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First published online February 18, 2009
doi: 10.1242/10.1242/jcs.030205

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Ancient animal ancestry for nuclear myosin

¹ Department of Physiology and Biophysics, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA
² Centre for Eukaryotic Evolutionary Microbiology, School of Biosciences, University of Exeter, Devon EX4 4QD, UK

View larger version (105K): [in this window] [in a new window]   Fig. 1. Myosin-I phylogeny. (A) Subsection of the myosin-I phylogeny (see Fig. 2 for the rest of the tree). The topology shown is a PHYML tree. Posterior probability/PHYML bootstrap (100 replicates)/SH test values are marked on nodes that are directly discussed in the text. All other topology support values are marked using black or white circles depending on topology support (see key). The latest possible acquisition of NMI phenotype is marked (blue triangle). Vertebrate ortholog sets are marked with gray blocks and labeled according to the annotation convention established by Gillespie et al. (Gillespie et al., 2001). Orange triangles and lines mark duplications that occurred in the ancestral vertebrate branch. Branches of the phylogenetic tree are labeled with species followed by a combination of GenBank accession number or DOE JGI gene annotation code (given in rounded parentheses) and/or followed by the annotation name given by Odronitz and Kollmar (Odronitz and Kollmar, 2007) (given in square parentheses) if available. Non-equivalent higher taxonomic groupings are labeled. Red ovals marked `N' are sequences with two alternative putative start sites, suggesting the presence of an N-terminal candidate nuclear-retention peptide. (B) Alignment of putative NMI N-terminal-extension peptides. The putative NMI N-terminal-extension peptide identified here in Ciona (DOE JGI identifier 240514–chr_04q) is aligned with additional NMI isoforms identified by Kahle et al. (Kahle et al., 2007) in Homo (XP_0238385), Bos (NP_776821), Mus (AAG02570), Gallus (NP_001006220), Xenopus (ENSXETP00000049503), Danio (XP_695924) and Tetraodon (GSTENT00022181001).

View larger version (66K): [in this window] [in a new window]   Fig. 2. Subsection of the myosin-I phylogeny showing additional vertebrate-specific duplications, bringing the total to nine vertebrate myosin-I paralogs. Phylogeny is labeled as described in Fig. 1A. We have extended the vertebrate ortholog annotation convention established by Gillespie et al. (Gillespie et al., 2001) to include one additional ortholog group (MYO1I), labeled using a gray box.

View larger version (44K): [in this window] [in a new window]   Fig. 3. Myosin-I isoforms in C. intestinalis. (A) Sequence alignment of the myosin-I-tail-region epitope that is recognized by the anti-pan-myosin-I-tail antibody. (B) Western blot analysis of total cell extract from C. intestinalis and NIH-3T3 cells using the anti-pan-myosin-I-tail antibody (left panel) and the anti-NMI-peptide antibody (right panel). Both antibodies recognize a protein with the appropriate molecular mass (120 kD) in NIH-3T3 and C. intestinalis whole-cell extracts. Molecular-mass markers are shown in kD on the left. (C) Confocal images of C. intestinalis hemocytes showing predominantly nuclear staining by the anti-NMI-peptide antibody, a combination of distinctive cytoplasmic staining with punctate nuclear staining by the anti-pan-myosin-I-tail antibody, and exclusively cytoplasmic staining by the anti--tubulin antibody. Left column: cells stained with the indicated antibodies. Middle column: cells stained with DAPI to visualize nuclei. Right column: merged images of the first and second columns. When the cell shape was not obvious (top and bottom rows), phase-contrast images were included into the merged images. The size of the scale bar is indicated in each panel individually.

View larger version (56K): [in this window] [in a new window]   Fig. 4. Identification of a myosin-I isoform in nuclei of C. intestinalis cells. (A) Sucrose gradient showing isolation of C. intestinalis nuclei. Layer 4, broken nuclei; layer 5, intact nuclei; layer 6, cytoplasmic debris. For a complete description of each layer, see Hinegardner (Hinegardner, 1962). (B) Western blot analysis of individual layers using the indicated antibodies. Relevant molecular-mass markers are shown in kD on the left. (C) Identification of NMI in C. intestinalis by immunoprecipitation. C. intestinalis nuclear extract was incubated with antibodies to the myosin-I tail or with nonspecific IgG. Proteins bound to the antibody were precipitated and analyzed by immunoblotting using either the NMI-specific peptide antibody (left panel) or the anti-pan-myosin-I-tail antibody (right panel). Both antibodies recognize the same two bands in the fraction precipitated with the anti-pan-myosin-I-tail antibody. No bands were recognized when nonspecific IgG was used. The relevant molecular-mass marker is indicated in kD on the right.

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