Functional fine-tuning between RecBCD and RecQ enzymes.

<p><b>Upper row:</b> DSB processing by the RecBCD-WT complex (RecBCD protein not shown) results in RecA-loaded 3’ DNA overhangs. In contrast, RecB¹⁰⁸⁰CD produces aberrant long, partially RecA-filled 5’ and 3’ overhangs. <b>Middle row:</b> The complex DNA unwinding, directed invasion disruption and shuttling activities of the RecQ-WT enzyme (triangle) enable efficient disruption of IR invasions (green) in the <i>recBCD-WT</i>, but low efficiency (yellow) in the <i>recB</i>^<i>1080</i><i>CD</i> background. <b>Lower row:</b> The enhanced (highly processive) DNA unwinding, but compromised directed invasion disruption and/or shuttling activities of RecQ* and RecQ-dH enzymes (triangle) are inefficient (yellow) for IR disruption in the <i>recBCD-WT</i> background. However, these activities become efficient (green) in the <i>recB</i>^<i>1080</i><i>CD</i> background where HR is more likely proceed through joint DNA molecules of aberrant structure.</p

RecQ and RecB functional defects exert cumulative effects on the repair of nitrofurantoin-induced DNA damage.

<p><b>(A)</b> Dose-dependent NIT survival of <i>E</i>. <i>coli</i> strains (means ± SE), fitted by a standard dose-response model (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192483#pone.0192483.s002" target="_blank">S2 Eq</a></b>). Symbols for low survival values at high NIT doses (all measured up to 10 μg/mL) are mostly obscured by those of other strains with similar values. <b>(B)</b> Fitted log <i>LD</i>₅₀ values of strains. Means ± SE of individual best-fits to biological replicates are shown. Asterisks indicate significant difference from the WT value (ANOVA, Tukey’s post-hoc test, <i>p</i> < 0.05). Double asterisks indicate further significant difference from values with single asterisk. Multiple WT controls are shown alongside mutant values determined in the same experimental run. Data for non-<i>recB1080</i> strains are from ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192483#pone.0192483.ref015" target="_blank">15</a>]. Sample sizes and determined parameters are listed in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192483#pone.0192483.s007" target="_blank">S3 Table</a></b>.</p

RecB and RecQ defects do not affect cell growth, whereas the <i>recB1080</i> mutation markedly impairs cell division under stress-free conditions.

<p><b>(A)</b> Growth (<i>OD</i>₆₀₀) curves of indicated <i>E</i>. <i>coli</i> strains, fitted by the modified Gompertz model (solid lines, <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192483#pone.0192483.s001" target="_blank">S1 Eq</a></b>) (21). Means ± SE for <i>n</i> = 5 biological replicates are shown. Error bars are within symbols where not visible. Inset: cell growth monitored via changes in CFU/ml over time (means ± SE for <i>n</i> = 3). Determined parameters and analysis results are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192483#pone.0192483.s003" target="_blank">S1 Fig</a></b> and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192483#pone.0192483.s006" target="_blank">S2 Table</a></b>. <b>(B)</b> Example images of methylene blue stained cells (see also <b>A panel in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192483#pone.0192483.s004" target="_blank">S2 Fig</a></b>). <b>(C)</b> Fraction of the total cell area found in large elongated structures (with individual area > 4 μm²; see also <b>Panel D in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192483#pone.0192483.s004" target="_blank">S2 Fig</a></b>). Percentages for the number of large cells (> 4 μm²) and other determined parameters are listed in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192483#pone.0192483.s007" target="_blank">S3 Table</a></b>. Means ± SE for <i>n</i> = 3 biological replicates are shown. Asterisks indicate significant difference from the WT value (ANOVA, Tukey’s post-hoc test, <i>p</i> < 0.05).</p

Loss of RecQ function further sensitizes <i>recB1080</i> cells to UV-induced lesions.

