1083/jcb.200506026. PubMed DOI PMC

Sitia R, Rubartelli A. The unconventional secretion of IL-1β: handling a dangerous weapon to optimize inflammatory responses. Seminars in Cell & Developmental Biology. 2018;83:12–21. doi: 10.1016/j.semcdb.2018.03.011. PubMed DOI

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. 2022a;11:e75545. doi: 10.7554/eLife.75545. PubMed DOI PMC

Sparn C, Meyer A, Saleppico R, Nickel W. Unconventional secretion mediated by direct protein self-translocation across the plasma membranes of mammalian cells. Trends in Biochemical Sciences. 2022b;47:699–709. doi: 10.1016/j.tibs.2022.04.001. PubMed DOI

Steringer JP, Bleicken S, Andreas H, Zacherl S, Laussmann M, Temmerman K, Contreras FX, Bharat TAM, Lechner J, Müller H-M, Briggs JAG, García-Sáez AJ, Nickel W. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-dependent oligomerization of fibroblast growth factor 2 (FGF2) triggers the formation of a lipidic membrane pore implicated in unconventional secretion. Journal of Biological Chemistry. 2012;287:27659–27669. doi: 10.1074/jbc.M112.381939. PubMed DOI PMC

Steringer JP, 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:e28985. doi: 10.7554/eLife.28985. PubMed DOI PMC

Steringer JP, Nickel W. A direct gateway into the extracellular space: unconventional secretion of FGF2 through self-sustained plasma membrane pores. Seminars in Cell & Developmental Biology. 2018;83:3–7. doi: 10.1016/j.semcdb.2018.02.010. PubMed DOI

Temmerman K, Ebert AD, Müller HM, 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. doi: 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. Journal of Lipid Research. 2009;50:1245–1254. doi: 10.1194/jlr.D800043-JLR200. PubMed DOI PMC

Tinevez JY, Perry N, Schindelin J, Hoopes GM, Reynolds GD, Laplantine E, Bednarek SY, Shorte SL, Eliceiri KW. TrackMate: an open and extensible platform for single-particle tracking. Methods. 2017;115:80–90. doi: 10.1016/j.ymeth.2016.09.016. PubMed DOI

Torrie GM, Valleau JP. Monte Carlo free energy estimates using non-Boltzmann sampling: application to the sub-critical Lennard-Jones fluid. Chemical Physics Letters. 1974;28:578–581. doi: 10.1016/0009-2614(74)80109-0. DOI

Torrie GM, Valleau JP. Nonphysical sampling distributions in Monte Carlo free-energy estimation: umbrella sampling. Journal of Computational Physics. 1977;23:187–199. doi: 10.1016/0021-9991(77)90121-8. DOI

Urlinger S, Baron U, Thellmann M, Hasan MT, Bujard H, Hillen W. Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. PNAS. 2000;97:7963–7968. doi: 10.1073/pnas.130192197. PubMed DOI PMC

Verlet L. Computer “experiments” on classical fluids: I. thermodynamical properties of lennard-jones molecules. Physical Review. 1967;159:98–103. doi: 10.1103/PhysRev.159.98. DOI

Wang C, Schueler-Furman O, Baker D. Improved side-chain modeling for protein-protein docking. Protein Science. 2005;14:1328–1339. doi: 10.1110/ps.041222905. PubMed DOI PMC

Wang C, Bradley P, Baker D. Protein-protein docking with backbone flexibility. Journal of Molecular Biology. 2007;373:503–519. doi: 10.1016/j.jmb.2007.07.050. PubMed DOI

Wang W, Yang D, Chen F, Pang Y, Huang S, Ge Y. Clustering with orthogonal autoencoder. IEEE Access. 2019;7:62421–62432. doi: 10.1109/ACCESS.2019.2916030. DOI

Widengren J, Mets U, Rigler R. Fluorescence correlation spectroscopy of triplet states in solution: a theoretical and experimental study. The Journal of Physical Chemistry. 1995;99:13368–13379. doi: 10.1021/j100036a009. DOI

Ye Y. Regulation of protein homeostasis by unconventional protein secretion in mammalian cells. Seminars in Cell & Developmental Biology. 2018;83:29–35. doi: 10.1016/j.semcdb.2018.03.006. PubMed DOI PMC

Young TS, Ahmad I, Yin JA, Schultz PG. An enhanced system for unnatural amino acid mutagenesis in E. coli. Journal of Molecular Biology. 2010;395:361–374. doi: 10.1016/j.jmb.2009.10.030. PubMed DOI

Zacherl S, La Venuta G, Müller H-M, Wegehingel S, Dimou E, Sehr P, Lewis JD, Erfle H, Pepperkok R, Nickel W. A direct role for ATP1A1 in unconventional secretion of fibroblast growth factor 2. The Journal of Biological Chemistry. 2015;290:3654–3665. doi: 10.1074/jbc.M114.590067. 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. PNAS. 2006;103:15479–15484. doi: 10.1073/pnas.0605997103. PubMed DOI PMC

Zhang M, Schekman R. Cell biology: unconventional secretion, unconventional solutions. Science. 2013;340:559–561. doi: 10.1126/science.1234740. PubMed DOI

Zhang M, Liu L, Lin X, Wang Y, Li Y, Guo Q, Li S, Sun Y, Tao X, Zhang D, Lv X, Zheng L, Ge L. A translocation pathway for vesicle-mediated unconventional protein secretion. Cell. 2020;181:637–652. doi: 10.1016/j.cell.2020.03.031. PubMed DOI

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