Additional file 2: of Are online symptoms checkers useful for patients with inflammatory arthritis?

Dataset used for this study. (XLSX 70 kb

Keks are Trk-like receptors expressed in the CNS.

<p>(A) Modular composition of TrkB, TrkB-T1, Dror, Otk and <i>Drosophila</i> LIGs. (B) Amongst the LIGs, Keks are closer to the Trks than any other mammalian or <i>Drosophila</i> LIGs, adapted from the phylogeny of Mandai et al.[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006968#pgen.1006968.ref022" target="_blank">22</a>]. (C,D) mRNA distribution in embryos: <i>CG15744</i>, <i>lambik</i> and <i>CG16974</i> are not expressed in the VNC (arrows) above background, but <i>lambik</i> is in PNS and <i>CG16974</i> in muscle precursors (arrowheads); <i>kek-1</i>, <i>kek-2</i> and <i>kek-6</i> transcripts are found in the VNC, and <i>kek5GAL4>tdTomato</i> drives expression in VNC and PNS (right) neurons. (E) Over-expression of <i>keks</i>– most prominently <i>kek2</i> and <i>6</i> -in all neurons with <i>elavGAL4</i> rescued the cold semi-lethality of <i>DNT1</i>^<i>41</i> <i>DNT2</i>^{<i>e03444</i>} double mutants, n = 52–313 pupae. Chi-square and Bonferroni multiple comparisons correction. *p<0.05, ***p<0.001. For statistical details see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006968#pgen.1006968.s006" target="_blank">S1 Table</a>.</p

Kek-6 is expressed pre-synaptically in motoneurons and binds post-synaptic DNT2.

<p>(A) In Kek-6^GFP larval VNCs, GFP colocalises with the neuronal marker HB9 (arrows show examples). (B) Kek-6^GFP was found in third instar larval muscle 6/7 NMJ and synaptic boutons (dotted rectangle: higher magnification, right). (C) Kek-6^GFP was found in the motoneuron axonal terminal (arrows), and in pre-synaptic bouton lumen (dotted rectangle: higher magnification, right), not colocalising with the post-synaptic marker anti-Dlg (arrows).(D) Kek-6>FlyBow was localized to CNS axons and dendrites (arrows), and cell bodies of the RP3,4,5 motoneuron clusters (ventral and transverse views, arrows). (E) Illustration. (F) Kek-6>FlyBow was also distributed along the motoneuron axons, NMJ terminal (arrow) and synaptic boutons (arrows). (G-K) Over-expression of GFP tagged full-length DNT2 in muscle <i>(MhcGAL4>UAS-DNT2-FL-GFP)</i> revealed: (G) DNT2-GFP distribution within the pre-synaptic bouton lumen (arrows), boutons labeled post-synaptically with anti-Dlg; (H-K) DNT2-GFP along the motoraxon (labeled with anti-FasII) and within the pre-synaptic bouton lumen (arrows).</p

<i>kek6</i> and <i>DNT2</i> mutants have smaller NMJs and impaired locomotion.

