Cold exposure reveals two populations of microtubules in pulmonary endothelia

Microtubules are composed of α-tubulin and β-tubulin dimers. Microtubules yield tubulin dimers when exposed to cold, which reassemble spontaneously to form microtubule fibers at 37°C. However, mammalian neurons, glial cells, and fibroblasts have cold-stable microtubules. While studying the microtubule toxicity mechanisms of the exotoxin Y from Pseudomonas aeruginosa in pulmonary microvascular endothelial cells, we observed that some endothelial microtubules were very difficult to disassemble in the cold. As a consequence, we designed studies to test the hypothesis that microvascular endothelium has a population of cold-stable microtubules. Pulmonary microvascular endothelial cells and HeLa cells (control) were grown under regular cell culture conditions, followed by exposure to an ice-cold water bath and a microtubule extraction protocol. Polymerized microtubules were detected by immunofluorescence confocal microscopy and Western blot analyses. After cold exposure, immunofluorescence revealed that the majority of HeLa cell microtubules disassembled, whereas a smaller population of endothelial cell microtubules disassembled. Immunoblot analyses showed that microvascular endothelial cells express the microtubule cold-stabilizing protein N-STOP (neuronal stable tubule-only polypeptides), and that N-STOP binds to endothelial microtubules after cold exposure, but not if microtubules are disassembled with nocodazole before cold exposure. Hence, pulmonary endothelia have a population of cold-stable microtubules

Filamin A is a phosphorylation target of membrane but not cytosolic adenylyl cyclase activity

Transmembrane adenylyl cyclase (AC) generates a cAMP pool within the subplasma membrane compartment that strengthens the endothelial cell barrier. This cAMP signal is steered toward effectors that promote junctional integrity and is inactivated before it accesses microtubules, where the cAMP signal causes phosphorylation of tau, leading to microtubule disassembly and barrier disruption. During infection, Pseudomonas aeruginosa uses a type III secretion system to inject a soluble AC, ExoY, into the cytosol of pulmonary microvascular endothelial cells. ExoY generates a cAMP signal that disrupts the endothelial cell barrier. We tested the hypothesis that this ExoY-dependent cAMP signal causes phosphorylation of tau, without inducing phosphorylation of membrane effectors that strengthen endothelial barrier function. To approach this hypothesis, we first discerned the membrane compartment in which endogenous transmembrane AC6 resides. AC6 was resolved in caveolin-rich lipid raft fractions with calcium channel proteins and the cell adhesion molecules N-cadherin, E-cadherin, and activated leukocyte adhesion molecule. VE-cadherin was excluded from the caveolin-rich fractions and was detected in the bulk plasma membrane fractions. The actin binding protein, filamin A, was detected in all membrane fractions. Isoproterenol activation of ACs promoted filamin phosphorylation, whereas thrombin inhibition of AC6 reduced filamin phosphorylation within the membrane fraction. In contrast, ExoY produced a cAMP signal that did not cause filamin phosphorylation yet induced tau phosphorylation. Hence, our data indicate that cAMP signals are strictly compartmentalized; whereas cAMP emanating from transmembrane ACs activates barrier-enhancing targets, such as filamin, cAMP emanating from soluble ACs activates barrier-disrupting targets, such as tau

Pseudomonas aeruginosa exotoxin Y-mediated tau hyperphosphorylation impairs microtubule assembly in pulmonary microvascular endothelial cells.

Pseudomonas aeruginosa uses a type III secretion system to introduce the adenylyl and guanylyl cyclase exotoxin Y (ExoY) into the cytoplasm of endothelial cells. ExoY induces Tau hyperphosphorylation and insolubility, microtubule breakdown, barrier disruption and edema, although the mechanism(s) responsible for microtubule breakdown remain poorly understood. Here we investigated both microtubule behavior and centrosome activity to test the hypothesis that ExoY disrupts microtubule dynamics. Fluorescence microscopy determined that infected pulmonary microvascular endothelial cells contained fewer microtubules than control cells, and further studies demonstrated that the microtubule-associated protein Tau was hyperphosphorylated following infection and dissociated from microtubules. Disassembly/reassembly studies determined that microtubule assembly was disrupted in infected cells, with no detectable effects on either microtubule disassembly or microtubule nucleation by centrosomes. This effect of ExoY on microtubules was abolished when the cAMP-dependent kinase phosphorylation site (Ser-214) on Tau was mutated to a non-phosphorylatable form. These studies identify Tau in microvascular endothelial cells as the target of ExoY in control of microtubule architecture following pulmonary infection by Pseudomonas aeruginosa and demonstrate that phosphorylation of tau following infection decreases microtubule assembly

ExoY activity causes a decrease in microtubules in PMVECs.

<p>[<b>A</b>] PMVECs infected with <i>P.aeruginosa</i> expressing either non-functional K81M mutant ExoY (ExoY^K81M; center) or wild type ExoY (right) were observed following processing for anti-tubulin immunofluorescence microscopy. Uninfected cells are also shown (left). Bar = 10 µm. [<b>B</b>] Levels of polymerized tubulin (P) and unpolymerized soluble tubulin (S) were quantified by immunoblot analysis using antibody against α-tubulin. Extracts obtained from untreated cells (Ctr) and from PMVECs infected with <i>P. aeruginosa</i> expressing either ExoY^K81M or ExoY⁺ are shown. The ratio of polymerized tubulin to soluble tubulin was significantly less in cells containing wild type ExoY⁺ [0.29±0.06 <i>vs.</i> 0.44±0.05 (ExoY^K81M) and 0.49±0.08 (Ctr); n = 5; P<0.05 compared to both ExoY^K81M and untreated control].</p

ExoY activity does not noticeably affect microtubule assembly from PMVEC centrosomes.

<p>PMVECs were infected with <i>P. aeruginosa</i> expressing either ExoY^K81M (middle panel) or ExoY⁺ (left panel). Untreated cells (Ctr) and infected cells were extracted to remove soluble proteins and then purified rat brain tubulin was added and microtubule nucleation was initiated by incubating at 37°C for 15 min. The preparations then were fixed and labeled with antitubulin antibodies. Centrosome nucleation of microtubules is shown by arrows. Bar = 10 µm.</p