ABSTRACT An important function of the cell is the lysosomal degradation of proteins that are coming from within the cell or from the extra cellular environment. This function requires proteolytic enzymes that are present in the lysosomes. Upon synthesis, carbohydrate moieties (including mannose 6-phosphate) are attached to these enzymes, followed by a targeting step involving the intracellular transport from the trans-Golgi network to the lysosome. This transport step is mediated by the mannose 6-phosphate receptor (MPR). Immunofluorescence microscopy has suggested that the intermediate transport elements that carry the MPR/enzyme complex from the trans-Golgi network to an endosomal compartment may consist of tubules.
My aim is to determine whether the MPR is indeed transported in tubules (rather than vesicles) using a recently developed correlative live-cell imaging and electron microscopy technique which capitalizes the advantages of both forms of microscopy: protein movement and morphological analysis at the ultra-structural level. Moreover, the transport elements will be further characterized by immuno-labelling them for proteins that are of particular interest, such as adaptor proteins and molecular motor proteins. For the proteins that will be found on the tubules their role in transport element dynamics will be analysed in further detail by interference studies.
Using the same correlative technique together with markers specific for the distinct endosomal compartments, the endosomal compartment with which t he transport elements fuse will be identified. Identification and characterization of the MPR containing transport elements using the correlative technique will unequivocally increase our insight in the cell's capability to move membrane proteins around in a highly structural and organized way without disturbing the identity and integrity of the different organelles.
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