Cdi and Ssh signaling is involved in the regulation of Twinstar in response to decreased levels of CoA.

<p>(<b>A</b>) <i>Drosophila</i> LIM kinases, dLimk, Center divider (Cdi), and Slingshot phosphates (Ssh) regulate levels of p-Tsr in S2 cells. Cells were treated with dsRNA against dLimk, Cdi, Ssh or with control dsRNA and the levels of Tsr and p-Tsr were assayed using Western blot analysis. Tubulin was used as a loading control. (<b>B</b>) dLimk, Cdi or Ssh were down-regulated for 3 days after which cells were subcultured and left untreated or HoPan was added to the cell culture medium. After additional 48 hr cell lysates were assayed for levels of Tsr and levels of p-Tsr. (<b>C</b>) Quantification of the levels of p-Tsr as illustrated in B. Fold change after HoPan addition is indicated above the bars, n.s: not significant.</p

HoPan induces accumulation of phospho-cofilin and affects neurite formation in neuronal differentiation <i>in vitro</i>.

<p>(<b>A</b>) Undifferentiated and retinoic-acid (RA) differentiated SHSY-5Y cells were lysed and levels of total and phosphorylated-cofilin were assayed using Western blot analysis with specific antibodies. Representative blots are shown. GAPDH was used as an additional loading control. Levels of p-cofilin were quantified from 2 independent experiments. The p-cofilin/cofilin ratio was set as 1 in mock treated cells. (<b>B</b>) RA-differentiated SHSY-5Y cells were additionally treated with HoPan or HoPan and pantethine. Levels of p-cofilin were assayed and quantified as in A. (<b>C</b>) Control SHSY-5Y cells were differentiated with RA, plated on poly-lysine and analyzed after 48 hours for their morphology (using rhodamin-phalloidin marking F-actin, red) and differentiation status (using β-III-Tubulin staining, green). In RA-treated cultures undifferentiated and differentiated cells are visible. Undifferentiated cells showed very short filopodia and did not express β-III-Tubulin (indicated as arrowheads). 48 hours after seeding many differentiated cells showed an elongated shape, neurite growth and high expression of β-III-Tubulin (indicated as arrows). (<b>D–F</b>) Control (D), HoPan (E) or HoPan plus pantethine (F) treated cells were differentiated with RA and plated on poly-lysine. 48 hours after seeding cells were fixed and stained for β-III-tubulin, representative parts of cell morphology and β-III-Tubulin staining of the different conditions are shown. The percentage of neurite-bearing cells was calculated for each condition. Scale bars represent 25 µm (<b>C–F</b>). (<b>G</b>) Quantification of cells showing β-III-Tubulin positive neurites is presented.</p

The morphology and viability of cultured human neuroblastoma SHSY-5Y cells is affected upon PANK inhibition.

<p>SHSY-5Y cells were cultured in medium supplemented with 0,5 mM HoPan. (<b>A</b>) After 6 days in culture the cell morphology was visualized with an inverted microscope. Representative images of control (mock treated) and HoPan treated cells are shown. (<b>B</b>) Cell viability after 0–6 days in culture with HoPan was assayed by a Trypan blue exclusion test. Viability of control untreated cells was set as 100% at each time point.</p

Inhibition of dPANK activity in S2 cells induces an increase in phospho-Twinstar levels.

<p>Immunoblots of whole cell extracts were incubated with an antibody against Tsr and p-Tsr. Tubulin was used as a loading control. (<b>A</b>) Cells were treated with control dsRNA or dsRNA to downregulate dPANK/Fbl protein levels and medium was supplemented with pantethine when indicated. The graph illustrates the quantified levels of p-Tsr relative to untreated control cells. (<b>B</b>) S2 cells were cultured in the presence of pantethine, HoPan or both compounds. The graph illustrates the quantified levels of p-Tsr relative to untreated control cells.</p

Impaired Vps13 function leads to defects in protein homeostasis.

