Immunohistochemical studies on the distribution of cellular myosin II isoforms in brain and aorta

Abstract

The distribution of nonmuscle myosin isoforms in brain and aorta was studied by using polyclonal antibodies against two synthetic peptides selected from a region near the carboxyl terminus of bovine brain (peptide IIB) and human macrophage (peptide IIA) myosin. Immunoblots of brain homogenates and purified myosin showed two major bands stained by anti-peptide IIB (MIIB1 and MIIB2) and a minor band stained by anti-peptide IIA (MIIA2). Polyclonal anti-human platelet myosin antibodies did not react with MIIB isoforms. In cryosections from bovine, rat, and mouse brains, anti-peptide IIB stained most neuronal cells. In bovine cryosections, glial staining was also observed. In contrast, anti-peptide IIA and anti-platelet myosin antibodies primarily stained blood vessels. In bovine aorta, the anti-peptide antibodies recognized four bands, MIIB3, MIIB4, MIIA1, and MIIA2. Only MIIA2 was recognized by anti-human platelet myosin antibodies. In bovine aorta cryosections, anti-peptide IIB stained smooth muscle cells in tunica intima and tunica media but did not stain endothelial cells. Anti-peptide IIA stained smooth muscle cells in the tunica media, and endothelial cells of vaso vasorum but not of aorta. Only polyclonal anti-platelet myosin antibodies stained the endothelial cells of aorta tunica intima. These results indicate that multiple isoforms of cellular myosins exist in mammals, that these isoforms are expressed in a cell specific manner, and that the major myosin isoforms isolated from whole brain originate from neurons and, at least in bovine brain, from glia, but not from blood vessels.

Myosin IIC: A Third Molecular Motor Driving Neuronal Dynamics

“…Homogenates of Neuro-2A cells were processed from four wells (ϳ5 ϫ 10 5 cells) for SDS gel electrophoresis (7% acrylamide gels with reduced concentrations of bis-acrylamide) (Murakami and Elzinga, 1992). Proteins were transferred to polyvinylidene difluoride (PVDF) membrane by standard procedures (Towbin et al, 1979), and lanes were cut for subsequent separate incubation with antibodies against myosins IIA, IIB, and IIC.…”

Differential Expression and Functions of Cortical Myosin IIA and IIB Isotypes during Meiotic Maturation, Fertilization, and Mitosis in Mouse Oocytes and Embryos

“…Furthermore, they are differentially expressed in neurons and cultured cells. Each nonmuscle isoform may perform different cellular tasks, e.g., growth cone protrusion versus retraction (Sun and Chantler, 1991;Cheng et al, 1992;Miller et al, 1992;Murakami and Elzinga, 1992;Maupin et al, 1994;Rochlin et al, 1995).…”

“…Furthermore, they are differentially expressed in neurons and cultured cells. Each nonmuscle isoform may perform different cellular tasks, e.g., growth cone protrusion versus retraction (Sun and Chantler, 1991;Cheng et al, 1992;Miller et al, 1992;Murakami and Elzinga, 1992;Maupin et al, 1994;Rochlin et al, 1995).Notwithstanding the literature on microfilaments in mammalian oocytes and embryos, little is known about myosin II distribution or functioning in mammalian gametes. Myosin II is reported to be homogeneously distributed in the cortex of immature rat oocytes (Amsterdam et al, 1976).…”

Localization of myosin IIB at the leading edge of growth cones from rat dorsal root ganglionic cells

“…DRGs wcrc homogabd with IO mM Tris-HCI (pH 7.0), 5 mM EGTA, 5 mM EDTA, 5 mM 2-mercaptocthanol, 0.1 mM PMSF, 0.1 mM benzamidine-HCI, and 0.1 mgml OT leupeptin, pcpstatin A, and aprotinin. The homogenates (20 E(g total protein for each lane) were applied ror clectrophorcsis on 7% SDS-polyacrylamidc gels followed by immunoblotting, as dcscribed [9,IO]. Cultured cells grown on cover-glasses were pcrmatcd with cold methanol (-20°C), immediately fixed with 4% butTered paraformaldchyde and preincubated with LO% normal goat serum in phosphate-bulfercd saline.…”