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Published on: February 25, 2015
Depletion of calpain2 accelerates epithelial barrier establishment and reduces growth factor-induced cell scattering
Jan Rasl1, Josef Caslavsky1, Josipa Grusanovic2
1Laboratory of Cell Signalling Institute of Microbiology of the Czech Academy of Sciences, 142 00 Prague, Czech Republic.
Abstract:
Calpain2 is a conventional member of the non-lysosomal calpain protease family that has been shown to affect the dynamics of focal and cell-cell adhesions by proteolyzing the components of adhesion complexes. Here, we inactivated calpain2 using CRISPR/Cas9 in epithelial MDCK cells. We show that depletion of calpain2 has multiple effects on cell morphology and function. Calpain2-depleted cells develop epithelial shape, however, they cover a smaller area, and cell clusters are more compact. Inactivation of calpain2 enhanced restoration of transepithelial electrical resistance after calcium switch, decreased cell migration, and delayed cell scattering induced by HGF/SF. In addition, calpain2 depletion prevented morphological changes induced by ERK2 overexpression. Interestingly, proteolysis of several calpain2 targets, including E-cadherin, β-catenin, talin, FAK, and paxillin, was not discernibly affected by calpain2 depletion. Taken together, these data suggest that calpain2 regulates the stability of cell-cell and cell-substratum adhesions indirectly without affecting the proteolysis of these adhesion complexes.
Insights
Calpain2 inactivation in epithelial cells alters cell shape and adhesion stability. Depletion of calpain2 impacts cell migration and scattering without affecting proteolysis of key adhesion proteins.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Calpain2 is a protease family member regulating cell adhesion dynamics.
- Its role in epithelial cell morphology and function requires further elucidation.
Purpose of the Study:
- To investigate the function of calpain2 in epithelial cell adhesion and morphology.
- To determine the impact of calpain2 depletion on cell-cell and cell-substratum interactions.
Main Methods:
- CRISPR/Cas9 gene editing was used to inactivate calpain2 in MDCK cells.
- Assays included measurements of cell area, cluster compactness, transepithelial electrical resistance, cell migration, and cell scattering.
Main Results:
- Calpain2-depleted cells exhibited an epithelial shape with smaller areas and more compact clusters.
- Transepithelial electrical resistance recovery was enhanced, while cell migration and scattering were reduced.
- Proteolysis of known calpain2 targets like E-cadherin and β-catenin was not significantly affected.
Conclusions:
- Calpain2 plays an indirect role in regulating the stability of cell-cell and cell-substratum adhesions.
- Calpain2's function in adhesion dynamics is independent of its proteolytic activity on specific target proteins.
