12 research outputs found
Development of innovative automated solutions for the assembly of multifunctional thermoplastic composite fuselage
No full textIn this study, the development of innovative tooling and end-effector systems for the assembly of a multifunctional thermoplastic fuselage is presented. The increasing demand for cleaner and new aircraft requires utilising novel materials and technologies. Advanced thermoplastic composites provide an excellent material option thanks to their weldability, low density, low overall production cost, improved fracture toughness and recyclability. However, to fully appreciate their potentials in weight, cost and production rate, new manufacturing approaches and techniques are needed. Hence, this project develops three end-effector solutions to demonstrate the feasibility of assembling a full-scale multifunctional integrated thermoplastic lower fuselage shell, including the integration of fully equipped floor and cargo structure. The developed assembly solution comprises three individual yet well-integrated tooling systems that allow housing the skin and assembly; picking, placing and welding of the assembly parts, i.e. clips and stringers; and welding of frames and floor beam sub-assemblies. The process of developing these systems from the end-user requirements, technical challenges, tooling and end-effectors design and manufacturing process are detailed in this paper
Zebrafish <i>ninl</i> knockdown causes loss of axonemes and outer segments, opsin mislocalization and vesicle/vacuole accumulation.
No full text<p>(<b>a-b</b>) Paraffin sections stained with Hematoxylin/Eosin of control (<b>a</b>) and <i>ninl</i> knockdown larvae (<b>b</b>) demonstrating shortened outer segments and grossly preserved retinal lamination in the morphants. (<b>c-d’</b>) Bodipy-stained cryosections highlight the shortened (brackets <b>c’-d’</b>) and dysmorphic outer segments of ninl knockdown larvae (<b>d</b> and <b>d’</b>) compared to the long cone- or rod-shaped outer segments of controls (<b>c</b> and <b>c’</b>). (<b>e-f</b>) Axonemes and connecting cilia marked with anti-acetylated alpha-tubulin and anti-Ift88 antibodies are severely shortened and reduced in numbers in <i>ninl</i> knockdown larvae (arrowhead in f). (<b>g-h’</b>) Immunofluorescence with anti-opsin antibody 4D2 demonstrates mislocalization of opsins within the cell body in <i>ninl</i> knockdown larvae (arrow in <b>h’</b>) compared to controls (<b>g</b>) where opsins are restricted to the outer segment. (<b>i</b>) Quantification of the intracellular opsin accumulation in <i>ninl</i> morphant photoreceptors compared to control: each single datapoint in the scatter graph displays the averaged mean grey value from one larva. The mean value and the Standard Error of the Mean (SEM) are displayed as bars. The difference is statistically significant (*** = p<0.0001, Student’s <i>t</i>-test). (<b>j-l’</b>) Transmission electron microscopy of control (<b>j</b>) and <i>ninl</i> knockdown larvae (<b>k-l’</b>) demonstrates absent or shortened and dysmorphic outer segments (OS) and accumulation of large vacuoles (v, arrow in <b>l’</b>) and smaller vesicular structures (bracket in <b>k”</b> and white arrowheads in <b>l’</b>) in morphants. Black arrowheads point to the connecting cilium in k and k”. k’ and k” are the boxed areas in k and l’ is the boxed area in l. (<b>m</b>) Quantification of the % of photoreceptors displaying these phenotypes. Absolute numbers of photoreceptors are also indicated. Error bars indicate 95% Confidence Intervals. The differences between morphant (red bars) and controls (blue bars) are statistically significant (*** = p<0.0001, Fisher’s exact test). Larvae in all panels are 4 dpf old. Scale bars are 30 μm in <b>a-b</b>, 15 μm in <b>c-d and g-h</b>, 3 μm in <b>c’-d’ and g’-h’</b>, 4 μm in <b>e-f</b>, 0.5 μm in <b>j-k and l</b> and 150nm in <b>k’-k”</b> and <b>l’</b>. OS outer segment, CC connecting cilium, m mitochondria, n nucleus, v vacuole.</p
<i>dzank1</i> and <i>ninl</i> knockdown leads to accumulation of vesicles and vacuoles in zebrafish photoreceptors.
