A nuclear isoform of myosin I beta that contains a unique 16-amino acid amino-terminal extension has been identified. An affinity-purified antibody to the 16-amino acid peptide demonstrated nuclear staining. Confocal and electron microscopy revealed that nuclear myosin I beta colocalized with RNA polymerase II in an alpha-amanitin- and actinomycin D-sensitive manner. The antibody coimmunoprecipitated RNA polymerase II and blocked in vitro RNA synthesis. This isoform of myosin I beta appears to be in a complex with RNA polymerase II and may affect transcription.
We produced and affinity-purified polyclonal antibodies to adrenal myosin I. These antibodies recognize adrenal myosin I by Western blot analysis (116 kDa) and inhibit the actin-activated ATPase activity of purified adrenal myosin I. They also recognize a 120-kDa protein in extracts prepared from many different cell lines. Fluorescence microscopy demonstrated the presence of immunoreactive material in the perinuclear region, the leading edges, and the nuclei of 3T3 cells. Fluorescence microscopy also demonstrated nuclear staining in mouse oocytes at the germinal vesicle stage and in the pronuclei during fertilization. Confocal and immunoelectron microscopy confirmed the intranuclear localization. Electron microscopy also demonstrated staining of structures in nucleoli that are thought to be associated with rDNA transcription. Western blot analyses revealed the presence of the 120-kDa protein in extracts prepared from nuclei that are apparently free of cytosolic contamination. The same nuclear protein binds 125 I-calmodulin and is photoaffinity labeled with [␣-32 P]ATP. The 120-kDa protein was partially purified from twice washed nuclei using ammonium sulfate fractionation and gel filtration chromatography. Column fractions containing 120-kDa protein as revealed by Western blot analysis also contain K ؉ -EDTA ATPase activity. The 120-kDa protein was also shown to bind actin in the absence, but not the presence, of ATP. Since K ؉ -EDTA ATPase activity, actin, and ATP binding are defining features of the members of the myosin superfamily of proteins, we propose that the 120-kDa protein is a previously undescribed myosin I isoform that is an intranuclear actin-based molecular motor.Myosin I is a single-headed, monomeric, actin-activated ATPase (1). First described in Acanthamoeba castellanii (1), myosin I is now known to be widely distributed in metazoan cells (1-12). As additional myosin I proteins have been identified, it has become clear that there are at least four different subclasses of myosin I (1). All myosin I proteins consist of a 110 -150-kDa heavy chain and 1-6 light chains located in the neck region between the head and tail (1). This light chain has been shown to be calmodulin in vertebrate myosin I proteins (1). Immunofluorescence studies of mammalian cells have shown that myosin I is diffusely distributed throughout the entire cytoplasm and that it concentrates near cortical surfaces and in the perinuclear region (12, 13). Although evidence for specific roles of myosin I proteins in metazoan cells is lacking, there is speculation, based on localization studies, that myosin I proteins are molecular motors involved in plasma membrane extension (12, 13), vesicle and organelle transport (14), and mechanochemical regulation of calcium channels in hair cells (15). To investigate the role of myosin I in mammalian cells, we produced and affinity-purified polyclonal antibodies to adrenal myosin I. These antibodies recognize a 120-kDa protein that is found in the cytoplasm and the nucleus. Moreover, we present...
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