4.1 Reagents
Cal-520™ AM (2.5mM; Abcam, Cambridge, UK), Yoda1 (1 mM; Cayman Chemical Company, US), Latrunculin A (0.5 mM; Abcam, UK), CK666 (20 mM; Sigma-Aldrich, MO, US), Jasplakinolide (0.5 mM; Life Technologies, California, US), and Y-27632(50 mM; Sapphire Bioscience, Australia) was dissolved in dimethyl sulfoxide (DMSO) (2.5 mM) and stored at -30°C. SPY650-FastAct (Spirochrome, Switzerland) was dissolved in 50 µL anhydrous DMSO and store at -30°C as company directed. GsMTx4 (250 µM; Abcam, UK) was dissolved in Mili-Q water and stored at -30°C. GdCl3 (5mM; Sigma-Aldrich, US) was prepared in Mili-Q and stored at 4°C.
Tyrode’s buffer (12 mM NaHCO3, 10 mM HEPES, 0.137 M NaCl, 2.7 mM KCl, 5.5 mM D-glucose, pH 7.2) supplemented with 1 mM CaCl2 was prepared and stored at room temperature. Carbonate/bicarbonate buffer (C-buffer; 2.1 g Na2CO3, 2.65 g NaHCO3 in 250 mL H2O, pH 8.5) was stored at 4°C fridge. Clexane (10,000 U mL− 1, Sigma-Aldrich, USA) was stored at room temperature. Full list of the chemicals and reagents can be found in the Supplementary Material.
4.2 Micropipette fabrication
A borosilicate glass capillary tube (G-1; Narishige International, US) was mounted on the micropipette puller (model P-1000; Sutter Instruments Co., US). For detailed operation procedures, please refer to the previously published protocols30,44. Since the taper length of conventional angled micropipette is too short to be inserted into our homemade glass chamber30,45,46, we then developed a multi-stage pulling in the program to fabricate the angled micropipette with the enough taper length (Fig. S10; Table 1). To fabricate parallel opening micropipettes (θ = 0°), two close-end micropipettes would then be fabricated by the preset pulling function in the micropipette puller (Heat: 505; Pull: 150; Velocity: 75; Time: 200; Pressure: 400; Ramp 500). Tips of micropipettes were cut and fashioned to diameters of 1–2 µm using a MicroForge (Model MF-900; Narishige International, US) equipped with a 20x eyepiece objective for RBC fMPA assay or 2.5 µm diameter for HEK293T fMPA assay. The pulling programs would result in micropipettes with opening orifice d = 1 µm. Then the pulled micropipettes were cut to desired orifice with the MicroForge.
Table 1
Parameters in Sutter P-1000 micropipette puller. All parameters are defined based on the application, slight modification is required for every new setup. Ramp value was measured based the heating filament in the puller.
Neck angle θ
|
Stage
|
Heat
|
Pull
|
Velocity
|
Time
|
Pressure
|
Repetition
|
0°
|
1
|
Ramp + 10
|
150
|
75
|
250
|
500
|
once
|
|
1
|
Ramp + 14
|
0
|
90
|
255
|
|
once
|
5°
|
2
|
Ramp + 14
|
0
|
32
|
255
|
670
|
twice
|
|
3
|
Ramp + 4
|
0
|
37
|
255
|
|
once
|
|
1
|
Ramp + 14
|
0
|
90
|
255
|
|
once
|
10°
|
2
|
Ramp + 14
|
0
|
32
|
255
|
670
|
twice
|
|
3
|
Ramp + 4
|
0
|
29
|
255
|
|
once
|
4.3 RBC collection and labeling
We conducted all human blood collection procedures from healthy volunteers following approval from the University of Sydney's Human Research Ethics Committee (HREC 2014/244). Detailed RBC isolation procedures can be found in our previous studies30,44,47. Briefly, we diluted 5 µL of Clexane treated whole blood in 1 mL of C-buffer and centrifuged it at 900×g for 1 min at room temperature. We discarded the supernatant and repeated the washing step with C-buffer and Tyrodes' buffer + 0.5% BSA. We sensitized the PIEZO1 by adding 0.5 µM Yoda1 to the tube before the fMPA. Alternatively, we added 50 µM GdCl3 or 2.5 µM GsMTx4 for 30 mins to suppress the PIEZO1 activity.
