Mapping the mechanics and macromolecular organization of hyaluronan-rich cell coats

The hyaluronan (HA)-rich pericellular coat (PCC) enveloping most mammalian cells plays a vital role in biological processes such as cell adhesion, proliferation, motility and embryogenesis. In particular its presence on chondrocytes, which live in the load-bearing cartilage, has a wide range of implications in diseases such as osteoarthritis, highlighting its mechanical role in living organisms. Despite its significance, the macromolecular organization of the cell coat remains speculative. In order to obtain a more detailed spatial picture of highly hydrated PCCs, we present two independent but complementary non-invasive techniques for the position-resolved analysis of the cell coat's mechanical and structural properties. Position-dependent microrheology provides a micromechanical map of the PCC that reveals a gradient of increasing elastic stiffness towards the plasma membrane on model rat chondrocyte cells (RCJ-P). This gradient can be correlated with the relative distribution of HA, which is inferred using an eGFP-labelled neurocan-binding domain, a small fluorescent molecule that binds to HA. The spatial variation of the HA concentration profile is consistent with the position-dependent elasticity. Combining these approaches sheds light on the molecular architecture of the PCC

Determination of the changes in contact area during osmotic stimulation of adhered primary hepatocytes.

<p>a) The dark patterns in the RICM image of two different wavelengths (470 nm shown here) correspond to cell patches with close surface contact. The original relative contact area (i) changes considerably during hypoosmotic stimulation (ii) and does not return to its original area afterwards (iii). A rapid decrease in relative contact area can be observed after returning to normoosmotic media (iv) followed by the stabilization of the relative contact area (v). b) All areas defined as relative contact areas in the consensus of both wavelengths are represented here as black areas. Images taken at 1 fps in both wavelength simultaneously enable a kinetic analysis: c) The tremendously quick reaction of the hepatocytes to the osmotic stimuli is evident as well as the stable plateaus (compared to the initial relative contact area (i)). The first plateau of this sample (iii) increases by ∼50% and the second (v) by ∼14%). Scale bars correspond to 10 µm.</p

Analysis of DW-RICM images to determine the contact area of hepatocytes.

<p>a) and b) show the RICM interferograms for the two different wavelengths used, the histogram of the background intensities (dotted line) and cell intensities (solid line) as well as the contact area determined from the use of the background intensity as a cutoff. For this, the mean intensity I_B and standard deviation (σ) of the background were determined and all areas of at least 4×4 pixels with an intensity of less than (I_B - ^σ/₂) were defined as contact patches. In c) the consensus of the contact patches observed in the two different wavelengths corresponding to the contact area is shown with regions of high variability in the two wavelengths emphasized. Scale bars corresponds to 20 µm.</p

Relative contact area changes in the observed plateau phases.

<p>Cells plated on fibronectin (light gray, n = 111) and collagen I (dark gray, n = 98) show different relative contact areas during the calibration phase (plateau i), the plateau phase after the hypoosmolar stimulation (plateau iii) and after the return to normal media (plateau v). (*p≤0.05 with respect to initial relative contact area i), error bars show SEM.</p

RICM also enables the tracking of changes in the cell’s membrane dynamics.

<p>The membrane dynamics can be visualized by the variance of the RICM intensity over 5 seconds at the midpoint of each of the plateaus i, iii, and v. Fluctuations are calculated over a 2×2 pixel area to reduce noise and are visualized in false color with blue being small fluctuations and red larger fluctuations. Corresponding contact images are shown below for comparison. Image on the right shows a magnified area of plateau i with the contact patches marked visualizing lower membrane fluctuations in the areas identified as surface contacts. Scale bar corresponds to 20 µm.</p

Adhesion Maturation of Neutrophils on Nanoscopically Presented Platelet Glycoprotein Ibα

Neutrophilic granulocytes play a fundamental role in cardiovascular disease. They interact with platelet aggregates <i>via</i> the integrin Mac-1 and the platelet receptor glycoprotein Ibα (GPIbα). <i>In vivo</i>, GPIbα presentation is highly variable under different physiological and pathophysiological conditions. Here, we quantitatively determined the conditions for neutrophil adhesion in a biomimetic <i>in vitro</i> system, which allowed precise adjustment of the spacings between human GPIbα presented on the nanoscale from 60 to 200 nm. Unlike most conventional nanopatterning approaches, this method provided control over the local receptor density (spacing) rather than just the global receptor density. Under physiological flow conditions, neutrophils required a minimum spacing of GPIbα molecules to successfully adhere. In contrast, under low-flow conditions, neutrophils adhered on all tested spacings with subtle but nonlinear differences in cell response, including spreading area, spreading kinetics, adhesion maturation, and mobility. Surprisingly, Mac-1-dependent neutrophil adhesion was very robust to GPIbα density variations up to 1 order of magnitude. This complex response map indicates that neutrophil adhesion under flow and adhesion maturation are differentially regulated by GPIbα density. Our study reveals how Mac-1/GPIbα interactions govern cell adhesion and how neutrophils process the number of available surface receptors on the nanoscale. In the future, such <i>in vitro</i> studies can be useful to determine optimum therapeutic ranges for targeting this interaction

