Final Activity Report Summary - MURMAR2004 (Defining the transport pathway of the receptor-mediated sorting of lysosomal enzymes by correlative live-cell imaging and electron microscopy) Proper functioning of the cell requires controlled lysosomal degradation of its own and foreign macromolecules. The importance of this degradation is demonstrated by diseases where the lysosomal function is impaired. The proteolytic enzymes that mediate lysosomal degradation are soluble proteins traversing the biosynthetic pathway before their targeting to the lysosome. Mannose 6-phosphate receptors (MPRs) and sortilin are two receptors known to mediate the transport of distinct subsets of lysosomal enzymes. Although trafficking of the MPR has been extensively studied important aspects of MPR have remained unaddressed.In this project, we aimed to identify the structural and morphological characteristics of the transport carriers that mediate the transport of the lysosomal enzyme receptors between the trans-Golgi network (TGN) and the endosomes.Based on real-time confocal fluorescence microscopical analysis of fluorescent-tagged CI-MPR, Waguri et al proposed that the CI-MPR leaves the TGN via dynamic tubular elements that accommodate the anterograde trafficking of the receptor (Waguri et al., 2003). The limited resolution of such a light microscopical analysis does not allow the definition of whether these carriers are real tubules or trains of vesicles, and where the carriers enter the endosomal system. It is also not known in what vehicles the MPRs recycle back to the TGN.We have applied a combination of light microscopy and ultrastructural morphological analysis of these elements by a correlative technique developed by the Utrecht lab for PC12 cells (Oorschot et al., 2002). We first adapted and improve this technique to our cellular model and scientific question (e.g. nature of the carrier's tubules versus vesicles). The immuno-EM analysis using fixative as well as the GRAB correlative approach (fixative free; Grabenbauer et al., 2005) have shown that the app. 1micrometre long tubular structures emerging from the TGN seen by light microscopy are vesicular elements sometimes following microtubules. It was occasionally possible to observe an alignment of CI-MPR positive vesicles for a short distance by immuno-EM or very short tubules (<100 nm) with the GRAB technique.All the correlative light and electron microcopy employed are of such a high technical complexity that they were not always applicable. Indeed, we could not exclude that several chemical steps in the procedure do not affect the tubular dynamics. In addition, the 3D-organisation of these tubules also hampered to obtain a realistic picture of them. For all these reasons the host laboratory is in a process of improving these approaches by developing a technique combining correlative light microcopy with the 3D-tomography (allowing the cell / organelles 3D reconstruction).In addition to better characterise the morphology of the carriers; we studied their molecular composition. So far, mannose 6-phosphate receptors and sortilin (a VPS10-D family protein) are the only known proteins to mediate the transport of distinct subsets of lysosomal enzymes. Both types of receptors share sequence similarity in their cytoplasmic tail, responsible for the interaction with proteins of the transport machinery at the TGN such as GGAs (Golgi-localised, gamma-ear containing, ADP-ribosylation factor binding). This suggests that sortilin and the MPRs share the same TGN to endosome / lysosome transport pathway.We combined quantitative immunogold-electron microscopy (EM), EM tomography, molecular and biochemical approaches to study the transport machinery and carriers of those receptors that are currently known to be involved in lysosomal protein sorting: i.e. the Cation-dependent (CD) and -independent (CI) MPRs and the unrelated protein sortilin, which was only recently implicated in the transport of lysosomal proteins.We localised endogenous sortilin in HepG2 cells and found it predominantly in the TGN and endosomes. In the TGN, sortilin co-localised with MPRs in the same clathrin-coated vesicles. In endosomes, sortilin and MPRs concentrated in sorting nexin 1 (SNX1)-positive buds and vesicles. SNX1-depletion by siRNA resulted in decreased pools of sortilin in the TGN and an increase in lysosomal degradation. These data indicate that sortilin and MPRs recycle to the TGN in SNX1-dependent carriers, which we named Endosome-to-TGN transport carriers (ETCs). Notably, ETCs emerge from Early endosomes (EE), lack recycling plasma membrane proteins and by 3D electron-tomography exhibit unique structural features. Hence, ETCs are distinct from hitherto described EE-derived membranes involved in recycling. Our data emphasise an important role of EEs in recycling to the TGN and indicate that different specialised exit events occur on the same EE vacuole. Thus our studies identified a novel sorting pathway that specifically recycles lysosomal protein receptors from EEs the TGN and conceptually change our interpretation of the morpho-functional design of EEs.Overall, our work adds to the growing interest in the process of endosomal sorting by identifying and characterising a novel transport carrier, the ETCs. Moreover, to the best of our knowledge our study is the first report demonstrating the involvement of a retromer component in the sorting of endogenous sortilin. Hence, we describe a novel endosomal sorting domain, we position this along the endo-lysosomal maturation axis and among the other endosomal sub-domains, and we allocate a specific function to it.