<p><b>(A)</b> UV survival (mean ± SE) of <i>E</i>. <i>coli</i> strains. Means ± SE for <i>n</i> = 5 biological replicates are shown. Low survival values (< 10⁻⁴) for <i>recB1080 recQ-dWH</i> and <i>recB1080 ΔrecQ</i> strains at UV doses above 10 J/m² could not be accurately determined. Due to this condition and the generally low UV survival values measured in the <i>recB1080</i> background, the standard dose-response model (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192483#pone.0192483.g002" target="_blank">Fig 2B</a></b>) could not be applied. <b>(B)</b> Log relative survival values at 10 J/m² UV irradiation (means ± SE). Asterisks indicate significant difference from the WT value (ANOVA, Tukey’s post-hoc test, <i>p</i> < 0.05). Double asterisks indicate further significant difference from each of the <i>recB1080 recQ*</i> and <i>recB1080 recQ-dH</i> strains (for <i>recB1080 recQ-dWH</i>), or from each of the <i>recB1080</i>, <i>recB1080 recQ*</i> and <i>recB1080 recQ-dH</i> strains (for <i>recB1080 ΔrecQ</i>). Multiple WT controls are shown alongside mutant values determined in the same experimental run. Data for non-<i>recB1080</i> strains are from ref.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192483#pone.0192483.ref015" target="_blank">15</a>].</p

NDK-1 functions in DTCs.

<p>NDK-1::GFP shows expression in distal tip cells (DTCs) in TTV2 (<b>B, D</b>) and TTV3 (<b>F</b>) translational reporter lines in L3 (<b>A, B</b>), L4 (<b>C, D</b>) larvae and adults (<b>E, F and I</b>). <b>A</b>, <b>C</b>, and <b>E</b> are the corresponding DIC images of <b>B</b>, <b>D</b>, and <b>F</b>, respectively. DTC locations are indicated with arrows. We note that on panel <b>D</b> GFP expression in DTC (marked by an arrow) is overshadowed by intense expression of autofluorescent granules in the intestine. <b>E, F and I</b>: gonads were isolated from adult animals. <b>H</b>: DIC image of an adult wild-type (N2) gonad arm. <b>G</b>: Variation and distribution of DTC migration phenotypes observed in <i>ndk-1</i>(<i>ok314</i>) mutants (see details in the main text). The migratory path of DTCs is marked by dashed lines. <b>I</b>: In TTV3 lines carrying the integrated transgene, NDK-1::GFP expression is also observed in gonadal sheath cells. DIC, fluorescent and merged images derive from the gonadal loop region of a transgenic animal. On the DIC panel white arrows show dying cells, which are surrounded by sheath cells strongly expressing NDK-1::GFP (white arrows on the fluorescent and merged panels).</p

Site of action of NDK-1 in the analogous processes of engulfment and DTC migration.

<p><b>A, B</b>: Cell corpse engulfment and DTC migration are similar processes. In each case, the surface membrane of a cell (black) extends along the surface of another cell (hatched). The small arrows near the black cells indicate the directions of cell-surface extension. <b>B</b>: Only the relevant parts of body muscles are shown <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092687#pone.0092687-Wu1" target="_blank">[19]</a>. <b>C</b>: Schematic review of DTC migration (based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092687#pone.0092687-Meighan1" target="_blank">[21]</a>). DTCs are located on the distal edges of the gonad primordium and start to migrate in L2. They migrate along the ventral surface (dashed line) of the hermaphrodite in L2 (first or ventral phase). Then they turn to the dorsal side during L3 (second or ventral to dorsal phase). A second turn redirects migration along the dorsal surface toward the center of the nematode during L4 (third or dorsal phase). The end of the migration is dorsal to the vulva, resulting in the mirror image U-shaped gonad of the adult. The developmental stage is indicated at right of each diagram. <b>D</b>: Signaling pathways in engulfment and DTC migration (based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092687#pone.0092687-Meighan1" target="_blank">[21]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092687#pone.0092687-Hurwitz1" target="_blank">[22]</a>. Common genes are blue, green colour indicates the factors involved only in DTC migration, genes in purple boxes play a role only in engulfment. We suggest that NDK-1/NM23 acts downstream of CED-10/Rac in the processes of DTC migration and engulfment of apoptotic corpses. NDK-1 shows a genetic interaction with DYN-1/Dynamin.</p

