Dietary fatty acids fine-tune Piezo1 mechanical response

This is the Source Data underlying Figs. 1d, 2e, 4a, 5e, g, 6b, and Supplementary Figs. 1g, 2c, 7a of the Nature Communications Paper "Dietary fatty acids fine-tune Piezo1 mechanical response"

Liquid chromatography–mass spectrometry

Control and fatty acid-treated N2A cells and HMVECs were cultured according to manufacturer’s protocols up to 2–4 million cells. Cells were rinsed with PBS three times and frozen in liquid N2. Total and free fatty acids were extracted and quantified at the Lipidomics Core Facility at Wayne State University. Membrane (i.e., esterified) fatty acids were determined by subtracting free from total fatty acids and normalized by the number of cells in the culture.

1d. MA membrane content fold change in N2A cells treated with MA 100 µM for 18 h or 10 µM each day for 5 days, as determined by LC-MS

2e. AA, EPA, and DHA membrane content fold change in N2A cells treated with AA, EPA, and DHA 100 µM for 18 h, as determined by liquid chromatography-mass spectrometry (LC-MS)

4a. Stacked bar chart illustrating the membrane fatty acid distribution in N2A cells and HMVEC, as determined by LC-MS. Bars are mean ± SD. n is denoted above the x-axis. Mann–Whitney test.

5e. EPA membrane content fold change of EPA-treated HMVEC supplemented with 100 µM for 18 h and 50 µM each day for 3 days, as determined by LC-MS.

5g. MA, AA, and DHA membrane content fold change in MA, AA, and DHA (100 µM)-treated HMVEC as determined by LC-MS

6b. Stacked bar chart illustrating the membrane fatty acid distribution in N2A cells, HMVEC, and LA (100 µM)-treated N2A cells, as determined by LC-MS. Bars are mean ± SD. One-way ANOVA and Bonferroni test.

Supplementary Fig. 1g. PDA membrane content fold change in N2A cells treated with PDA 100 µM for 18 h, as determined by LC-MS.

Supplementary Fig. 2c. EPA membrane content fold change in EPA (10 µM each day for five days)-treated N2A cells, as determined by LC-MS.

Abstract

Mechanosensitive ion channels rely on membrane composition to transduce physical stimuli into electrical signals. The Piezo1 channel mediates mechanoelectrical transduction and regulates crucial physiological processes, including vascular architecture and remodeling, cell migration, and erythrocyte volume. The identity of the membrane components that modulate Piezo1 function remain largely unknown. Using lipid profiling analyses, we here identify dietary fatty acids that tune Piezo1 mechanical response. We find that margaric acid, a saturated fatty acid present in dairy products and fish, inhibits Piezo1 activation and polyunsaturated fatty acids (PUFAs), present in fish oils, modulate channel inactivation. Force measurements reveal that margaric acid increases membrane bending stiffness, whereas PUFAs decrease it. We use fatty acid supplementation to abrogate the phenotype of gain-of-function Piezo1 mutations causing human dehydrated hereditary stomatocytosis. Beyond Piezo1, our findings demonstrate that cell-intrinsic lipid profile and changes in the fatty acid metabolism can dictate the cell’s response to mechanical cues.