No full text<p>Transmission electron microscopy of control (a), <i>ninl</i> knockdown larvae (b,d) and <i>dzank1</i> knockdown larvae (c,e). <i>Ninl</i> and <i>dzank1</i> morphants (b-e) demonstrate absent or shortened and dysmorphic outer segments (OS), whereas connecting cilia (CC) are still intact (arrowheads in b-c,h), and accumulation of vesicular structures (v), highlighted in the boxed area in e (e’). Sub-effective concentration of <i>ninl</i> (f) and <i>dzank1</i> (g) MO-injection leads to a normal phenotype compared to control MO-injected larvae (a) and uninjected controls. Injection of combined sub-effective concentrations of <i>dzank1</i> and <i>ninl</i> (h) MO leads to increased vesicle accumulation in OS (h’) and inner segment (h”). (i) Analysis of the presence of vesicles in inner segments of photoreceptor of cells (%). On the Y—axis the different classes are indicated (minimum of 6 eyes per group). (* <i>P</i><0.01 and *** <i>P</i><0.001 Student’s <i>t</i>-test). Larvae in all panels are 4 dpf. Scale bars represent 0.5 μm. OS: outer segment, CC: connecting cilium, m: mitochondria, n: nucleus, v: vesicular structures, G: Golgi system, L: lysosome.</p
Genetic interaction between <i>ninl</i> and <i>cc2d2a</i>.
No full text<p>(<b>a-d</b>) Partial <i>ninl</i> knockdown enhances the cystic kidney phenotype of <i>cc2d2a</i> mutants. (<b>a-c</b>) Glomerulus and proximal pronephric tubules highlighted in the transgenic line Tg(wt1b-EGFP). (<b>a</b>) Injection of a low dose of <i>ninl</i> atgMO (0.75 ng/nl) causes no cysts in wild-type larvae. (<b>b</b>) <i>cc2d2a</i>-/- larvae display small dilatations of the proximal tubules (arrow) in ~40% of cases. (<b>c</b>) Injection of this low dose of <i>ninl</i> atgMO in the <i>cc2d2a</i>-/- background leads to large dilatations of the proximal tubules and glomerular space (arrow) in 89% of mutants. <b><i>g</i></b> glomerulus, <b><i>p</i></b> pancreas. (<b>d</b>) Quantification of the glomerular + proximal tubular area displayed as a scatter plot, demonstrating a significant increase in proximal pronephric area in <i>cc2d2a</i>-/- larvae injected with low-dose <i>ninl</i> atgMO. The bars represent the mean and standard error of the mean (SEM) for each treatment group and each datapoint is an individual fish. (<b>e-g’</b>) Immunohistochemistry with anti-opsin antibody (4D2, green) on retinal cryosections of 4dpf <i>cc2d2a</i>-/- uninjected larvae (<b>f-f”</b>) and <i>cc2d2a</i>-/- larvae injected with subphenotypic doses of <i>ninl</i> MO (<b>g’g”’</b>), that cause no mislocalization in wild-type fish (<b>e-e’</b>), demonstrates that partial <i>ninl</i> knockdown increases the mislocalization of opsins (<b>e’-g’</b>). (<b>h</b>) Quantification of the mean intracellular fluorescence displayed as a scatter plot shows significant increase in intracellular fluorescence in <i>cc2d2a</i>-/- larvae injected with low dose of <i>ninl</i> atgMO. The bars represent the mean and standard error of the mean (SEM) for each treatment group and each datapoint represents the mean intracellular fluorescence from 10 photoreceptors in one individual fish. Cell membrane and outer segments are stained with bodipy (red in <b>e-</b>g). Nuclei are counterstained with DAPI. Scale bars are 100 μm in (a-c) and 4 μm in (e-g’). (<b>i</b>) Pedigree of a consanguineous family with one affected boy (UW48-3) and 4 unaffected siblings. UW48-3 carried a homozygous missense <i>CC2D2A</i> mutation as well as a frameshift mutation in <i>NINL</i> leading to premature truncation. (<b>j</b>) Pedigree of a family where the affected individual (UW36-3) carries the same homozygous <i>CC2D2A</i> mutation as in (<b>i</b>) but no additional rare deleterious