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D. A. Dartt, L. M. H. Jonsson; Characterization of the A-Kinase Anchoring Proteins (AKAP), AKAP150, WAVE1 and WAVE2, in Rat Lacrimal Gland and in Cultured Myoepithelial Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1910.
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© ARVO (1962-2015); The Authors (2016-present)
To detect and characterize AKAP150, WAVE1 and WAVE2 in lacrimal gland and in cultured myoepithelial cells from lacrimal gland.
Exorbital lacrimal glands were removed from Sprague-Dawley rats. To isolate myoepithelial cells, lacrimal glands were subjected to three rounds of collagenase digestion. Cells from the second and third round were grown on culture dishes in RPMI 1640 supplemented with 10% fetal bovine serum, until passaged onto coverslips and fixed in 4% paraformaldehyde. Western blot analysis was performed to detect the presence of AKAP150, WAVE1 and WAVE2 in whole lacrimal gland, acinar lysate and myoepithelial cell lysate. Samples were lysed in buffer containing 9.1 mM dibasic sodium phosphate, 1.7 mM monobasic sodium phosphate 150 mM NaCl pH 7.4, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS. To determine the localization of AKAP150, WAVE1 and WAVE2, immunofluorescence microscopy was performed on fixed lacrimal gland sections and on cultured myoepithelial cells fixed on coverslips. Antibodies specific to the different AKAPs, Protein Kinase A Regulatory subunit II (PKA RII), alpha-smooth muscle actin, phalloidin, and a mitochondria marker (OxPhos Complex V inhibitor protein) were used.
AKAP150, WAVE1 and WAVE2 were present in whole lacrimal gland, acinar cells and myoepithelial cells as distinct bands at molecular weight 150 kDa, 84 kDa and 64 kDa respectively. AKAP150, WAVE1 and WAVE2 were located at the basal membrane and in the cytosol of the acinar cells and colocalized with PKA RII in both acinar as well as myoepithelial cells. WAVE1 also colocalized with alpha-smooth muscle actin on fibers in myoepithelial cells and WAVE2 colocalized with mitochondria in lacrimal gland acinar and myoepithelial cells.
AKAP150, WAVE1 and WAVE2 are present in distinct locations in lacrimal gland acinar and myoepithelial cells, reflecting a potential differential function in these cells.
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