Mutation of basic residues in the neck of NM1/Myo1c abolishes its nuclear import.

<p>U2OS cells were transfected with full length NM1-V5/His (<b>A</b>), NM1-V5/His lacking the second IQ motif (<b>B</b>), and NM1-V5/His with point mutation of basic amino acids within the NLS into alanines (<b>C</b>). Below the pictures are schematic representations of constructs used. Color coding is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030529#pone-0030529-g002" target="_blank"><b>Fig. 2</b></a>. Cells were fixed 48 hours post transfection and labeled with anti-V5 antibody, pictures were obtained using wide-field microscope, scale bar: 10 µm (<b>D</b>) U2OS cells transiently transfected with Myo1c-V5/His show nuclear localization of the protein Picture is a single confocal plane, obtained by confocal microscope. Scale bar: 10 µm. (<b>E</b>) Nuclear and cytosolic extracts were prepared from liver of either wild type (WT) or NM1 knock-out (KO) mice. Equal amount of protein was resolved using SDS-PAGE and electro-transferred to nitrocellulose. Membrane was probed with anti-NM1, anti-Myo1c, anti hnRNP C1/C2 and GAPDH antibody. Signal was detected using LI-COR Odyssey infrared imaging system. (<b>F</b>) U2OS cells were transiently transfected with Myo1c-V5/His. 24 hours after transfection cells were treated with nocodazole or aphidicolin to stall the cells either in G2/M or in G1/S phase of cell cycle. After the release from the block cells were cultivated for another 24 hours. Samples were taken in indicated timepoints. Cells were labeled with antibody to V5 tag, patterns counted and divided into three groups according to the localization of fluorescent proteins. More than 100 cells were counted in each timepoint, expreriment was repeated twice with similar result.</p

Identification of NM1 interacting proteins in the cytosol.

<p>Digitonin extract from suspension HeLa cells was incubated with recombinant Str-IQ12-His peptide containing N-terminal OneStrep tag (IQ12) and Streptactin beads as a control for background binding. Bound proteins were resolved on 4–20% SDS-PAGE gel and stained with SimplyBlue. Mass spectrometric analysis of the protein bands that co-purified with bait (arrows) identified importin 5 and heat shock protein 90 beta (HSP90) (<b>A</b>). SimplyBlue stained 4–20% SDS-PAGE gel with proteins that interacted with Str-GFP-NM1-(Q123.T) and Str-GFP as a control in digitonin extract of HEK293T cells. The arrows show positions of bands that contained proteins identified using mass spectrometry as importin 5, importin 7, importin-β1, HSP90 beta and calmodulin (<b>B</b>). Proteins that co-immunoprecipitate with antibody to endogenous NM1 from HeLa extracts were resolved using SDS-PAGE and tranferred onto nitrocelulose membrane. Membrane was probed with with anti-NM1, anti-importin 5 (IPO5), anti-importin 7 (IPO7), anti-importin-β1 (KPNB1). Rabbit polyclonal antibody against GFP was used as a control for backgroung binding (<b>C</b>). N-terminally Strep tagged GFP-NM1-(Q123.T) ^NLSwt (wt), GFP-NM1-(Q123.T) ^NLSmut (mut) and GFP as negative control (nc) were expressed in HEK293T cells. Cells were extracted with buffer containing digitonin (digi) to obtain soluble cytosol; pellet was re-extracted with the same buffer containing 1% Triton X-100 (triton). Bound proteins were resolved on SDS-PAGE, transferred to nitrocelulose. Membrane was incubated with antibody to importin 5 ans GFP (<b>D</b>). Beads containing Str-GFP-NM1-(Q123.T) and Str-GFP-SV40 NLS and associated proteins were eluted first with buffer containing GTP-loaded RanQ69L or buffer alone and then with biotin containig buffer that liberated Strep-tagged bait proteins from the column. Proteins eluted from the beads were resolved on SDS-PAGE and transferred to nitrocelulose membrane. GFP, importin 5 and importin-β1 signals were detected using specific antibodies (<b>E</b>). Signal from secondary antibodies was detected using LI-COR Odyssey infrared imaging system.</p

Overexpression of calmodulin influences the nuclear import of NM1.

<p>U2OS cells were co-transfected with GFP-PK constructs containing IQ domains, and calmodulin. Calmodulin was visualized using specific antibody (<b>A</b>,<b>B</b>,<b>C</b>). Scale bar 10 µm. HEK293T cells electroporated with the same constructs as in (<b>A</b>,<b>B</b>,<b>C)</b>. Whole cell extracts were subjected to immunoprecipitation with anti-GFP nanobody. Bound proteins were resolved on SDS-PAGE and transferred to nitrocelulose. GFP and CaM were visualized using specific antibodies (<b>D</b>). (<b>E</b>) Comparison of IQ1 and IQ2 sequences. The consensus IQ motif is shown below. The NM1 NLS sequence is highlighted in red.</p

Preparation of NM1 knock-out cassette.

