Sub-micron cell indentation with the optical trap and AFM.

<p><b>A)</b> Cell indentation measured with AFM. The force was limited to 140 pN. The indentation (black) and retraction (grey) curves are not identical, but show a considerable amount of hysteresis. <b>B)</b> When the indentation force is limited to 75 pN the difference between the curves is reduced. <b>C)</b> At a force of 25 pN, close to the intrinsic noise of the AFM cantilever, hysteresis between the indentation and retraction curves cannot be clearly distinguished. <b>D)</b> Cell indentation measured with the optical trap. The high force resolution allows the controlled application of forces of less than 10 pN. The indentation (red) and retraction (orange) curves look identical with no obvious hysteresis. <b>E)</b> The relative amount of energy lost between indentation and retraction curves (mean ± s.e.m) was obtained by numerically calculating the area that is enclosed by the indentation and retraction curve, and dividing this by the area under the indentation curve. Only those measurements were analyzed that showed no sticking of the bead to the cell (which is clearly visible as a negative force during retraction). Each point represents measurements on 7 to 15 different cells. Both optical trap (red) and AFM (black) measurements that were performed at forces of up to 30 pN show that less than 15% of the indentation energy is lost. At higher forces this increased to almost 50%. <b>F)</b> The cell indentation was estimated for all indentation curves that were used for E). Data is shown as mean ± s.e.m. Measurements performed at the lowest forces (<30 pN) resulted at indentations of 0.2 µm, which increases to 0.8 µm at 150 pN. The contact point was defined as the position in the indentation curve where the force reaches a value below 0 pN (starting from the maximum force). The error in this method depends on the noise during the force measurement and will lead to an underestimation of the real indentation. Since the force noise in the AFM measurements is larger as compared to optical trapping also the error in the estimated indentation is larger.</p

Cell stretching experiments. A)

<p>Beads that got stuck during cell indentation were used as a handle to stretch the cell. The stretching curve (orange) is steeper than the indentation curve. <b>B)</b> The Young’s modulus that is estimated from the stretching experiments is about twice as high (239 Pa) as the modulus we calculated from the indentation experiments (100 Pa).</p

Experimental setups to measure the cell response in vertical direction. A)

<p>AFM: The cantilever is moved up and down with the z-piezo. When the AFM tip touches and indents the cell, the cantilever will bend. The amount of bending is measured via a laser beam that is reflected onto a split photodiode. Its electrical signal is linear proportional with the applied force on the cell. <b>B)</b> Optical trap: A laser beam, emitted from a single mode fibre, is coupled into the optical path of a standard upright microscope via a dichroic mirror and focused into the sample by the objective. The vertical position of the trap is controlled by a z-piezo that moves the objective up and down. The inset figure shows a bead that is trapped in the focus, and pushed into a cell. To monitor the displacement of the bead from the centre of the trap, the laser light is collected by the condenser, coupled out of the optical path via a second dichroic mirror and cast onto a photodiode. Both scale bars are 10 µm.</p

Crystal structure of human P2.

<p>(a) Superimposed structures of human wtP2 (cyan) and P2-P38G (green) before neutron scattering experiments and wtP2 after the experiment (magenta). Pro38 is indicated with the red arrow. (b) The P38G mutation site. Note how the main-chain hydrogen bonding (dashed lines) remains conserved at the edge of the β sheet also in the mutant. (c) Locations of the residues with side-chain methyl groups in P2 are shown as sticks. (d) A top view of the barrel-shaped P2 with bound fatty acid and stationary water molecules at cryo temperatures in the high-resolution crystal structure [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128954#pone.0128954.ref011" target="_blank">11</a>].</p

Simulated and experimental hydrogen MSDs.

<p>(a) The averaged MSD of i) hydrogens in CH₃-groups (top group of curves with higher MSD values) and ii) hydrogens in CH₂- and CH-groups (bottom group with lower MSD values) for all the four systems studied by MD simulations. Data for P2 without palmitate are shown in black (wild type) and red (P38G mutant). The results for P2 with palmitate are depicted in green (wild type) and blue (P38G). (b) Experimentally determined MSD contributions from methyl (upper panels) and non-methyl hydrogens (lower panels) for wtP2 (red) and P2-P38G (blue) using the bimodal model. Left: IN13; right: IN16. Solid (wtP2) and dashed (P2-P38G) lines represent the low-temperature harmonic vibrational contributions.</p

Temperature dependence of the normalised elastic scattering intensities.

<p>Data from wtP2 were acquired on IN16 (left), IN13 (middle), and IN6 (right). Data are shown for the temperatures 20, 100, 150, 200, 240, 260, 280, and 300 K (from top to bottom). Solid lines represent the best fitting to IN16 and IN13 data using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128954#pone.0128954.e004" target="_blank">Eq 4</a> and to IN6 data using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128954#pone.0128954.e001" target="_blank">Eq 1</a>. Insets: magnification of the fitting region using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128954#pone.0128954.e001" target="_blank">Eq 1</a>.</p

Temperature dependence of the MSD.

<p>Data from wtP2 (red) and P2-P38G (blue) were evaluated from the fit of data acquired on IN6 (top), IN13 (middle), and IN16 (bottom) using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128954#pone.0128954.e001" target="_blank">Eq 1</a>. The low-temperature fits using the Einstein harmonic oscillator function (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128954#pone.0128954.e005" target="_blank">Eq 5</a>) are shown as solid (wtP2) and dashed lines (P2-P38G).</p

Folding and dynamics of P2.

<p>(a) CD spectra of wtP2 (red) and P2-P38G (blue) in solution. Solid lines represent the samples before neutron scattering experiments and dashed lines the samples after these experiments at 293 K. Dashed dotted lines are denatured samples at 363 K. The melting curves at 201 (dashed line) and 217 nm (solid line) as a function of temperature are shown in the inset. (b) Root-mean square fluctuation (RMSF) per residue analyzed for all the four simulated systems from the final 2500 ns of the simulation trajectory. Data for P2 without palmitate are shown in black (wild type) and red (P38G mutant). The results for P2 with palmitate are depicted in green (wild type) and blue (P38G). Large RMSF values represent regions with high flexibility. The most flexible parts of the protein are found at the two loops opposing the lid. (c) A visualisation in VMD [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128954#pone.0128954.ref042" target="_blank">42</a>] of the RMS fluctuations in the P38G mutant with the palmitate chain inside the binding pocket. The yellow color corresponds to the mutated amino acid Gly38. The most flexible parts of the protein are pictured in bright magenta color. The colour bar at the bottom describes the range of RMSF values (light blue for low, violet for high).</p

Crystallographic data collection and refinement statistics.

<p>^a The numbers in parentheses refer to the highest-resolution shell.</p><p>^b CC_1/2 is defined as the correlation coefficient between two random half-datasets [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128954#pone.0128954.ref041" target="_blank">41</a>]</p><p>^c Validation was carried out using Molprobity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128954#pone.0128954.ref028" target="_blank">28</a>]</p><p>Crystallographic data collection and refinement statistics.</p

Functional assays.

<p>(a) Overexpressed wtP2 (top) and P2-P38G (bottom) both form stacked membrane domains in cell culture. The scale bar is 10 μm. Cell surface glycoproteins (visualised by concanavalin A; red) are depleted from the stacked membrane domains containing fluorescent P2 (green). (b) wtP2 (red) and P2-P38G (blue) both induce vesicle aggregation, which can be observed as increasing turbidity.</p