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

Neck domain of NM1 contains the NLS.

<p>U2OS cell transfected with a panel of truncation constructs of full length NM1 (<b>A–F</b>) and IQ domains fused to GFP-PK (<b>G–L</b>). Cells were fixed 48 hours post transfection. Below the pictures are schematic representations of the truncations affecting various NM1 domains as well as the GFP-PK phusions. Pictures (<b>A–F</b>) were acquired using confocal microscope, single confocal planes are shown. Pictures (<b>G–L</b>) were photographed using wide-field fluorescent microscope. Scale bar: 10 µm.</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