Spatial confinement induces reciprocating migration of epidermal keratinocytes and forms triphasic epithelia
Curation statements for this article:-
Curated by eLife
eLife Assessment
In this manuscript the authors established a novel three-dimensional culture system for stratified epithelia that allows epithelial cells to undergo epithelial-to-mesenchymal transition (EMT) and subsequent mesenchymal-to-epithelial transition (MET) while migrating through a membrane with 3.0-µm micropores, and, thus, provides a valuable tool to study EMT and possibly wound regeneration or metastasis. Furthermore, a set of experiments provides solid data suggesting that TGF beta signaling and actin polymerization promote movement of epithelial cells into the pores, while Piezo1 and Keratin 6 prevent keratinocyte migration and EMT.
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
Abstract
Epithelial cells undergo epithelial–mesenchymal transition (EMT) during migration and regain their epithelial phenotype in the post-migration phase (mesenchymal– epithelial transition; MET). We established an experimental system that reproduced three-dimensional triphasic epithelia, i.e., the original epithelium, its EMT, and MET. Keratinocytes (KCs), skin epithelial cells, placed on a microporous membrane migrated through 3.0-µm or larger micropores. The 3.0-µm-pored membrane induced an epithelial structure with three states: stratified KCs above the membrane, KCs showing EMT within the micropores, and a new stratified epithelium under the membrane. The membrane with larger micropores failed to maintain the triphasic epithelia. Live imaging revealed that KCs moved in a reciprocating manner, with actin-rich filopodia-like KC structures extending into and out of the 3.0-µm micropores, while the cells migrated unidirectionally into larger micropores. Piezo1 and keratin 6 were identified as negative modulators of KC entry to and exit from the 3.0-µm micropores. These results demonstrate that non-cancerous epithelial cells migrate through confined spaces in a reciprocating manner, which might help form triphasic epithelia, recapitulating wound healing processes.
Article activity feed
-
eLife Assessment
In this manuscript the authors established a novel three-dimensional culture system for stratified epithelia that allows epithelial cells to undergo epithelial-to-mesenchymal transition (EMT) and subsequent mesenchymal-to-epithelial transition (MET) while migrating through a membrane with 3.0-µm micropores, and, thus, provides a valuable tool to study EMT and possibly wound regeneration or metastasis. Furthermore, a set of experiments provides solid data suggesting that TGF beta signaling and actin polymerization promote movement of epithelial cells into the pores, while Piezo1 and Keratin 6 prevent keratinocyte migration and EMT.
-
Reviewer #1 (Public review):
Summary:
The study describes the migration of epidermal keratinocytes through porous membranes and observes a unique size selection whereby only on 3-micron membrane are keratinocytes able to migrate and reform an intact epidermis. The authors propose that the model replicates three cell states of the intact epidermis, EMT, and MET. They also show that this response depends on the actin cytoskeleton and Piezo1, and the migration could be stimulated with TGFbeta ligands.
Strengths:
Strengths of the study include the establishment of a simple yet robust in vitro model that captures all three cell states, which could be useful for future investigation of wound healing or metastasis. There is also good characterisation of the pore size effects, providing some interesting observations about the physical …
Reviewer #1 (Public review):
Summary:
The study describes the migration of epidermal keratinocytes through porous membranes and observes a unique size selection whereby only on 3-micron membrane are keratinocytes able to migrate and reform an intact epidermis. The authors propose that the model replicates three cell states of the intact epidermis, EMT, and MET. They also show that this response depends on the actin cytoskeleton and Piezo1, and the migration could be stimulated with TGFbeta ligands.
Strengths:
Strengths of the study include the establishment of a simple yet robust in vitro model that captures all three cell states, which could be useful for future investigation of wound healing or metastasis. There is also good characterisation of the pore size effects, providing some interesting observations about the physical regulation of keratinocyte migration. The images and presentation are clear.
Weaknesses:
(1) Some of the terminology would benefit from better definition or refinement. Triphasic suggests different physical behaviours (e.g. liquid-liquid phase separation) rather than cellular properties. Perhaps it would be better to refer to these as cell states or to describe the model more specifically as an invasion or EMT model. Likewise, the term 'reciprocating' implies two-way communication, but it is used to describe two-way migration or oscillating migration. Here, perhaps oscillatory would be clearer.
(2) The quantification and statistical analysis of key results could be improved. Notably, quantification of immunostaining in Figures 1 and 2 would strengthen core findings, and greater detail is needed on the sample sizes and number of experiments used for statistical analysis. These details are missing or only appear to N=1 in some places.
