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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

Keratinocytes efficiently break symmetry in the polarization assay. (A) Representative cell undergoing polarization assay. The first image in the time series shows the rounded morphology of the cell after being subjected to 8% DMSO–containing experiment medium for 30 min and marks the starting point (0 min) for the time quantification. At image 3, the cell has completely spread, with a lamellipodium-like membrane structure around the cell body. Symmetry breaking is visible at 45 min and completed by 60 min. (B) Classification of cell morphologies after cells broke symmetry and bar chart of morphologies for supplemented and unsupplemented medium. The number of cells quantified is indicated above each column. (C) Quantification of the time it takes for symmetry breaking after the DMSO is removed. Single dots represent single cells. The red line is the average value for each condition. (D) Rose plots representing the direction of symmetry breaking for random (left) and EF-controlled (right) polarization. The length of the gray pie slices corresponds to the number of cells belonging to the particular angular slice. The percentages are given for four different quarters, represented by dashed lines. Without the extracellular cue, cells are randomly polarizing. When cells are subjected to EF, 70% define their polarity axes with their lamellipodia facing the cathode (–). The p value is calculated with Student’s t-test. **p < 0.01.
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Figure 1: Keratinocytes efficiently break symmetry in the polarization assay. (A) Representative cell undergoing polarization assay. The first image in the time series shows the rounded morphology of the cell after being subjected to 8% DMSO–containing experiment medium for 30 min and marks the starting point (0 min) for the time quantification. At image 3, the cell has completely spread, with a lamellipodium-like membrane structure around the cell body. Symmetry breaking is visible at 45 min and completed by 60 min. (B) Classification of cell morphologies after cells broke symmetry and bar chart of morphologies for supplemented and unsupplemented medium. The number of cells quantified is indicated above each column. (C) Quantification of the time it takes for symmetry breaking after the DMSO is removed. Single dots represent single cells. The red line is the average value for each condition. (D) Rose plots representing the direction of symmetry breaking for random (left) and EF-controlled (right) polarization. The length of the gray pie slices corresponds to the number of cells belonging to the particular angular slice. The percentages are given for four different quarters, represented by dashed lines. Without the extracellular cue, cells are randomly polarizing. When cells are subjected to EF, 70% define their polarity axes with their lamellipodia facing the cathode (–). The p value is calculated with Student’s t-test. **p < 0.01.

Mentions: Keratinocytes plated on a collagen type I surface acquire a distinct front–rear polarity with clearly defined leading and lagging edges. This kind of polarization is achieved via spontaneous symmetry breaking after attachment and spreading. To synchronize symmetry breaking in a group of single cells, we performed an assay that we named the “polarization assay” (Figure 1A). We rounded cells by incubating them in experiment medium containing 8% dimethyl sulfoxide (DMSO) for 30 min, followed by a medium wash. DMSO treatment leads to temporary lamellipodial loss and cell rounding, from which cells recover quickly after washout (Keren et al., 2008; Sanger et al., 1980). As cells regain volume, they spread uniformly on the substrate, producing a lamellipodium-like membrane structure encircling the cell body. Ninety-six percent of keratinocytes broke symmetry after spreading, and this was followed by migratory activity (Figure 1B and Supplemental Movie S1). Cells required an average of 48 min to break symmetry after the DMSO washout (Figure 1C). To test the external requirements for polarization, we removed the supplements recombinant EGF and bovine pituitary extract from the medium at the time of DMSO washout or 5 h before the experiment. No statistically significant difference in obtaining a stable polarity axis was observed, and the time needed to achieve symmetry breaking remained similar (Figure 1C).


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)

Keratinocytes efficiently break symmetry in the polarization assay. (A) Representative cell undergoing polarization assay. The first image in the time series shows the rounded morphology of the cell after being subjected to 8% DMSO–containing experiment medium for 30 min and marks the starting point (0 min) for the time quantification. At image 3, the cell has completely spread, with a lamellipodium-like membrane structure around the cell body. Symmetry breaking is visible at 45 min and completed by 60 min. (B) Classification of cell morphologies after cells broke symmetry and bar chart of morphologies for supplemented and unsupplemented medium. The number of cells quantified is indicated above each column. (C) Quantification of the time it takes for symmetry breaking after the DMSO is removed. Single dots represent single cells. The red line is the average value for each condition. (D) Rose plots representing the direction of symmetry breaking for random (left) and EF-controlled (right) polarization. The length of the gray pie slices corresponds to the number of cells belonging to the particular angular slice. The percentages are given for four different quarters, represented by dashed lines. Without the extracellular cue, cells are randomly polarizing. When cells are subjected to EF, 70% define their polarity axes with their lamellipodia facing the cathode (–). The p value is calculated with Student’s t-test. **p < 0.01.
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Related In: Results  -  Collection

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Figure 1: Keratinocytes efficiently break symmetry in the polarization assay. (A) Representative cell undergoing polarization assay. The first image in the time series shows the rounded morphology of the cell after being subjected to 8% DMSO–containing experiment medium for 30 min and marks the starting point (0 min) for the time quantification. At image 3, the cell has completely spread, with a lamellipodium-like membrane structure around the cell body. Symmetry breaking is visible at 45 min and completed by 60 min. (B) Classification of cell morphologies after cells broke symmetry and bar chart of morphologies for supplemented and unsupplemented medium. The number of cells quantified is indicated above each column. (C) Quantification of the time it takes for symmetry breaking after the DMSO is removed. Single dots represent single cells. The red line is the average value for each condition. (D) Rose plots representing the direction of symmetry breaking for random (left) and EF-controlled (right) polarization. The length of the gray pie slices corresponds to the number of cells belonging to the particular angular slice. The percentages are given for four different quarters, represented by dashed lines. Without the extracellular cue, cells are randomly polarizing. When cells are subjected to EF, 70% define their polarity axes with their lamellipodia facing the cathode (–). The p value is calculated with Student’s t-test. **p < 0.01.
Mentions: Keratinocytes plated on a collagen type I surface acquire a distinct front–rear polarity with clearly defined leading and lagging edges. This kind of polarization is achieved via spontaneous symmetry breaking after attachment and spreading. To synchronize symmetry breaking in a group of single cells, we performed an assay that we named the “polarization assay” (Figure 1A). We rounded cells by incubating them in experiment medium containing 8% dimethyl sulfoxide (DMSO) for 30 min, followed by a medium wash. DMSO treatment leads to temporary lamellipodial loss and cell rounding, from which cells recover quickly after washout (Keren et al., 2008; Sanger et al., 1980). As cells regain volume, they spread uniformly on the substrate, producing a lamellipodium-like membrane structure encircling the cell body. Ninety-six percent of keratinocytes broke symmetry after spreading, and this was followed by migratory activity (Figure 1B and Supplemental Movie S1). Cells required an average of 48 min to break symmetry after the DMSO washout (Figure 1C). To test the external requirements for polarization, we removed the supplements recombinant EGF and bovine pituitary extract from the medium at the time of DMSO washout or 5 h before the experiment. No statistically significant difference in obtaining a stable polarity axis was observed, and the time needed to achieve symmetry breaking remained similar (Figure 1C).

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