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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.


GPCR signaling in directional polarization. (A) Quantification of cell morphologies for inhibition of Gαi (PTX), Gβγ (gallein), purinergic signaling (suramin [also GPCR inhibition] and PPADS), and M1 muscarinic GPCR (telenzepine). None of the inhibitors, except for gallein to an extent, showed a significant effect on cell morphology. The number of cells quantified is indicated above each column. (B, C) Rose plots representing the direction of symmetry breaking with indicated inhibitors and doses. Whereas inhibiting Gαi with PTX treatment randomizes polarization, Gβγ inhibition reverses the direction of polarization toward the anode. At the receptor level, suramin reverses the direction of polarization, and PPADS leads to randomization of direction. Inhibiting the M1 muscarinic GPCR has no effect on the direction of polarization.
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Figure 4: GPCR signaling in directional polarization. (A) Quantification of cell morphologies for inhibition of Gαi (PTX), Gβγ (gallein), purinergic signaling (suramin [also GPCR inhibition] and PPADS), and M1 muscarinic GPCR (telenzepine). None of the inhibitors, except for gallein to an extent, showed a significant effect on cell morphology. The number of cells quantified is indicated above each column. (B, C) Rose plots representing the direction of symmetry breaking with indicated inhibitors and doses. Whereas inhibiting Gαi with PTX treatment randomizes polarization, Gβγ inhibition reverses the direction of polarization toward the anode. At the receptor level, suramin reverses the direction of polarization, and PPADS leads to randomization of direction. Inhibiting the M1 muscarinic GPCR has no effect on the direction of polarization.

Mentions: GPCRs comprise another class of receptors that have been associated with electrotaxis in various cell types (Zhao et al., 2002a; Cao et al., 2013). Among them is the purinergic P2Y receptor family, which has recently also been implicated in keratinocyte electrotaxis (Riding and Pullar, 2015). We used several compounds to inhibit both GPCR and P2 receptor signaling: pertussis toxin (PTX) for Gαi inhibition, gallein for Gβγ inhibition, telenzepine for the muscarinic M1 subclass of GPCRs, suramin and pyridoxal-phosphate-6-azophenyl-2’,4’-disulfonic acid (PPADS) for P2Y and P2X receptor inhibition. In addition to its antipurinergic effect, suramin also impairs general G protein coupling to GPCRs (Beindl et al., 1996). None of these treatments impaired the ability for spontaneous polarization (Figure 4A). PTX led to a reduction of cathodal polarization, and a significant portion of cells showing a randomized response. Whereas telenzepine had no effect on directionality, PPADS led to a similar randomization of polarization as PTX. Strikingly, treatment with suramin and gallein completely reversed the direction of symmetry breaking from the cathode to the anode (Figure 4, B and C).


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)

GPCR signaling in directional polarization. (A) Quantification of cell morphologies for inhibition of Gαi (PTX), Gβγ (gallein), purinergic signaling (suramin [also GPCR inhibition] and PPADS), and M1 muscarinic GPCR (telenzepine). None of the inhibitors, except for gallein to an extent, showed a significant effect on cell morphology. The number of cells quantified is indicated above each column. (B, C) Rose plots representing the direction of symmetry breaking with indicated inhibitors and doses. Whereas inhibiting Gαi with PTX treatment randomizes polarization, Gβγ inhibition reverses the direction of polarization toward the anode. At the receptor level, suramin reverses the direction of polarization, and PPADS leads to randomization of direction. Inhibiting the M1 muscarinic GPCR has no effect on the direction of polarization.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 4: GPCR signaling in directional polarization. (A) Quantification of cell morphologies for inhibition of Gαi (PTX), Gβγ (gallein), purinergic signaling (suramin [also GPCR inhibition] and PPADS), and M1 muscarinic GPCR (telenzepine). None of the inhibitors, except for gallein to an extent, showed a significant effect on cell morphology. The number of cells quantified is indicated above each column. (B, C) Rose plots representing the direction of symmetry breaking with indicated inhibitors and doses. Whereas inhibiting Gαi with PTX treatment randomizes polarization, Gβγ inhibition reverses the direction of polarization toward the anode. At the receptor level, suramin reverses the direction of polarization, and PPADS leads to randomization of direction. Inhibiting the M1 muscarinic GPCR has no effect on the direction of polarization.
Mentions: GPCRs comprise another class of receptors that have been associated with electrotaxis in various cell types (Zhao et al., 2002a; Cao et al., 2013). Among them is the purinergic P2Y receptor family, which has recently also been implicated in keratinocyte electrotaxis (Riding and Pullar, 2015). We used several compounds to inhibit both GPCR and P2 receptor signaling: pertussis toxin (PTX) for Gαi inhibition, gallein for Gβγ inhibition, telenzepine for the muscarinic M1 subclass of GPCRs, suramin and pyridoxal-phosphate-6-azophenyl-2’,4’-disulfonic acid (PPADS) for P2Y and P2X receptor inhibition. In addition to its antipurinergic effect, suramin also impairs general G protein coupling to GPCRs (Beindl et al., 1996). None of these treatments impaired the ability for spontaneous polarization (Figure 4A). PTX led to a reduction of cathodal polarization, and a significant portion of cells showing a randomized response. Whereas telenzepine had no effect on directionality, PPADS led to a similar randomization of polarization as PTX. Strikingly, treatment with suramin and gallein completely reversed the direction of symmetry breaking from the cathode to the anode (Figure 4, B and C).

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.