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Activation of a chimeric soluble adenylyl cyclase reorganizes microtubules near the cell periphery sufficient to disrupt the endothelial cell barrier
Author(s) -
Prasain Nutan,
Alexeyev Mikhail,
Stevens Troy
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.a1432-a
Subject(s) - forskolin , microbiology and biotechnology , adenylyl cyclase , microtubule , cytosol , microtubule polymerization , adcy10 , endothelial stem cell , chemistry , transfection , biology , cell culture , tubulin , signal transduction , adcy9 , biochemistry , in vitro , genetics , enzyme
The Pseudomonas aeruginosa toxin, ExoY, is injected through a type III secretion system into the cytosol of endothelial cells, where it associates with microtubules and acts as a soluble adenylyl cyclase (sAC) to produce cAMP. Although ExoY generates cytosolic cAMP pool that disrupts the endothelial barrier, it is unclear whether cytosolic cAMP reorganizes microtubules necessary to disrupt barrier function. We sought to determine whether activation of a soluble AC reorganizes microtubules necessary to induce endothelial cell gaps. To test this idea, retrovirus was prepared to express a green fluorescent protein fused to α‐tubulin in pulmonary microvascular endothelial cells (PMVECs). These cells were then infected with an adenovirus expressing a mammalian soluble AC construct (sAC I/II). sACI/II is catalytically inactive, unless it is stimulated by forskolin. Live cell confocal imaging was performed in the presence or absence of forskolin. Forskolin activates both endogenous transmembrane ACs and the sACI/II in live cells. Forskolin did not disrupt the PMVEC barrier or alter microtubule organization in control cells. In contrast, forskolin reorganized peripheral microtubule networks that were sufficient to induce gap formation in sACI/II expressing cells. Thus, sACI/II generates a cytosolic cAMP pool that reorganizes microtubules important for inter‐endothelial cell gap formation. Supported by HL‐60024 & HL‐66299.