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Spontaneous and electric field-controlled front-rear polarization of human keratinocytes.

Saltukoglu D, Grünewald J, Strohmeyer N, Bensch R, Ulbrich MH, Ronneberger O, Simons M - Mol. Biol. Cell (2015)

Bottom Line: By contrast, we found a crucial role for extracellular pH as well as G protein coupled-receptor (GPCR) or purinergic signaling in the control of directionality.Overall our work puts forward a model in which the EF uses distinct polarization pathways.The cathodal pathway involves GPCR/purinergic signaling and is dominant over the anodal pathway at neutral pH.

View Article: PubMed Central - PubMed

Affiliation: Center for Systems Biology, University of Freiburg, 79104 Freiburg, Germany Renal Division, University Hospital Freiburg, 79106 Freiburg, Germany Spemann Graduate School of Biology and Medicine, University of Freiburg, 79104 Freiburg, Germany BIOSS Centre for Biological Signalling Studies, University of Freiburg, 79104 Freiburg, Germany.

No MeSH data available.


Related in: MedlinePlus

The role of cytoskeletal regulators in spontaneous and directional polarization. (A) Quantification of polarization phenotypes for inhibition of cytoskeletal force generators (myosin II [blebbistatin] and ROCK [Y-27362] for contractile rear and Arp2/3 complex [CK-666] for protrusive front) and inhibition of small GTPases Cdc42 (ZCL-278) and Rac1 (NSC-23766). Inhibiting the Arp2/3 complex, Cdc42, and Rac1 leads to the loss of symmetry breaking, whereas ROCK and myosin II inhibition generates cells with more than one lamellipodium. The EF significantly increases the percentage of cells breaking symmetry in the Arp2/3 and Rac1 inhibition conditions. The number of cells quantified is indicated above each column. The p value is calculated with Student’s t test. **p < 0.01. (B) Rose plots representing the direction of symmetry breaking of polarizing cells with the indicated inhibitors. (C) Automated boundary detection from phase contrast videos for the representative cells with the indicated treatments. Time progression is represented from blue to red. In Cdc42-inhibited cells, EF does not induce any protrusions toward the cathode, and the percentage of cells breaking symmetry does not increase with EF. Rac1-inhibited cells show small, unsustained, and random boundary protrusions without EF. Two examples are shown for Rac1-inhibited cells with EF. In the left example, the cell clearly polarizes to the cathode. In the right example, small protrusions are driven toward the cathode and suppressed at the anodal side.
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Figure 2: The role of cytoskeletal regulators in spontaneous and directional polarization. (A) Quantification of polarization phenotypes for inhibition of cytoskeletal force generators (myosin II [blebbistatin] and ROCK [Y-27362] for contractile rear and Arp2/3 complex [CK-666] for protrusive front) and inhibition of small GTPases Cdc42 (ZCL-278) and Rac1 (NSC-23766). Inhibiting the Arp2/3 complex, Cdc42, and Rac1 leads to the loss of symmetry breaking, whereas ROCK and myosin II inhibition generates cells with more than one lamellipodium. The EF significantly increases the percentage of cells breaking symmetry in the Arp2/3 and Rac1 inhibition conditions. The number of cells quantified is indicated above each column. The p value is calculated with Student’s t test. **p < 0.01. (B) Rose plots representing the direction of symmetry breaking of polarizing cells with the indicated inhibitors. (C) Automated boundary detection from phase contrast videos for the representative cells with the indicated treatments. Time progression is represented from blue to red. In Cdc42-inhibited cells, EF does not induce any protrusions toward the cathode, and the percentage of cells breaking symmetry does not increase with EF. Rac1-inhibited cells show small, unsustained, and random boundary protrusions without EF. Two examples are shown for Rac1-inhibited cells with EF. In the left example, the cell clearly polarizes to the cathode. In the right example, small protrusions are driven toward the cathode and suppressed at the anodal side.

Mentions: As a next step, we inhibited force generators of the actin network. The rear contractile system was inhibited with blebbistatin (nonmuscle myosin II inhibitor) or Y-27632 (Rho kinase [ROCK] inhibitor), and the protrusive front system was inhibited with CK-666 (Arp2/3 complex inhibitor). Both blebbistatin and Y-27632 significantly increased the percentage of cells with more than one lamellipodial structure but did not interfere with their symmetry-breaking ability (Figure 2A and Supplemental Figure S4B). The efficiency of Y-27632 treatment was confirmed by testing the reduction of doubly phosphorylated myosin regulatory light chain (ppMRCLC2) compared with untreated cells via immunoblotting (Supplemental Figure S4A). By contrast, Arp2/3 complex inhibition led to only 44 and 16% (using 100 or 200 μM CK-666, respectively) of cells being able to break symmetry, compared with 96% of cells in the control case (Figure 2A). Moreover, migration tracks were decreased in length by CK-666 treatment (Supplemental Figure S3B). Morphologically, inhibition of the Arp2/3 complex resulted in unpolarized cells with filopodia-like boundaries (Supplemental Figure S3B). Phalloidin staining revealed that the actin cytoskeleton was disorganized upon CK-666 treatment (Supplemental Figure S3C). In particular, the transverse arcs were reduced, and the direction of actin bundles was perpendicular to the cell periphery rather than parallel. Neither the reduction in polarization nor the loss of transverse arcs was seen with the inactive CK-666 analogue CK-689 (Supplemental Figure S3, A and C). Together these results demonstrate that Arp2/3 complex–dependent cytoskeleton rearrangements and protrusive forces at the cell periphery are key for breaking symmetry in keratinocytes, whereas myosin II– and ROCK-based contractility is not required.


