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.
Ochoa
Cristhiaan D
CD
Departments of Pharmacology, University of South Alabama, Mobile, Alabama 36688, USA.
Stevens
Troy
T
Balczon
Ron
R
D002478:Cells, Cultured; D003080:Cold Temperature; D004727:Endothelium / Q000166:cytology / Q000502:physiology*; D006367:HeLa Cells / Q000166:cytology / Q000502:physiology; D006801:Humans; D008168:Lung / Q000166:cytology / Q000502:physiology*; D008833:Microcirculation / Q000502:physiology; D008870:Microtubules / Q000502:physiology*; D009474:Neurons / Q000502:physiology; D011652:Pulmonary Circulation / Q000502:physiology; D014404:Tubulin / Q000502:physiology
D014404:Tubulin