Unit: Membrane Trafficking and Pathogenesis
Director: Chiara ZURZOLO
By using an imaging and FRET approach in living cells combined with a classical biochemical approach our laboratory has two major objectives :
to understand the molecular requirements for apical sorting of GPI-anchored proteins in polarized epithelial cells.
to characterize the exocytic and endocytic trafficking of the normal and abnormal prion proteins in epithelial and neuronal cells in order to identify the intracellular site of conversion.
One of the most important properties of epithelia is cell polarity, which results in the formation of distinct apical and basolateral domains of the plasma membrane. These distinct domains enable the cells to perform their vectorial activities. The mechanisms responsible for the generation and maintenance of epithelial polarity have been only partially characterized. Elucidation of the molecular basis of cell polarity is therefore a fundamental goal in mammalian cell biology. We have focused our attention on studying the mechanism of apical sorting of GPI-anchored proteins and the role of lipid microdomains (or rafts) and proteins and lipid segregation at the level of the TGN in two model epithelial cell lines, MDCK and FRT cells, which have different sorting phenotypes. In these cells we are also studying the exocytic and endocytic transport of the prion protein, PrPc, and its pathological mutants, in order to understand the role of intracellular trafficking and rafts in the function and the pathological conversion of this cellular protein to the infectious scrapie.
Two major projects are ongoing in my unit:
1) Mechanism of GPI-anchored protein sorting to the plasma membrane
2) Intracellular trafficking of PrPc and its mutants: correlation with TSE pathogenesis
1. Mechanism of GPI-anchored protein sorting to the plasma membrane
Rafts are membrane microdomains enriched in glycosphingolipids (GSLs) and cholesterol which have been proposed to function in the last Golgi cisterna, the TGN, as a sorting platform for the apical delivery of plasma membrane proteins. This is particularly evident in the case of GPI-anchored proteins that are sorted to the apical membrane in several epithelial cell lines and associate with rafts (or DRMs, Detergent-Resistant Microdomains) during their transport to the plasma membrane. Our knowledge of how this occurs is only rudimentary: e.g., what determines the association of these proteins with rafts, whether there are different kinds of rafts for different GPI-anchored proteins, or whether a TGN receptor that sorts apical and basolateral proteins into different post-Golgi vesicles is involved, are not yet known.
In this project we are using both microscopical and biochemical approaches to analyze the role of raft domains in sorting and trafficking of GPI-anchored proteins and to characterize the molecular components of the machinery.
For this purpose we had already obtained stable clones of FRT and MDCK cells expressing different chimeric proteins fused to GFP and set up an assay to study transport in basal conditions using a microscopy-based imaging system and deconvolution software. We had found that GFP-GPI is basolateral in FRT cells and apical in MDCK cells, whereas a GFP-TM construct containing the transmembrane and cytosolic domains of the LDL (low density lipoprotein) receptor is basolateral in both cells. We have further utilized this approach to characterize the kinetics of the basolateral and apical trafficking of these fluorescent markers and the characteristics of the post TGN carriers. We have followed the kinetics of emptying of the Golgi apparatus and plasma membrane arrival after block at 20°C and we have been able to determine the size and velocity of post-TGN intermediates arriving at the apical and basolateral plasma membranes in FRT and MDCK cells. This work is still in progress and has been done both using the CID at Pasteur and in collaboration with Dr Fogarty at the University of Massachussets (USA).
We and others have demonstrated that raft-association depends on the GPI anchor. However this event is not sufficient to determine apical sorting, which we believe occurs following "concentration and stabilization" of the protein in these microdomains probably through interaction via protein ectodomains. By using a biochemichal approach we found that only apical and not basolateral GPI-anchored proteins form high molecular weight oligomers. We believe that stabilization in rafts could occur because of protein oligomerization. We plan to analyse these oligomers by mass spectrometry to eventually identify a putative sorter molecule.
