Adherens junctions are cadherin-dependent structures that mediate intercellular
signaling and structural integrity of the endothelial barrier. Formins are a highly
conserved family of cytoskeletal remodeling proteins whose activity has been implicated
in regulating adherens junction formation in other cell-types. Therefore, we tested the
hypothesis that formin activity is essential for adherens junction assembly in endothelial
cells. A small-molecule formin inhibitor (smiFH2) was used to determine the effect of
formin inhibition on junction formation using an in vitro vascular permeability assay. We
determined that smiFH2 treatment caused a dose-dependent inhibition of junction
formation. We used siRNAs to knockdown expression of the seven formins shown to be
expressed in TIME cells and determined that individual knockdown of FHOD1, FHOD3
and Dia1 significantly increased the permeability of the endothelial monolayer.
Interestingly, FMNL2 knockdown actually potentiated barrier function. Knockdown of
the remaining formins had little or no effect on junction formation. Knockdown of
FHOD3 had the greatest inhibitory effect on junction assembly; VE-cadherin protein
levels were decreased in FHOD3-depleted cells. The FHOD3 knockdown cells were also
elongated in comparison to controls and formed thin linear adherens junctions and few
focal adherens junctions. In contrast, the morphology of FMNL2-depleted cells did not
appear obviously different from controls. In conclusion, our results suggest that multiple
formins play diverse roles in adherens junction formation and maintenance in endothelial
cells.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/34274 |
| Date | January 2016 |
| Creators | Mumal, Iqra |
| Contributors | Copeland, John |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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