variants. (<b>k</b>) Pedigree of a family where the affected individual (UW07-3) carries compound heterozygous <i>C5ORF42</i> frameshift mutations and a nonsense mutation in <i>NINL</i>. (<b>l</b>) Pedigree of a family where the affected individual (UW57-3) carries compound heterozygous <i>TMEM67</i> mutations and a missense <i>NINL</i> mutation. The phenotype of the affected individuals is detailed in <i>italic</i> on each pedigree under the corresponding mutations. <i>MTS</i> Molar Tooth Sign, <i>DD</i> Developmental Delay, <i>ESRF</i> End-Stage Renal Failure.</p
The ciliopathy protein CC2D2A Associates with NINL and functions in RAB8-MICAL3-regulated vesicle trafficking
No full textCiliopathies are a group of human disorders caused by dysfunction of primary cilia, ubiquitous microtubule-based organelles involved in transduction of extra-cellular signals to the cell. This function requires the concentration of receptors and channels in the ciliary membrane, which is achieved by complex trafficking mechanisms, in part controlled by the small GTPase RAB8, and by sorting at the transition zone located at the entrance of the ciliary compartment. Mutations in the transition zone gene CC2D2A cause the related Joubert and Meckel syndromes, two typical ciliopathies characterized by central nervous system malformations, and result in loss of ciliary localization of multiple proteins in various models. The precise mechanisms by which CC2D2A and other transition zone proteins control protein entrance into the cilium and how they are linked to vesicular trafficking of incoming cargo remain largely unknown. In this work, we identify the centrosomal protein NINL as a physical interaction partner of CC2D2A. NINL partially co-localizes with CC2D2A at the base of cilia and ninl knockdown in zebrafish leads to photoreceptor outer segment loss, mislocalization of opsins and vesicle accumulation, similar to cc2d2a-/- phenotypes. Moreover, partial ninl knockdown in cc2d2a-/- embryos enhances the retinal phenotype of the mutants, indicating a genetic interaction in vivo, for which an illustration is found in patients from a Joubert Syndrome cohort. Similar to zebrafish cc2d2a mutants, ninl morphants display altered Rab8a localization. Further exploration of the NINL-associated interactome identifies MICAL3, a protein known to interact with Rab8 and to play an important role in vesicle docking and fusion. Together, these data support a model where CC2D2A associates with NINL to provide a docking point for cilia-directed cargo vesicles, suggesting a mechanism by which transition zone proteins can control the protein content of the ciliary compartment
<i>ninl</i> and <i>dzank1</i> knockdown results in epinephrine-induced melanosome retraction delay.
No full text<p>Control MO-injected larvae (10 ng/nl; n = 20) at 5 dpf (a-a”‘), <i>ninl</i> morphant (2 ng/nl; n = 20) (b-b”‘) and <i>dzank1</i> morphant (6 ng/nl; n = 20) (c-c”‘). White Box denotes the area at higher magnification (40x) (a’-c”). (a-c’) Melanosome pattern of the different larvae before treatment, (a”-c”), 5 min after epinephrine addition and (a”‘-c”‘) 20 min after epinephrine addition, t represents time in minutes. (d) Graphical representation with error bars (standard deviation) demonstrating a significant delay of epinephrine-induced melanosome retrograde trafficking compared with wild-type and control MO-injected (10 ng/nl) larvae. Treatment (control, <i>ninl</i>- or <i>dzank1</i>-morphants) is noted on the <i>x</i>-axis and average response time in minutes is noted on the Y-axis. ***: P<0.001 (two-tailed, unpaired Student’s <i>t</i>-test).</p
NINL and DZANK1 co-localize at the base of cilia in RPE cells and rat retina.
