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

Gupta, Anjali; Lu, Danqin; Balasubramanian, Harikrushnan; Zhang, Chi; Wohland, Thorsten

doi:10.1016/j.jlr.2022.100220

Cited by 4 publications

(9 citation statements)

References 64 publications

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“…The freed receptors interact with each other to form oligomers. Other studies have also reported an increase in EGFR oligomerization and phosphorylation after cholesterol depletion using both biochemical (cross-linking, immunoprecipitation) and biophysical (FCCS, SMP, immunoelectron microscopy) techniques (5,26,(60)(61)(62)(63)(64)(65)(66).…”

Section: Egfr Oligomerization After Cholesterol Depletion Using Mβcdmentioning

confidence: 90%

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The dependence of EGFR oligomerization on environment and structure: A camera-based N&B study

Balasubramanian

Sankaran

Goh

et al. 2022

Preprint

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Number and Brightness analysis (N&B) is a fluorescence spectroscopy technique to quantify protein oligomerization. Accurate results, however, rely on a good knowledge of non-fluorescent states of the fluorescent labels, especially of fluorescent proteins (FP), which are widely used in biology. FPs have been characterized for confocal but not camera-based N&B, which allows in principle faster measurements over larger areas. Here, we calibrate camera-based N&B implemented on a total internal reflection fluorescence (TIRF) microscope for various fluorescent proteins by determining their propensity to be fluorescent. We then apply camera-based N&B in live CHO-K1 cells to determine the oligomerization state of the epidermal growth factor receptor (EGFR), a transmembrane receptor tyrosine kinase that is a crucial regulator of cell proliferation and survival with implications in many cancers. EGFR oligomerization in resting cells and its regulation by the plasma membrane microenvironment is still under debate. Therefore, we investigate the effects of extrinsic factors, including membrane organization, cytoskeletal structure, and ligand stimulation, and intrinsic factors, including mutations in various EGFR domains, on the receptor's oligomerization. Our results demonstrate that EGFR oligomerization increases with removal of cholesterol or sphingolipids, or the disruption of GM3-EGFR interactions, indicating raft association. However, oligomerization was not significantly influenced by the cytoskeleton. Mutations in either I706/V948 residues or E685/E687/E690 residues in the kinase and juxtamembrane domains, respectively, led to a decrease in oligomerization, indicating their necessity for EGFR dimerization. Finally, EGFR phosphorylation is oligomerization-dependent involving the extracellular domain (550-580 residues). Coupled with biochemical investigations, camera-based N&B indicates that EGFR oligomerization and phosphorylation is the outcome of several molecular interactions involving the lipid content and structure of the cell membrane and multiple residues in the kinase, juxtamembrane, and extracellular domains.

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Section: Egfr Oligomerization After Cholesterol Depletion Using Mβcdmentioning

confidence: 90%

“…Numerous studies have reported that a part of the EGFR population is trapped in lipid domains (5,26,(60)(61)(62)(63)(64)(65)(66). Since lipid domains are enriched in cholesterol (67), cholesterol depletion disrupts these domains.…”

Section: Egfr Oligomerization After Cholesterol Depletion Using Mβcdmentioning

confidence: 99%

The dependence of EGFR oligomerization on environment and structure: A camera-based N&B study

Balasubramanian

Sankaran

Goh

et al. 2022

Preprint

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show abstract

Section: Resultsmentioning

confidence: 88%

“…Numerous studies have reported that a part of the EGFR population is trapped in lipid domains ( 6 , 33 , 74 , 75 , 76 , 77 , 78 , 79 , 80 ). Since lipid domains are enriched in cholesterol ( 81 ), cholesterol depletion disrupts these domains.…”

Section: Resultsmentioning

confidence: 99%

The dependence of EGFR oligomerization on environment and structure: A camera-based N&B study

Balasubramanian¹,

Sankaran²,

Pandey³

et al. 2022

Biophysical Journal

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“…Recent studies have suggested that 1-heptanol could interfere with cell functions opposite to ursolic acid. This alkanol was used to evaluate the preference of proteins or lipids for Lo-like or Ld-like domains [ 31 ]. The authors of this study showed that green fluorescent protein (GFP) linked to the GPI anchor expressed in SH-SY5Y cells, increased lateral diffusion in the presence of 1-heptanol, and formed segregated clusters, which was in contrast to the control cells.…”

Section: Introductionmentioning

confidence: 99%

Enhanced Membrane Fluidization and Cholesterol Displacement by 1-Heptanol Inhibit Mast Cell Effector Functions

Bugajev

Dráberová

Utekal

et al. 2023

Cells

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Signal transduction by the high-affinity IgE receptor (FcεRI) depends on membrane lipid and protein compartmentalization. Recently published data show that cells treated with 1-heptanol, a cell membrane fluidizer, exhibit changes in membrane properties. However, the functional consequences of 1-heptanol-induced changes on mast cell signaling are unknown. This study shows that short-term exposure to 1-heptanol reduces membrane thermal stability and dysregulates mast cell signaling at multiple levels. Cells treated with 1-heptanol exhibited increased lateral mobility and decreased internalization of the FcεRI. However, this did not affect the initial phosphorylation of the FcεRI-β chain and components of the SYK/LAT1/PLCγ1 signaling pathway after antigen activation. In contrast, 1-heptanol inhibited SAPK/JNK phosphorylation and effector functions such as calcium response, degranulation, and cytokine production. Membrane hyperfluidization induced a heat shock-like response via increased expression of the heat shock protein 70, increased lateral diffusion of ORAI1-mCherry, and unsatisfactory performance of STIM1-ORAI1 coupling, as determined by flow-FRET. Furthermore, 1-heptanol inhibited the antigen-induced production of reactive oxygen species and potentiated stress-induced plasma membrane permeability by interfering with heat shock protein 70 activity. The combined data suggest that 1-heptanol-mediated membrane fluidization does not interfere with the earliest biochemical steps of FcεRI signaling, such as phosphorylation of the FcεRI-β chain and components of the SYK/LAT/PLCγ1 signaling pathway, instead inhibiting the FcεRI internalization and mast cell effector functions, including degranulation and cytokine production.

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

Cited by 4 publications

References 64 publications

The dependence of EGFR oligomerization on environment and structure: A camera-based N&B study

The dependence of EGFR oligomerization on environment and structure: A camera-based N&B study

The dependence of EGFR oligomerization on environment and structure: A camera-based N&B study

Enhanced Membrane Fluidization and Cholesterol Displacement by 1-Heptanol Inhibit Mast Cell Effector Functions

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