Rapid plasma membrane isolation via intracellular polymerization-mediated biomolecular confinement.

Autor: Lin CL; Institute of Biomedical Sciences, Academia Sinica. 128 Academia Road, Sec. 2, Taipei 11529, Taiwan., Fang ZS; Institute of Biomedical Sciences, Academia Sinica. 128 Academia Road, Sec. 2, Taipei 11529, Taiwan., Hsu CY; Institute of Biomedical Sciences, Academia Sinica. 128 Academia Road, Sec. 2, Taipei 11529, Taiwan., Liu YH; Institute of Biomedical Sciences, Academia Sinica. 128 Academia Road, Sec. 2, Taipei 11529, Taiwan., Lin JC; Institute of Biomedical Sciences, Academia Sinica. 128 Academia Road, Sec. 2, Taipei 11529, Taiwan., Yao BY; Institute of Biomedical Sciences, Academia Sinica. 128 Academia Road, Sec. 2, Taipei 11529, Taiwan., Li FA; Institute of Biomedical Sciences, Academia Sinica. 128 Academia Road, Sec. 2, Taipei 11529, Taiwan., Yen SB; Institute of Biomedical Sciences, Academia Sinica. 128 Academia Road, Sec. 2, Taipei 11529, Taiwan; Taiwan International Graduate Program in Molecular Medicine, National Yang Ming Chiao Tung University and Academia Sinica, Taipei, Taiwan., Chang YC; Institute of Cellular and Organismic Biology, Academia Sinica, No. 128, Sec. 2, Taipei 11529, Taiwan., Hu CJ; Institute of Biomedical Sciences, Academia Sinica. 128 Academia Road, Sec. 2, Taipei 11529, Taiwan; Research Center for Nanotechnology and Infectious Diseases, Taipei, Taiwan. Electronic address: chu@ibms.sinica.edu.tw.
Jazyk: angličtina
Zdroj: Acta biomaterialia [Acta Biomater] 2024 Jan 01; Vol. 173, pp. 325-335. Date of Electronic Publication: 2023 Nov 23.
DOI: 10.1016/j.actbio.2023.11.026
Abstrakt: Plasma membrane isolation is a foundational process in membrane proteomic research, cellular vesicle studies, and biomimetic nanocarrier development, yet separation processes for this outermost layer are cumbersome and susceptible to impurities and low yield. Herein, we demonstrate that cellular cytosol can be chemically polymerized for decoupling and isolation of plasma membrane within minutes. A rapid, non-disruptive in situ polymerization technique is developed with cell membrane-permeable polyethyleneglycol-diacrylate (PEG-DA) and a blue-light-sensitive photoinitiator, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP). The photopolymerization chemistry allows for precise control of intracellular polymerization and tunable confinement of cytosolic molecules. Upon cytosol solidification, plasma membrane proteins and vesicles are rapidly derived and purified as nucleic acids and intracellular proteins as small as 15 kDa are stably entrapped for removal. The polymerization chemistry and membrane derivation technique are broadly applicable to primary and fragile cell types, enabling facile membrane vesicle extraction from shorted-lived neutrophils and human primary CD8 T cells. The study demonstrates tunable intracellular polymerization via optimized live cell chemistry, offers a robust membrane isolation methodology with broad biomedical utility, and reveals insights on molecular crowding and confinement in polymerized cells. STATEMENT OF SIGNIFICANCE: Isolating the minute fraction of plasma membrane proteins and vesicles requires extended density gradient ultracentrifugation processes, which are susceptible to low yield and impurities. The present work demonstrates that the membrane isolation process can be vastly accelerated via a rapid, non-disruptive intracellular polymerization approach that decouples cellular cytosols from the plasma membrane. Following intracellular polymerization, high-yield plasma membrane proteins and vesicles can be derived from lysis buffer and sonication treatment, respectively. And the intracellular content entrapped within the polymerized hydrogel is readily removed within minutes. The technique has broad utility in membrane proteomic research, cellular vesicle studies, and biomimetic materials development, and the work offers insights on intracellular hydrogel-mediated molecular confinement.
Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: CMJ Hu, ZS Fang, CL Lin, CY Hsu, and JC Lin are listed as inventors on a related patent filing.
(Copyright © 2023 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)
Databáze: MEDLINE
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