Adhesion Maturation of Neutrophils on Nanoscopically Presented Platelet Glycoprotein Ibα

Neutrophilic granulocytes play a fundamental role in cardiovascular disease. They interact with platelet aggregates <i>via</i> the integrin Mac-1 and the platelet receptor glycoprotein Ibα (GPIbα). <i>In vivo</i>, GPIbα presentation is highly variable under different physiological and pathophysiological conditions. Here, we quantitatively determined the conditions for neutrophil adhesion in a biomimetic <i>in vitro</i> system, which allowed precise adjustment of the spacings between human GPIbα presented on the nanoscale from 60 to 200 nm. Unlike most conventional nanopatterning approaches, this method provided control over the local receptor density (spacing) rather than just the global receptor density. Under physiological flow conditions, neutrophils required a minimum spacing of GPIbα molecules to successfully adhere. In contrast, under low-flow conditions, neutrophils adhered on all tested spacings with subtle but nonlinear differences in cell response, including spreading area, spreading kinetics, adhesion maturation, and mobility. Surprisingly, Mac-1-dependent neutrophil adhesion was very robust to GPIbα density variations up to 1 order of magnitude. This complex response map indicates that neutrophil adhesion under flow and adhesion maturation are differentially regulated by GPIbα density. Our study reveals how Mac-1/GPIbα interactions govern cell adhesion and how neutrophils process the number of available surface receptors on the nanoscale. In the future, such <i>in vitro</i> studies can be useful to determine optimum therapeutic ranges for targeting this interaction

Adhesion Maturation of Neutrophils on Nanoscopically Presented Platelet Glycoprotein Ibα

Neutrophilic granulocytes play a fundamental role in cardiovascular disease. They interact with platelet aggregates <i>via</i> the integrin Mac-1 and the platelet receptor glycoprotein Ibα (GPIbα). <i>In vivo</i>, GPIbα presentation is highly variable under different physiological and pathophysiological conditions. Here, we quantitatively determined the conditions for neutrophil adhesion in a biomimetic <i>in vitro</i> system, which allowed precise adjustment of the spacings between human GPIbα presented on the nanoscale from 60 to 200 nm. Unlike most conventional nanopatterning approaches, this method provided control over the local receptor density (spacing) rather than just the global receptor density. Under physiological flow conditions, neutrophils required a minimum spacing of GPIbα molecules to successfully adhere. In contrast, under low-flow conditions, neutrophils adhered on all tested spacings with subtle but nonlinear differences in cell response, including spreading area, spreading kinetics, adhesion maturation, and mobility. Surprisingly, Mac-1-dependent neutrophil adhesion was very robust to GPIbα density variations up to 1 order of magnitude. This complex response map indicates that neutrophil adhesion under flow and adhesion maturation are differentially regulated by GPIbα density. Our study reveals how Mac-1/GPIbα interactions govern cell adhesion and how neutrophils process the number of available surface receptors on the nanoscale. In the future, such <i>in vitro</i> studies can be useful to determine optimum therapeutic ranges for targeting this interaction

Adhesion Maturation of Neutrophils on Nanoscopically Presented Platelet Glycoprotein Ibα

Neutrophilic granulocytes play a fundamental role in cardiovascular disease. They interact with platelet aggregates <i>via</i> the integrin Mac-1 and the platelet receptor glycoprotein Ibα (GPIbα). <i>In vivo</i>, GPIbα presentation is highly variable under different physiological and pathophysiological conditions. Here, we quantitatively determined the conditions for neutrophil adhesion in a biomimetic <i>in vitro</i> system, which allowed precise adjustment of the spacings between human GPIbα presented on the nanoscale from 60 to 200 nm. Unlike most conventional nanopatterning approaches, this method provided control over the local receptor density (spacing) rather than just the global receptor density. Under physiological flow conditions, neutrophils required a minimum spacing of GPIbα molecules to successfully adhere. In contrast, under low-flow conditions, neutrophils adhered on all tested spacings with subtle but nonlinear differences in cell response, including spreading area, spreading kinetics, adhesion maturation, and mobility. Surprisingly, Mac-1-dependent neutrophil adhesion was very robust to GPIbα density variations up to 1 order of magnitude. This complex response map indicates that neutrophil adhesion under flow and adhesion maturation are differentially regulated by GPIbα density. Our study reveals how Mac-1/GPIbα interactions govern cell adhesion and how neutrophils process the number of available surface receptors on the nanoscale. In the future, such <i>in vitro</i> studies can be useful to determine optimum therapeutic ranges for targeting this interaction