Abstract
Lateral heterogeneity of cholesterol distribution in cell plasma membrane was revealed by a complex study using microfluorimetry, immunofluorescence microscopy, and time-of-flight secondary ion mass spectrometry (TOF-SIMS). Glioblastoma cells U87MG exhibit a high motility, and cell movements are accompanied by redistribution of proteins along the membrane surface. The formation of the protein caveolin-1 clusters at one of the plasma membrane edges was detected by confocal microscopy on cells labeled with antibodies against caveolin 1. Using two-photon excitation fluorescence of a membrane probe 4-dimethylaminochalcone, membrane areas of about 200 × 200 nm were examined on glioblastoma cells. Simultaneous detection of the decay kinetics and the fluorescence spectrum revealed the presence of regions with an increased cholesterol concentration in the membrane at the poles of the live migrating cell. TOF-SIMS provided direct data with high spatial resolution indicating colocalization of cholesterol and caveolin 1 and confirmed previously published data on the association of caveolin 1 complexes with cholesterol clusters. Thus, three independent methods of cell membrane analysis testify that localization of cholesterol-enriched membrane regions correlates with morphological features of moving glioblastoma cells.
Similar content being viewed by others
REFERENCES
Ridley A.J., Schwartz M.A., Burridge K., Firtel R.A., Ginsberg M.H., Borisy G., Parsons J.T., Horwitz A.R. 2003. Cell migration: Integrating signals from front to back. Science. 302 (5651), 1704–1709.
Vicente-Manzanares M., Webb D.J., Horwitz A.R. 2005. Cell migration at a glance. J. Cell Sci. 118 (21), 4917–4919.
Grande-García A., Echarri A., de Rooij J., Alderson N.B., Waterman-Storer C.M., Valdivielso J.M., del Pozo M.A. 2007. Caveolin-1 regulates cell polarization and directional migration through Src kinase and Rho GTPases. J. Cell. Biol. 177 (4), 683–694.
Tomassian T., Humphries L.A., Liu S.D., Silva O., Brooks D.G., Miceli M.C. 2011. Caveolin-1 orchestrates TCR synaptic polarity, signal specificity, and function in CD8 T cells. J. Immunol. 187 (6), 2993–3002.
Lange Y., Ye J., Steck T.L. 2004. How cholesterol homeostasis is regulated by plasma membrane cholesterol in excess of phospholipids. Proc. Natl. Acad. Sci. USA. 101 (32), 11 664–11 667.
Borst J.W., Visser N.V., Kouptsova O., Visser A.J. 2000. Oxidation of unsaturated phospholipids in membrane bilayer mixtures is accompanied by membrane fluidity changes. Biochim. Biophys. Acta, 1487 (1), 61–73.
Henson P.M., Bratton D.L., Fadok V.A. 2001. The phosphatidylserine receptor: A crucial molecular switch? Nat. Rev. Mol. Cell Biol. 2 (8), 627–633.
Kagan V.E., Tyurin V.A., Jiang J., Tyurina Y.Y., Ritov V.B., Amoscato A.A., Osipov A.N., Belikova N.A., Kapralov A.A., Kini V., Vlasova I.I., Zhao Q., Zou M., Di P., Svistunenko D.A., Kurnikov I.V., Borisenko G.G. 2005. Cytochrome c acts as a cardiolipin oxygenase required for release of proapoptotic factors. Nat. Chem. Biol. 1, 223.
Rao C.S., Chung T., Pike H.M., Brown R.E. 2005. Glycolipid transfer protein interaction with bilayer vesicles: Modulation by changing lipid composition. Biophys. J. 89 (6), 4017–4028.
Ivkov V.G., Berestovskii G.N. 1981. Dinamicheskaya struktura lipidnogo bisloya (Dynamic structure of the lipid bilayer). Moscow: Nauka.
Sengupta P., Holowka D., Baird B. 2007. Fluorescence resonance energy transfer between lipid probes detects nanoscopic heterogeneity in the plasma membrane of live cells. Biophys. J. 92 (10), 3564–3574.
Gidwani A., Holowka D., Baird B. 2001. Fluorescence anisotropy measurements of lipid order in plasma membranes and lipid rafts from RBL-2H3 mast cells. Biochemistry. 40 (41), 12 422–12 429.
Korlach J., Baumgart T., Webb W.W., Feigenson G.W. 2005. Detection of motional heterogeneities in lipid bilayer membranes by dual probe fluorescence correlation spectroscopy. BBA – Biomembranes. 1668 (2), 158–163.
Lange Y., Tabei S.M.A., Ye J., Steck T.L. 2013. Stability and stoichiometry of bilayer phospholipid–cholesterol complexes: Relationship to cellular sterol distribution and homeostasis. Biochemistry. 52 (40), 6950–6959.
Sokolov A., Radhakrishnan A. 2010. Accessibility of cholesterol in endoplasmic reticulum membranes and activation of SREBP-2 switch abruptly at a common cholesterol threshold. J. Biol. Chem. 285 (38), 29480–29490.
Lange Y., Steck T.L. 2016. Active membrane cholesterol as a physiological effector. Chem. Phys. Lipids. 199, 74–93.
Di Vizio D., Adam R.M., Kim J., Kim R., Sotgia F., Williams T., Demichelis F., Solomon K.R., Loda M., Rubin M.A., Lisanti M.P., Freeman M.R. 2008. Caveolin-1 interacts with a lipid raft-associated population of fatty acid synthase. Cell Cycle. 7 (14), 2257–2267.
Lajoie P., Goetz J.G., Dennis J.W., Nabi I.R. 2009. Lattices, rafts, and scaffolds: Domain regulation of receptor signaling at the plasma membrane. J. Cell. Biol. 185 (3), 381.