No full text<p>(a-c) Centrosomal co-localization of NINL<sup>isoB</sup> and DZANK1 in hTERT-RPE1 cells. When expressed alone, mRFP-NINL<sup>isoB</sup> (red signal in, a-a” and c-c”) was localized to both centrioles at the base of the cilia marked with GT335 (green signal in a’-a” and red signal in b-b”), whereas eYFP–DZANK1 was localized to the basal body and to the microtubule network of the cell (green signal in b-b”). After co-expression of NINL<sup>isoB</sup> and DZANK1, both proteins were localized at the basal body of the cilia in the centrosome (cilia marked by GT335, cyanid signal), supporting an interaction between the two proteins (c-c”). Nuclei were stained with DAPI (blue signal). (d-f) Co-localization of endogenous NINL<sup>isoB</sup> and DZANK1 in rat retina. (d-e) Co-immunostaining of NINL and DZANK1 in radial cryo-sections of adult (P20) rat retina with anti-NINL antibodies (green signal; d) and anti-DZANK1 antibodies (red signal; e) showing co-localization (yellow signal: f) in the inner segment (IS) and in the region of the connecting cilium (CC). (d’-f’) show details of the subcellular (co-) localization of NINL and DZANK1 in, respectively, d, e and f. Scale bars represent 10 μm (a-c”), 5 μm (f) and 1 μm (f’).</p
Impaired Ush2a transport in <i>dzank1</i> and <i>ninl</i> morphants.
No full text<p>(a-d) Overview of Ninl and Ush2a localization in cryo-sections of 4 dpf zebrafish retina using anti-Ninl (red signal), anti-Ush2a (green signal) and the basal body marker anti-Centrin (cyanid signal). In all images the nuclei are counterstained with DAPI (blue signal). (a-a”“‘) Un-injected wildtype, (b-b”“‘) 10 ng/nl control MO-injected, (c-c”“‘) 2 ng/nl <i>ninl</i> MO-injected and (d-d”“‘) 6 ng/nl <i>dzank1</i> MO-injected zebrafish retinas. (a’-d’) Anti-Ush2a staining (green signal) is strongly reduced in <i>dzank1</i> and <i>ninl</i> morphants (c’-d’), while Ush2a is clearly present in wildtype and control MO-injected larvae. (a”-d”). Specific Ninl-immunofluorescence (red signal) was largely abolished in <i>ninl</i> morphants and reduced in <i>dzank1</i> morphants. (a”‘-d”‘) Centrin (cyanide signal) was observed in wildtype, un-injected control and in both <i>ninl</i> and <i>dzank1</i> morphants. (a”“-d”“) Co-localization of Ush2a and Ninl (yellow signal) was observed in wildtype and control MO-injected larvae. (a”“‘-d”“‘) Co-localization of Ush2a and Centrin (yellow signal) was seen in all images (WT, Control Oligo, <i>ninl</i> and <i>dzank1</i> morphants), despite strong reduction of Ush2a immunofluorescence in <i>ninl</i> and <i>dzank1</i> morphants. Scale bars represent 15 μm, except for (a-d) in which the scale bars represent 50 μm.</p
Protein-protein interaction studies.