<p>(A) Percentage of isogenic control and <i>Vps13</i> mutant flies that eclosed at increasing temperatures. (B) Percentage of homozygous <i>Vps13</i> mutant flies and excision line flies that eclosed at 29°C. (C) Percentage of flies of various genotypes that eclosed at 29°C. Two independent deficiency lines (lacking a genomic area containing the <i>Vps13</i> gene) were crossed with <i>Vps13/ CyO</i> heterozygous flies. Eclosion rate of the following genotypes was analyzed: <i>Vps13/+</i>, <i>Df #7535/+</i>, <i>Vps13/Df #7535</i>, <i>Df #7534/+</i> and <i>Vps13/Df #7534</i>. (D) Percentage of <i>Vps13</i> flies that eclosed at 22°C on food with increasing concentrations of L-canavanine. (E) Western blot analysis of lysates of 1 day old control and <i>Vps13</i> mutant fly heads. Ubiquitylated proteins, K48 ubiquitylated proteins and K63 ubiquitylated proteins were detected. All quantifications show the mean and SEM of at least three independent experiments per condition. For statistical analysis a two-tailed students T-test was used in combination with a Welch’s correction if necessary. P<0.05 is *, P<0.01 is ** and P<0.001 is ***.</p

Central nervous system of larval and adult <i>Vps13</i> mutants contain protein aggregates.

<p>(A) Ventral nerve cords of control, <i>Vps13</i> mutant, <i>Vps13/Df #7534</i> and <i>Vps13/Df #7535</i> third instar larvae were stained for ubiquitylated proteins and DAPI. The presence of DAPI indicates areas where nuclei of neuronal cell bodies or glial cells are located. DAPI negative regions represent areas mainly containing axonal and synaptic structures [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170106#pone.0170106.ref033" target="_blank">33</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170106#pone.0170106.ref035" target="_blank">35</a>]. The areas in the grey boxes are shown below as higher magnification images. The scale bar indicates 50 μm. (B) Quantification of the number of ubiquitylated protein puncta in the ventral nerve cord. (C) Staining of 1 day old adult control brains using DAPI. The grey box denotes the area in the brain where the two antennal lobes are located. The presence of DAPI indicates areas where nuclei of neuronal or glial cell bodies are located. The center area which is negative for DAPI contains axons and synaptic structures [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170106#pone.0170106.ref034" target="_blank">34</a>]. The scale bar indicates 50 μm. (D) Quantification of the number of puncta of ubiquitylated proteins in the antennal lobes derived from 1 day old isogenic controls, <i>Vps13</i> mutants and excision line 3. (E) Staining of brains derived from 1 day old controls, <i>Vps13</i> mutants and excision line 3 flies for ubiquitylated proteins, Ref(2)p and DAPI. The scale bar indicates 20 μm Arrows indicate colocalization of Ref(2)P and Ubiquitin positive foci. All quantifications show the mean and SEM of at least three independent experiments per condition. For statistical analysis a two-tailed students T-test was used in combination with a Welch’s correction if necessary. P<0.05 is *, P<0.01 is ** and P<0.001 is ***.</p

<i>Vps13</i> mutant flies show a decreased life span, age dependent impairment of locomotor function and neurodegeneration.

<p>(A) Life span analysis of isogenic control and <i>Vps13</i> mutant flies. (B) The fraction of dead flies of total flies used,observed within the indicated time intervals. (C) Life span curve of <i>Vps13</i> mutant flies and three excision lines. (D) Climbing behavior was analyzed by determining the percentage of isogenic control and <i>Vps13</i> mutant flies (4 and 17 days old) able to climb 5 cm against gravity within 15 seconds. Mean and SEM are plotted (n = 5). For statistical analysis a two-tailed students T-test was used. P<0.001 is ***. (E) Fly heads (20 day old) of control and homozygous <i>Vps13</i> mutant flies were fixed, dehydrated and embedded in epon. Sections, visualizing a cross section of the complete brain, were stained with toluidine blue. The scale bar indicates 50 μm.(F) Higher magnification images of the boxed area’s in Fig E. The central complex is denoted with a dotted line. The scale bar indicates 25 μm.</p

<i>Vps13</i>^{<i>c03628</i>} encodes for a truncated Vps13 protein.

