Direct measurement of TRPV4 and PIEZO1 activity reveals multiple mechanotransduction pathways in chondrocytes
- Max Delbruck Center for Molecular Medicine, Germany
- School of Medical Sciences, University of New South Wales, Australia
Figures
Figure 1 with 1 supplement
Primary, murine chondrocyte culture.
(A) Transcript levels of the transcription factor Sox9 in just harvested chondrocytes, dedifferentiated cells (post 7 days in monolayer culture) and redifferentiated chondrocytes (recovered from 2D …
Figure 1—figure supplement 1
Schematic diagram of the isolation and culture of primary murine chondrocytes.
(A) Knees and femoral heads were isolated from 4- to 5-day-old mouse pups to obtain chondrocytes. A fraction of the isolated cells were encapsulated in alginate to maintain their differentiation …
Figure 2
Mechanoelectrical transduction currents in primary cells isolated from mouse cartilage.
(A) Deflection stimuli applied via cell-matrix contact points. Left panel: cartoon of pillar array experiment, stimuli are applied by deflecting a pilus subjacent to a cell that is concurrently …
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Figure 2—source data 1
Electrophysiological characteristics of WT chondrocytes and WT dedifferentiated cells.
Chondrocytes were isolated from C57Bl/6 mice. For each sample (chondrocyte phenotype and dedifferentiated phenotype) the number of litters, recorded cells and number of cells that respond to pillar deflections are shown along with the total number of stimulation points (corresponding to the number of distinct pili deflected) and the total number of measurements (i.e. individual deflections). For each recorded current, the latency and the current amplitude were measured, and the activation time constant and current decay were obtained from a mono-exponential fit of the data. The mean ± s.e.m. and the median are displayed for each kinetic parameter.
- https://doi.org/10.7554/eLife.21074.006
Figure 3
Chondrocytes and dedifferentiated cells display distinct mechanosenstivity to substrate deflections.
(A) Stimulus-response graph of deflection-gated currents in chondrocytes (red circles) and dedifferentiated cells (cyan squares). Measurements from an individual cell were binned according to …
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Figure 3—source data 1
Statistical comparison of mechanoelectrical transduction currents, chondrocytes vs dedifferentiated cells.
(A) Statistical comparison of deflection-gated mechanoelectrical transduction responses. For each individual cell, currents were binned in the indicated size ranges (in nm) and the current amplitudes within each bin averaged and then averaged across cells. Bins were subsequently tested for normal distribution and subsequently compared with a Student’s t-test (parametric data sets) or a Mann Whitney test (non-parametrical data). The p values are shown for significant comparisons, ‘NS’ indicates no significant differences and ‘NA’ is shown when all measurements within a bin were equal to zero. The number of compared points is shown in brackets. An ordinary two-way ANOVA was used to compare the cellular response over the range of stimuli, reported are the p value and F statistic (including DFn, DFd). (B) Statistical comparison of stretch-gated mechanoelectrical transduction in chondrocytes. Chondrocytes were isolated from WT mice, expanded and encapsulated in alginate. After deposition on coverslips for measurement, cells were analyzed using HSPC. For each condition, the number of litters, recorded membrane patches and maximal current (pA) are shown. Data are displayed as mean ± s.e.m. Conditions were compared with Student’s t-test (parametric data sets), and the p values are shown for significant comparisons, ‘NS’ indicates no significant.
- https://doi.org/10.7554/eLife.21074.008
Figure 4 with 1 supplement
Substrate-deflection gated currents are mediated by PIEZO1 and TRPV4.
(A) Fraction of chondrocytes that responded with at least with one mechanically gated current in response to deflection stimuli. Knockdown of Piezo1 resulted in significantly fewer responsive cells …
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Figure 4—source data 1
Electrophysiological characteristics of WT, Trpv4-/- and miRNA-treated chondrocytes.
(A) Electrophysiological characteristics of WT, Trpv4-/- and miRNA-treated chondrocytes. Chondrocytes were isolated from C57Bl/6 and Trpv4-/- mice, expanded, transfected (in the case of Scrambled and Piezo1 miRNA constructs) and encapsulated in alginate. For each condition, the number of litters, recorded cells and number of cells that respond to pillar deflections are shown. The total number of stimulation points (corresponding to the number of distinct pili deflected) and the total number of measurements (i.e. individual deflections) are displayed. For each recorded current, the latency and the current amplitude were measured, and the activation time constant and current decay were obtained from a mono-exponential fit of the data. The mean ± s.e.m. and the median are displayed for each kinetic parameter. (B) Statistical comparison of deflection-gated mechanoelectrical transduction responses. For each individual cell, currents were binned in the indicated size ranges (in nm) and the current amplitudes within each bin averaged and then averaged across cells. Bins were tested for normal distribution and subsequently compared with a Student’s t-test (parametric data sets) or a Mann-Whitney test (non-parametrical data). The p values are shown for significant comparisons, ‘NS’ indicates no significant differences and ‘NA’ is shown when all measurements within a bin were equal to zero or data were not enough to perform the comparison. The number of compared points is shown in brackets. An ordinary two-way ANOVA was used to compare the cellular response over the range of stimuli, reported are the p value and F statistic (including DFn, DFd).
