Figure 5. Arp2/3- and Formins-Dependent Actin Architectures Regulate MT-Dependent Protrusions that Promote the CG Response.

(A) Contractile (+DMSO) and actomyosin contraction inhibited (+Blebb) cells under control conditions (top row), Arp2/3 inhibition (+CK666), MTs disruption (+Nocodazole) or Formins inhibition (+SMIFH2). See Figure S4 for individual channels and cross-sections of protrusions into nanogrooves.

(B) Population views and mean values for lengths along and widths across nanolines for the conditions outlined in (A).

(C) (Top) 3D reconstructions of cell protrusion along compliant (2.3kPa) and stiff (50kPa) collagen-coated PAA nanogroove substrates. (Bottom) Stereometric view.

(D) Lengths and widths for conditions in (C). Note that cells on both stiffnesses produce MT-rich in-groove protrusions, while on stiff substrates on-ridge lamellipodial protrusions are more robust.

(E) Schematic and plot of metrics capturing in-groove protrusive invasiveness that decreases from Formins inhibition and increases from Arp2/3-inhibition, where Formins and Arp2/3 regulate the transition between ventral and/or dorsal SFs and transverse arcs to regulate in-groove MTs that promote directed protrusion and the response to CG (see Figure S5).

(F) Schematic of competitive dynamics between on-ridge lamellipodial and in-groove MT-driven apical nanogroove-guided protrusions. Data are mean ± SD; ns, no significant difference; *p < 0.05, ^**p < 0.001