IMPDH activity in the presence of nucleotides.

<p>Activity of purified His-IMPDH proteins following 20 min pre-incubation with 1 mM or 5 mM nucleotides (ATP, AMP, XMP) and normalised to control, no addition, activity. Shown is the mean ± SEM (n = 3, **<i>p</i><0.01, ***<i>p</i><0.001 <i>cf.</i> to the control activity).</p

Immuno-EM of IMPDH localisation.

<p>Representative electron micrographs of CHO cells treated with (A) vehicle or (B and C) 2 µM MPA for 4 h. Cells were fixed, processed and labelled with anti-panIMPDH antibody and gold-labelled anti-mouse secondary antibody, as outlined in methods. Plasma membrane (PM), Nucleus (N) and mitochondria (M) are indicated. Scale bars = 0.5 µm.</p

Investigating a role for the Bateman domain in IMPDH clustering.

<p>(A) Superimposed structure of IMPDH2 (1B3O; yellow) with IMPDH1 (1JCN; red). Ligands have been removed for clarity. N labels the N-terminus. (B) Micrographs of CHO cells transiently expressing HA-IMPDH proteins (as labelled), treated with vehicle (control) or 1 µM MPA for 4 h. Cells were fixed and labelled for HA (HA-IMPDH; green) and nuclei were counterstained with DAPI (blue). Representative of at least four independent experiments. (C) Representative micrographs of CHO cells transiently expressing IMPDH1/IMPDH2 chimera (as labelled). Cells were fixed and stained for HA (HA-IMPDH; green) and nuclei were counterstained with DAPI (blue). Scale bars = 10 µm. (D) Table shows qualitative scoring of chimera subcellular distribution pattern according to the similarity to IMPDH1 (macrostructure formation) or IMPDH2 (diffuse) distribution. Assignments were based on three independent experiments. Shown by the schematics are the IMPDH1 (red) and IMPDH2 (yellow) regions of chimera constructs, with internal numbering referring to IMPDH1 sequence boundary and the striped boxes indicating the CBS dimer.</p

R224P mutation affects ATP mediated protease protection and affects spontaneous clustering.

<p>(A) Representative Coomassie stained gel of a protease protection assay with His-IMPDH1 proteins. INM stands for IMP, NAD and MPA. (B) Quantitation of remaining full-length protein from protease protection assay presented as percent protection. Shown is the mean ± SEM and the number of experiments (n) is indicated below the graph. (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 <i>cf.</i> the control (digested), # <i>p</i><0.01 <i>cf.</i> IMPDH1. (C) Representative micrographs of CHO cells transiently expressing HA-IMPDH1, HA-IMPDH1 R224P or HA-IMPDH1 D226N. Cells were fixed and stained for HA (HA-IMPDH; green) and nuclei were counterstained with DAPI (blue). Representative of at least four independent experiments. Scale bar = 10 µm. (D) CHO cells transiently expressing HA-IMPDH proteins, as indicated, were treated with either vehicle, 2 µM MPA for 4 h or 2 µM MPA for 4 h and supplemented with 100 µM guanosine for the final 2 h. Cells were fixed and stained with anti-HA antibody. From random fields, 50–80 labelled healthy cells were counted, in a blinded manner, and the classification of the subcellular distribution of the protein classified as diffuse (blue), in spicules (red) or macrostructures (green). Representative of two independent experiments.</p

IMPDH1 directly binds ATP.

<p>(A) Representative ATP binding experiment with His-IMPDH proteins, shows [³²P] ATP bound to His-IMPDH1 only in the presence of 5 µM cold ATP. Data represents mean counts ± SD. (B) Specific binding was determined by expressing the counts per reaction as a fold over the non-specific counts present in the BSA sample. Shown is the mean specific binding ± SEM (fold over non-specific binding) from three to six independent experiments (***<i>p</i><0.001 compared to the control).</p

Analysis of IMPDH macrostructures in cryofixed material by correlative light and electron microscopy.

<p>HeLa cells selected for stable low expression of HA-IMPDH2-GFP were treated with 2 µM MPA for 4 h prior to high pressure freezing, freeze-substitution, and embedding in resin at low temperature. Semi-thin sections were viewed by fluorescence light microscopy to detect GFP-labelled macrostructures (arrows, upper left panel) and then ultrathin sections of the same areas were prepared for viewing in the TEM. Sections were immuno-gold labelled for detection of GFP. The panels show the same area (boxed) at increasing magnification to show the ultrastructure of the IMPDH macrostructures. Note the filamentous striated nature of the labelled elements in the cryofixed freeze substituted sample. Mitochondria (M) are indicated. Scale bars = 200 nm.</p

Optical sections of IMPDH macrostructures.

<p>Representative confocal z-series of HeLa cells treated with 1 µM MPA for 4 h. Cells were fixed, permeabilised and labelled with the anti-panIMPDH antibody (green) and nuclei were counterstained with DAPI (blue). (A) Montage of images from apical (top left) to basal (bottom right) are spaced by increments of 0.539 µm on the z-axis. Scale bar = 20 µm. (B) Stacked images were viewed with Zeiss Zen 2007 software using the transparency rendering mode in the 3-dimension function. The plane of the cells was rotated to view macrostructures wrapping around the nucleus and the tops of the “donut” macrostructures. Axis' are coloured to orientate to the top left image in A.</p

Nucleotides protect IMPDH in an isoform-specific manner via the Bateman domain.

<p>(A) Representative Coomassie stained SDS-PAGE gel of a protease protection assay experiment, performed as outline in methods, with His-IMPDH2. INM stands for IMP, NAD and MPA. Molecular weight marker sizes are indicated. (B) Quantitation of remaining full-length protein from protease protection assay with His-IMPDH2 and His-IMPDH1 presented as percent protection. Shown is the mean ± SEM and the number of experiments (n) is indicated below the graph. (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 <i>cf.</i> the control (digested), # <i>p</i><0.01 IMPDH1 <i>cf.</i> IMPDH2). (C) Representative gel of a protease protection assay experiment with His-Core2 protein. (D) Quantitated results as per B. Shown is the mean ± SEM from five independent experiments. (***<i>p</i><0.001 <i>cf.</i> the control (digested), ## <i>p</i><0.001 IMP, NAD and MPA <i>cf.</i> adenosine nucleotides).</p

MPA induced clustering of HA-IMPDH2-GFP in live cells.

<p>Images are maximum intensity projections from confocal z-series of live HeLa HA-IMPDH2-GFP cells treated with 2 µM MPA. Selected frames, from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051096#pone.0051096.s010" target="_blank">Video S1</a>, are presented with the time captured relative to the addition of MPA recorded at the top right (h∶mm∶ss). Data is representative of three independent experiments. (A) Field of cells with low HA-IMPDH2-GFP expression, which is initially diffuse throughout the cytosol and with time HA-IMPDH2-GFP clusters into spicules and macrostructures. Scale bar = 20 µm. (B) Selected frames within subset region shown in A, corresponds to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051096#pone.0051096.s011" target="_blank">Video S2</a>, which highlight emergence of macrostructures, movement while undergoing a 180° clockwise rotation of in the X-Y plane (indicated by yellow arrow) and end-to-end fusion of spicules (indicated by yellow asterisk). Scale bar = 10 µm.</p