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) focusiong 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 intercellular transport of the prion protein, PrPC, and its infectious form PrPSc (scrapie), which are both GPI-anchored. This will enable us to identify the intracellular site and the mechanism of pathological of conversion as well as the mechanism of spreading of the infection to the brain.

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. We have shown that:

a. GPI-APs are sorted via a direct pathway to the apical domain of living cells (Paladino, 2006)

b. Distinct v-SNAREs regulate apical delivery in polarized epithelial cells (Pocard, 2007)

c. Oligomerization of GPIAPs is a specific requirement for apical sorting (Paladino, 2004, 2007)

d. The lipid composition of AP and BL detergent resistant domains is similar (Tivodar, 2006)

e. N- and O- glycans are not directly involved in the oligomerization and apical sorting (Catino, 2008)

f. GPI-signals affect oligomerisation, raft partition and apical sorting (Lebreton 2008, Paladino 2008)

g. GPI-APs are organized in homoclusters forming choleseterol-dependent heteroclusters (under revision)

2) Intracellular and intercellular trafficking of PrPC and PrPSc : the site of conversion and the mechanisms of 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. Furthermore it is not known how prion spread from the intestin to (and within) the brain in the infectious form of the disease. We are analysing both these aspects using both biochemical and single-cell imaging approaches. We have shown that :

a. Different mutants of the prion protein have different intracellular pathways (Campana 2006, 2007)

b. The proteasome is not involved in the degradation of PrP mutants (Campana, 2006)

c. Wild-type and mutant PrPs partition and interact into detergent resistant membranes  (Schiff , 2008)

d. Both raft-dependent and clathrin-dependent pathways are involved in PrPC endocytosis (Sarnataro, 2009)

e. Doppel and PrPC co-immunoprecipitate in detergent-resistant membrane domains (Caputo, 2009)

f. Prions hijack tunnelling nanotubes for intercellular spread  (Gousset, 2009)

g. The recycling endosomes are a site for prion conversion (Marijanovic, 2009)

We are currently analysing the molecular mechanisms of conversion and spreading.

Keywords: Intracellular trafficking, prion , conversion site, GPI-anchored proteins, rafts, protein sorting, epithelial and neuronal cells

- Paladino S, Pocard T, Catino MA, & Zurzolo C (2006) . « GPI-anchored proteins are directly targeted to the apical surface in fully polarized MDCK cells. » J Cell Biol., 172:1023-34

- 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., 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

- Gousset K, Schiff E, Langevin C, Marijanovic Z, Caputo A, Browman D, Chenouard N, de Chaumont F, Martino A, Enninga J, Olivo-Marin JC, Männel D & Zurzolo C (2009). «  Prions hijack tunnelling nanotubes for intercellular spread. » Nature Cell Biology, 11(3):328-36

- Marijanovic Z, Caputo A, Campana V & Zurzolo C (2009), " Identification of the intracellular site of prion conversion. " PLoS Pathog., 5(5):e1000426