Hyaluronic acid selective anchoring to the cytoskeleton: An atomic force microscopy study

Abstract

The hyaluronic acid component of the glycocalyx plays a role in cell mechanotransduction by selectively transmitting mechanical signals to the cell cytoskeleton or to the cell membrane. The aim of this study was to evaluate the mechanical link between the hyaluronic acid molecule and the cell cytoskeleton by means of atomic force microscopy single molecule force spectroscopy. Hyaluronic acid molecules on live cells were targeted with probes coated with hyaluronic acid binding protein. Two different types of events were observed when the detachment of the target molecule from the probe occurred, suggesting the presence of cytoskeleton- and membrane-anchored molecules. Membrane-anchored molecules facilitated the formation of tethers when pulled. About 15% of the tested hyaluronic acid molecules were shown to be anchored to the cytoskeleton. When multiple molecules bonded to the probe, specific detachment patterns were observed, suggesting that a cytoskeletal bond needed to be broken to improve the ability to pull tethers from the cell membrane. This likely resulted in the formation of tethering structures maintaining a cytoskeletal core similar to the ones observed for cells over-expressing HA synthases. The different observed rupture events were associated with separate mechanotransductive mechanisms in an analogous manner to that previously proposed for the endothelial glycocalyx. Single cytoskeleton anchored rupture events represent HA molecules linked to the cytoskeleton and therefore transmitting mechanical stimuli into the inner cell compartments. Single membrane tethers would conversely represent the glycocalyx molecules connected to areas of the membrane where an abundance of signalling molecules reside.

Fig 1. Protocol summary for rupture event localisation and classification.

Each force spectroscopy curve (1) was analysed separately. After contact point fitting, the first derivative of the force was calculated (2). A threshold was set to locate peaks in the derivative (3) correspondent to rupture events (4). A data interval of 50 nm prior to the rupture event was fitted with a line (5); the slope was evaluated to classify the rupture event as cytoskeleton anchored or membrane tether.

Fig 2. Example of rupture events.

From left to right, representative AFM raw force spectroscopy data are shown for single cytoskeleton anchored ruptures (red asterisks), single membrane tether ruptures (green asterisks) and multiple ruptures, respectively.

Fig 3. Histogram of the extend baseline slope to define the rupture classification threshold.

The histogram of the slope of all extend baseline data is shown. The red line represents a normal distribution fit (μ = -0.002 pN/nm and σ = 0.103 pN/nm).

Fig 4. Box plot of the rupture distance from the contact point for the study and control samples.

Box plot of rupture distance from the contact point for the study sample (Sample 1—HABP/HA, solid line) and the control samples (Sample 2—BSA/HA, Sample 3—untreated/HA, Sample 4—HABP/HAase, dashed line). The rupture distance from the contact point for the study sample was statistically significantly higher suggesting specificity of the bond between HABP and HA (Kruskal–Wallis test by ranks, p < .01).

Fig 5. Classified rupture events in the study sample.

Scatter plot of rupture events in Sample 1 (HABP/HA). The x-axis represents the distance from the contact point, the y-axis the slope of the curve before rupture. The rupture events classified as cytoskeleton anchored are shown in red, as membrane tethers in green.

Fig 6. Histogram of the membrane tether force.

Probability histogram of the membrane tether force in Sample 1 (HABP/HA). The median value is equal to 16 pN, the IQR to 10 pN.

Fig 7. Box plot of the multiple rupture events slope.

Box plot of the slope of multiple rupture events. The slope of the first (I) event is statistically significantly different from the slope of the second to sixth (II-VI) rupture events (Kruskal–Wallis test by ranks, p < .01). The size of samples VII-X was too small for accurate statistical analysis as less than 1% of data were in these samples.

Fig 8. Force-dependent bond lifetime.

The calculated force-dependent lifetime of the system for the cytoskeleton anchored (red circles) and the membrane tether (green circles) rupture events is shown. Calculated points did not collapse on a master curve as expected for single-exponential molecule kinetics.