Furthermore in order to analyse the role of the GPI anchor and of the ectodomain in the sorting event we have transfected different GPI-anchored and transmembrane (TM) proteins in MDCK and FRT cells. By chemical cross-linking and co-immunoprecipitation protocols we have preliminary results suggesting that two different GPI-proteins are in the same complex, from which TM proteins are excluded. A parallel approach to analyse proximity of GPI and TM proteins at the cell surface and in the TGN (during their sorting) is by analysing FRET. To this aim we will use both classical FRET (using the CFP/YFP pair) and anisotropy studies (using GFP). In the last months we have prepared CFP/YFP and GFP constructs fused to different GPI anchors (from PLAP, DAF, FR, and PrP). We are currently transfecting them in pairs in order to obtain stable clones and to analyse FRET and/or anisotropy.
In addition, to see whether there are some important differences in the GPI anchor between apical and basolateral GPI proteins we have purified different apical and basolateral GPI-anchored proteins from FRT cells and will study the structure of the anchors in collaboration with the laboratory of Dr. M. Ferguson in Dundee.
Intracellular trafficking of PrPc and its mutants: correlation with TSE pathogenesis
Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative disorders of humans and animals that are unique because they can be either infectious, genetic or sporadic in origin. All three forms result from a post-translational alteration in the conformation of a host-encoded membrane glycoprotein called PrPC, which denotes the cellular isoform of the prion protein. Conversion of this normal isoform from an -helical, readily proteolysed surface protein, to the scrapie isoform, PrPSc, a highly protease resistant, ß-pleated sheet-containing hydrophobic aggregate is not only diagnostic of TSE infection, but is widely taken to be the defining infectious event and source of the subsequent neuropathology .
It is our contention that the conversion of prion protein from PrPC to the pathogenic PrPSc isoform is strongly influenced by the intracellular trafficking of the protein in the cells. Epithelial cells are very similar to neuronal cells, but are better characterized for their intracellular trafficking and endocytosis, so they represent an ideal model to study the intracellular trafficking of PrPc and its mutants. Furthermore, in neuroblastoma cells PrPC associates with TX100-insoluble membrane microdomains and the depletion of cholesterol reduces PrPC degradation and inhibits PrPSc generation. By extraction in cold TX100 we found that PrPc is mainly insoluble, indicating that association with detergent-resistant microdomains (DRM) is not sufficient for apical sorting of GPI-proteins. In the last months we have further characterized the DRM asssociation of PrPc during pulse chase experiments and found that one of the immature PrPc isoforms already associates with DRM in the endoplasmic reticulum. Perturbation of this association by depleting the cells of cholesterol leads to protein misfolding, therefore suggesting that DRM association is necessary for the correct folding of PrP. Because TSE is a result of PrP misfolding this issue appears to be very relevant for understanding the mechanisms of PrP misfolding. We are currently testing DRM-association and its effect on folding of different PrP mutants. In collaboration with Tony Futerman at the Weizmann Institute we are analyzing the lipid composition of DRMs associated with the normal and mutated forms of PrP.
We have also analyzed the exocytic pathway of single and double glycosylation mutants and of a mutant lacking the GPI anchor. Interestingly the glycosylation mutant A182T, which is pathological (i.e., found in familial forms of CJD), displays important differences in sorting and DRM association compared to the wild-type protein. We have just received the P3 facilities and begun our project on the infected cells to study PrPSc trafficking and possibly to attempt to identify the site of conversion. We have prepared wild-type and mutant PrP-GFP constructs that we plan to use to identify the site of conversion in living cells.
In the last 6 months we have made some progress in our understanding of the internalization route followed by PrP. It has been shown that PrPC can be internalized either via clathrin-coated pits or via caveolae. Following endocytosis, PrP is recycled back to the cell surface after processing. Study of the endocytic recycling pathway is of interest because it may be the route along which the critical conversion of PrPC to PrPSc takes place, and because one physiological function of PrPC might be to facilitate uptake of an as yet unidentified extracellular ligand, by analogy with receptors responsible for uptake of transferrin and low-density lipoprotein.
To test the involvement of caveolae and coated pits in the endocytosis of PrPc isoforms we have used FRT cells which do not express caveolin and do not have caveola, as well as FRT cells transfected with cav1, which do form caveolae. We found that PrPc is internalized in both cell lines, but is never found in coated pits. From these and other data we believe that PrP is internalized via a non-coated pits non-caveolae internalization pathway, which is, however, raft-dependent. We are currently charachterizing the requirements of this internalization pathway.
Keywords: intracellular trafficking, rafts, protein sorting, prion, conversion site