(3) There is an attempt to analyse the underlying molecular mechanisms, but these studies lack depth and detail. For example, it is not clear how actin, keratins, and piezo1 communicate to regulate cell migration. Are they acting directly on EMT genes such as SNAI1 or through changes in cell mechanics and cell-cell adhesions? Likewise, is TGF-beta signalling active in the system (e.g. nuclear pSMAD during cell migration)? As a result, the new biological insight is somewhat limited and confirms much of what is known about these pathways in keratinocyte migration.
-
Reviewer #2 (Public review):
Summary:
The manuscript by Nohara et al. presents a novel 3D assay that allows for stratification of epithelia, active EMT through small pores, and active MET. They show that 3um pores allow for keratinocytes to sample the pore through filopodia and up-regulate EMT genes to transverse the pores to the other side of the membrane where EMT genes are downregulated as the cells re-establish stratified epithelia. The TGFbeta pathway and actin polymerization promote the movement of cells into the pores and Piezo1 and KRT6 actively block this movement. This work provides a novel 3D assay that is likely to become a benchmark to analyze these processes using a more complex system than other current culture-specific EMT and MET assays.
Strengths:
The strengths of the manuscript include the foundational analysis of the …
Reviewer #2 (Public review):
Summary:
The manuscript by Nohara et al. presents a novel 3D assay that allows for stratification of epithelia, active EMT through small pores, and active MET. They show that 3um pores allow for keratinocytes to sample the pore through filopodia and up-regulate EMT genes to transverse the pores to the other side of the membrane where EMT genes are downregulated as the cells re-establish stratified epithelia. The TGFbeta pathway and actin polymerization promote the movement of cells into the pores and Piezo1 and KRT6 actively block this movement. This work provides a novel 3D assay that is likely to become a benchmark to analyze these processes using a more complex system than other current culture-specific EMT and MET assays.
Strengths:
The strengths of the manuscript include the foundational analysis of the pathways involved in establishing the tri-phasic epithelium. The authors have incorporated live imaging, drug studies, KO analysis, and RNA sequencing to show the relevant pathways involved.
Weaknesses:
While the authors provide strong evidence that the tri-phasic epithelium represents the EMT process, the MET process is largely relegated to the absence of EMT genes. It would be interesting to know how the stratified MET epithelia submerged in the media is similar or different from the stratified epithelia at the air-liquid interface.
-
Reviewer #3 (Public review):
Summary:
The authors established an experimental system that reproduced three-dimensional triphasic epithelia, i.e., the original epithelium, its EMT, and MET. Keratinocytes (KCs), skin epithelial cells, placed on a microporous membrane migrated through 3.0-um or larger micropores. The 3.0-um-pored membrane induced an epithelial structure with three states: stratified KCs above the membrane, KCs showing EMT within the micropores, and a new stratified epithelium under the membrane. The membrane with larger micropores failed to maintain this triphasic epithelium. Live imaging revealed that KCs moved in a reciprocating manner, with actin-rich filopodia-like KC structures extending into and out of the 3.0-um micropores, while the cells migrated unidirectionally into larger micropores. KO of Piezo1 and keratin 6 …
Reviewer #3 (Public review):
Summary:
The authors established an experimental system that reproduced three-dimensional triphasic epithelia, i.e., the original epithelium, its EMT, and MET. Keratinocytes (KCs), skin epithelial cells, placed on a microporous membrane migrated through 3.0-um or larger micropores. The 3.0-um-pored membrane induced an epithelial structure with three states: stratified KCs above the membrane, KCs showing EMT within the micropores, and a new stratified epithelium under the membrane. The membrane with larger micropores failed to maintain this triphasic epithelium. Live imaging revealed that KCs moved in a reciprocating manner, with actin-rich filopodia-like KC structures extending into and out of the 3.0-um micropores, while the cells migrated unidirectionally into larger micropores. KO of Piezo1 and keratin 6 increased KC entry to and exit from the 3.0-um micropores. Their results demonstrate that benign keratinocytes migrate through confined spaces in a reciprocating manner, which might help form triphasic epithelia, recapitulating wound healing processes.
Strengths:
Careful observation of the behaviour of keratinocytes on the different-sized pores. CrispR-Cas9 gene editing to KO Piezo 1 and keratin 6 isoforms in HaCaT keratinocytes.
Weaknesses:
There is no analysis of the matrix produced by the keratinocytes on the different pore sizes as this may influence migration.
HaCaT cells are quite different from normal keratinocytes in terms of migration. Pilcher et al. PMID: 9182674
-
