Myosin II and Arp2/3 cross-talk governs intracellular hydraulic pressure and lamellipodia formation

Figures

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  FIGURE 1:. Increasing intracellular osmotic pressure is sufficient to trigger disassembly of lamellipodia. (A) Primary human dermal fibroblasts plated on 2D glass surfaces were treated with isosmotic, hyperosmotic, or hypo-osmotic media and stained for F-actin (magenta) and cortactin (green) to visualize lamellipodial protrusions (LM, yellow arrowheads). While lamellipodia are abundant under iso- and hyperosmotic conditions, treatment with hypo-osmotic media prevented cortactin-positive lamellipodia formation (n ≥ 28, N = 3). Bars, 10 µm. Quantified in B. (C) Direct measurement of intracellular cytoplasmic pressure (Pic) in cells treated as in A reveals pressure significantly increases upon exposure to hypo-osmotic media (n = 30, N = 3). *, P < 0.0001 vs. control.

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  FIGURE 2:. RhoA signaling increases intracellular pressure and can act upstream of Rac1 to inhibit lamellipodia formation. (A) Expression of constitutively active GFP-RhoA (RhoA-CA) in primary human dermal fibroblasts prevents cortactin-positive lamellipodia (LM, yellow arrowheads) from forming on 2D glass surfaces. Treatment with 25 µM blebbistatin to inhibit myosin II activity did not restore lamellipodia formation in RhoA-CA expressing cells (n ≥ 20, N = 3). Bars, 10 µm. Quantified in B. *, P < 0.0001 vs. untransfected. (C) The intracellular pressures of cells treated as in A. Expression of RhoA-CA was sufficient to significantly increase the cytoplasmic pressure in cells on glass in a manner partially dependent on NMII activity (n ≥ 17, N = 3). *, P < 0.0001 vs. control. **, P < 0.04 vs. RhoA-CA. (D) Direct measurements of intracellular pressure from untransfected and GFP-RhoA-CA transfected cells plated on 2D glass either untreated or treated with 1 mM acetazolamide. The increased pressure in cells expressing RhoA-CA is significantly reduced following treatment with AZ (n = 30, N = 3). *, P < 0.0001 vs. control. **, P < 0.0001 vs. RhoA-CA. (E) Cell volume measurements from…

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  FIGURE 3:. Lamellipodia can form in high-pressure cells expressing constitutively active RhoA. (A) Primary fibroblasts were plated on glass and transiently transfected with GFP-tagged Rac1-CA, CFP-tagged RhoA-CA, or both together and then fixed and stained with rhodamine-phalloidin to visualize F-actin. While Rac1-CA and RhoA-CA solitary expression induced lamellipodia (LM, yellow arrowheads) and stress fibers, respectively, expression of both together generated abundant lamellipodia and stress fibers (n = 15, N = 3). (B) Mean F-actin intensity was highest in the cotransfected cells suggesting actin monomer availability is not limiting under these conditions. *, P < 0.003 vs. Rac1-CA. (C) The extent of lamellipodia formation (LM, yellow arrowheads) in control and cells transiently transfected as indicated was verified by staining fixed cells for the lamellipodial marker cortactin (n ≥ 7, N = 3). Quantified in D. *, P < 0.007 vs. control. (E) Intracellular pressure measurements of control cells or cells transiently transfected as indicated. Expression of RhoA-CA significantly increases cytoplasmic pressure and coexpression of Rac1-CA with RhoA-CA reduces pressure (n = 18, N = 3). *, P <…

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  FIGURE 4:. Lamellipodia dynamics are slowed in high-pressure cells. (A) Fluorescent images of a lamellipodium found in primary fibroblasts transiently transfected with either GFP-tagged constitutively active Rac1 alone (Rac1-CA) or in combination with mScarlet constitutively active RhoA (RhoA-CA; top panels). Kymograph analysis (bottom panels) of protrusion dynamics in these cells reveals that lamellipodia protrusion and retraction are both significantly slowed in cotransfected cells (n ≥ 95, N = 3). Bars, 5 µm. Quantified in B and C. *, P < 0.009.