<p>(A) Plotted trajectories of filmed larvae, and (B) histograms of percentage frames at each speed analysed with FlyTracker. Kruskal-Wallis p<0.0001 and ***p<0.001 post-hoc Dunn test, n≥22344 frames. (C-E) Speed distribution classified into arbitrary categories. (C) Mutants spend more time at the lowest speeds than controls, generally do not crawl at the higher speeds (pale grey, left), but like controls can reach the highest speeds for a small fraction of time. (D) Wild-type larvae are hardly at speed = 0, contrary to the mutants. (E) All genotypes can achieve the highest speeds, but none spend much time crawling at these speeds. (F) NMJs (left, with higher magnification details of areas indicated by asterisks) and box-plot graphs (right) showing: <i>kek-6</i>^{<i>–/–</i>}and <i>DNT2</i>^{<i>–/–</i>}single mutants and <i>kek-6</i>^{<i>–/–</i>}<i>DNT2</i>^<i>-/-</i>double mutants have fewer Dlg+ boutons, smaller HRP+ axonal terminals (normalized to muscle area, MSA), and less complex NMJs with reduced axonal branching. Dlg: Kruskal-Wallis p<0.0001, and *p<0.05, **p<0.01, ***p<0.001 post-hoc Dunn; HRP: One Way ANOVA p<0.0001, and **p<0.01, ***p<0.001 post-hoc Dunnett. <i>kek-6</i> and <i>DNT2</i> single mutants, but not the double mutants, have increased active zone density (Brp+/HRP+axonal length). Brp: Kruskal-Wallis p = 0.0012, and **p<0.01, ***p<0.001 Dunn’s post-hoc. <i>Kek-6</i> mutants have reduced Synapsin, Mann-Whitney U test ***p<0.001. For statistical details, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006968#pgen.1006968.s006" target="_blank">S1 Table</a>. N = 30–113 hemisegments. Mutant genotypes throughout figures: Control: <i>yw/+;</i> Mutants: <i>kek-6</i>^{<i>–/–</i>}: <i>kek6</i>^<i>34</i><i>/Df(3R)6361; DNT2</i>^{<i>–/–</i>}: <i>DNT2</i>^<i>37</i><i>/Df(3L)6092; kek-6</i>^{<i>–/–</i>}<i>DNT2</i>^{<i>–/–</i>}: <i>kek6</i>^<i>34</i><i>Df(3L)6092/</i> Df(3R)6361 <i>DNT2</i>^<i>37</i>.</p

VAP33A functions downstream of Kek-6.

<p>(A) Confocal images of NMJs from segments A3-4, muscle 6/7. (B-E) Box-plot graphs. (B) <i>VAP33A</i>^<i>G0231</i> mutants have reduced bouton number, Mann-Whitney U test ***p<0.001. (C,D) Pre-synaptic over-expression of <i>VAP33A</i> rescues bouton number in (C) <i>kek-6</i>^{<i>–/–</i>}mutants and (D) <i>DNT2</i>^{<i>–/–</i>}single mutants, Kruskal-Wallis p<0.0001 and *p<0.05, ***p<0.001 post-hoc Dunn for both. (E) <i>kek-6</i>^{<i>–/–</i>}<i>DNT2</i>^{<i>–/–</i>}double mutants rescue the bouton number phenotype caused by <i>VAP33A</i> gain of function, Kruskal-Wallis p<0.0001 and **p<0.01, ***p<0.001 post-hoc Dunn. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006968#pgen.1006968.s006" target="_blank">S1 Table</a>. N = 23–48 hemisegments. MN = motoneuron, <i>D42GAL4</i> (D) or <i>Toll-7GAL4</i> (E); Neurons = <i>elavGAL4</i>. Rescue genotypes: (C) <i>UASVAP33A/+; D42GAL4 kek6</i>^<i>34</i><i>/Df(3R)6361</i>. (D) <i>UASVAP33A/+; elavGAL4 Df(3L)6092/DNT2</i>^<i>37</i>.. (E) <i>UASVAP33A/Toll-7GAL4; kek6</i>^<i>34</i><i>Df(3L)6092/ Df(3R)6361 DNT2</i>^<i>37</i>.</p

Retrograde DNT2 binds pre-synaptic Kek-6 activating CaMKII and regulating structural synaptic plasticity.

<p>(A) Illustration of Kek-6 compared to Trk isoforms. DNT2 binds Kek-6, which functions via CaMKII and VAP33A downstream. (B) Pre-synaptic motoneuron terminal at the NMJ: DNT2 is produced at the muscle and secreted, binds pre-synaptic Kek-6, functioning via CaMKII and VAP33A downstream. DNT2 also binds Toll-6 which can interact with Toll-6. (C) The concerted functions of DNT2 and its two receptors Kek-6 and Toll-6 regulates NMJ growth and synaptic structure. Kek-6 functions via CaMKII and VAP33A downstream, the mechanism downstream of Toll-6 at the NMJ has not been investigated in this work. Red arrows: positive regulation by Kek-6; blue arrows: positive regulation by Toll-6. (D-F) Summary of the experimental evidence provided, green arrows indicate up- or down-regulation as a result or loss or gain function genotypes. Altering the levels of DNT2, Kek-6 and Toll-6 affects locomotion, NMJ growth and synaptic structure. Importantly, loss of both kek-6 and Toll-6 prevents homeostatic compensation of active zones, and whereas gain of function for kek-6 or Toll-6 is not sufficient to increase NMJ size, over-expression of DNT2 can. The data suggest that Kek-6 and Toll-6 function in concert as a receptor complex for DNT2, to regulate structural synaptic plasticity.</p

