This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. MHC Class 11 is an essential protein in the immune system; its absence leads to a severe immune deficiency. Specialized antigen presenting cells (APCs) express this transmembrane protein to present peptides of degraded proteins (antigens) to CD4+ T (helper) cells. For the presentation of peptides the MHC Class 11 heterodimer contains a specialized domain called the peptide-binding groove, which is able to bind a peptide with a length of 9 to 14 amino acids. Inside the cell MHC Class 11 is loaded with peptide and transported to the plasma membrane by a yet not fully understood mechanism, either directly after peptide loading or after cell stimulation, depending on the celltype. At the plasma mebrane binding with (T-cell) CD4 can lead to an (primary) immune response. MIICs1 (MHC Class 11 positive compartments) are the organelles in which antigen degradation and peptide loading takes place. They primarily consist of endosomes, multivesicular bodies (late endosomes) and lysosomes. In dendritic cells the internal vesicles of late endosomes function as a storage compartment for MHC Class II. After cell stimulation these vesicles fuse with the limiting membrane, leading to more efficient peptide loading and transport of MHC Class 11 to the plasma membrane. During this back fusion the organelles change dramatically in morphology and become tubular. Vesicles that bud from these tubules may mediate transport to the cell surface. More knowledge about the changes in morphology during maturation of dendritic cells could provide further insights into how MHC Class 11 is transported to the plasma membrane. Morphology could also reveal how internal vesicles are formed. High-pressure freezing is a very suitable technique to capture very fast processes in the cell. By generating a pressure of 2100 bar a biological sample with a maximal thickness of about 200 micrometers can be vitrified (frozen without ice crystals) within 30 ms, using liquid nitrogen 4. The frozen samples can be embedded in resins by freeze substitution, a technique that allows dehydration and embedding of cells at very low temperatures to prevent ice crystal formation. When the specimen is embedded in resin it can be used for ultra-thin or semi-thick sectioning and electron microscopy. By making an automated tilt series of the sections with a Tecnai20 (FEI/Philips) microscope it is possible to reconstruct the 3D structure of organelles. A tilt series is a collection of images of the organelle of interest tilted by different rotation angles between -70 and 70 degrees. By using the above techniques we hope to reveal the 3-D morphology of MlICs in D1 cells (mouse spleen derived dendritic cells 5) during their different phases of maturation and to visualize the formation of internal vesicles. The computer programs I will use for reconstruction, visualization and modelling will be Imod, Priism, Xvoxtrace and Synu.
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