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  FIGURE 5:. RhoA signaling acts downstream from Rac1 to inhibit lamellipodia formation in cells with high osmotic pressure. Primary fibroblasts transiently transfected with GFP-tagged Rac1-CA, alone or in combination with CFP-tagged RhoA-CA (A), or CFP-tagged RhoA-DN (B) and treated as indicated. Cortactin staining reveals the presence or absence of lamellipodia (LM, yellow arrowheads) in the imaged cells. Expression of RhoA-DN prevents the loss of lamellipodia in Rac1-CA coexpressing cells treated with hypo-osmotic media relative to RhoA-CA (n ≥ 18, N = 3). Bars, 10 µm. Quantified in C. *, P < 0.007 vs. Rac1-CA.

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  FIGURE 6:. NMII activity is required to increase intracellular pressure and prevent lamellipodia formation following Arp2/3 inhibition. (A) Primary fibroblasts on 2D glass were either untreated (control) or treated with 25 µM blebbistatin (NMII inh.), 100 µM CK-666 (Arp2/3 inh.), or both together (NMII inh. + Arp2/3 inh.) for 60 min before fixing and staining for actin (magenta) and cortactin (green). Arp2/3 inhibition removes lamellipodia (LM, yellow arrowheads), while inhibiting both Arp2/3 and NMII prevented the removal of lamellipodia from the cell periphery (n ≥ 12, N = 3). Bars, 10 µm. Quantified in B. *, P < 0.0001 vs. control. (C) Intracellular pressure measurements of live cells treated as in A (n = 30, N = 3). Arp2/3 inhibition increases intracellular pressure through myosin II activity. *, P < 0.0001 vs. control. (D) Primary human fibroblasts were plated on 2D glass and treated as indicated for 60 min at 37°C. Latrunculin-A (Lat-A) treatment significantly reduced intracellular pressure compared with control cells, while treatment with 50 and 100 µM blebbistatin (BS) had no effect (n = 30, N = 3). *, P = 0.0001 vs. control.

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  FIGURE 7:. The lamellipodia formed during the coinhibition of Arp2/3 and NMII contain branched actin networks but are slower to protrude and retract. (A) Platinum replica electron microscopy (PREM) images of untreated primary fibroblasts, or cells treated with 25 µM blebbistatin (NMII inh.), 200 µM CK-666 (Arp2/3 inh.), or both together (NMII inh. + Arp2/3 inh.) for 60 min before fixation (n = 27, N = 3). Bars, 500 nm (top panels) and 50 nm (bottom panels). Networks of F-actin branching at 70° (pseudocolored orange) were abundant in all conditions except following solitary Arp2/3 inhibition. Quantified in B and C. *, P < 0.0001 vs. control. (D) Quantification of fibroblast velocity on 2D glass when treated as in A. NMII inhibition partially rescued cell velocity in CK-666 treated cells (n = 40, N = 3). *, P < 0.03. (E) Fluorescent images of a lamellipodium found in primary fibroblasts transiently transfected with lifeact-mScarlet and either untreated (control) or treated simultaneously with CK-666 and blebbistatin (top panels). Kymograph analysis (bottom panels) of protrusion dynamics in these cells reveals that lamellipodia protrusion is significantly slowed when Arp2/3 and myosin II are…

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  FIGURE 8:. RhoA signaling can govern lamellipodia formation by acting both upstream of and downstream from Rac1. The switch between lamellipodia-dependent and -independent protrusions is governed by at least two cross-talk pathways connecting RhoA activation to the inhibition of Rac1 signaling. RhoA activity can act upstream of Rac1 to prevent the activation of Rac1 and the formation of lamellipodia. This inhibitory cross-talk may activate or inhibit a Rac1 GAP or GEF, respectively, as demonstrated for the RhoA-Rac1 cross-talk governing the amoeboid–mesenchymal transition (Sanz-Moreno et al., 2008). The second pathway prevents Rac1-mediated lamellipodia formation through reciprocal cross-talk between Arp2/3 and NMII. Both pathways lead to the generation of intracellular hydraulic pressure and the formation of lamellipodia-independent protrusions downstream from RhoA activity.