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. 2022 Jun;63(6):100220.
doi: 10.1016/j.jlr.2022.100220. Epub 2022 Apr 28.

Heptanol-mediated phase separation determines phase preference of molecules in live cell membranes

Anjali Gupta  1 , Danqin Lu  2 , Harikrushnan Balasubramanian  1 , Zhang Chi  3 , Thorsten Wohland  4
Affiliations

Heptanol-mediated phase separation determines phase preference of molecules in live cell membranes

Anjali Gupta et al. J Lipid Res. 2022 Jun.

Abstract

The localization of many membrane proteins within cholesterol- and sphingolipid-containing microdomains is essential for proper cell signaling and function. These membrane domains, however, are too small and dynamic to be recorded, even with modern super-resolution techniques. Therefore, the association of membrane proteins with these domains can only be detected with biochemical assays that destroy the integrity of cells require pooling of many cells and take a long time to perform. Here, we present a simple membrane fluidizer-induced clustering approach to identify the phase-preference of membrane-associated molecules in individual live cells within 10-15 min. Experiments in phase-separated bilayers and live cells on molecules with known phase preference show that heptanol hyperfluidizes the membrane and stabilizes phase separation. This results in a transition from nanosized to micronsized clusters of associated molecules allowing their identification using routine microscopy techniques. Membrane fluidizer-induced clustering is an inexpensive and easy to implement method that can be conducted at large-scale and allows easy identification of protein partitioning in live cell membranes.

Keywords: MFIC; alcohols; assay; clustering; epidermal growth factor receptor; fluidizers; heptanol; membranes; phase separation; phases.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Fig. 1
Fig. 1
Heptanol induces hyperfluidization and domain segregation in model and live cell membranes. Samples were treated with 5 mM heptanol. Representative TIRF images and the corresponding average diffusion coefficients (D) of different samples are shown here. A, B: Rhodamine-PE–labeled DOPC:DPPC:Chol (4:4:3) supported lipid bilayer before and after heptanol treatment. The D increases from 1.21 ± 0.11 μm2/s to 3.51 ± 1.03 μm2/s following heptanol treatment. C, D: DiI-C18–labeled SH-SY5Y cells before and after the heptanol treatment. The D increases from 2.51 ± 0.56 μm2/s to 4.34 ± 1.54 μm2/s following heptanol treatment. E, F: GFP-GPI expressing SH-SY5Y cells before and after the heptanol treatment. The D increases from 0.83 ± 0.06 μm2/s to 1.72 ± 0.47 μm2/s following heptanol treatment. Experiments were repeated at least four times. The scale bars represent 5 μm. DiI-C18, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DPCC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine.
Fig. 2
Fig. 2
Heptanol phase separates molecules based on their domain preference. Representative TIRF images of probe organization on SH-SY5Y cells before and after 5 mM heptanol treatment. A: Rhodamine 18 (R18). B: Cholera toxin B (CTxB). C: allL-trLAT. D: WT-trLAT. Experiments were repeated at least three times. The scale bars represent 5 μm. trLAT, transmembrane domain of linker for activation of T-cells.
Fig. 3
Fig. 3
Domain preference of signaling related proteins as determined by heptanol-induced phase separation. Representative TIRF images of protein organization on SH-SY5Y cells before and after 5 mM heptanol treatment. A: K-Ras. B: H-Ras. C: IL-2Rɑ. Experiments were repeated at least three times. The scale bars represent 5 μm.
Fig. 4
Fig. 4
Heptanol-induced phase separation of EGFR. Representative TIRF images of EGFR transfected SH-SY5Y cells under various conditions. A: EGFR-mEGFP in resting transfected cells. B: EGFR-mEGFP after 5 mM heptanol treatment. C: EGFR antibody-Alexa 488 and EGFR-mApple channels after labeling of resting cells with EGFR antibody. D: EGFR antibody-Alexa 488 and EGFR-mApple channels after labeling of the cells with EGFR antibody followed by 5 mM heptanol treatment. Experiments were repeated at least three times. The scale bars represent 5 μm. EGFR, epidermal growth receptor factor.
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