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Immobilization of Phosphatidylinositides Revealed by Bilayer Leaflet Decoupling

  • Simou Sun
    Simou Sun
    Department of Chemistry, Penn State University, University Park, Pennsylvania 16802, United States
    More by Simou Sun
  • , 
  • Chang Liu
    Chang Liu
    Department of Chemistry, Penn State University, University Park, Pennsylvania 16802, United States
    More by Chang Liu
  • , 
  • Danixa Rodriguez Melendez
    Danixa Rodriguez Melendez
    Department of Chemistry, University of Puerto Rico at Cayey, Cayey, Puerto Rico 00737, United States
    More by Danixa Rodriguez Melendez
  • , 
  • Tinglu Yang
    Tinglu Yang
    Department of Chemistry, Penn State University, University Park, Pennsylvania 16802, United States
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  • , and 
  • Paul S. Cremer*
    Paul S. Cremer
    Department of Chemistry, Penn State University, University Park, Pennsylvania 16802, United States
    Department of Biochemistry and Molecular Biology, Penn State University, University Park, Pennsylvania 16802, United States
    *[email protected]
    More by Paul S. Cremer
    Orcidhttp://orcid.org/0000-0002-8524-0438
Cite this: J. Am. Chem. Soc. 2020, 142, 30, 13003–13010
Publication Date (Web):July 20, 2020
https://doi.org/10.1021/jacs.0c03800
Copyright © 2020 American Chemical Society
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    Abstract

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    Phosphatidylinositol 4,5-bisphosphate (PIP2) has a significantly lower mobile fraction than most other lipids in supported lipid bilayers (SLBs). Moreover, the fraction of mobile PIP2 continuously decreases with time. To explore this, a bilayer unzipping technique was designed to uncouple the two leaflets of the SLB. The results demonstrate that PIP2 molecules in the top leaflet are fully mobile, while the PIP2 molecules in the lower leaflet are immobilized on the oxide support. Over time, mobile PIP2 species flip from the top leaflet to the bottom leaflet and become trapped. It was found that PIP2 flipped between the leaflets through a defect-mediated process. The flipping could be completely inhibited when holes in the bilayer were backfilled with bovine serum albumin (BSA). Moreover, by switching from H2O to D2O, it was shown that the primary interaction between PIP2 and the underlying substrate was due to hydrogen bond formation, which outcompeted electrostatic repulsion. Using substrates with fewer surface silanol groups, like oxidized polydimethylsiloxane, led to a large increase in the mobile fraction of PIP2. Moreover, PIP2 immobilization also occurred when the bilayer was supported on a protein surface rather than glass. These results may help to explain the behavior of PIP2 on the inner leaflet of the plasma membrane, where it is involved in attaching the membrane to the underlying cytoskeleton.

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.0c03800.

    • Materials and methods section; fluorescence images of PIP2 SLBs at 0 and 20 h; mobile fraction of 0.5 BODIPY-PC/99.5 POPC SLB over 20 h; FRAP recovery profiles of PIP2 SLBs with and without bound PH domains; protein titration results at different equilibration times; mobile fraction changes over 20 h for SLBs containing 0.5 TF-PIP2/0.5 DOPIP2/99 POPC; linescan profiles of unzipping results with SLBs containing 0.5 BODIPY-PC/99.5 POPC and 0.5 TR-DHPE/99.5 POPC; AFM profiles of unzipped lipid monolayers; FRAP recovery profile of an unzipped lipid monolayer; unzipping results for POPC SLBs containing TR-DHPE only in one leaflet made by the LB-LS method; unzipping results for PIP2 SLBs at 0, 200, and 1200 min; unzipping results for PC SLBs at 0 and 1200 min; fluorescence images of defects in PIP2 SLBs; FRAP recovery profiles for PIP2 SLBs before and after BSA backfilling; AFM profile of the BSA coating on glass; mobile fraction changes over 20 h for PIP2 SLBs with and without 1 μM EDTA; mobile fraction changes over 20 h for PIP2 SLBs with 100 mM NaCl and with 600 mM NaCl; FRAP recovery profiles of PIP2 SLMs in H2O and D2O; fluorescence images of PIP2 SLBs on BSA-coated glass; mobile fraction of PIP2 SLBs on BSA-coated glass in a D2O buffer at 0 and 20 h; effects of streptavidin backfilling into the defects on PIP2 SLBs supported by biotin-BSA coated glass; mobile fraction changes over 20 h for SLBs containing 1 mol % POPA, 1 mol % PI4P, and 1 mol % PIP2; schematic demonstration of a proposed bilayer unzipping mechanism; and table containing a summary of diffusion constants for TF-PIP2 in SLBs and SLMs under a variety of conditions (PDF)

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    Cited By

    This article is cited by 5 publications.

    1. Grant J. Myres, Jay P. Kitt, Joel M. Harris. Inter-Leaflet Phospholipid Exchange Impacts the Ligand Density Available for Protein Binding at Supported Lipid Bilayers. Langmuir 2022, 38 (22) , 6967-6976. https://doi.org/10.1021/acs.langmuir.2c00526
    2. Tun Naw Sut, Abdul Rahim Ferhan, Soohyun Park, Dong Jun Koo, Bo Kyeong Yoon, Joshua A. Jackman, Nam-Joon Cho. Modulating noncovalent and covalent forces to control inverse phosphocholine lipid self-assembly on inorganic surfaces: Nanoarchitectonic design principles. Applied Materials Today 2022, 29 , 101618. https://doi.org/10.1016/j.apmt.2022.101618
    3. Yundan Ruan, Pritam Guha, Shun-Li Chen, Qunhui Yuan, Wei Gan. Observing the structural variations on binary complex vesicle surfaces and the influence on molecular transportation. Chemical Physics 2021, 548 , 111250. https://doi.org/10.1016/j.chemphys.2021.111250
    4. R. T. Coones, R. J. Green, R. A. Frazier. Investigating lipid headgroup composition within epithelial membranes: a systematic review. Soft Matter 2021, 17 (28) , 6773-6786. https://doi.org/10.1039/D1SM00703C
    5. Rajasree Kundu, Amitava Chandra, Ankona Datta. Fluorescent Chemical Tools for Tracking Anionic Phospholipids. Israel Journal of Chemistry 2021, 61 (3-4) , 199-216. https://doi.org/10.1002/ijch.202100003
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