A Membrane-Bound Biosensor Visualizes Shear Stress-Induced Inhomogeneous Alteration of Cell Membrane Tension

Abstract

Cell membrane is the first medium from where a cell senses and responds to external stress stimuli. Exploring the tension changes in cell membrane will help us to understand intracellular force transmission. Here, a biosensor (named MSS) based on fluorescence resonance energy transfer is developed to visualize cell membrane tension. Validity of the biosensor is first verified for the detection of cell membrane tension. Results show a shear stress-induced heterogeneous distribution of membrane tension with the biosensor, which is strengthened by the disruption of microfilaments or enhancement of membrane fluidity, but weakened by the reduction of membrane fluidity or disruption of microtubules. These findings suggest that the MSS biosensor is a beneficial tool to visualize the changes and distribution of cell membrane tension. Besides, cell membrane tension does not display obvious polar distribution, indicating that cellular polarity changes do not first occur on the cell membrane during mechanical transmission.

Keywords: Biophysics; Membrane Architecture; Molecular Biology; Sensor.

Graphical abstract

Figure 1

MSS Plasmid Could Visualize the Changes of Membrane Tension (A) Tension sensor module contains a linker sequence (GPGGA)₈ inserted into two fluorescent proteins. When force extends the elastic linker, FRET efficiency decreases, otherwise it increases. (B) MSS sensor consists of tension sensor module and membrane-bound sequences Lyn and K-Ras. KMSS is a Head-less control sensor of MSS. (C and D) (C) The representative YPet/ECFP emission ratio images and (D) their average time courses of FRET biosensors in HeLa cells after exposure to hypertonic (MSS, n = 10; KMSS, n = 11) and hypotonic solutions (MSS, n = 7; KMSS, n = 12), respectively. (E) Average normalized YPet/ECFP emission ratio of KMSS at 0 min (baseline), MSS at 15 min in hypertonic solution (sucrose) and MSS at 15 min in hypotonic solution (H₂O). *p < 0.05 significantly different from baseline. All error bars represent SEM. Scale bar, 20 μm. See also Figure S1.

Figure 2

The Overall Changes of Membrane Tension under Different Shear Stresses (A and B) (A) The representative YPet/ECFP emission ratio images and (B) their average time courses of MSS in HeLa cells after exposure to the shear stress of 0.5 (n = 13), 2 (n = 14), and 4 Pa (n = 11) and KMSS in HeLa cells (served as control) after exposure to the shear stress of 2 Pa (n = 9). (C) Average normalized YPet/ECFP emission ratio of FRET biosensors at 15 min under different shear stresses. *p < 0.05 significantly different from control. All error bars represent SEM. Scale bar, 10 μm.

Figure 3

Shear Stress Changes Membrane Tension (A and B) (A) YPet/ECFP emission ratio of different cellular regions in timescale and (B) the fitting curve (points for sample data and solid lines for fitting) at 0 and 15 min under shear stress of 0.5, 2, or 4 Pa. (C and D) (C) Comparisons of normalized minimum YPet/ECFP emission ratio and (D) normalized upstream variation range and downstream variation range between different shear stress groups at 15 min *p < 0.05 compared with 0.5 Pa group. #p > 0.25 between groups of UVR and DVR. All error bars represent SEM. See also Figures S2 and S3 and Tables S1 and S2.

Figure 4

The Overall Changes of Membrane Tension under Different Cell Membrane Fluidities (A and B) (A) The representative YPet/ECFP emission ratio images and (B) their average time courses of MSS in HeLa cells pretreated for 15 min with 45 mmol/L BA (n = 10) or 3 hr with 0.1 mmol/L of Cho (n = 11) under shear stress of 2 Pa. (C) Average normalized YPet/ECFP emission ratio of FRET biosensors at 15 min under different cell membrane fluidities. *p < 0.05 significantly different from 2 Pa group. All error bars represent SEM. Scale bar, 10 μm.

Figure 5

High Cell Membrane Fluidity Enlarges Membrane Tension (A and B) (A) YPet/ECFP emission ratio of different cellular regions in timescale and (B) the fitting curve (points for sample data and solid lines for fitting) at 0 and 15 min for cells pretreated for 15 min with 45 mmol/L BA or 3 hr with 0.1 mmol/L of Cho under shear stress of 2 Pa. (C and D) (C) Comparisons of normalized minimum YPet/ECFP emission ratio and (D) normalized upstream variation range and downstream variation range between different membrane fluidities group at 15 min *p < 0.05 compared with 2 Pa group. #p > 0.3 between groups of UVR and DVR. All error bars represent SEM. See also Figures S2 and S3 and Tables S1 and S2.

Figure 6

The Overall Changes of Membrane Tension under Different Disruptions of Cytoskeleton (A and B) (A) The representative YPet/ECFP emission ratio images and (B) their average time courses of MSS in HeLa cells pretreated for 30 min with 1 μg/mL of CytoD (n = 13), 15 min with 5 μg/mL Noco (n = 18), or 1 hr with 1 μg/ml ML-7 (n = 10) under shear stress of 2 Pa. (C) Average normalized YPet/ECFP emission ratio of MSS at 15 min with different pretreatments. *p < 0.05 significantly different from control. All error bars represent SEM. Scale bar, 10 μm.

Figure 7

The Membrane Tension Depends on Not Only Microtubules but Also Actin Filaments (A and B) (A) YPet/ECFP emission ratio of different cellular regions in timescale and (B) the fitting curve (points for sample data and solid lines for fitting) at 0 and 15 min for cells pretreated for 30 min with 1 μg/mL of CytoD, 15 min with 5 μg/mL Noco, or 1 hr with 1 μg/ml ML-7 under shear stress of 2 Pa. (C and D) (C) Comparisons of normalized minimum YPet/ECFP emission ratio and (D) normalized upstream variation range and downstream variation range at 15 min with different pretreatments. *p < 0.05 compared with 2 Pa group. #p > 0.11 between groups of UVR and DVR. All error bars represent SEM. See also Figures S2 and S3 and Tables S1 and S2.