- https://doi.org/10.7554/eLife.21074.010
Figure 4—figure supplement 1
Normalized transcript levels of Piezo1, Piezo2 and Trpv4 in primary chondrocytes.
Knees and femoral heads were isolated from litters of C57Bl/6 mouse pups to obtain isolated chondrocytes. The chondrocytes from one litter were pooled to obtain mRNA, which was retro-transcribed to …
Figure 5
TRPV4 directly mediates deflection-gated currents in primary chondrocytes.
(A) Representative traces of currents gated by pillar deflections before, during and after the wash out of the TRPV4 antagonist GSK205 (10 µM, 3 min). (B) Quantification of the inhibition of the …
Figure 6 with 2 supplements
Murine chondrocytes display a stretch-sensitive current dependent on PIEZO1 but not TRPV4.
(A) Comparison of maximal current induced by membrane stretch in outside-out patches isolated from chondrocytes. HSPC experiments were performed in membrane patches isolated from chondrocytes that …
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Figure 6—source data 1
Statistical comparison of stretch-gated mechanoelectrical transduction in chondrocytes.
Chondrocytes were isolated from WT and Trpv4-/- mice, expanded, transfected (in the case of Scrambled and Piezo1 miRNA constructs) and encapsulated in alginate. For each condition, the number of litters, recorded membrane patches and maximal current (pA) are shown. Data are displayed as mean ± s.e.m. Conditions were compared with Student’s t-test (parametric data sets) and the p values are shown for significant comparisons, ‘NS’ indicates no significant.
- https://doi.org/10.7554/eLife.21074.014
Figure 6—figure supplement 1
The P50 measured in WT and Trpv4-/- chondrocytes using HSPC is not significantly different.
Stimulus-response curves obtained from outside-out patches pulled from WT or Trpv4-/- chondrocytes and stimulated with positive pressure steps (10-150 mmHg). Individual normalized responses were …
Figure 6—figure supplement 2
WT chondrocytes respond to the TRPV4 agonist GSK101 but not chondrocytes isolated from a Trpv4-/- mouse.
(A) WT chondrocytes respond to the application of TRPV4 agonist GSK1016790A (GSK101). Intensiometric epifluorescent imaging was used to demonstrate the presence of functional TRPV4 in WT, murine …
Figure 7 with 2 supplements
TRPV4 is efficiently gated by substrate deflections.
(A) HEK-293 cells were used as a heterologous system to test stretch- indentation- and deflection-mediated currents. In the left panel is a cartoon of the HSPC experiment (stretch), in the center, …
Figure 7—figure supplement 1
Mechanical stimulation of cell-attached patches in HEK-293 cells overexpressing PIEZO1 or TRPV4.
(A) Schematic of the cell-attached configuration together with the HSPC. (B) Example traces of mechanically gated currents elicited by HEK-293 cells expressing PIEZO1, TRPV4 or eGFP as a negative …
Figure 7—figure supplement 2
Mechanical indentation of HEK cells overexpressing PIEZO1 or TRPV4.
(A) Schematic of mechanical indentation in whole-cell patch clamp configuration. (B) Example traces of indentation-mediated currents from HEK-293 cells expressing PIEZO1, TRPV4 or LifeAct-mCherry as …
Figure 8 with 1 supplement
Deflection-mediated activation of TRPV4.
(A) The deflection-gated current observed in HEK-293 cells expressing TRPV4 is reversibly blocked by the TRPV4-specific antagonist, GSK205 (10 μM, 3 min) (B) TRPV4 is more sensitive to substrate …
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Figure 8—source data 1
Electrophysiological characteristics of HEK-293 cells overexpressing either TRPV4 or PIEZO1.
(A) Electrophysiological characteristics of HEK-293 cells overexpressing either TRPV4 or PIEZO1. HEK-293 cells were transiently transfected with a plasmid encoding either TRPV4 or PIEZO1. For each condition the number of transfections, total number of recorded cells and number of stimuli are indicated. For each recorded current, the latency and the current amplitude were measured, and the activation time constant and current decay were obtained from a mono-exponential fit. Every kinetic parameter is shown as mean ± s.e.m. (B) Statistical comparison of deflection-gated mechanoelectrical transduction responses. For each individual cell, currents were binned in the indicated size ranges (in nm) and the current amplitudes within each bin averaged and then averaged across cells. Bins were subsequently tested for normal distribution and subsequently compared with a Student’s t-test (parametric data sets) or a Mann Whitney test (non-parametrical data). The p values are shown for significant comparisons, ‘NS’ indicates no significant differences. The number of compared points is shown in brackets. An ordinary two-way ANOVA was used to compare the cellular response over the range of stimuli, reported are the p value and F statistic (including DFn, DFd).
- https://doi.org/10.7554/eLife.21074.021
Figure 8—figure supplement 1
PLA2 is not involved in the activation by pillar-deflection of TRPV4
(A) Scheme of the TRPV4 signaling pathway activated after hypotonic stress compared with TRPV4 activation by pillar displacement. After the hypotonic change, PLA2 produces arachidonic acid (AA), …
Author response image 1
(A) Indentation of Neuro2A cells. Neuro2A cells were indented using a glass probe and resulting mechanically-gated currents were monitored using whole-cell patch-clamp. Indentation stimuli were …