Hailstones D., Sleer L.S., Parton R.G., Stanley K.K. 1998. Regulation of caveolin and caveolae by cholesterol in MDCK cells. J. Lipid. Res. 39 (2), 369–379.
Gularyan S.K., Petrukhin A.N., Zolotavvin P.N., Svetlichnyi V.Yu., Dobtretsov G.E., Sarkisov O.M. 2006. Fluorescent probe 4-dimethylaminochalkon as a detector of structural differences of subcellular organelles in situ. Biol. Membrany (Rus.). 23 (6), 503–509.
Svetlichnyi V.Yu., Dobtretsov G.E., Gularyan S.K., Merola F., Syreishchikova T.I. 2007. The influence of cholesterol on polar groups of the lipid bilayer: Studies using fluorescent probe 4-dimethylaminochalkon. Biol. Membrany (Rus.). 24 (3), 266–272.
Chen Y., Qin J., Cai J.Y., Chen Z.W. 2009. Cold induces micro- and nano-scale reorganization of lipid raft markers at mounds of T-cell membrane fluctuations. Plos One. 4 (4), e5386.
Navarro A., Anand-Apte B., Parat M.O. 2004. A role for caveolae in cell migration. FASEB J. 18 (15), 1801–1811.
Ivkov V.G., Berestovskii G.N. 1982. Lipidnyi bisloy biologicheskikh membran (Lipid bilayer of biological membranes). Moscow: Nauka.
Vladimirov Yu.A., Dobtretsov G.E. 1980. Fluorestsentnye zondy v issledovanii biologicheskilh membran (Fluorescent probes in studies of biological membranes). Moscow: Nauka.
Gularyan S.K., Dobtretsov G.E., Svetlichnyi V.Yu. 1996. Studies of the lipid spatial structure in human blood leukocyte by the method of non-irradiating energy transfer. Biol. Membrany (Rus.). 13, 588–597.
Ashcroft R.G., Coster H.G.L., Laver D.R., Smith J.R. 1983. The effects of cholesterol inclusion on the molecular organisation of bimolecular lipid membranes. BBA – Biomembranes, 730 (2), 231–238.
Cevc G., Watts A., Marsh D. 1981. Titration of the phase transition of phosphatidylserine bilayer membranes. Effects of pH, surface electrostatics, ion binding, and head-group hydration. Biochemistry. 20 (17), 4955–4965.
Gularyan S.K., Dobtretsov G.E., Polyak B.M., Svetlichnyi V.Yu., Zhukhlistova N.E., Krasovitskii B.M., Kormilova L.I., Zavodnik V.E. 2006. Fluorescent probe 4-dimethylaminochalkon: Interaction with the medium according to the data of quantum-chemical calculations. Izv. RAN. Ser. Khimich. (Rus.). 10, 1674–1679.
Bakhshiev N.G., Gularyan S.K., Dobtretsov G.E., Kirillova A.Yu., Svetlichnyi V.Yu. 2006. Solvatochromia and solvatofluorochromia of th band of the intramolecular transfer of the charge in electron spectra of the 4-dimethylaminochalkon solutions. Optika i Spektroskopia (Rus.). 100 (5), 700–708.
Gulin A., Pavlyukov M.S., Gusev S.A., Malakhova Yu.N., Buzin A.I., Chvalun S.N., Aldarov K.G., Klinov D.V., Gularyan S.K., Nadtochenko V.A. 2017. Applicability of TOF-SIMS for the assessment of lipid composition of cell membrane structures. Biochem. (Moscow) Suppl. Series A: Membr. Cell Biology. 34 (3), 215–222.
Gulin A., Nadtochenko V., Astafiev A., Pogorelova V., Rtimi S., Pogorelov A. 2016. Correlating microscopy techniques and ToF-SIMS analysis of fully grown mammalian oocytes. Analyst. 141 (13), 4121–4129.
Gómez-Moutón C., Lacalle R.A., Mira E., Jiménez-Baranda S., Barber D.F., Carrera A.C., Martínez-A C., Mañes S. 2004. Dynamic redistribution of raft domains as an organizing platform for signaling during cell chemotaxis. J. Cell. Biol. 164 (5), 759–768.
Komura N., Suzuki K.G., Ando H., Konishi M., Koikeda M., Imamura A., Chadda R., Fujiwara T.K., Tsuboi H., Sheng R., Cho W., Furukawa K., Furukawa K., Yamauchi Y., Ishida H., Kusumi A., Kiso M. 2016. Raft-based interactions of gangliosides with a GPI-anchored receptor. Nat. Chem. Biol. 12 (6), 402–410.
ACKNOWLEDGMENTS
The work was supported by the Russian Foundation for Basic Research (project nos. 16-04-00660, 17-29-06056, and 18-29-01027). Part of the TOF-SIMS measurements was performed at the expense of the subsidy issued to the IBCh RAS in support of the State task, theme 0082-2018-0005 (code AAAA-A18-118020690203-8), with the use of the instruments of the Center of the Collective Equipment ICP RAS (no. 506 694).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
The authors declare that they have no conflict of interest. This article does not contain any studies involving animals or human participants performed by any of the authors.
Additional information
Translated by A. Dunina-Barkovskaya
Rights and permissions
About this article
Cite this article
Pavlyukov, M.S., Gulin, A.A., Astafiev, A.A. et al. Lateral Heterogeneity of Cholesterol Distribution in Cell Plasma Membrane: Investigation by Microfluorimetry, Immunofluorescence, and TOF-SIMS. Biochem. Moscow Suppl. Ser. A 13, 50–57 (2019). https://doi.org/10.1134/S1990747818040098
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1134/S1990747818040098