![Figure 6. Matrix elastic behavior governs the mode of normal 3D migration. (A) HFFs can remodel trypsinized CDM (middle), whereas the structure of the untreated (left) or the trypsinized (tryp) and cross-linked CDM (XL; right) is unaffected by HFF migration. CFP-Rac1 is shown in green, and Alexa Fluor 633–labeled matrix is in gray. Bars, 40 µm. (B) Cross-linked 1.7 mg/ml collagen (middle) and 8.6 mg/ml collagen (right) are not remodeled during HFF migration. GFP-actin is shown in green, and collagen is shown in gray (reflection). Bars, 10 µm. (C) Matrix stiffness (Young’s modulus [E]) of the indicated modified matrices. (D) Strain-stiffening (Ehigh/Emed) behavior of the indicated native and modified matrices. The dashed red line indicates a value of Ehigh/Emed corresponding to 1 (linear elasticity). (C and D) *, P < 0.05 versus trypsinized CDM; **, P < 0.05 versus cross-linked collagen. (E–G) Trypsinization and chemical cross-linking redistribute Rac1 activity and switch the mode of cell migration. (E) Maximally projected confocal stacks of HFFs expressing the Rac1 biosensor migrating in the trypsinized CDM (top), trypsinized and cross-linked CDMs (middle), or cross-linked collagen (bottom). The Fc images, representing the total activity of each GTPase, were pseudocolored according to the 16-color scale. Arrowheads indicate regions of intracellular signaling. Bars, 5 µm. (F) Mean PI of active Rac1 in HFFs migrating in the indicated ECM environments. *, P < 0.05 versus 3D trypsinized CDM. (G) The percentage of lobopodia-bearing HFFs migrating inside the CDM or collagen treated as indicated. *, P < 0.001 versus untreated CDM; **, P < 0.001 versus trypsinized CDM; ***, P < 0.007 versus untreated collagen. (H) 8.6 mg/ml collagen supports lamellipodia-based 3D cell migration. (top) A representative image of cortactin enrichment at the leading edge of cells migrating in 8.6 mg/ml collagen (arrow). (bottom) The mean cortactin intensity profile measured from 13 cells, with three measurements per cell. Cells are oriented with their leading edge toward the top right (A and B) or the right (E and H) of the figure. a.u., arbitrary unit.](https://cdn.rupress.org/rup/content_public/journal/jcb/197/3/10.1083_jcb.201201124/6/jcb_201201124_fig6.jpeg?Expires=1773583006&Signature=w39ey7GcpMowKUmhAc3xkGeGlgw5ovjt7ADuPGMr4JJqxutxcLLB1g-0KysQKvj1yIqbaHMVx29lppRc7WM287nU8xrlVVS26vRyYYOF6Bc3lfndMW91wH4Qs07Lp68utPJLLFKGtt5dJbWjm-ikFh2Yuh8nn7sAOdBN7wE4LoqF4CHqDwaocM08TElB5gi1HWBtkZ4mx-Ooi6kVtwYVIYTg98SC1Jvbzmhjp4h7~UByci49ofZUmHdBrfvMYlID~i-r2EzTfUS2Pu4Bx99PG5Ud0RMWg2-wNW7su69nf-mVcN-bI7G~lWwet2RpKiU8JLPblPTZvWzt4XT8oog6Xg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Matrix elastic behavior governs the mode of normal 3D migration. (A) HFFs can remodel trypsinized CDM (middle), whereas the structure of the untreated (left) or the trypsinized (tryp) and cross-linked CDM (XL; right) is unaffected by HFF migration. CFP-Rac1 is shown in green, and Alexa Fluor 633–labeled matrix is in gray. Bars, 40 µm. (B) Cross-linked 1.7 mg/ml collagen (middle) and 8.6 mg/ml collagen (right) are not remodeled during HFF migration. GFP-actin is shown in green, and collagen is shown in gray (reflection). Bars, 10 µm. (C) Matrix stiffness (Young’s modulus [E]) of the indicated modified matrices. (D) Strain-stiffening (Ehigh/Emed) behavior of the indicated native and modified matrices. The dashed red line indicates a value of Ehigh/Emed corresponding to 1 (linear elasticity). (C and D) *, P < 0.05 versus trypsinized CDM; **, P < 0.05 versus cross-linked collagen. (E–G) Trypsinization and chemical cross-linking redistribute Rac1 activity and switch the mode of cell migration. (E) Maximally projected confocal stacks of HFFs expressing the Rac1 biosensor migrating in the trypsinized CDM (top), trypsinized and cross-linked CDMs (middle), or cross-linked collagen (bottom). The Fc images, representing the total activity of each GTPase, were pseudocolored according to the 16-color scale. Arrowheads indicate regions of intracellular signaling. Bars, 5 µm. (F) Mean PI of active Rac1 in HFFs migrating in the indicated ECM environments. *, P < 0.05 versus 3D trypsinized CDM. (G) The percentage of lobopodia-bearing HFFs migrating inside the CDM or collagen treated as indicated. *, P < 0.001 versus untreated CDM; **, P < 0.001 versus trypsinized CDM; ***, P < 0.007 versus untreated collagen. (H) 8.6 mg/ml collagen supports lamellipodia-based 3D cell migration. (top) A representative image of cortactin enrichment at the leading edge of cells migrating in 8.6 mg/ml collagen (arrow). (bottom) The mean cortactin intensity profile measured from 13 cells, with three measurements per cell. Cells are oriented with their leading edge toward the top right (A and B) or the right (E and H) of the figure. a.u., arbitrary unit.
