|Membrane Traffic and Pathogenesis|
|HEAD||Dr Chiara Zurzolo / firstname.lastname@example.org|
|MEMBERS||Dr Duncan Browman / Melle Anna Caputo / Melle Maddalena Costanzo / Dr Gousset Karine / Dr Gianni Guizzunti / Melle Naga Sailaja Imjeti / Dr Christelle Langevin / Dr Stéphanie Lebreton / Melle Marie Lemesle / Dr Zrinka Marijanovic / Mr Mickaël Marin / Melle Ludovica Marzo
One of the most important properties of epithelia is cell polarity, which enable cells to perform their vectorial activities. We study the mechanism of apical sorting of GPI-anchored proteins (GPI-APs) focusing our attention on the role of membrane microdomains (or rafts), and protein and lipid segregation in this event. We are also studying the intracellular and the extracellular trafficking of the prion protein, PrPC, and its infectious form PrPSc (scrapie), as well as pathological mutants associated with hereditary forms of TSEs. This will enable us to identfy the intracellular site of the pathological conversion and the mechanism of the spreading of the infection. The major projects of the Unit are the following:
1) Mechanism of GPI-anchored protein sorting to the plasma membrane
GPI-APs are sorted to the apical membrane in several epithelial cell lines and associate with rafts during their transport to the plasma membrane. Our knowledge of how this occurs is only rudimentary. In this project we are using both microscopic 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. Starting from our original observation that rafts are not sufficient for sorting of GPI-APs, but oligomerization is required (Paladino et al, 2004), in the last few years we have shown that oligomerization is a specific requirement for GPI-AP sorting in different epithelia (Paladino et al, 2007). Therefore we have subsequently analyzed the respective involvement in this sorting event of the lipid anchor and the surrounding lipid environment as well as of the protein ectodomain. Specifically we found that different GPI-attachment signals affect the oligomerisation of GPI-anchored proteins and their apical sorting (Paladino et al, 2008), however we also demonstrated that the lipid composition of apical and basolateral detergent resistant domains is similar (Tivodar et al, 2006). Interestingly by biochemical analysis and live FRAP approach we could show that both actin and cholesterol are involved in the compartmentalization of apical proteins in different membrane domains both at the plasma membrane and Golgi levels (Lebreton et al 2008). Regarding the role of the ectodomain we demonstrated that both N- and O- glycans are not directly involved in the oligomerization and apical sorting of GPI-proteins and a N-glycosilated factor might be involved (Catino et al, 2008). Based on these data our current hypothesis is that differences in the lipid anchor might regulate the affinity for rafts of different GPI-APs, therefore regulating the rate of oligomerization and apical sorting. However the situation id more complicated by the fact that also the ectodomain of the protein can be involved in the oligomerization process by specific interactions with factors putatively enriched in rafts. Regarding the mechanism of trasport and fusion of GPI-APs with the apical membrane we have shown that GPI-APs are sorted via a direct pathway to the apical domain of living cells (Paladino et al, 2006) and that distinct v-SNAREs regulate apical delivery in polarized epithelial cells (Pocard et al, 2007). We are currently continuing these studies both towards the characterization of the apical pathway and the identification of specific factors involved in the sorting event.
2) Prions trafficking : intracellular site of pathological conversion and mechanisms of intercellular spreading
Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative disorders of humans and animals of either infectious, genetic or sporadic origin. They result from a post-translational alteration in the conformation of a host-encoded GPI-AP called PrPC into to the scrapie isoform PrPSc. This conformational transition is thought to be catalyzed by a specific physical interaction between endogenous PrPC and PrPSc, which is the principal component of the transmissible agent (or prion). The intracellular compartment where PrPC - PrPSc conversion occurs and how this process leads to neurological dysfunction are still unknown. Analyzing the intracellular trafficking of PrPC will be critical to understand how and where it is converted to PrPSc. We have analyzed the biosynthetic/ degradative pathway, exocytic/endocytic trafficking of PrPC and of some mutants responsible for the hereditary diseases (Campana et al, 2005, Campana et al, 2007). Numerous data in the literature suggest that a specific membrane environment (eg., cholesterol-enriched rafts) could be important for the conversion process in the infectious forms of the disease. We hypothesized that they could also be relevant in the case of hereditary diseases, indeed our recent data indicate that the reft environment might stabilize the proper folding of PrPC and may protect PrP mutants from conversion (Campana et al, 2005, Campana et al, 2006, Sarnataro et al, 2006). On the other hand we have shown that that co-expression of different PrP mutants with the wild-type protein affected their reciprocal trafficking. Interestingly we observed a perfect intracellular co-localization of the wild type protein with the mutant and an increase in their association to DRMs compared with the single transfected cells. These data, confirmed by a FRET approach, indicate that PrPC could interact with PrP mutants interfering with their trafficking, which could be of relevance for the pathogenesis and the progression of the pathological hereditary diseases (Schiff et al , 2008). By selectively blocking different intracellular pathways we are currently studying the site of the pathological conversion. In the mean time we have produced a fluorescent form of PrPSc to analyze its intercellular spreading by live imaging.
Keywords: Intracellular trafficking, prion , conversion site, intercellular spreading, GPI-anchored proteins, rafts, protein sorting, epithelial and neuronal cells
- Paladino S, Lebreton S, Tivodar S, Campana V, Tempre R & Zurzolo C (2008) « Different GPI-attachment signals affect the oligomerisation of GPI-anchored proteins and their apical sorting. » J Cell Sci. 2008 Dec 15;121(Pt 24):4001-7.
- Lebreton S, Paladino S & Zurzolo C (2008) « Selective roles for cholesterol and actin in compartmentalization of different proteins in the Golgi and plasma membrane of polarized cells. » J Biological Chemistry , 283(43)29545-53
- Schiff E, Campana V, Tivodar S, Lebreton S, Gousset K & Zurzolo C (2008) « Co-expression of wild-type and mutant PrPs alters their partitioning into detergent resistant membranes. » Traffic, 9(7) 1101.1115(15)
Pocard T., Le Bivic A., Galli T. & Zurzolo C. (2007) « Distinct v-SNAREs regulate direct and indirect apical delivery in polarized epithelial cells. » J Cell Sci., 120 : 3309-3320.
Paladino, S., T. Pocard, M. A. Catino, & Zurzolo C. (2006) « GPI-anchored proteins are directly targeted to the apical surface in fully polarized MDCK cells. » J Cell Biol 172:1023-34.
Activity Reports 2009 - Institut Pasteur
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