Spontaneous and electric field-controlled front-rear polarization of human keratinocytes.

Saltukoglu D, Grünewald J, Strohmeyer N, Bensch R, Ulbrich MH, Ronneberger O, Simons M - Mol. Biol. Cell (2015)

The role of cytoskeletal regulators in spontaneous and directional polarization. (A) Quantification of polarization phenotypes for inhibition of cytoskeletal force generators (myosin II [blebbistatin] and ROCK [Y-27362] for contractile rear and Arp2/3 complex [CK-666] for protrusive front) and inhibition of small GTPases Cdc42 (ZCL-278) and Rac1 (NSC-23766). Inhibiting the Arp2/3 complex, Cdc42, and Rac1 leads to the loss of symmetry breaking, whereas ROCK and myosin II inhibition generates cells with more than one lamellipodium. The EF significantly increases the percentage of cells breaking symmetry in the Arp2/3 and Rac1 inhibition conditions. The number of cells quantified is indicated above each column. The p value is calculated with Student’s t test. **p < 0.01. (B) Rose plots representing the direction of symmetry breaking of polarizing cells with the indicated inhibitors. (C) Automated boundary detection from phase contrast videos for the representative cells with the indicated treatments. Time progression is represented from blue to red. In Cdc42-inhibited cells, EF does not induce any protrusions toward the cathode, and the percentage of cells breaking symmetry does not increase with EF. Rac1-inhibited cells show small, unsustained, and random boundary protrusions without EF. Two examples are shown for Rac1-inhibited cells with EF. In the left example, the cell clearly polarizes to the cathode. In the right example, small protrusions are driven toward the cathode and suppressed at the anodal side.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4666133&req=5

Figure 2: The role of cytoskeletal regulators in spontaneous and directional polarization. (A) Quantification of polarization phenotypes for inhibition of cytoskeletal force generators (myosin II [blebbistatin] and ROCK [Y-27362] for contractile rear and Arp2/3 complex [CK-666] for protrusive front) and inhibition of small GTPases Cdc42 (ZCL-278) and Rac1 (NSC-23766). Inhibiting the Arp2/3 complex, Cdc42, and Rac1 leads to the loss of symmetry breaking, whereas ROCK and myosin II inhibition generates cells with more than one lamellipodium. The EF significantly increases the percentage of cells breaking symmetry in the Arp2/3 and Rac1 inhibition conditions. The number of cells quantified is indicated above each column. The p value is calculated with Student’s t test. **p < 0.01. (B) Rose plots representing the direction of symmetry breaking of polarizing cells with the indicated inhibitors. (C) Automated boundary detection from phase contrast videos for the representative cells with the indicated treatments. Time progression is represented from blue to red. In Cdc42-inhibited cells, EF does not induce any protrusions toward the cathode, and the percentage of cells breaking symmetry does not increase with EF. Rac1-inhibited cells show small, unsustained, and random boundary protrusions without EF. Two examples are shown for Rac1-inhibited cells with EF. In the left example, the cell clearly polarizes to the cathode. In the right example, small protrusions are driven toward the cathode and suppressed at the anodal side.
Mentions: As a next step, we inhibited force generators of the actin network. The rear contractile system was inhibited with blebbistatin (nonmuscle myosin II inhibitor) or Y-27632 (Rho kinase [ROCK] inhibitor), and the protrusive front system was inhibited with CK-666 (Arp2/3 complex inhibitor). Both blebbistatin and Y-27632 significantly increased the percentage of cells with more than one lamellipodial structure but did not interfere with their symmetry-breaking ability (Figure 2A and Supplemental Figure S4B). The efficiency of Y-27632 treatment was confirmed by testing the reduction of doubly phosphorylated myosin regulatory light chain (ppMRCLC2) compared with untreated cells via immunoblotting (Supplemental Figure S4A). By contrast, Arp2/3 complex inhibition led to only 44 and 16% (using 100 or 200 μM CK-666, respectively) of cells being able to break symmetry, compared with 96% of cells in the control case (Figure 2A). Moreover, migration tracks were decreased in length by CK-666 treatment (Supplemental Figure S3B). Morphologically, inhibition of the Arp2/3 complex resulted in unpolarized cells with filopodia-like boundaries (Supplemental Figure S3B). Phalloidin staining revealed that the actin cytoskeleton was disorganized upon CK-666 treatment (Supplemental Figure S3C). In particular, the transverse arcs were reduced, and the direction of actin bundles was perpendicular to the cell periphery rather than parallel. Neither the reduction in polarization nor the loss of transverse arcs was seen with the inactive CK-666 analogue CK-689 (Supplemental Figure S3, A and C). Together these results demonstrate that Arp2/3 complex–dependent cytoskeleton rearrangements and protrusive forces at the cell periphery are key for breaking symmetry in keratinocytes, whereas myosin II– and ROCK-based contractility is not required.

Bottom Line: By contrast, we found a crucial role for extracellular pH as well as G protein coupled-receptor (GPCR) or purinergic signaling in the control of directionality.Overall our work puts forward a model in which the EF uses distinct polarization pathways.The cathodal pathway involves GPCR/purinergic signaling and is dominant over the anodal pathway at neutral pH.

View Article: PubMed Central - PubMed

Affiliation: Center for Systems Biology, University of Freiburg, 79104 Freiburg, Germany Renal Division, University Hospital Freiburg, 79106 Freiburg, Germany Spemann Graduate School of Biology and Medicine, University of Freiburg, 79104 Freiburg, Germany BIOSS Centre for Biological Signalling Studies, University of Freiburg, 79104 Freiburg, Germany.

No MeSH data available.


Related in: MedlinePlus