Self‐assembly of lipid rafts revealed by fluorescence correlation spectroscopy in living breast cancer cells

Lipids and proteins in the plasma membrane are laterally heterogeneous and formalised as lipid rafts featuring unique biophysical properties. However, the self-assembly mechanism of lipid raft cannot be revealed even its physical properties and components were determined in specific physiological processes. In this study, two-photon generalised polarisation imaging and fluorescence correlation spectroscopy were used to study the fusion of lipid rafts through the membrane phase and the lateral diffusion of lipids in living breast cancer cells. A self-assembly model of lipid rafts associated with lipid diffusion and membrane phase was proposed to demonstrate the lipid sorting ability of lipid rafts in the plasma membrane. The results showed that the increased proportion of slow subdiffusion of GM1 -binding cholera toxin B-subunit (CT-B) is accompanied with an increased liquid-ordered domain during the β-estradiol-induced fusion of lipid rafts. And slow subdiffusion of CT-B was vanished with the depletion of lipid rafts. Whereas the dialkylindocarbocyanine (DiIC18 ) diffusion was not specifically regulated by lipid rafts. This study will open up a new insight for uncovering the self-assembly of lipid rafts in specific pathophysiological processes. This article is protected by copyright. All rights reserved.

[1]  Akihiro Kusumi,et al.  Confined diffusion of transmembrane proteins and lipids induced by the same actin meshwork lining the plasma membrane , 2016, Molecular biology of the cell.

[2]  Daniel S. Banks,et al.  Anomalous diffusion of proteins due to molecular crowding. , 2005, Biophysical journal.

[3]  Daniel S. Banks,et al.  Characterizing anomalous diffusion in crowded polymer solutions and gels over five decades in time with variable-lengthscale fluorescence correlation spectroscopy. , 2016, Soft matter.

[4]  K. Gaus,et al.  Quantitative imaging of membrane lipid order in cells and organisms , 2011, Nature Protocols.

[5]  S. Mayor,et al.  The mystery of membrane organization: composition, regulation and roles of lipid rafts , 2017, Nature Reviews Molecular Cell Biology.

[6]  E. Ikonen,et al.  Functional rafts in cell membranes , 1997, Nature.

[7]  Raphael Zidovetzki,et al.  Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies. , 2007, Biochimica et biophysica acta.

[8]  S. Hell,et al.  Direct observation of the nanoscale dynamics of membrane lipids in a living cell , 2009, Nature.

[9]  K. Gaus,et al.  FSCS Reveals the Complexity of Lipid Domain Dynamics in the Plasma Membrane of Live Cells , 2018, Biophysical journal.

[10]  Y. J. Wang,et al.  Controlling Anomalous Diffusion in Lipid Membranes. , 2017, Biophysical journal.

[11]  N. Saini,et al.  Lipid rafts in immune signalling: current progress and future perspective , 2016, Immunology.

[12]  Satyajit Mayor,et al.  Actomyosin dynamics drive local membrane component organization in an in vitro active composite layer , 2016, Proceedings of the National Academy of Sciences.

[13]  I. Levental,et al.  The Continuing Mystery of Lipid Rafts. , 2016, Journal of molecular biology.

[14]  S. Hell,et al.  STED microscopy detects and quantifies liquid phase separation in lipid membranes using a new far-red emitting fluorescent phosphoglycerolipid analogue. , 2013, Faraday discussions.

[15]  E. Gratton,et al.  Modes of diffusion of cholera toxin bound to GM1 on live cell membrane by image mean square displacement analysis. , 2015, Biophysical journal.

[16]  Amitabha Chattopadhyay,et al.  Effect of cholesterol on lateral diffusion of fluorescent lipid probes in native hippocampal membranes. , 2006, Chemistry and physics of lipids.

[17]  E. Gratton,et al.  Two-photon fluorescence microscopy of laurdan generalized polarization domains in model and natural membranes. , 1997, Biophysical journal.

[18]  Hédi Soula,et al.  Anomalous versus slowed-down Brownian diffusion in the ligand-binding equilibrium. , 2012, Biophysical journal.

[19]  C. Vitale,et al.  Membrane lipid rafts and estrogenic signalling: a functional role in the modulation of cell homeostasis , 2015, Apoptosis.

[20]  E. Bongarzone,et al.  Recent progress on lipid lateral heterogeneity in plasma membranes: From rafts to submicrometric domains. , 2016, Progress in lipid research.

