Exogenous MT1-MMP expression abolishes compartmentalized pressure in primary human fibroblasts. (A) Morphology of primary human dermal fibroblasts expressing GFP-MT1MMP. GFP-MT1MMP is cytoplasmic in intracellular vesicles and at the plasma membrane (Video 5). Bar, 10 µm. (B) The expression of total MT1MMP in HT1080 cells transiently transfected with GFP-MT1MMP relative to untransfected cells (n = 15, N = 3). *, P < 0.0001 versus untransfected cells. (C) The anterior cytoplasmic intracellular pressure (P_ic) in primary human dermal fibroblasts, either transfected with GFP or GFP-MT1MMP and untreated, or transfected with GFP-MT1MMP and treated with 10 µM GM6001 (n = 15, N = 3). *, P < 0.001 versus GFP. (D) The velocity of HT1080 cells transfected with either GFP or GFP-MT1MMP ± GM6001 treatment moving through CDM (n = 30, N = 3). P = 0.44. Error bars indicate SEM. (E) Model. Our data suggest mesenchymal tumor cells use MMP activity to enlarge constricting pores and move the nucleus through the fibrillar 3D matrix without activating the nuclear piston mechanism. When MMP activity is low, either in tumor cells or primary human fibroblasts migrating through 3D ECM, cells rely on robust cell-matrix adhesions and actomyosin contractility to pull the nucleus forward and increase pressure in the anterior cytoplasmic compartment. This nuclear piston mechanism may represent an alternative strategy to move the rigid nucleus through a confining 3D matrix. a.u., arbitrary units.