All procedures involving the use of mice were approved by the University of Sydney Animal Ethics Committee (Project # 2018/1461). Similar to the previously described methods24,41, we generated RBC-specific PIEZO1 knockout mice (Piezo1-KORBC) with erythropoietin receptor (EpoR) Cre recombinase48. Subsequently, we introduced the Piezo1flox alleles to the EpoR-Cre mice. The first generation of this crossbreeding resulted in all traits being heterozygous. The next step involved back-crossing these heterozygous mice with homozygous Piezo1flox animals to produce the Piezo1-KORBC experimental subjects, where the EpoR-Cre induced recombination specifically in the erythroid lineage, knocking out the Piezo1 gene. All male mice with C57BL/6J background and carrying and EpoR-Cre were bred and sourced from Australian BioResources (ABR; Moss Vale, NSW, Australia). Piezo1flox mice were obtained from Jackson Laboratories (JAX stock 029213).
To collect mouse RBCs, we anaesthetized mice (6 to 8 weeks old) with either sodium pentobarbitone (60 mg kg− 1) or ketamine/xylazine (150/15 mg kg− 1). We then collected mouse blood via the inferior vena cava as previously described49, isolated the RBCs, and maintained them at a concentration of 1×107 mL− 1. For the fMPA assay, we supplemented 150 µL washed RBCs with 1:150 Cal-520 AM and incubated at room temperature for 1 hr. To label F-actin, we added 1:1000 SPY650-FastAct to the cell mixture 1 hr before the calcium intensiometric dye. We maintained the concentration of dye probes throughout all experiments.
4.4 Cell culture, harvesting and labeling.
We cultured HEK293T cells with PIEZO1 wildtype (WT), knockout (KO; kindly gifted from Dr Ardem Patapoutian)31, and overexpressing (OE; kindly gifted from Dr Philip Gottlieb)50 in RPMI Medium 1640 (Thermo Fisher Scientific, USA) supplemented with 10% Fetal Bovine Serum (FBS; Thermo Fisher Scientific, USA) and 1% Penicillin/Streptomycin (PS; Thermo Fisher Scientific). We maintained cells at 5% CO2 and 37°C.
Before experiments, we detached cells using TrypLETM Express (Thermo Fisher Scientific) and transferred them into a 96 well cell culture plate (Thermo Fisher Scientific) at approximately 50% confluency in the RPMI medium. We incubated the cells at 37°C overnight. For the fMPA on suspended cells, we added 1:000 Cal-520 AM and SPY650-FastAct dye to the wells and incubated them in a 37°C incubator for 1 hr. To disrupt the F-actin network, we concurrently treated cells with 2.5 µM LatA for 2 hrs, 100 µM CK-666 for 2 hrs, 10 µM Jasp for 30 min, 30 µM Y-27632 for 30 min, or 1:500 DMSO for 2 hrs at 37°C. We then harvested the cells using TrypLE Express (Thermo Fisher Scientific) and spun them down at ~ 200×g for 5 min. We removed the supernatant and resuspended the cell pellets in Tyrodes' buffer supplemented with the same dye and drug concentration.
To perform fMPA on attached cells, we harvested cells from wells and seeded them on a 24×40 mm cover glass coated with 50 µg mL− 1 human fibronectin (FN; Thermo Fisher). We covered the cover glass with RPMI medium and placed it in a 37°C incubator overnight to allow the cells to adhere firmly. Before the fMPA, we supplemented the medium with dye probes and incubated it further for 1 hr. We then gently replaced the liquid with Tyrodes' buffer.
4.5 Fluorescent micropipette aspiration assay (fMPA)
A detailed protocol for the fluorescent micropipette aspiration (fMPA) assay was outlined in our previous study30. Briefly, a prepared glass micropipette was mounted into a micropipette holder (Narishige International, US), which was connected to a high-speed pneumatic pressure clamp device (HSPC; ALA Scientific Instruments, US). Two coverslip slices were adhered to the top and bottom of the chamber holder respectively, with the cell solution introduced between them. The chamber was subsequently positioned on a custom manual stage. By utilizing the µMp-3 Triple Axis Micromanipulator (SENSAPEX, Finland), the micropipette was gently introduced into the chamber from the side at an angle of approximately 10°. The aspiration pressure, ranging from ∆p = -5 to -40 mmHg, was controlled using a custom LabVIEW program (National Instrument, US).
