The nuclear piston mechanism requires integrin-mediated, cell-matrix adhesion, actomyosin contractility, vimentin, nesprin 3, and lamin A. (A and B) Primary human dermal fibroblasts migrating in 3D CDM use the nuclear piston and compartmentalized pressure. Inhibition of β1 and αV integrins is sufficient to revert the intracellular pressure (Pic) to low and uniform, without completely disrupting cell adhesion (n = 16, N = 3). *, P < 0.001 versus control cells. Bars, 10 µm. (C) The velocity of HT/MT1 cells migrating in 3D CDM either untreated or treated with GM6001, integrin inhibitors (Int. Inh.), or blebbistatin in combination with GM6001 (n = 45, N = 3). *, P < 0.001 versus control cells. (D) Cell morphology of the HT/MT1 cells treated as indicated. Cells can remain polarized with clear leading and trailing edges. Bars, 10 µm. (E) The increased compartmentalized pressure generated by protease inhibition is consistent with the nuclear piston mechanism in lobopodial primary human fibroblasts in that it requires β1 and αV integrins in combination with actomyosin contractility (n = 18, N = 3). *, P < 0.001 versus the anterior compartment. (F) Quantification of the blots represented in Fig. S3 A demonstrating the specificity of siRNA-mediated knockdown of nesprin 3, vimentin, or lamin A using a pool of siRNA sequences (N = 3). *, P < 0.01 versus the control. (G) MMP inhibition does not increase compartmentalized pressure in HT1080 cells in 3D CDM after nesprin 3, vimentin, or lamin A siRNA-mediated protein knockdown (n = 15, N = 3). *, P < 0.001 versus the anterior compartment. Error bars indicate SEM. MyoII inh., myosin II inhibitor.
