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, 117 (5), 2506-2512

Pressure Sensing Through Piezo Channels Controls Whether Cells Migrate With Blebs or Pseudopods

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Pressure Sensing Through Piezo Channels Controls Whether Cells Migrate With Blebs or Pseudopods

Nishit Srivastava et al. Proc Natl Acad Sci U S A.

Abstract

Blebs and pseudopods can both power cell migration, with blebs often favored in tissues, where cells encounter increased mechanical resistance. To investigate how migrating cells detect and respond to mechanical forces, we used a "cell squasher" to apply uniaxial pressure to Dictyostelium cells chemotaxing under soft agarose. As little as 100 Pa causes a rapid (<10 s), sustained shift to movement with blebs rather than pseudopods. Cells are flattened under load and lose volume; the actin cytoskeleton is reorganized, with myosin II recruited to the cortex, which may pressurize the cytoplasm for blebbing. The transition to bleb-driven motility requires extracellular calcium and is accompanied by increased cytosolic calcium. It is largely abrogated in cells lacking the Piezo stretch-operated channel; under load, these cells persist in using pseudopods and chemotax poorly. We propose that migrating cells sense pressure through Piezo, which mediates calcium influx, directing movement with blebs instead of pseudopods.

Keywords: Dictyostelium; Piezo; blebbing; cell migration; chemotaxis.

Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Uniaxial load causes cells to move using blebs instead of pseudopods. (A) Diagram of the cell squasher used to apply uniaxial loads to cells moving under an agarose overlay (36). The load is applied using a plunger on the bridge between two wells cut into the agarose, one containing cells and the other, the chemoattractant cyclic AMP, which attracts the cells under the agarose toward it. (B) Distinction between pseudopods and blebs. At the Left are shown cells expressing an F-actin reporter, and at the Right, kymographs taken along the lines indicated at the Left. (Scale bar: 10 µm.) (C) Uniaxial load causes cells to migrate using blebs. The cells are migrating under an overlay of 0.5% agarose to which increasing uniaxial loads are applied. Blebs are indicated by white stars. (Scale bar: 10 µm.) (D) Blebbing of migrating cells increases with increasing load or overlay stiffness. (E) Cell speed decreases under increasing load or stiffness of the agarose overlay. The data are represented as mean ± SD for n ≥ 30 cells for each case with measurements made for about 30 min, starting 8 to 10 min after load was applied. The stiffness of the agarose overlays is as follows: 0.5% = 6.6 kPa; 0.75% = 11.9 kPa; 1% = 20.5 kPa; and 2% = 73.6 kPa. Aggregation-competent Ax2 cells expressing the ABD120-GFP reporter for F-actin and migrating toward cyclic AMP in KK2MC are used throughout.
Fig. 2.
Fig. 2.
Uniaxial load causes a rapid switch to bleb-driven migration. (A) Rapid induction of blebbing by uniaxial loading of migrating cells. Frames from a movie timed with respect to the start of loading (t = 0); blebs are indicated by an asterisk (*). (Scale bar: 10 μm.) (B) Time course of bleb induction by load. At the Top is shown a typical loading regime with a small up-tick as the plunger first touches the agarose followed by a 15-s ramping of load to 400 Pa. At the Bottom is shown the bleb frequency, with blebs binned into 1-s intervals and scored at the time they first appear (typically, they are fully expanded in one frame of the movie). Aggregation-competent Ax2 cells expressing the F-actin reporter ABD120-GFP were filmed at 2 frames per s under a 0.5% agarose overlay (n = 17 cells).
Fig. 3.
Fig. 3.
Uniaxial load causes cytoskeletal reorganization. (A) Coronin, an F-actin binding protein, relocates under load from pseudopods to the actin scars left behind by blebs. Quantification of the coronin localization from the cell edge. Data are represented as mean ± SD for n ≥ 40 cells for each case; one-way ANOVA, ***P < 0.005. (B) Paxillin patches, thought to mediate adhesion to the substratum, disperse under load. Quantification of the number of paxillin patches in the cell under different loading conditions. Data are represented as mean ± SD for n ≥ 20 cells for each case; one-way ANOVA, ***P < 0.005. Load was applied to aggregation-competent Ax2 cells expressing either coronin–GFP or GFP–paxillin and migrating toward cyclic AMP, under an overlay of 0.5% agarose. (Scale bar: 10 µm.)
Fig. 4.
Fig. 4.
Myosin II is rapidly recruited to the cell cortex in response to load. (A) Load causes myosin II to be recruited to the cell cortex. Blebs are indicated by an asterisk (*). (Scale bar: 10 µm.) (B) Quantification of cortical enrichment of myosin II under load. Data are represented as mean ± SD for n ≥ 20 cells for each case; one-way ANOVA, *P < 0.0005. (C) Time course showing that myosin II is rapidly recruited to the cell cortex under load. Data are given as mean ± SEM; n = 10 cells; one-way ANOVA, P < 0.005. Cortical enrichment is calculated by measuring the ratio of fluorescence intensity of membrane and cytoplasm around the cell. Ax2 cells expressing myosin II–GFP (GFP–MhcA) and chemotaxing to cyclic AMP under 0.5% agarose gels were used throughout. In time courses, load is applied at t = 0.
Fig. 5.
Fig. 5.
Extracellular calcium and the Piezo stretch-operated channel are required for cells to respond to load. (A) Illustration of typical responses to load of cells either in calcium-free medium, or lacking the Piezo channel (PzoA cells, strain HM1812). Compare to Ax2 controls in Figs. 1C and 2A. (Scale bar: 10 µm.) (B) Quantification of the blebbing response to load of cells either in calcium-free medium, or lacking the Piezo channel. Data are mean ± SD for n ≥ 15 cells tracked before and after applying load in each case. Cells, either Ax2 parental or Piezo-null mutant (PzoA, strain HM1812), were incubated under agarose made with the standard KK2MC buffer, which has 100 µM calcium, or this buffer lacking calcium and supplemented with 200 μM EGTA. A load of 400 Pa was applied as indicated. (C) Quantification of the cortical enrichment of myosin II in PzoA cells under a load of 400 Pa. The cortical enrichment of RFP–myosin II is calculated by measuring the ratio of fluorescence intensity of membrane and cytoplasm around the cell. Cells are chemotaxing to cyclic AMP under 0.5% agarose gels. The data are mean ± SD for n ≥ 20 cells analyzed for each case; ***P < 0.0005 for wild-type cells and P > 0.5 for Piezo-null cells, Mann–Whitney U test and one-way ANOVA.
Fig. 6.
Fig. 6.
The Piezo channel is required for the calcium response to load and for efficient chemotaxis. (A) Loading causes a transient increase in cytosolic calcium, which depends on extracellular calcium and the Piezo channel. Changes in cytoplasmic calcium were detected by microscopy using the Cameleon FRET-based sensor. The normalized ratio of YFP (535 nm)/CFP (485 nm) indicates the cytosolic calcium concentration. Aggregation-competent cells under 0.5% agarose were subjected to a load of 400 Pa as indicated (n = 15 cells). (B) Piezo-null cells are defective in chemotaxis to cyclic AMP when constrained under agarose. Representative tracks of wild-type (WT) (strain Ax2, R.R.K. laboratory) and Piezo-null cells (PzoA cells, strain HM1812) chemotaxing toward a cyclic-AMP source in an under-agarose assay. Agarose overlays of different stiffness (0.5% and 2% agarose with Young’s modulus of 6.6 and 73.6 kPa, respectively) were used. (C) Table of the chemotactic parameters obtained by tracking cells in an under-agarose assay. Speed was calculated by dividing the accumulated distance by total time. Persistence is defined as the ratio between Euclidian distance and accumulated distance, and chemotactic index is defined as the cosine of the angle between net distance traveled in the direction of the gradient and the Euclidian distance. Data are represented as mean ± SEM from measurements obtained for n ≥ 50 cells on at least three different days; P < 0.005, Mann–Whitney U test and one-way ANOVA.
Fig. 7.
Fig. 7.
Biophysical representation of the changes in cell geometry and pressure upon application of external load and the proposed link to Piezo activation. (A) Illustration of two mechanisms for applying compressive load to a cell. (Left) External loading is imposed from the upper boundary and transmitted through the gel and cells to the substrate. (Right) Elastic overlays would generate stress when deformed to accommodate a cell between them and a rigid substrate, and so apply load to the cell. (B) In a spherical cell, the Laplace equation links the hydrostatic pressure, P0, in the cytosol, the radius, r0, of the cell, and its cortical tension T. (C) Application of external load to a spherical cell leads to its flattening and change to a pancake shape. In this case, the top surface is approximately flat, and the external load σ must be balanced by a higher cell hydrostatic pressure P. However, the load is not acting along the periphery of the cell; there, Laplace law now relates the internal pressure P with cortical tension and increased local curvature, therefore relating external load σ to the cell height. We propose the increased cytosolic pressure, resulting from loading and squashing the cell, activates the Piezo channels by increasing tension in the plasma membrane.

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