Utilización de herramientas de simulación en la robótica industrial

Análisis de herramienta de simulación utilizada en el entorno industrial y estudio de las ventajas de la programación "offline" en un entorno productivo. Programación de una célula de trabajo industrial y ampliación posterior de la misma utilizando la herramienta de simulación industrial

Over-expression of APμ1 perturbed the Rab5A compartment morphology.

<p><b>A, B</b>- Confocal images showing the localization of Rab5A (A, green), Rab7 (B, green) and cMycμ1 (A, B, red) in the parental strain RH and DDμ1 parasites treated with shield-1 (+S) for 16 hours. A zoom of the Golgi area indicated by the white frame in each image is shown as an inset on the right. Bar: 2μm. <b>C-</b> SIM images showing the localization of proROP4 (red) and Rab5A-HA (green) proteins in control RH parental strain (upper panel) and cMycμ1 over-expressing parasites (lower panel) treated with shield-1 (+S) for 16 hours. Bars: 2μm. <b>D-</b> SIM images showing the co-distribution of Rab5A-positive vesicles surrounding proROP4 vesicular compartments in RH parental strain (upper panel), while TGN-distant preROP compartments were negative for Rab5A staining (arrows). Induced DDμ1 parasites exhibited Rab5A-positive enlarged vesicular compartments (lower panel, arrow) empty of proROP4 proteins (red). Bars: 500 nm. <b>E-</b> Left: SIM image of DDμ1 parasites induced with shield-1 showing proRO4 proteins (red) contained in vesicles with a strong Rab5A (green) signal at their limiting membrane illustrated by the intensity profile of each signal (graph). Right: Histogram depicting the percentage of proROP4-positive vacuoles showing a Rab5A signal at their limiting membrane in RH and DDμ1 parasites treated with shield-1 (+S). Data are presented as average ± SD (n = 50 parasites), ***p<0.001 (Student’s t-test).</p

List of proteins identified by mass spectrometry following the IP of μ1-HA proteins in KI parasites expressing μ1-HA under the endogenous promotor.

<p>The detailed list is included in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006331#ppat.1006331.s002" target="_blank">S2 Fig</a>. The parental strain RHΔKU80 was used as a control for non-specific binding to the anti-HA antibody-coated beads. The table indicates the number of “unique peptides / spectra” for each identified protein in two biological independent assays (IP1 and IP2).</p

Correlative Light Electron Microscopy (CLEM) images illustrating the organization of the basal pole (arrow heads) and the residual body (arrows) in control and APμ1-KO parasites.

<p><b>A-D</b>: Non-YFP control vacuoles (<b>C</b>: zoom of the region indicated by a white frame in <b>B)</b>. Note the typical organization in a rosette-like structure and the correct morphology of the parasites. <b>E-H</b>: YFP-positive APμ1-KO parasites (detected in the region 3T shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006331#ppat.1006331.s010" target="_blank">S10 Fig</a>). Bars: 500 nm.</p

Dual role of the <i>Toxoplasma gondii</i> clathrin adaptor AP1 in the sorting of rhoptry and microneme proteins and in parasite division - Fig 10

<p>Transmission electron microscopy images showing the formation of mature rhoptries (Rh) and micronemes (Mi) anchored at the apical pole in control parasites (<b>A</b>) and the normal distribution of parasites in rosette-like structures (<b>B</b>). Bars: 500nm. (<b>C</b>)- Zoom of the posterior end of the control parasites showing the tight constriction of the basal pole with a thin continuity to the residual body (arrowhead). In DDμ1 parasites induced with shield-1 (+S) for 24 hours (D-I), apically positioned rhoptries could not be detected in contrast to micronemes (<b>D</b>) or they were found dispersed in the cytoplasm (<b>E,</b> arrow). Numerous giant lucent vesicles (V) were also observed (D, G, H). In addition, the parasites were found disorganised within the vacuole with a distorted morphology (G-I) particularly at the basal pole (F, G and I, arrow), which appeared deformed and elongated (arrows in F) despite the detection of the residual body (F and I, arrowhead). In addition, some parasites seemed to remain attached by discrete lateral contact sites (H, arrow). Bars: 500nm. Mi: micronemes, Rh: rhoptries, PV: parasitophorous vacuole, V: vesicles.</p

The unique ENTH-domain containing protein of <i>T</i>. <i>gondii</i> is a key partner of APμ1.

<p>A- Western blot showing the expression of the cMyc-tagged EpsL protein at the expected size of 66 kDa in single KI parasites (lane 2), and together with μ1-HA (49 kDa) in double knock-in parasites (lane 3). The parental strain (RHΔKU80) is shown in lane 1. SORTLR was used as a loading control. B- Images illustrating the co-localization of EpsL-cMyc (green) with μ1-HA (red) (arrows), acquired with confocal microscopy (upper panel) or SIM (lower panel). Bars: 2μm. C- Co-immunoprecipitation of μ1-HA with EpsL-cMyc in double KI parasites (lanes 2) using anti-cMyc antibodies. No binding of the μ1-HA protein on anti-cMyc coated beads was detected in single KI μ1-HA expressing parasites (lane 1). D- Reverse co-immunoprecipitation of EpsL-cMyc with μ1-HA in double KI parasites using anti-HA antibodies. No binding of EpsL-cMyc protein with anti-HA antibody coated beads was detected in the single KI EpsL-cMyc expressing parasites. E, F- A GST-pull down experiment with the GST-tagged gamma appendage ear (GAE) and GST-tagged beta appendage ear (BAE) domains of <i>Tg</i>AP1 was performed in presence of a total lysate from EpsL-cMyc/ <i>μ</i>1-HA double KI parasites. E: SDS-PAGE gel stained with coomassie blue showing that a similar quantity of GST, GST-GAE and GST-BAE (asterisks) was bound on the gluthation beads used in the assay shown in F. F: WB analysis demonstrated the preferential binding of EpsL-cMyc to the ear domain of the γ sub-unit (GAE). A weak interaction of SORTLR and the μ1 sub-unit with the BAE domain was also detected. GST alone was used as a control. FT: Flow-Through.</p