DOI: 10.5061/dryad.gtht76hq5
title: Data for: The energetics of rapid mechanotransduction

description: Cells throughout the human body detect mechanical forces. While it is
 known that the rapid (millisecond) detection of mechanical forces is
 mediated by force-gated ion channels, a detailed quantitative
 understanding of cells as sensors of mechanical energy is still lacking.
 Here, we combine atomic force microscopy with patch-clamp
 electrophysiology to determine the physical limits of cells expressing the
 force-gated ion channels Piezo1, Piezo2, TREK1, and TRAAK. We find that,
 depending on the ion channel expressed, cells can function either as
 proportional or non-linear transducers of mechanical energy, detect
 mechanical energies as little as ~100 fJ, and with a resolution of up to
 ~1 fJ. These specific energetic values depend on cell size, channel
 density, and cytoskeletal architecture. We also make the surprising
 discovery that cells can transduce forces either nearly instantaneously
 (&lt;1 ms), or with substantial time delay (~10 ms). Using a chimeric
 experimental approach and simulations we show how such delays can emerge
 from channel-intrinsic properties and the slow diffusion of tension in the
 membrane. Overall, our experiments reveal the capabilities and limits of
 cellular mechanosensing and provide insights into molecular mechanisms
 that different cell types may employ to specialize for their distinct
 physiological roles.
description: AFM data was collected on a
 modified Digital Instruments Bioscope and passed through a breakout box to
 be integrated with electrophysiology data in a HEKA EPC10 amplifier.
 Details regarding the collection of raw data are given below. Raw data
 were preprocessed in batches using the package EphysAFM using the preprocess module. The preprocessed output is saved into an Arrow file ending in the suffix "_preprocessed.arrow" This data was then further summarized for downstream analysis using the module summarize in the same package. Details for package use can be found in the repository. <strong>AFM Stimulation of HEK293T Cells</strong> All stimuli were applied at 5 s intervals. Prior to initial contact, a coarse step-motor moved the cantilever into contact with the cell in 1-5 µm steps. Contact was determined by a sudden increase in the photodetector signal. Following contact, the step-motor moved the cantilever towards the cell in 0.2-1 µm increments until mechanotransduction currents were observed. Stimuli were applied at a rate of 40 μm/s for a total distance of ~6 μm. The speed was chosen based on initial validation experiments in HEK293T cells overexpressing Piezo1 as it was the slowest speed that was able to elicit robust mechanotransduction currents of the speeds tested (10 μm/s, 20 μm/s, 40 μm/s, 80 μm/s, and 160 μm/s). <strong>Electrophysiology</strong> All whole-cell recordings were performed at room temperature using an EPC10 amplifier and Patchmaster software (HEKA Elektronik). Data were sampled at 25 kHz and filtered at 2.9 kHz using an 8-pole Bessel filter. Series resistance was compensated 20-65%. Thin-walled borosilicate glass pipettes (1.5 mm OD, 1.17 mm ID; Sutter Instrument Company, Novato, CA) were pulled and wrapped in parafilm to reduce pipette capacitance.  The final pipette resistance was 2.0-6.0 MΩ when filled with pipette solution (in mM: 133 CsCl, 0.5 EGTA, 10 HEPES, 1 MgCl<sub>2</sub>, 4 MgATP, 0.4 Na<sub>2</sub>GTP for Piezo1, Piezo2, and YFP or 133 KCl, 0.5 EGTA, 10 HEPES, 1 MgCl<sub>2</sub>, 4 MgATP, and 0.4 Na<sub>2</sub>GTP for TRAAK and TREK1). The bath solution for experiments involving Piezo1, Piezo2, and YFP contained in mM: 150 NaCl, 1 MgCl<sub>2</sub>, 2.5 CaCl<sub>2</sub>, 10 HEPES, 10 Glucose, and 3 KCl), and the bath solution for experiments involving TRAAK or TREK1 contained in mM: 140 NaCl, 3 MgCl<sub>2</sub>, 1 CaCl<sub>2</sub>, 10 HEPES, 3 KCl, 10 Glucose, and 10 TEA-Cl. For TREK1 experiments using RbCl-based solutions the bath solution consisted of in mM: 140 NaCl, 3 MgCl<sub>2</sub>, 1 CaCl<sub>2</sub>, 10 HEPES, 30 KCl, 10 Glucose, and 10 TEA-Cl. The pipette solution for these experiments consisted of in mM: 133 RbCl, 1 MgCl<sub>2</sub>, 10 HEPES, 0.5 EGTA, 4 MgATP, and 0.4 Na2GTP. For bath solutions pH was adjusted to 7.4 and for pipette solutions 7.2 using the hydroxide of the dominant cationic species. Osmolality was adjusted to ~310 for bath solutions and ~290 for pipette solutions with sucrose when necessary. The internal solution was allowed three minutes to dialyze prior to recording to promote GTP-mediated run-up of Piezo currents (Jia et al., 2013). Recording sessions for individual coverslips were limited to one hour.
description: All data was preprocessed and summarized using a package
 developed in Python. The full reproducible environment along with
 installation and usage instructions can be found at EphysAFM.