For concurrent imaging of the calcium signal and transmitted light, a customized light path was integrated into the Olympus IX83 inverted microscope, equipped with a 60× dry objective (NA 0.7). A pE-300 (CoolLED, UK) LED light source served as the fluorescence light source, emitting 488 nm excitation light which was reflected by the first dichroic mirror to excite the calcium dye probes. The 510 nm emission light from Cal-520 was passed through the first dichroic mirror, reflected by the second dichroic mirror (long pass: 560 nm), and captured by the ultrasensitive sCMOS Prime 95B camera (Teledyne Photometrics, US). For the transmitted light path, a 760 nm long-pass filter was placed in front of the light source to facilitate transmission through all DMs, and the resultant images were captured by a Manta GigE G-040 CMOS camera (Allied Vision, Germany). Both cameras were controlled by µManager.
Olympus FV3000 confocal microscope with two high sensitivity detectors (HSD) was utilized to image three channel concurrently (i.e., transmitted, 510 nm fluorescence, and 674 nm fluorescence). The chamber was placed on our designed manual stage integrated with the micropipette manipulator. A 40× silicon oil-immersion objective (NA 1.25) was used while the images were illuminated using the silver-coated resonance scanning mirrors at 10 fps. A 1.6× magnification was implemented during acquisition using the Olympus FV31S-SW software.
4.6 Fluorescence intensity analysis and F-actin accumulation tracking
The fluorescence intensity of the aspirated cell over time was quantified using Imaris 9.0.1 (Oxford Instruments). Background signals were subtracted from all measurements. The normalized Ca2+ intensity change was then calculated using the following equation:
Where Fmax,absolute is the absolute maximum intensity of the aspirated cell, F0,absolute is the absolute intensity of the cell at the resting state, and Fb is the background intensity.
Taking the tip of the micropipette as the boundary, a small 20×20 pixels ROI was drawn around the micropipette neck to examine the F-actin accumulation time tacc. Aspirated cell’s F-actin signal inside the ROI Fa,neck is tracked over the whole aspiration process. Then a ratio between the neck and whole cell signal Fa,cell was derived:
$${ratio}_{\text{F}-\text{a}\text{c}\text{t}\text{i}\text{n}}=\frac{{F}_{\text{a}, \text{n}\text{e}\text{c}\text{k}}}{{F}_{\text{a},\text{c}\text{e}\text{l}\text{l}}}$$
2
If the ratio increased more than 50% and was stabilized for more than 1 min, we define the F-actin accumulated. The time taken for F-actin to accumulate, tacc was recorded by measuring the interval between the timepoint that aspiration started, tpressure and the timepoint that ratio increased to the threshold, t50%:
$${t}_{\text{a}\text{c}\text{c}}={t}_{50\%}-{t}_{\text{p}\text{r}\text{e}\text{s}\text{s}\text{u}\text{r}\text{e}}$$
3
4.7 Patch-clamp recording
HEK293T PIEZO1-OE cells were harvested and placed in a recording chamber containing 145 mM NaCl, 3m M KCl, 1 mM MgCl2 and 10 mM HEPES (pH 7.2, adjusted using NaOH) for patch-clamp analysis. A density of ~ 10,000 cells were seeded on coverslips for patch-clamp analysis on attached cells. HSPC was employed to apply negative pressure.
Micropipettes were fashioned similarly to those used in the fMPA assay, yielding an electrode resistance of 1.5–2.5 MΩ. EGTA was added to control level of Ca2+ inside the micropipette. Single-channel PIEZO1 currents were then amplified using the AxoPatch 200B amplifier (Molecular Devices, US). All data were acquired at a sampling rate of 10 kHz with 1–2 kHz filtration, using the Digidata 1550A Data Acquisition System (Molecular Devices, US). Analysis was conducted with pCLAMP10 software (Molecular Devices, US). Boltzmann plots were derived by fitting open probability Po ~ I/Imax versus negative pressure using the following function:
where ∆p is the negative pressure generated by the HSPC, P1/2 is the pressure at which Po = 0.5, and α is the slope of the plot reflecting the channel mechanosensitivity26.