Developmentally regulated autophagy is required for eye formation in <i>Drosophila</i>

<p>The compound eye of the fruit fly <i>Drosophila melanogaster</i> is one of the most intensively studied and best understood model organs in the field of developmental genetics. Herein we demonstrate that autophagy, an evolutionarily conserved selfdegradation process of eukaryotic cells, is essential for eye development in this organism. Autophagic structures accumulate in a specific pattern in the developing eye disc, predominantly in the morphogenetic furrow (MF) and differentiation zone. Silencing of several autophagy genes (<i>Atg</i>) in the eye primordium severely affects the morphology of the adult eye through triggering ectopic cell death. In <i>Atg</i> mutant genetic backgrounds however genetic compensatory mechanisms largely rescue autophagic activity in, and thereby normal morphogenesis of, this organ. We also show that in the eye disc the expression of a key autophagy gene, <i>Atg8a</i>, is controlled in a complex manner by the anterior Hox paralog Lab (Labial), a master regulator of early development. <i>Atg8a</i> transcription is repressed in front of, while activated along, the MF by Lab. The amount of autophagic structures then remains elevated behind the moving MF. These results indicate that eye development in <i>Drosophila</i> depends on the cell death-suppressing and differentiating effects of the autophagic process. This novel, developmentally regulated function of autophagy in the morphogenesis of the compound eye may shed light on a more fundamental role for cellular self-digestion in differentiation and organ formation than previously thought.</p> <p><b>Abbreviations</b>: αTub84B, α-Tubulin at 84B; <i>Act5C, Actin5C</i>; AO, acridine orange; Atg, autophagy-related; Ato, Atonal; CASP3, caspase 3; Dcr-2; Dicer-2; Dfd, Deformed; DZ, differentiation zone; eGFP, enhanced green fluorescent protein; EM, electron microscopy; <i>exd, extradenticle; ey, eyeless</i>; FLP, flippase recombinase; FRT, FLP recognition target; <i>Gal4</i>, gene encoding the yeast transcription activator protein GAL4; GFP, green fluorescent protein; GMR, Glass multimer reporter; Hox, homeobox; <i>hth, homothorax; lab, labial</i>; L3F, L3 feeding larval stage; L3W, L3 wandering larval stage; lf, loss-of-function; MAP1LC3, microtubule-associated protein 1 light chain 3; MF, morphogenetic furrow; PE, phosphatidylethanolamine; PBS, phosphate-buffered saline; PI3K/PtdIns3K, class III phosphatidylinositol 3-kinase; PZ, proliferation zone; Ref(2)P, refractory to sigma P, RFP, red fluorescent protein; RNAi, RNA interference; RpL32, Ribosomal protein L32; RT-PCR, reverse transcription-coupled polymerase chain reaction; S.D., standard deviation; SQSTM1, Sequestosome-1, Tor, Target of rapamycin; TUNEL, terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay; UAS, upstream activation sequence; qPCR, quantitative real-time polymerase chain reaction; <i>w, white</i></p

<i>ndk-1</i>(<i>ok314</i>);<i>dyn-1</i>(<i>ky51</i>) double mutants are lethal.

<p><b>A</b>: In the F1 progeny of <i>ndk-1</i>(-)<i>/+</i> heterozygotes only 9.2% homozygous Pvl, Ste adults can be observed instead of the expected 25%, since 15.8% of <i>ndk-1</i>(<i>ok314</i>) homozygotes die as embryos at 25°C. At the restrictive temperature (25°C) 50% of <i>dyn-1</i>(<i>ky51</i>) single mutants die as embryos. At 25°C, in the F1 progeny of <i>dyn-1(ky51);ndk-1(ok314)/+</i> animals we got decreased brood size and we did not notice any Pvl, Ste animals, suggesting that the double mutants are not viable. <b>B</b>: 3-fold stage homozygous <i>ndk-1</i>(<i>ok314</i>) embryo shows late embryonic lethality with persistent cell corpses. Arrows indicate apoptotic corpses.</p

Western blot analysis and migration assay of transfected MDA-MB-231T cells.