[21]  P. Schwille,et al.  Partitioning, diffusion, and ligand binding of raft lipid analogs in model and cellular plasma membranes. , 2012, Biochimica et biophysica acta.

[22]  E. Gratton,et al.  Laurdan generalized polarization fluctuations measures membrane packing micro-heterogeneity in vivo , 2012, Proceedings of the National Academy of Sciences.

[23]  E. Betzig,et al.  Self-organizing actin patterns shape membrane architecture but not cell mechanics , 2017, Nature Communications.

[24]  E Gratton,et al.  Phase fluctuation in phospholipid membranes revealed by Laurdan fluorescence. , 1990, Biophysical journal.

[25]  H. Waldmann,et al.  Lateral Organization of Host Heterogeneous Raft-like Membranes Altered by the Myristoyl Modification of Tyrosine Kinase c-Src. , 2017, Angewandte Chemie.

[26]  Yong Zhou,et al.  Ras nanoclusters: Versatile lipid-based signaling platforms. , 2015, Biochimica et biophysica acta.

[27]  Jon D. Wright,et al.  Soluble klotho regulates TRPC6 calcium signaling via lipid rafts, independent of the FGFR‐FGF23 pathway , 2019, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[28]  G. Brezesinski,et al.  Self-assembly of lipid domains in the extracellular leaflet of the plasma membrane and models thereof , 2016 .

[29]  F. Mollinedo,et al.  Lipid rafts as major platforms for signaling regulation in cancer. , 2015, Advances in biological regulation.

[30]  T. Wohland,et al.  The Secreted Signaling Protein Wnt3 Is Associated with Membrane Domains In Vivo: A SPIM-FCS Study. , 2016, Biophysical journal.

[31]  James H. Davis,et al.  Fluorescent probe partitioning in giant unilamellar vesicles of 'lipid raft' mixtures. , 2010, The Biochemical journal.

[32]  E. Gratton,et al.  Cholesterol modifies water concentration and dynamics in phospholipid bilayers: a fluorescence study using Laurdan probe. , 1994, Biophysical journal.

[33]  S. Andò,et al.  Differential insulin-like growth factor I receptor signaling and function in estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cells. , 2001, Cancer research.

[34]  Xiaolu Yang,et al.  CD317 Activates EGFR by Regulating Its Association with Lipid Rafts. , 2019, Cancer research.

[35]  C. Eggeling,et al.  Laurdan and Di-4-ANEPPDHQ probe different properties of the membrane , 2016, bioRxiv.

[36]  C. Eggeling,et al.  Spectral Imaging to Measure Heterogeneity in Membrane Lipid Packing , 2015, Chemphyschem : a European journal of chemical physics and physical chemistry.

[37]  Eleanor Stride,et al.  Spectral imaging toolbox: segmentation, hyperstack reconstruction, and batch processing of spectral images for the determination of cell and model membrane lipid order , 2017, BMC Bioinformatics.

[38]  P. Schwille,et al.  Lateral membrane diffusion modulated by a minimal actin cortex. , 2013, Biophysical journal.

[39]  L. Sklar,et al.  Synthetic estrogen derivatives demonstrate the functionality of intracellular GPR30. , 2007, ACS chemical biology.

[40]  Petra Schwille,et al.  Fluorescence correlation spectroscopy relates rafts in model and native membranes. , 2004, Biophysical journal.

[41]  Astrid Magenau,et al.  Sub-resolution lipid domains exist in the plasma membrane and regulate protein diffusion and distribution , 2012, Nature Communications.

[42]  T. Hellweg,et al.  Protein diffusion in a bicontinuous microemulsion: inducing sub-diffusion by tuning the water domain size. , 2017, Soft matter.

[43]  S. Xie,et al.  Use of quantitative optical imaging to examine the role of cholesterol-rich lipid raft microdomains in the migration of breast cancer cells , 2018 .

[44]  T. Koller,et al.  Diffusion of lipids and GPI-anchored proteins in actin-free plasma membrane vesicles measured by STED-FCS , 2016, bioRxiv.

[45]  L. Pike Rafts defined: a report on the Keystone symposium on lipid rafts and cell function Published, JLR Papers in Press, April 27, 2006. , 2006, Journal of Lipid Research.

[46]  G. Sethi,et al.  Oleuropein induces apoptosis via abrogating NF‐κB activation cascade in estrogen receptor–negative breast cancer cells , 2018, Journal of cellular biochemistry.

[47]  Christopher B. Stanley,et al.  Lipid Rafts: Buffers of Cell Membrane Physical Properties. , 2019, The journal of physical chemistry. B.