Pressure sensing through Piezo channels controls whether cells migrate with blebs or pseudopods

Significance Cells migrating within the body perform vital functions in development and for defense and repair of tissues. In this dense environment, cells encounter mechanical forces and constraints not experienced when moving under buffer, and, accordingly, many change how they move. We find that gentle squashing, which mimics mechanical resistance, causes cells to move using blebs—a form of projection driven by fluid pressure—rather than pseudopods. This behavior depends on the Piezo stretch-operated ion channel in the cell membrane and calcium fluxes into the cell. Piezo is highly conserved and is required for light touch sensation; this work extends its functions into migrating cells.

cells were brought to aggregation-competence in KK2MC at 2x10 7 cells/ml with shaking at 180 rpm. After 1 hour cells were pulsed with 90 nM final cyclic-AMP, every 6 minutes for another 4.5 hours. Aggregation-competence was confirmed morphologically by the formation of small clumps. These cells were then imaged in round glass-bottom dishes (35 mm dish with a 10 mm glass bottom, MatTek corporation) using an inverted laser-scanning confocal microscope (Zeiss 780 or 710) with a 63x/1.4 NA oil-immersion objective. The images were collected using Zen2010 software (Zeiss) and processed using Fiji or Image J. A modified version of underagarose assay was used, as described previously (3)(4)(5). A thin layer of SeaKem GTG agarose (Lonza Biochemicals) in KK2MC (different concentrations to prepare gels of 4 different stiffnesses chiefly 0.5%, 0.75%, 1% and 2% w/v) of height 2 mm was poured in a preheated round glass-bottom dish (MatTek). Two parallel rectangular troughs of sizes 4 mm by 8 mm and 1 mm by 5 mm were cut in the gel, once it solidified. A gradient of cyclic-AMP was formed in the chamber by adding 5 µM cyclic-AMP to the larger well and leaving it for about 30 minutes. Once the gradient formed, 2x10 5 cells/ml were added to the smaller trough and allowed to migrate. Load was applied using a cell squasher, as described previously (5), once the cells had migrated under the gel (about 40 minutes). Briefly, the plunger was bought in close contact with the surface of the gel by manually positioning it using micrometers and subsequently, a command to apply the load was given. A log file recorded the applied load and the position of the plunger.

Chemotaxis assay
Chemotactic ability of aggregation-competent cells was analysed by under-agarose assay, as described above (3,4). The migration of aggregation-competent cells under an overlay of agarose (0.5% or 2% w/v) was imaged by low-magnification 20X objective on Zeiss 710 confocal microscope. The images were collected every 20 seconds for 20 minutes.

Blebbing assay
This assay was adapted as previously described (6). Aggregation competent cells at 2x10 5 /ml in 200 µl KK2MC were allowed to attach to a glass-bottom dish for 20 minutes, after which they were imaged by confocal microscope. 16 µl of 50 µm cyclic-AMP was added to the buffer while recording cells at 2fps. 5 Blebs and pseudopodia were identified by their morphological characteristics.

Image analysis
Additionally, they were confirmed using kymographs where blebs could be seen as fast projections, devoid of actin polymerization at the leading edge whereas pseudopodia were identified by their characteristic slow actin dynamics ( Figure 1B).
In the movies where load was applied dynamically, blebs were scored at their first occurrence and the total number of blebs was binned in 1 second or 1 minute intervals.
Speed of cells was calculated by automated tracking using QUIMP plug-in in Image J (7). The centroid of cells was tracked at each frame of the movie to calculate the total distance travelled by the cell and divided by total time to calculate their speed.
Cell height was measured by reconstruction of the z-stacks (taken at 0.4 m increments) using Imaris (Bitplane). Z-axis elongation which occurs due to mismatch in the refractive index (8) was corrected by comparing the true and apparent height of a fluorescent bead of known height (9.7 µm diameter Fluospheres; Molecular probes). The stacks were corrected by dividing them by 1.97 (giving a true z-axis increment of 0.203 m). Statistical analysis of cell height was done using One Way ANOVA and Tukey's means comparison test in Origin software (Originlabs). Cell volume was calculated from the reconstructed stacks by calculating the volume of individual voxels occupied by the cells. Surface area was measured by calculating the total area of voxels on the outer surface. Fluorescence intensity was measured as the total florescence intensity of all relevant voxels.
Cell volume by FXM method was measured as described previously in (9).
Aggregation competent Dictyostelium cells were confined using PDMS micropillars of heights 6 m (corresponding to no load condition), 4 m and 3 m (corresponding to 6 the cell height under load or 2% agarose). The media around the cells contained 1mg/ml FITC-dextran which was displaced by the cells and the drop in fluorescence intensity was quantified using a custom MATLAB program, to measure the volume of the cells.
Cells were tracked using Manual Tracking plug-in in Image J with a centre correction option. The tracks were further analysed for chemotaxis by the Chemotaxis plug-in for ImageJ (NIH). The tracks were re-plotted with their origin as starting point.
Euclidian distance was calculated as the difference between the starting and end points of the cell track, while accumulated distance followed the whole track. Speed was calculated by dividing accumulated distance by total time. Persistence is defined as the ratio between Euclidian distance and accumulated distance and chemotactic index as the cosine of the angle between net distance travelled in the direction of the gradient and the Euclidean distance.
The distribution and localization of myosin was analysed using a MATLAB plug-in kindly provided by Douwe Veltman and described in (10).
with the ratio of two terms calculated as: where S: position around cell contour L: perimeter The first term of the series is mean value of the function while the second one is the trigonometric function. The ratio of the first two terms of the series evaluates the difference in the peaks of the described signal. The more heterogeneous the signal, the larger is the gap between the peaks and as a result, larger is the ratio. In The intensity of the scar is fitted with a sigmoid curve to obtain its half-life. The sigmoid curve used for fitting is: and critical time obtained from the fit is given by