<p>(A) Schematic representation of the <i>Vps13</i> gene and the genomic localization, RNA and protein is depicted. The epitopes of the polyclonal Vps13 antibodies (Vps13 NT and Vps13 #62) are indicated.(B) Relative levels of <i>Vps13</i> mRNA in control and <i>Vps13</i> mutant flies were determined by Q-PCR. Mean and SEM (n = 2) are plotted. (C) Western blot analysis of Vps13 protein in control and <i>Vps13</i> mutant fly heads using the Vps13 #62 antibody. β-Actin was used as a loading control. (D) Western blot analysis of the level of Vps13 protein in control and <i>Vps13</i> mutant fly head extracts analyzed with the Vps13 NT antibody. α-tubulin was used as a loading control. (E) Lysates of the heads of control flies and whole control flies were analyzed for Vps13 levels. α-tubulin was used as a loading control. (F) Lysates of the heads of control flies, <i>Vps13</i> mutant flies and three excision lines were analyzed for Vps13 levels. Human VPS13A was detected in samples of Hek293 cells. <i>Drosophila</i> samples and human samples were run on the same gel, separated by a lane containing the molecular weight standards, after transfer of the membrane, the marker lane was split to detect human and Drosophila VPS13 separately using species specific antibodies. The marker lane was used to align the blots after antibody detection. α-tubulin was used as a loading control.</p

Overexpression of HsVps13A rescues phenotypes of <i>Vps13</i> mutants.

<p>(A) Samples from fly heads of <i>Actin-GAL4 / +</i> (as a control) and <i>Actin-GAL4 / UAS-HsVps13A</i> (HsVps13A expressing) flies were separated into a membrane and cytosol fraction and analyzed by Western blot for HsVps13A levels. EGFR and GAPDH used as controls for membrane and cytosolic proteins, respectively. (B) Eclosion rate of <i>Vps13</i> mutant flies a <i>Actin-GAL4/+</i> (control) or <i>Actin-GAL4/UAS-HsVp13A (HsVps13A expressing)</i> background at 25°C. (C) Ubiquitylated proteins from samples of 1 day old fly head extracts <i>of Vps13/CyO; Actin-GAL4/+</i> (as a control), <i>Vps13/ Vps13; Actin-GAL4/+</i> (representing homozygous mutants) and <i>Vps13/ Vps13; Actin-GAL4/UAS-HsVps13A</i> (representing homozygous mutants expressing human VPS13A). (D) Representative picture of ubiquitylated protein staining of the third instar larval ventral nerve cord of <i>Vps13/CyO; Actin-GAL4/+</i> (as a control), <i>Vps13/ Vps13; Actin-GAL4/+</i> and <i>Vps13/ Vps13; Actin-GAL4/UAS-HsVps13A</i>. Arrows indicate accumulations of ubiquitylated positive structures. The scale bar indicates 50 μm and 12,5 μm in the enlargement. (E) Quantification of the number of puncta in third instar larval ventral nerve cord of the experiment presented in Fig 6D. (F) Life span curve of <i>Vps13/ Vps13; Actin-GAL4/+</i> and <i>Vps13/ Vps13; Actin-GAL4/UAS-HsVps13A</i>. All quantifications show the mean and SEM of at least three independent experiments per condition. For statistical analysis a two-tailed students T-test was used in combination with a Welch’s correction if necessary. P<0.05 is *, P<0.01 is ** and P<0.001 is ***.</p

Vps13 co-fractionates with Rab7 and Rab5.

<p>(A) Western blot analysis of control fly head samples fractionated into a cytosolic and membrane fraction from postnuclear supernatant (PNS). EGFR was used as a membrane marker and GAPDH as a cytosolic marker. (B) Membrane fractions from control fly heads treated with 1 M KCl, Na₂CO₃ pH 11 or 6 M urea were centrifuged to separate the soluble and insoluble (membrane containing) fractions. The level of Vps13 was determined in these fractions. Markers for peripheral membrane proteins (GM130), integral membrane proteins (EGFR) and the cytosolic proteins (GAPDH) were used. The “<i>Vps13</i> lysate” lane contains a lysate derived from <i>Vps13</i> homozygous mutant fly heads, as expected no Vps13 is detected, demonstrating the specificity of the antibody against Vps13. (C) Membranes from control fly heads were fractionated on a sucrose gradient. Western blot analysis was performed to analyze the distribution of Vps13 in relation to markers associated with membranes of various organelles: Rab7 (late endosomes), Rab5 (early endosomes), GM130 (golgi), Lamp1 (lysosomes) and ATP5A (mitochondria). (D) Immunoisolation of membranes from fraction 14 of the sucrose gradient using Vps13 NT, Rab7 and Rab5 antibodies. (E) Quantification of the sucrose gradient fractionation of Fig 2C.</p