Supplemental Data CellA Genome-wide Multi-Dimensional RNAi Screen Reveals Pathways Controlling MHC Class II Antigen Presentation Petra Paul1,5, Tineke van den Hoorn1,5, Marlieke L.M. Jongsma1,5, Mark J. Bakker1, Rutger C. C. Hengeveld1, Peter Cresswell2, David A. Egan4, Marieke van Ham3, Anja ten Brinke3, Huib Ovaa1, Roderick L. Beijersbergen4, Coenraad Kuijl1 and Jacques Neefjes1,* MHC class II molecules (MHC-II) present peptides to T helper cells to facilitate immune responses and are strongly linked to autoimmune diseases. To unravel processes controlling MHC-II antigen presentation, we performed a genome-wide flow cytometry-based RNAi screen detecting MHC-II expression and peptide loading followed by additional high-throughput assays. All data sets were integrated to answer two fundamental questions: what regulates tissuespecific MHC-II transcription, and what controls MHC-II transport in dendritic cells?MHC-II transcription was controlled by nine regulators acting in feedback networks with higher-order control by signaling pathways, including TGFb. MHC-II transport was controlled by the GTPase ARL14/ARF7, which |
Supplemental Data JCB
Cholesterol sensor ORP1L contacts the ER protein VAP to control Rab7-RILP-p150 Glued and late endosome positioning.Rocha N, Kuijl C, van der Kant R, Janssen L, Houben D, Janssen H, Zwart W, Neefjes J. Division of Cell Biology, The Netherlands Cancer Institute, 1066CX Amsterdam, Netherlands. Late endosomes (LEs) have characteristic intracellular distributions determined by their interactions with various motor proteins. Motor proteins associated to the dynactin subunit p150(Glued) bind to LEs via the Rab7 effector Rab7-interacting lysosomal protein (RILP) in association with the oxysterol-binding protein ORP1L. We found that cholesterol levels in LEs are sensed by ORP1L and are lower in peripheral vesicles. Under low cholesterol conditions, ORP1L conformation induces the formation of endoplasmic reticulum (ER)-LE membrane contact sites. At these sites, the ER protein VAP (VAMP [vesicle-associated membrane protein]-associated ER protein) can interact in trans with the Rab7-RILP complex to remove p150(Glued) and associated motors. LEs then move to the microtubule plus end. Under high cholesterol conditions, as in Niemann-Pick type C disease, this process is prevented, and LEs accumulate at the microtubule minus end as the result of dynein motor activity. These data explain how the ER and cholesterol control the association of LEs with motor proteins and their positioning in cells. Supplemental: These CellProfiler modules and pipelines have been tested with CellProfiler_5811Bugfix and are provided as is. For more information regarding CellProfiler please visit www.cellprofiler.org Modules: MeasureCenterGravityIntensity.m Measure the center of gravity of an object based on the pixel intensities within the object. This center of gravity can be used e.g. in the MeasureRadialDistribution module.
Measures the radial distribution of one or more proteins within a cell. This module is a patched version of the one that is supplied by CellProfiler. When the centroid of the cell lays outsite the object itself (e.g. bean shaped nucleus) the original modules produces an error. This has been fixed in this module. for more information visit http://www.cellprofiler.org/forum/viewtopic.php?f=3&t=598
Used to speed up CellProfiler. Sometimes the resolution of an images is higher than necessary, resulting is waisted time in identifying objects. By resizing the image to a lower resolution this process can be sped up. The resulting label matrix however does not match the size of the high resolution images in which you want to measure your parameters. With this module you can resize your label matrix back to the high resolution image size. Note that the coordinates are not resized in the handles structure, only the label matrix image! Pipe Lines: Pipeline used to quantify distribution. The pipe line makes use of mask images in which each white area is a cell. These mask images can be generated in photoshop or matlab itself.
Supplemental Data Nature
Intracellular bacterial growth is controlled by a kinase network around PKB/AKT1.Kuijl C, Savage ND, Marsman M, Tuin AW, Janssen L, Egan DA, Ketema M, van den Nieuwendijk R, van den Eeden SJ, Geluk A, Poot A, van der Marel G, Beijersbergen RL,Overkleeft H, Ottenhoff TH, Neefjes J. Division of Tumor Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. With the emergence of multidrug resistant (MDR) bacteria, it is imperative to develop new intervention strategies. Current antibiotics typically target pathogen rather than host-specific biochemical pathways. Here we have developed kinase inhibitors that prevent intracellular growth of unrelated pathogens such as Salmonella typhimurium and Mycobacterium tuberculosis. An RNA interference screen of the human kinome using automated microscopy revealed several host kinases capable of inhibiting intracellular growth of S. typhimurium. The kinases identified clustered in one network around AKT1 (also known as PKB). Inhibitors of AKT1 prevent intracellular growth of various bacteria including MDR-M. tuberculosis. AKT1 is activated by the S. typhimurium effector SopB, which promotes intracellular survival by controlling actin dynamics through PAK4, and phagosome-lysosome fusion through the AS160 (also known as TBC1D4)-RAB14 pathway. AKT1 inhibitors counteract the bacterial manipulation of host signalling processes, thus controlling intracellular growth of bacteria. By using a reciprocal chemical genetics approach, we identified kinase inhibitors with antibiotic properties and their host targets, and we determined host signalling networks that are activated by intracellular bacteria for survival.
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