<p>MDA-MB-231T cells were stably transfected with, pcDNA3 (K1 and K2), pcDNA3/FLAG-<i>nm23-H1</i> (HA1 and HA2), pcDNA3/FLAG-<i>ndk-1</i> (CE1 and CE2) and pcDNA3/MYC-<i>nm23-H2</i> (HB1 and HB2). <b>A</b>: Western blot with anti-α-tubulin antibodies (loading control). <b>B</b>: Western blot with anti-FLAG- antibodies, visible band in HA1, HA2, CE1 and CE2 proves stable overexpression of introduced transgenes. <b>C</b>: Western blot with anti-MYC- antibodies, visible band in HB1 and HB2 (overexpression of NM23-H2). <b>D</b>: Migration assay. MDA-MB-231T cells stably transfected with one of the following constructs: pcDNA3 (K1 and K2), pcDNA3FLAG/<i>nm23</i>-H1 (HA1 and HA2), pcDNA3FLAG/<i>ndk-1</i> (CE1 and CE2) and pcDNA3/MYC-<i>nm23-H2</i> (HB1 and HB2) were tested for migration potential. The cells were stained with crystal violet and counted (the number of migrated cells were counted in four representative microscopic fields per each clone). The CE1 and CE2 clones as well as HA1, HA2, HB1 and HB2 exhibited significantly diminished migration potential compared to control (K1 and K2) clones (Student's t-test, p<0.05). The results are presented as an absolute number of migrated cells in 4 representative fields for every clone (±SD).</p

<i>ndk-1</i>(<i>ok314</i>);<i>abi-1</i>(<i>ok640</i>) double mutants show additive Ced phenotye.

<p><i>P_lim-7ced-1::gfp</i> transgenic worms treated by <i>ndk-1</i>(<i>RNAi</i>) (<b>D</b>) show an excess of apoptotic corpses in the germline compared to worms carrying the same transgene treated by control RNAi (<b>B</b>). <b>A</b>, <b>C</b> are corresponding DIC images of <b>B</b>, <b>D</b> respectively. Arrows indicate apoptotic germ cells. <b>E</b>: Increase of apoptotic germ cell death in <i>ndk-1</i>(<i>RNAi</i>) animals compared to the control, where p<i>_lim-7ced-1::gfp</i> transgenic worms were grown on control RNAi (e.g. <i>E. coli</i> HT115(DE3) carrying an empty vector). <b>F–K</b>: Monitoring apoptotic corpses in wild-type embryos (<b>F</b>), <i>ndk-1</i>(-) (<b>G</b>), <i>abi-1</i>(-) (<b>H</b>), <i>ced-10</i>(-) (<b>J</b>) single mutants and <i>ndk-1</i>(-);<i>abi-1</i>(-) (<b>I</b>), <i>ndk-1</i>(-);<i>ced-10</i>(-) (<b>K</b>) double mutants using DIC optics. Embryos slightly before or at the comma stage were scored. Each panel shows two focal planes (<b>F–K</b>). Arrowheads indicate apoptotic corpses. Panel <b>L</b> shows a summary of apoptotic corpses scored in <i>ndk-1</i>(<i>ok314</i>), <i>abi-1</i>(<i>ok640</i>), <i>ced-10</i>(<i>n1993</i>) single mutant and <i>ndk-1(ok314);abi-1(ok640)</i> and <i>ndk-1(ok314);ced-10(n1993)</i> double mutant embryos. <i>ndk-1</i>(-);<i>abi-1</i>(-) double mutants display an enhanced Ced phenotype compared to single mutants, however <i>ndk-1</i>(-);<i>ced-10</i>(-) doubles are reminiscent of <i>ced-10</i>(-) single mutants. Panel <b>M</b> is the graphic representation of panel <b>L</b>. <i>p</i>-values refer to comparisons of apoptotic cell corpse numbers between single and double engulfment mutants. * * * means <i>p</i><0.001; n.s. means not significant (<i>p</i> = 0.627).</p