Cooperative unfolding of distinctive mechanoreceptor domains transduces force into signals

  1. Lining Ju
  2. Yunfeng Chen
  3. Lingzhou Xue
  4. Xiaoping Du
  5. Cheng Zhu  Is a corresponding author
  1. Georgia Institute of Technology, United States
  2. Heart Research Institute, Australia
  3. The University of Sydney, Australia
  4. The Pennsylvania State University, United States
  5. University of Illinois at Chicago, United States
8 figures and 3 videos

Figures

Figure 1 with 2 supplements
BFP analysis of ligand binding kinetics and domain unfolding mechanics of platelet GPIbα.

(A) BFP micrograph. A micropipette-aspirated RBC with a probe bead attached to the apex (left) was aligned against a platelet held by an opposing micropipette (right). (B) BFP functionalization. The …

https://doi.org/10.7554/eLife.15447.003
Figure 1—figure supplement 1
BFP test cycle.

(A) Schematic of a force-clamp experiment mode. Position (horizontal axis) of the probe bead attached to the apex of the RBC aspirated by the fixed micropipette (left) goes through indicated stages …

https://doi.org/10.7554/eLife.15447.004
Figure 1—figure supplement 2
Fitting of the WLC model to the force-extension traces (Figure 1E insert) before (blue) and after (red) the observed GPIbα ramped unfolding event.

The unfolding length can be estimated directly (indicated) or calculated from the difference between the two best-fit contour lengths (318.6–279.3 = 39.3 nm). Both give comparable results.

https://doi.org/10.7554/eLife.15447.005
Figure 2 with 1 supplement
Identification and characterization of unfolding of LRRD and MSD.

(AC) Schematics of GPIbα on the platelet membrane (A), highlighting the folded (−) and unfolded (+) LRRD (B) and MSD (C). Different regions and binding sites for VWF-A1 and mAbs are indicated. (DG)…

https://doi.org/10.7554/eLife.15447.007
Figure 2—figure supplement 1
Statistical analysis on ramped unfolding length distribution.

(A,B) Nonparametric kernel density estimation using the software R with the given kernel ('gaussian' or 'epanechnikov') and bandwidth selected by Sheather and Jones (1991). Both estimates identify …

https://doi.org/10.7554/eLife.15447.008
Figure 3 with 1 supplement
Force- and ligand-dependent cooperative unfolding of GPIbα LRRD and MSD.

(AD) Frequency (A,B) and force (C,D) of LRRD (A,C) or MSD (B,D) unfolding events occurred in the ramping phase induced by pulling via A1WT (blue) or A1R1450E (red) with indicated preset clamped …

https://doi.org/10.7554/eLife.15447.010
Figure 3—source data 1

Statistics and cooperativity evaluation of the GPIbα domains unfolding.

(A) Decision rules for and statistical summary of GPIbα domain unfolding in force-clamp experiment mode. Criteria for deciding whether or not (+ or −) and which (LRRD, MSD, or both) GPIbα domain(s) was (were) unfolded are based on BFP profile signatures and the unfolding lengths. YES = observed, NO = not observed. NA = not applicable. (B) Related to Figure 3F–H. Evaluation of LRRD and MSD unfolding cooperativity. All probabilities were calculated from occurrence data in (A). Observed joint probabilities were compared to their predicted counterparts based on the assumption that LRRD and MSD unfolded independently. For example, in 'WT A1 vs. Platelet' under 25 pN: The probability of LRRD unfolding is P(LRRD) = 3.4% + 6.9% + 2.76% = 13.06%. The probability of MSD unfolding is P(MSD) = 7.6% + 13.8% + 6.9% + 2.76% = 31.06%. The probability of MSD ramped unfolding is P(MSD, ramp) = 7.6% + 2.76% = 10.36%. The probability of MSD clamped unfolding is P(MSD, clamp) = 13.8% + 6.9% = 20.7%.

https://doi.org/10.7554/eLife.15447.011
Figure 3—figure supplement 1
GC LRRD unfolding occurrence frequencies.

Frequencies of LRRD unfolding events occurred in the ramping phase induced by pulling GC via A1WT (blue) or A1R1450E (red) with 10, 25, 40 and 60 pN preset clamped forces.

https://doi.org/10.7554/eLife.15447.012
Figure 4 with 1 supplement
LRRD unfolding prolongs A1–GPIbα bond lifetime and facilitates MSD clamped unfolding.

