Evidence for the Presence of Myosin I in the Nucleus

Abstract

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...

Nuclear localisation of the G-actin sequestering peptide thymosin β4

“…Recently, the nuclear localisation of actin itself (Scheer et al, 1984;Gonsior et al, 1999), actin-related proteins (Arps) (Cairns et al, 1998;Harata et al, 1999;Harata et al, 2000) and a number of actin binding proteins (Nowak et al, 1997;Giesemann et al, 1999;Pestic-Dragovich et al, 2000;Skare et al, 2003) has been reported. Although the functions of nuclear actin are far from being fully understood, it has been proposed that it might be involved in chromatin remodelling (Zhao et al, 1998;Shen et al, 2003), mRNA processing and transport (Hofmann et al, 2001;Percipalle et al, 2001;Percipalle et al, 2002).…”

“…Lately, there has been increasing evidence for the presence of cytoskeletal proteins in the nucleus, such as actin itself (Scheer et al, 1984;Gonsior et al, 1999), actin-related proteins (Arps) (Cairns et al, 1998;Harata et al, 1999;Harata et al, 2000) and a number of different actin binding proteins (Nowak et al, 1997;Giesemann et al, 1999;Pestic-Dragovich et al, 2000;Skare et al, 2003). Although the functions of these proteins in the nucleus are still under investigation, there is evidence that they are involved in activities ranging from nuclear assembly and shape changes to DNA replication and transcription (Rando et al, 2000;Olave et al, 2002;Pederson and Aebi, 2002;Shumaker et al, 2003).…”

An Emerin “Proteome”: Purification of Distinct Emerin-Containing Complexes from HeLa Cells Suggests Molecular Basis for Diverse Roles Including Gene Regulation, mRNA Splicing, Signaling, Mechanosensing, and Nuclear Architecture

“…The nuclear and cytosolic isoforms are identical except for 16 extra residues at the N-terminus of the nuclear isoform (33,58). NMI is necessary for mRNA transcription in vitro, binds directly to the large subunit of RNA polymerase II, and is proposed to facilitate movement of the transcription machinery (33,59) or chromatin itself during transcription (47).…”

Ancient animal ancestry for nuclear myosin

“…NMI contains a unique N-terminal peptide, because the transcription start site for NMI is in an exon that is located upstream to the start site for cytoplasmic myosin IC (PesticDragovich et al, 2000). Functionally, NMI colocalizes with RNA polymerase I (Fomproix and Percipalle, 2004;Nowak et al, 1997) and RNA polymerase II (Fomproix and Percipalle, 2004;Nowak et al, 1997;Pestic-Dragovich et al, 2000), and co-purifies and coimmunoprecipitates with both polymerases (Pestic-Dragovich et al, 2000;Philimonenko et al, 2004). Additionally, NMI-specific antibodies inhibit transcription by these polymerases both in vivo and in vitro (Fomproix and Percipalle, 2004;Pestic-Dragovich et al, 2000;Philimonenko et al, 2004).…”