Generation of compartmentalized pressure by a nuclear piston governs cell motility in a 3D matrix

Abstract

Cells use actomyosin contractility to move through three-dimensional (3D) extracellular matrices. Contractility affects the type of protrusions cells use to migrate in 3D, but the mechanisms are unclear. In this work, we found that contractility generated high-pressure lobopodial protrusions in human cells migrating in a 3D matrix. In these cells, the nucleus physically divided the cytoplasm into forward and rear compartments. Actomyosin contractility with the nucleoskeleton-intermediate filament linker protein nesprin-3 pulled the nucleus forward and pressurized the front of the cell. Reducing expression of nesprin-3 decreased and equalized the intracellular pressure. Thus, the nucleus can act as a piston that physically compartmentalizes the cytoplasm and increases the hydrostatic pressure between the nucleus and the leading edge of the cell to drive lamellipodia-independent 3D cell migration.

Fig. 1

Actomyosin contractility governs intracellular pressure in 3D ECM. Comparison of the intracellular pressures (n ≥ 20 each) of lamellipodial cells on 2D CDM and in 3D collagen, untreated lobopodial cells in 3D CDM, or cells in CDM treated overnight with inhibitors of myosin II (25 µM blebbistatin, ROCK (10 µM Y-27632), or RhoA (10 µg/ml C3 transferase). N=3, *P < 0.001.

Fig. 2

Lobopodial fibroblasts are compartmentalized into high- and low-pressure zones. (A) Intracellular pressures were measured immediately in front of (green dot) and behind (red dot) the nucleus. Scale bar 5 µm. (B) Comparison of intracellular pressures in front and behind the nucleus of fibroblasts (n ≥ 25) migrating on 2D glass (2D lamellipodia), in 3D collagen (3D lamellipodia), or in 3D CDM (3D lobopodia). N=3. (C) A sub-population of PA-GFP was activated near the leading edge (dashed line) and rate of translocation was measured at the indicated regions of interest (ROI) immediately in front of and behind the nucleus in live cells. Scale bar 5 µm. (D) Time constants of PA-GFP accumulation show that the nucleus significantly slows the rate of PA-GFP translocation in 3D lobopodial versus 2D lamellipodial fibroblasts (n ≥ 17, N=3). (E) Myosin II inhibition results in immediate backward movement of the nucleus in lobopodial but not lamellipodial cells (quantified in the lower panel). n=10, N=3. Scale bar 3 µm. (F) Pressure rises transiently behind the nucleus following myosin II inhibition only in lobopodial cells (n ≥ 16, N=3). **P < 0.001 vs. front P_ic, *P < 0.01 vs. back P_ic. (G) Anterior inhibition of myosin II prevents forward movement of the nucleus (n=10, N=3). *P < 0.01.

Fig. 3

Nesprin 3 associates with vimentin and actomyosin, and it mediates independent movement of the nucleus. (A and B) Filamentous vimentin (A) and myosin IIA (B) are polarized to the anterior of lobopodial fibroblasts. n=10. Scale bars 10 µm. (C) Immunoprecipitation reveals that actin and myosin IIA form a myosin II ATPase-dependent complex with vimentin and nesprin 3 in primary fibroblasts (N=6). (D) Anterior inhibition of myosin II reduces forward pressure (n=22, N=3), and the leading edge retracts (n=27, N=5). (E–G) Instantaneous velocities of the nucleus (V_N) and trailing edge (V_TE) measured in lobopodial cells (n ≥ 14) expressing GFP-MLC2 and RFP-NLS (E, scale bar 5 µm) treated with control or nesprin 3 siRNA (F). Nesprin 3 knockdown reduces the independent movement of the nucleus relative to the trailing edge (G). N=3. (H) Myosin II activity and nesprin 3 are each required for efficient 3D migration (n ≥ 45, N=3). **P < 0.0001, *P < 0.05.

Fig. 4

Nesprin 3 compartmentalizes intracellular pressure to mediate lobopodial 3D migration. (A) Comparison of anterior and posterior intracellular pressure in cells treated with the indicated siRNAs (n≥ 23, N=3). (B) The average size of focal adhesions formed by cells treated with the indicated siRNAs and plated on 2D glass (n ≥ 13, N=3). (C) Cortactin localization in siRNA-treated cells. n=17. Arrowheads indicate local accumulation of the lamellipodial marker cortactin at the leading edge. (D) Quantification of lamellipodial cells in 3D CDM following siRNA treatments (n ≥ 33, N=3). Scale bars 10 µm. *P < 0.001.