(AC) Mean ± s.e.m. of MSD time-to-unfold (tu, A) and GPIbα bond lifetimes (tb, B,C) with A1WT (blue) or A1R1450E (red) were measured in the clamping phase at different forces in the absence (−) or …

https://doi.org/10.7554/eLife.15447.013
Figure 4—source data 1

MSD unfolding rates (ku) and the fraction (w1) and off-rates (k1, k2) of GPIbα dissociating from A1WT or A1R1450E under different forces.

w1 represents the fraction of binding events that dissociate with the off-rate k1. The fraction of events that dissociate with the off-rate of k2 is simply calculated as w2 = 1-w1. NA = not applicable.

https://doi.org/10.7554/eLife.15447.014
Figure 4—figure supplement 1
MSD time-to-unfold distribution for A1WT and 3D probability density surface plot for A1R1450E.

(A) Time-to-unfold (tu) distributions of A1WT MSD clamped unfolding with (red) and without (black) a preceding LRRD unfolding at 25 pN clamped force. The unfolding rate ku was calculated from the …

https://doi.org/10.7554/eLife.15447.015
Figure 5 with 2 supplements
Concurrent analysis of single-platelet Ca2+ flux and GPIb-mediated single-bond binding at 25 pN clamped force.

(A) Representative epi-fluorescence pseudo-colored images of intraplatelet Ca2+ of null (top row), α- (middle row), and β- (bottom row) types at indicated times. (B) Representative time courses of …

https://doi.org/10.7554/eLife.15447.016
Figure 5—figure supplement 1
Concurrent analysis of single-platelet Ca2+ flux and single-bond dissociation from GPIb at 25 pN clamped force.

(A,B) Concurrent measurement of single-platelet Ca2+ flux and kinetics of single GPIb bonds with anti-GPIbα (A) and anti-GPIbβ (B). Top: representative epi-fluorescence pseudo-colored images of …

https://doi.org/10.7554/eLife.15447.017
Figure 5—figure supplement 2
Specificity-sensitivity analysis of optimal threshold.

(A) Binary classifications of threshold performance outcomes on tmax–ΔImax data. (B) The receiver operating characteristic (ROC) plot: the true positive rate (i.e., sensitivity) against the false …

https://doi.org/10.7554/eLife.15447.018
GPIbα can sense different force waveforms and discriminate different ligands.

(AD) Force-ramp fBFP experiment mode. Individual ΔImax values and their mean ± s.e.m. (A,B, points), Ca2+ types (C,D, stacked bars, left ordinate), and mean ± s.e.m. of pre-Ca2+largest rupture …

https://doi.org/10.7554/eLife.15447.020
Figure 7 with 2 supplements
Correlation between GPIbα domain unfolding and Ca2+ triggering at 25 pN clamped force.

(AD) Individual ΔImax values and their mean ± s.e.m. (points, left ordinate) in platelets triggered by A1 (A,C) or WM23 (B,D) binding, which were segregated into groups with (+) or without (−) …

https://doi.org/10.7554/eLife.15447.021
Figure 7—figure supplement 1
Comparison of GPIbα bond lifetime and MSD clamped unfolding by A1WT and WM23.

The number of lifetime events (A), their average lifetimes (B), MSD unfolding occurrence frequency (C), expected number of MSD unfolding events (D), and calculated probabilities of observing at …

https://doi.org/10.7554/eLife.15447.022
Figure 7—figure supplement 2
Model of GPIb-mediated platelet mechanosensing.

For a circulating platelet distal to an injured site without physical contact, its GPIb signaling will not be triggered (A). Upon interacting with VWF, platelets tether and translocate on the …

https://doi.org/10.7554/eLife.15447.023
Author response image 1
Domain organization and antibody epitopes on GPIbα.
https://doi.org/10.7554/eLife.15447.024

Videos

Video 1
BFP experiment mimics platelet translocation on sub-endothelium.

This animation (produced by Adobe Flash; 12 fps) illustrates the resemblance between platelet translocation on the sub-endothelium (a collagen network covered with VWF on the surface, upper panel) …

https://doi.org/10.7554/eLife.15447.006
Video 2
Force-clamp experiment mode with a bond lifetime event.

The video consists of two parts in series. Part I is an animation (produced by Adobe Flash; 12 fps), and part II is a video recording of a representative fluorescence BFP experiment (recorded by a …

https://doi.org/10.7554/eLife.15447.009
Video 3
Force-ramp experiment mode with a bond rupture event.

Similar to Video 2, this video consists of two parts in series. In part I, the synchronized BFP illustration (upper panel), A1–GPIbα molecular interaction (middle panel) and 'Force vs. Time' signal …

https://doi.org/10.7554/eLife.15447.019

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