No full text<p>(a) Schematic protein structure of DZANK1. Two different deletion constructs of DZANK1, one consisting of the ZNF_RBZ domains (244-317aa) and the other of the ANK domains (595-681aa), were used for co-transformation in yeast. (b) Results of the co-transformation in yeast. Yeast transformants were selected on low (SD/-Trp/-Leu/-His) and high-stringency (SD/-Trp/-Leu/-His-/-Ade) medium with observed growth, indicating interaction of the tested bait and prey proteins. In addition, the β-galactosidase filter lift assay was performed. The USH2A_intracellular region was used as a positive control. Empty prey vector was used as a negative control. Yeast-two-hybrid analysis revealed a specific interaction between DZANK1 ZNF_RBZ domains and both NINL<sup>isoA/B</sup>. (c) GST pull-down assays, showing that Strep/FLAG-tagged DZANK1 was efficiently pulled down by GST-fused NINL<sup>isoB</sup>, but not by GST alone. The first lane shows 5% input of the protein lysate. (d) Co-immunoprecipitation of DZANK1 Full Length (FL) with NINL<sup>isoB</sup>, but not with LRRK2. The immunoblot (IB) in the top panel shows that HA-tagged NINL co-immunoprecipitated with Strep/FLAG-tagged DZANK1 (lane 2), whereas unrelated FLAG-tagged LRRK2 (lane 3) did not. The anti-HA immunoprecipitates are shown in the middle panel; protein input is shown in the bottom panel. (d’) Reciprocal IP experiments using anti-FLAG antibodies confirmed the co-immunoprecipitation of HA-tagged NINL<sup>isoB</sup> with Strep/FLAG-tagged DZANK1 (lane 2) and not with LRRK2 (lane 3) shown in the top panel. The anti-FLAG immunoprecipitations are shown in the middle panel; protein input is shown in the bottom panel. ANK: ankyrin repeat domain; ZNF_RBZ: Zinc Finger domain like in Ran-binding proteins; aa: amino acids.</p
Morphological, functional and epistatic effects of <i>ninl</i> and <i>dzank1</i> knockdown in zebrafish retina.
No full text<p>(a-c) Images of 4 dpf living zebrafish. Un-injected controls (WT) appear morphologically normal (a), while embryos injected with 2 ng of <i>ninl</i> atgMO display morphological defects, including ventrally curved body axis and small eyes (b). Embryos injected with 6 ng <i>dzank1</i> ex8 spMO resulted in expanded melanophores, small eyes and severe pericardial edema (c) (a’-c’) Retinal histology of 4 dpf zebrafish morphants examined by cryo-sections, where bodipy highlights the OS (red) and nuclei are stained with DAPI (blue) in all panels. Outer segments were shortened and dysmorphic in <i>ninl</i> and <i>dzank1</i> morphants compared to wildtype larvae. (d-g) <i>Ninl</i> interacts genetically with <i>dzank1</i>. Injection of sub-effective <i>dzank1</i> (1 ng/nl) MO (e’) or <i>ninl</i> (0.5 ng/nl) MO (f’) shows normal OS shape and length in morphologically normal appearing larvae, which could not be distinguished from un-injected embryos (WT) or control MO-injected larvae (d’). Combined injection of sub-effective concentrations of <i>ninl</i> (0.5 ng/nl) and <i>dzank1</i> (1 ng/nl) MO together results in almost complete absence of OS (g’). (h) Quantification of Outer Segment length, shown as a scatter graph where each datapoint represents the mean OS length in one larva, revealed a significantly decreased length of outer segments in <i>ninl</i> (2ng/nl), <i>dzank1</i> (6ng/nl) and <i>ninl/dzank1</i> double morphants as compared to controls. Bars represent the mean value for each treatment group with the Standard error of the mean (SEM) ***<i>P</i><0.0001, ** <i>P</i><0.001, unpaired Student’s t-test. (i) Analysis of the Opto Kinetic Response (OKR) showing severely decreased responses in larvae injected with 2 ng <i>ninl</i> atgMO or 6 ng <i>dzank1</i> ex8 spMO (*** p<0.0001, Student’s <i>t</i>-test). Scale bars represent 500 μm (a-c and e-h) and 15 μm (a’-c’ and e’-h’). RPE, retinal pigment epithelium; OS, Outer Segment; IS, Inner Segment; ONL, Outer Nuclear Layer; OPL, Outer Plexiform Layer.</p