4.8 3D confocal imaging
To prepare the adhered HEK293T cells for imaging, they were seeded into a fibronectin coated Lab-Tek™ II 8-well chamber coverglass (Thermo Fisher Scientific, US) at a density of ~ 5,000 cells well− 1 and incubated at 37°C a day prior to the experiment. The cells were then stained with SPY650-FastAct for 1 hr and Hoechst 33342 for 30 mins at 37°C before imaging. The Olympus FV3000 confocal microscope (Olympus Corporation, Japan), equipped with 2 High-Speed Disc (HSD) units, was used for imaging. The coverglass was placed in a stage top incubator (Tokai Hit, Japan) at 37°C with 5% CO2 supplementation. To excite Hoechst 33342 and SPY650-FastAct, lasers at 408 nm and 647 nm were used, respectively. The images were scanned at a 1024×1024 resolution and 0.4 µm z-step size.
4.9 Finite element analysis on membrane tension
The simulation of micropipette aspiration was conducted using ANSYS Mechanical (Ansys, US), a commercially available finite element analysis (FEM) software. Due to the negligible inertial forces, a quasistatic condition was applied to the simulations51. RBC and HEK293T cell were considered as a hyperelastic material using five constant Mooney-Rivlin mode. The strain energy function of Mooney Rivlin model is based on I1, I2 and I3 which are the first, the second, and the third deviatoric strain invariant, respectively:
$${I}_{1}={\lambda }_{1}^{2}+{\lambda }_{2}^{2}+{\lambda }_{3}^{2}$$
5
$${I}_{2}={\lambda }_{1}^{2}{\lambda }_{2}^{2}+{\lambda }_{1}^{2}{\lambda }_{3}^{2}+{\lambda }_{2}^{2}{\lambda }_{3}^{2}$$
6
$${I}_{3}={\lambda }_{1}^{2}{\lambda }_{2}^{2}{\lambda }_{3}^{2}$$
7
where λi (i = 1, 2, 3) are the square roots for Cauchy-Green strains tensor52. The Mooney-Rivlin energy function can be written as a power series of the three invariants from their undeformed states. Since the two, and three constant Mooney-Rivlin model have limitations in capturing high deformations, we have applied five parameter which is governed by:
$$W={C}_{10}\left({I}_{1}-3\right)+{C}_{01}\left({I}_{2}-3\right)+{C}_{20}{\left({I}_{1}-3\right)}^{2}+{C}_{11}\left({I}_{1}-3\right)\left({I}_{2}-3\right)+{C}_{02}{\left({I}_{1}-3\right)}^{2}+\frac{1}{{D}_{1}}{\left(J-1\right)}^{2}$$
8
where J is the determinant of the elastic deformation, C10, C01, C20, C11, C02 are material constants fitted from experimental data, and D1 is the material incompressibility parameter.
An axisymmetric boundary condition was applied which drastically reduced the computational cost53. Since the micropipette is significantly stiffer than red blood cell, the glass micropipette was considered as a rigid body. A fillet radius was applied to the tip of the micropipette to mimic the experimental setups and avoid high element distortion and the contacts. The frictionless boundary condition was applied to the cell and the adjacent micropipette wall since BSA was included during aspiration experiments. Pure penalty boundary was applied to the contact between the micropipette and the cell. RBC was considered as a sphere with a radius of 2.25 µm depending on its volume at resting state. For RBC aspiration modelling three sets of simulations were performed: with a parallel (θ = 0°) micropipette and with conical (θ = 5°, 10°) ones. The diameter of the micropipette tip were set to be 1 µm. A uniform pressure was applied to the tip of the RBC to mimic the experiments. For HEK293T aspiration simulation, a fixed displacement was applied to the tip of the cell, while the body was fixed when mechanical anchor was applied. In order to compare the results, von Mises stress was calculated for each studied case that can be calculated from
$${S}_{v}= \sqrt{\frac{{{\left({S}_{1}-{S}_{2}\right)}^{2}+{\left({S}_{1}-{S}_{3}\right)}^{2}+\left({S}_{1}-{S}_{2}\right)}^{2}}{2}}$$
9
where Si (i = 1, 2 ,3) are the three stress components in x, y, z direction. The values were normalized based on the maximum value of von-Mises of all conditions at each cell type. The normalized equivalent (von-Mises) stress fields are compared.
4.10 Statistics and reproducibility.
All statistical analyses were performed using Prism 9 (GraphPad Software, US). Welch’s t-test (unpaired, two-tailed, not assuming equal standard deviation) was employed to evaluate the statistical significance. In instances where more than two groups were compared, one-way Welch’s ANOVA was implemented. The threshold for statistical significance was set at p < 0.05. All data are represented as mean ± s.e.m. Aspiration measurements were performed independently three to five times on separate days.