Kek-6 functions downstream of DNT2.

<p>Confocal images of NMJs from A3-4 muscle 6/7 (left), and box-plot graphs (right), showing: (A) Over-expression of <i>kek-6</i> in motoneurons rescued the phenotypes of <i>kek-6</i> mutants. Dlg: One Way ANOVA p<0.0001 and post-hoc Bonferroni *p<0.05, ***p<0.001. HRP: One Way ANOVA p<0.001 and post-hoc Bonferroni **p<0.01, **p<0.01. (B) Left: Over-expression of <i>kek-6</i> in neurons rescued the phenotype of <i>DNT2</i> mutants. Dlg: Kruskal-Wallis p = 0.001, and post-hoc Dunn test **p<0.01, ***p<0.001. Right: <i>kek-6</i> loss of function rescued the increase in boutons caused by the muscle over-expression of <i>DNT2</i>. Dlg: Welch ANOVA p<0.01 and post-hoc Games-Howell *p<0.05, **p<0.01. (C) Over-expression of <i>kek-6</i> in motoneurons rescued the phenotypes of <i>kek-6 DNT-2</i> double mutants. Dlg: Kruskal-Wallis p = 0.001 and post-hoc Dunn’s test *p<0.05, **p<0.01. HRP: Welch ANOVA p = 0.000, post-hoc Games Howell **p<0.01. n = 29–101 hemisegments. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006968#pgen.1006968.s006" target="_blank">S1 Table</a>. GAL4 drivers: Muscle: <i>MhcGAL4</i>; Neurons: <i>elavGAL4;</i> MN: <i>D42 or Toll-7GAL4</i>. Controls: white boxes: yw/+; grey boxes: GAL4/+; mutant genotypes as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006968#pgen.1006968.g004" target="_blank">Fig 4</a>. Rescue genotypes: (A) <i>w; UASkek6RFP/+; Df(3R)6361/kek6</i>^<i>34</i><i>D42GAL4</i>. (B) <i>w; UASkek6RFP/+; elavGAL4 Df(3L)6092/ DNT2</i>^<i>37</i>; and <i>w; UASDNT2-FL/+; Df(3R)6361/kek6</i>^<i>34</i><i>D42GAL4</i>. (C) <i>w; Toll-7GAL4/UASkek6RFP; kek6</i>^<i>34</i><i>Df(3L)6092/ Df(3R)6361 DNT2</i>^<i>37</i>.</p

CaMKII functions downstream of Kek6 and DNT2 at the NMJ.

