Overview of NLR family members according to their domain organization.

<p>Protein name and synonyms (new synonyms in bold), accession number, and chromosomal location according to <a href="http://www.genenames.org/genefamily/nlr.php" target="_blank">http://www.genenames.org/genefamily/nlr.php</a> followed by domains as defined by FFAS. CARD, caspase activation and recruitment domain; PYD, pyrin; NACHT, domain present in NAIP, CIITA, HET-E, TP-1; NALP, NACHT-LRR-PYD-containing protein; WH, winged helix domain; SH, superhelical domain, LRR, leucine-rich repeats.</p

Key elements of Apaf-1 and NLR NACHT-WH-SH domains extracted from the multiple sequence alignment in Figure 1.

<p>Sequence identities shown are derived from pairwise alignments using FFAS between Apaf-1 and individual NLRs. WH His and WH cons show the conserved histidine and the conserved METEEV sequence patch, respectively which are located in the winged helix (WH) domain.</p

Conservation of interface residues in the acidic patch and the basic patch based on the multiple sequence analysis in Figure 5A and 5B.

<p>Bold residues contribute to the interface in NOD1/RICK and Apaf-1/Caspase9, respectively.</p

Figure 5

<p>A Multiple sequence alignment of NLR and Apaf-1 CARD domains. Acidic key residues participating in the CARD-CARD interface are indicated by red borders. Residues that belong to the basic patch of the CARD-CARD interface are indicated by blue borders. Nod2.1 and Nod2.2 refer to NOD2 CARD domain 1 and 2, respectively. B Multiple sequence alignment of NLR PYRIN domains. Patch of negatively charged residues from ASC2 in helices 1 and 4 and their corresponding residues in the PYRIN domain containing NLR proteins (red box). Patch of positively charged residues from ASC2 in helices 2 and 3 and their corresponding residues in the PYRIN containing NLR proteins (blue box).</p

Multiple sequence alignment of NLR NACHT-WH-SH domains and the Apaf-1 NACHT-WH-SH domain.

<p>Degree of conservation is shown as blue shading. The secondary structure of Apaf-1 is shown above the Apaf-1 sequence. Arrows underneath the alignment indicate domain boundaries. Conserved sequence features important for catalytic activity are shown in black boxes. Orange and magenta boxes depict interfaces residues while the orange ones contribute to interactions with the left partner and the magenta residues are thought to interact with the right partner in the oligomer. Green boxes indicate additional motifs as described in the text.</p

Figure 4

<p>A NLR oligomerization interface: Apaf-1 oligomer modeled on the basis of the NtrC1 heptamer crystal structure. For clarity only three NACHT domains are shown in ribbon representation with an ADP molecule depicted in sticks to highlight the nucleotide-binding site in each domain. Side-chains of residues in the oligomerization interfaces are shown in sticks color-coded according to the alignment in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002119#pone-0002119-g001" target="_blank">Figure 1</a>. The two interfaces form across the nucleotide-binding site of the NACHT domain including the GxP domain. B Model of NLR activation and inflammasome formation based on the Apaf-1 apoptosome.</p

Model of the NOD2 nucleotide-binding site with an ADP molecule and conserved sequence motifs Walker A, Walker B, Sensor 1, GxP, and WH-His shown in sticks.

<p>Model of the NOD2 nucleotide-binding site with an ADP molecule and conserved sequence motifs Walker A, Walker B, Sensor 1, GxP, and WH-His shown in sticks.</p

Network model of the hidden spindle hubs.

<p>Hidden spindle hubs (rectangular nodes) and associated known spindle proteins (pink circle nodes). Enriched functional classes related to spindle clusters are indicated – see Methods (black labels). For the spindle interacting proteins IDs see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031813#pone.0031813.s020" target="_blank">Table S10</a></b>.</p

Mitotic phenotype observed upon depletion by siRNA of the selected predicted spindle proteins.

<p>(<b>A</b>) HeLa S3 cells were treated for 48 h with control (GL2) or <i>KIAA0841</i>-and <i>Nup88</i> specific siRNAs, respectively, then fixed and stained with α-Tubulin (green). DNA was visualized using DAPI (blue). Bar = 10 µm. (<b>B</b>) HeLa S3 cells were treated for 48 h with control (<i>GL2</i>) and p59Fyn specific siRNAs, respectively, then fixed and stained with α-Tubulin (green). DNA was visualized using DAPI (blue). Bar = 10 µm. (<b>C–G</b>) Stills of representative movies of H2B-GFP expressing HeLa S3 cells treated with control (<i>GL2</i>), <i>KIAA0841</i>, <i>Nup88</i>, <i>p59Fyn</i> and <i>WD75</i> siRNAs for 36 h before filming. Time points are indicated in h:min.</p

Summary of the methods used in this study.

<p>The class, method, type and laboratory where the methods were developed is shown.</p