<p>(<b>A</b>) WT genomic locus of <i>Myo1c</i> gene. Short homology arm (SA), floxed part (FP), and long homology arm (LA) of appropriate length (0.9; 0.3; 1.7 kb respectively), were cloned to pEasyFlox vector carrying neomycin (NeoR) and thymidine kinase (ThK) selection genes (<b>B</b>). Black lines represent genomic sequences; red line represents sequences derived from pEasyFlox vector. (<b>B</b>). (<b>C</b>) Genomic loci of <i>Myo1c</i> gene with excision of exon -1; P1 – P6 represent different primers needed for genotyping of ES cells and knock-out mice, yellow triangles represent loxP recombination sites. (<b>D</b>) Genotyping of NM1 knock-out mice by PCR. P5 and P6 primers were used to distinguish between wild type (+/+), heterozygous (+/−) and knock-out (−/−) animals. (<b>E</b>) Western blot analysis of NM1 levels in NM1 wild type (+/+) and knock-out (−/−) mice. Fifteen micrograms of protein per lane was loaded, and probed for NM1. Equal loading was monitored by Coomassie Brilliant Blue staining of the band corresponding to actin.</p

Overall body and bone parameters in NM1 knock-out and wild type mice.

<p>Overall body and bone parameters in NM1 knock-out and wild type mice.</p

Myo1C is able to function in Pol I and Pol II transcription without changes in its expression level.

<p>(<b>A</b>) The level of nascent rRNA was decreased to 80% of WT levels in NM1/Myo1c knock-down cells (U2OS+C8). An overexpression of mouse NM1 (U2OS+C8+NM1) or Myo1c (U2OS+C8+M1c) resistant to shRNA causes restoration of Pol I transcription to almost endogenous levels. As a negative control were used U2OS cells with transduced empty pLKO1.1 vector (U2OS+NC). (<b>B</b>) Both NM1-Flag and Myo1c-Flag interact with Pol II. Extracts from cells overexpressing NM1-Flag and Myo1c-Flag were co-immunoprecipitated with Flag antibody and control IgG. Immunoprecipitates were analyzed by western blotting with antibodies against Flag, Pol II CTD subunit and Myo1c (tail domain recognizing both NM1 and Myo1c). (<b>C</b>) NM1 knock-out does not cause compensatory changes in expression of Myo1c. Expression of Myo1c was measured by RT-qPCR and compared relative to GAPDH expression. Data are presented as mean +/− SD. *** p<0.001.</p

Ratio between NM1 and Myo1c is nearby equal.

<p>(<b>A</b>) HeLa cells were fractionated into cytosolic and nuclear fractions. NM1 and Myo1c amounts were quantified using double fluorescent labeling of western blot membranes after normalization to NM1-GFP band. (<b>B</b>) Total amounts of NM1+Myo1c were compared in mouse skin fibroblasts derived from NM1 knock-out and NM1 wild type mouse. Beta actin signal was used as loading normalizer. (<b>C</b>) Total amounts of NM1+Myo1c were compared in lungs and stomach from NM1 knock out and NM1 wild type mouse. GAPDH signal was used as loading normalizer. (<b>D</b>) The graph shows the amount of NM1 and Myo1c after densitometric quantification of bands from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061406#pone-0061406-g004" target="_blank">figures 4B and 4C</a> showing the ratio between NM1 and Myo1c as compared to actin/GAPDH expression.</p

NM1 knock-out has no effect on cell proliferation and Pol I transcription.

<p>(<b>A</b>) Proliferation of NM1 KO cells (NM1 −/−) is not altered in comparison to WT cells (NM1 +/+). 1×10⁵ cells were seeded on plates (20% confluence; day 0) and let to grow for six days when number of cells was counted again (day 6). (<b>B</b>) Pol I transcription rate in NM1 KO (NM1 −/−) and WT (NM1 +/+) cells is equal. RNA from exponentially growing cells was isolated and expression of 45S pre-rRNA was measured by RT-qPCR. Expression of 45S pre-rRNA is compared relatively to GAPDH expression. Data are presented as mean +/− SD.</p

Red blood cells related phenotype in NM1 knock-out mice.

<p>Red blood cells related phenotype in NM1 knock-out mice.</p