<p>Confocal images showing A3-4 muscle 6/7 NMJs, labeled as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006968#pgen.1006968.g009" target="_blank">Fig 9</a>. (A) Reduced pCaMKII^T287 levels in <i>kek-6</i> mutants, and <i>kek-6 DNT2</i> double mutants. Kurskal-Wallis p = 0.0009, and post-hoc Dunn. (B-D) Over-expresison of activated <i>CaMKII</i>^<i>T287D</i> in motoneurons rescued the NMJ phenotypes of (B) <i>kek-6</i> mutants, Kruskal-Wallis p = 0.0081, post-hoc Dunn; (C) <i>DNT2</i> mutants, Kruskal-Wallis p = 0.0001, post-hoc Dunn, and (D) <i>kek-6 DNT2</i> double mutants. Welch ANOVA p<0.000, post-hoc Games-Howell. *p<0.05, **p<0.01, ***p<0.001 see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006968#pgen.1006968.s006" target="_blank">S1 Table</a>. N = 23–75 hemisegments. Genotypes: MN = motoneurons, <i>D42GAL4;</i> Neurons: <i>elavGAL4</i>. Different neuronal drivers were used due to genetic constraints. Control: wild-type: <i>yw/+;</i> grey boxes: <i>D42GAL4/+</i>. (A) Mutant genotypes as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006968#pgen.1006968.g004" target="_blank">Fig 4</a>; Rescues: (B) <i>w;UASCaMKII</i>^<i>T287D</i><i>/+; D42GAL4 kek6</i>^<i>34</i><i>/ Df(3R)6361</i>. (C) <i>w;UASCaMKII</i>^<i>T287D</i><i>/+; elavGAL4 Df(3L)6092/DNT2</i>^<i>37</i>. (D) <i>w; UASCaMKII</i>^<i>T287D</i><i>/Toll-7GAL4; kek6</i>^<i>34</i><i>Df(3L)6092/ Df(3R)6361 DNT2</i>^<i>37</i>.</p

Kek-6 and Toll-6 interact for NMJ structural homeostasis.

<p>(A) Toll-6GAL4>mCD8-GFP is distributed in FasII+ motoneuron axons (arrows) at the muscle 6/7 NMJ terminal. (B) Muscle 6/7 NMJs (left) and box-plot graphs (right) showing: <i>Toll-6</i>^{<i>MIO2127</i>}<i>/Df(3L)BSC578</i> mutants had fewer 1b boutons. <i>Toll-6</i>^{<i>–/–</i>}and <i>Toll-6</i>^{<i>MIO2127</i>}<i>Df(3R)6361/kek6</i>^<i>35</i> <i>Df(3L)BSC578</i> double mutants had smaller NMJs (HRP, Kruskal-Wallis p = 0.0001) with reduced branching, and reduced active zones (Brp, Kruskal-Wallis p = 0.0055), post-hoc Dunn for both *p<0.05, ***p<0.001. Pre-synaptic over-expression of <i>kek-6</i> in motoneurons in Toll-6^-/-mutants (<i>w; UASkek-6/+; Toll-6</i>^{<i>MIO2127</i>}<i>GAL4/ Df(3L)BSC578</i>) did not rescue NMJ size, but upregulated Brp+. Over-expression of activated <i>Toll-6</i>^<i>CY</i> did not affect NMJ size (HRP) but increased active zones. N = 34–46 hemisegments. (C) Co-immunoprecitation from co-transfected S2 cells: Precipitating Toll-6 and Toll-7 with anti-Flag brought down Kek-6 detected with anti-HA. IP: immuno-precipitation; WB: western blot; asterisk: co-IP. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006968#pgen.1006968.s006" target="_blank">S1 Table</a>. <i>MN = D42GAL4</i>. </p

Kek-6 and DNT2 can induce active zones and NMJ growth.

<p>Confocal images of NMJs from A3-4 muscle 6/7 (left, higher magnification deail of areas indicated by asterisks), and box-plot graphs (right), showing: (A) Over-expression of <i>kek6</i> in motoneurons had no effect on HRP+ NMJ size, but it increased Brp+ active zones. HRP: Student t test n.s. p = 0.07; Brp: Mann-Whitney U test ***p<0.001.(B) Over-expression of full-length DNT2 in muscle increased NMJ size (HRP) and active zones (Brp), revealing a retrograde function. HRP: Mann-Whitney U test *p<0.05; Brp: Student t test **p<0.01. (C) Over-expression of both full-length DNT2 and mature DNT2-CK in motoneurons induced active zone formation. Brp DNT2-CK: Student t test **p<0.01, and Brp DNT2-FL: Mann-Whitney U test ***p<0.001. n = 29–66 hemisegments. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006968#pgen.1006968.s006" target="_blank">S1 Table</a>. <i>MN = D42GAL4; Muscle = MhcGAL4</i>.</p