TY - JOUR
T1 - Junctional Localization of Septin 2 Is Required for Organization of Junctional Proteins in Static Endothelial Monolayers
AU - Kim, Joanna
AU - Cooper, John A.
N1 - Funding Information:
This work was funded by the National Institutes of Health (NIH) R35 GM118171 to Dr Cooper.
Funding Information:
We are extremely grateful to Olivia L. Mooren and Patrick McConnell for scoring the morphology of cell junctions as well as for advice and assistance. We are grateful to Michael D. Onken for helping to score junction morphology and for invaluable advice related to statistical analysis and to RNA sequence experiments and data analysis. The Genome Technology Access Center (GTAC) of Washington University School of Medicine provided advice and assistance with RNA sequencing. Light microscopy was performed in part through the use of the Washington University Center for Cellular Imaging (WUCCI) supported by the Washington University School of Medicine, The Children’s Discovery Institute of Washington University, and St Louis Children’s Hospital (CDI, CORE-2015-505) and National Institutes of Health (NIH)/National Institute of Neurological Disorders and Stroke (NINDS) NS086741.
Publisher Copyright:
© 2020 American Heart Association, Inc.
PY - 2021/1
Y1 - 2021/1
N2 - OBJECTIVE: Septin 2 is localized at junctions in human microvascular endothelial monolayers. The junctional localization of septin 2 is necessary for organization of cell-cell adhesion proteins of endothelial cells. APPROACH AND RESULTS: Septin 2 was depleted at junctions by suppression of expression using shRNA, treatment with inflammatory cytokine, TNF (tumor necrosis factor)-α, and ectopic overexpression of septin 2 phosphatidylinositol 4,5-bisphosphate binding mutant defect in interaction with plasma membrane. Under those conditions, organizations and expression levels of various junctional proteins were analyzed. Confocal images of immunofluorescence staining showed substantial disorganization of adherens junctional proteins, nectin-2 and afadin, TJP (tight junction protein), ZO (zonula occludens)-1, and intercellular adhesion protein, PECAM-1 (platelet-endothelial cell adhesion molecule-1). Immunoblots for those proteins did not show significant changes in expression except for nectin-2 that highly increased in expression. Significant differential gene expression profiles and biological pathway analysis by septin 2 suppression and by TNF-α treatment using RNA-seq showed common overlapping pathways. The commonalities in expression may be consistent with the similar effects on the overall organization of cell-cell adhesion proteins.
AB - OBJECTIVE: Septin 2 is localized at junctions in human microvascular endothelial monolayers. The junctional localization of septin 2 is necessary for organization of cell-cell adhesion proteins of endothelial cells. APPROACH AND RESULTS: Septin 2 was depleted at junctions by suppression of expression using shRNA, treatment with inflammatory cytokine, TNF (tumor necrosis factor)-α, and ectopic overexpression of septin 2 phosphatidylinositol 4,5-bisphosphate binding mutant defect in interaction with plasma membrane. Under those conditions, organizations and expression levels of various junctional proteins were analyzed. Confocal images of immunofluorescence staining showed substantial disorganization of adherens junctional proteins, nectin-2 and afadin, TJP (tight junction protein), ZO (zonula occludens)-1, and intercellular adhesion protein, PECAM-1 (platelet-endothelial cell adhesion molecule-1). Immunoblots for those proteins did not show significant changes in expression except for nectin-2 that highly increased in expression. Significant differential gene expression profiles and biological pathway analysis by septin 2 suppression and by TNF-α treatment using RNA-seq showed common overlapping pathways. The commonalities in expression may be consistent with the similar effects on the overall organization of cell-cell adhesion proteins.
KW - Adherens junctions
KW - Cadherins
KW - Cytokines
KW - Endothelial cells
KW - Nectins
UR - http://www.scopus.com/inward/record.url?scp=85098188176&partnerID=8YFLogxK
U2 - 10.1161/ATVBAHA.120.315472
DO - 10.1161/ATVBAHA.120.315472
M3 - Article
C2 - 33147991
AN - SCOPUS:85098188176
SN - 1079-5642
VL - 41
SP - 346
EP - 359
JO - Arteriosclerosis, thrombosis, and vascular biology
JF - Arteriosclerosis, thrombosis, and vascular biology
IS - 1
ER -