Enteropathogenic Escherichia coli (EPEC) Recruitment of PAR Polarity Protein Atypical PKCζ to Pedestals and Cell–Cell Contacts Precedes Disruption of Tight Junctions in Intestinal Epithelial Cells
Abstract
1. Introduction
2. Results
2.1. EPEC Disrupts PAR Polarity Complexes In Vivo and In Vitro
2.2. EspF, Via Its SNX9-Binding Domain, Impacts the Structural Organization of aPKCζ and F-actin at Pedestals
2.3. EspF and Its SNX9-Binding Domain Contribute to the Recruitment of Phosphorylated aPKCζ–T560 to Pedestals without Altering Kinase Activity
2.4. SNX9-Binding Domain of EspF Is Essential to Disrupt TJ Structure and Function in SKCO-15 Monolayers
2.5. Temporal Disruption of TJ Proteins Mediated by EPEC Infection
3. Discussion
4. Materials and Methods
4.1. Tissue Culture
4.2. Antibodies and Reagents
4.3. Bacterial Culture
4.4. Murine Infection
4.5. Immunofluorescence
4.6. Imaging
4.7. Western Blot Analysis
4.8. Generation of Tet-On System
4.9. Measurement of Transepithelial Electrical Resistance
4.10. PKC activity Assays
4.11. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Abbreviations
aPKCζ | Atypical protein kinase C zeta |
EPEC | Enteropathogenic Escherichia coli |
F-actin | Filamentous actin |
JAM-A S285 | Junctional adhesion molecule-A phosphorylated at Serine 285 |
JAM-A Y280 | Junctional adhesion molecule-A phosphorylated at Tyrosine 280 |
p-aPKCζ-T410 | aPKCζ phosphorylated at Threonine 410 |
p-aPKCζ-T560 | aPKCζ phosphorylated at Threonine 560 |
PS | aPKCζ pseudosubstrate inhibitor |
SNX9 | Sorting nexin 9 |
TJ | Tight junctions |
TER | Transepithelial electrical resistance |
ZO-1 | Zonula Occludens -1 |
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Tapia, R.; Kralicek, S.E.; Hecht, G.A. Enteropathogenic Escherichia coli (EPEC) Recruitment of PAR Polarity Protein Atypical PKCζ to Pedestals and Cell–Cell Contacts Precedes Disruption of Tight Junctions in Intestinal Epithelial Cells. Int. J. Mol. Sci. 2020, 21, 527. https://doi.org/10.3390/ijms21020527
Tapia R, Kralicek SE, Hecht GA. Enteropathogenic Escherichia coli (EPEC) Recruitment of PAR Polarity Protein Atypical PKCζ to Pedestals and Cell–Cell Contacts Precedes Disruption of Tight Junctions in Intestinal Epithelial Cells. International Journal of Molecular Sciences. 2020; 21(2):527. https://doi.org/10.3390/ijms21020527
Chicago/Turabian StyleTapia, Rocio, Sarah E. Kralicek, and Gail A. Hecht. 2020. "Enteropathogenic Escherichia coli (EPEC) Recruitment of PAR Polarity Protein Atypical PKCζ to Pedestals and Cell–Cell Contacts Precedes Disruption of Tight Junctions in Intestinal Epithelial Cells" International Journal of Molecular Sciences 21, no. 2: 527. https://doi.org/10.3390/ijms21020527
APA StyleTapia, R., Kralicek, S. E., & Hecht, G. A. (2020). Enteropathogenic Escherichia coli (EPEC) Recruitment of PAR Polarity Protein Atypical PKCζ to Pedestals and Cell–Cell Contacts Precedes Disruption of Tight Junctions in Intestinal Epithelial Cells. International Journal of Molecular Sciences, 21(2), 527. https://doi.org/10.3390/ijms21020527