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Method Article
Jeremy Logue
Logue Lab
Corresponding Author
Clare Waterman
Cell Biology and Physiology Center, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, 20892
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Within the confines of tissues, cells rarely migrate by a single mechanism. Recent reports have revealed a transition from a mesenchymal to a bleb-based form of motility occurs when cells are confined. Until now, control over the amount of confinement in in vitro tissue culture required the use of microfabrication techniques. This protocol describes seven easy steps to assemble a simple system that allows control of the degree of cell confinement and the adhesivity of the cellular microenvironment in culture conditions that allow analysis of cell behavior by high-resolution light microscopic imaging. This method requires only basic lab tissue culture equipment and thus should make studies on the effects of confinement on cellular processes including cell migration, division and differentiation, accessible to a broad range of researchers. We validate that the method can precisely control confinement and that high confinement of human melanoma A375 cells promotes leader bleb-based migration.
Keywords
Cell migration, confinement, cancer, bleb, leader bleb, cytoskeleton, microfabrication
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
The authors declare no competing financial interests.
This is a list of supplementary files associated with this preprint. Click to download.
- supplement0.avi
Movie S2 High confinement will promote positioning of the nucleus inside the leader bleb and leader bleb-based migration Central Z-plane confocal time-lapse movie of leader bleb-based migration in an A375 cell that was co-expressing Emerald-F-tractin (red, actin cytoskeleton) and FusionRed-H2B (green, nucleus), confined between PDMS and BSA coated glass maintained at h= 3.11 µm by polystyrene beads. Note that the speed of migration increases after the nucleus enters the leader bleb, like the “A2” phenotype previously described by Liu et al. (2015). Scale bar and elapsed time are shown.
- supplement0.avi
Movie S1 Low confinement of cells will not promote leader bleb-based migration Central Z-plane confocal time-lapse movie of continuous blebbing in an A375 cell that was co-expressing Emerald-F-tractin (red, actin cytoskeleton) and FusionRed-H2B (green, nucleus), confined between PDMS and BSA coated glass maintained at h=5.15 µm by polystyrene beads. Scale bar and elapsed time are shown.
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Associated Publications
c-Src activity is differentially required by cancer cell motility modes
by Jeremy S. Logue, Alexander X. Cartagena-Rivera, and Richard S. Chadwick
Oncogene (08 February, 2018)
An open repository of community-contributed protocols sponsored by Nature Research.
Learn more about Protocol Exchange
Method Article
Jeremy Logue
Logue Lab
Corresponding Author
Clare Waterman
Cell Biology and Physiology Center, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, 20892
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 12 Feb, 2018
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Within the confines of tissues, cells rarely migrate by a single mechanism. Recent reports have revealed a transition from a mesenchymal to a bleb-based form of motility occurs when cells are confined. Until now, control over the amount of confinement in in vitro tissue culture required the use of microfabrication techniques. This protocol describes seven easy steps to assemble a simple system that allows control of the degree of cell confinement and the adhesivity of the cellular microenvironment in culture conditions that allow analysis of cell behavior by high-resolution light microscopic imaging. This method requires only basic lab tissue culture equipment and thus should make studies on the effects of confinement on cellular processes including cell migration, division and differentiation, accessible to a broad range of researchers. We validate that the method can precisely control confinement and that high confinement of human melanoma A375 cells promotes leader bleb-based migration.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
The authors declare no competing financial interests.
This is a list of supplementary files associated with this preprint. Click to download.
- supplement0.avi
Movie S2 High confinement will promote positioning of the nucleus inside the leader bleb and leader bleb-based migration Central Z-plane confocal time-lapse movie of leader bleb-based migration in an A375 cell that was co-expressing Emerald-F-tractin (red, actin cytoskeleton) and FusionRed-H2B (green, nucleus), confined between PDMS and BSA coated glass maintained at h= 3.11 µm by polystyrene beads. Note that the speed of migration increases after the nucleus enters the leader bleb, like the “A2” phenotype previously described by Liu et al. (2015). Scale bar and elapsed time are shown.
- supplement0.avi
Movie S1 Low confinement of cells will not promote leader bleb-based migration Central Z-plane confocal time-lapse movie of continuous blebbing in an A375 cell that was co-expressing Emerald-F-tractin (red, actin cytoskeleton) and FusionRed-H2B (green, nucleus), confined between PDMS and BSA coated glass maintained at h=5.15 µm by polystyrene beads. Scale bar and elapsed time are shown.
Loading...
Associated Publications
c-Src activity is differentially required by cancer cell motility modes
by Jeremy S. Logue, Alexander X. Cartagena-Rivera, and Richard S. Chadwick
Oncogene (08 February, 2018)