Project 1: GPI protein sorting
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.
One of our aims is to characterize the sorting, transport and arrival of GPI-anchored and transmembrane (TM) proteins to the plasma membrane of polarized epithelial cells.
Using fast confocal imaging based on the spinning-disc technology (Paladino et al., 2006) we analyze the site of sorting of apical and basolateral proteins and follow their segregation in living polarized cells seeded on filters. To this aim we have stably transfected two-colour GFP mutants fused to different apical and basolateral markers in MDCK cells. By using software of 3D reconstruction/tracking we will finely determined the areas of the Golgi where the two proteins bud and will track the post-TGN carriers in 3D allowing us to better understand where and when apical and basolateral GPI-APs are sorted. Furthermore, by using specific software of fluorescence quantization (e.g., volocity quantitation, matlab etc) we will investigate the properties of post-TGN carriers (as size, speed, content of fluorescent molecules).
We have shown that association with rafts domains is not sufficient for apical sorting of GPI-APs which need to form high molecular weight (HMW) oligomeric complexes in the Golgi (Paladino et al., 2004; Paladino et al., 2007). The molecular machinery involved in this sorting event is investigated by using the above-described imaging approach in conditions of perturbation of lipids domains and or proteins components. We are also setting an RNAi screen to fish putative factors.
Fig. 1. GFP-GPI labelled Golgi and vesicles (top panel). 3D reconstruction of Golgi vesicles budding (center panel). Apical and Basolateral GFP/Cherry linked proteins at the Golgi (lower panel).
We are also studying the spatial organization and raft association of GPI-linked proteins both at the plasma membrane and in the TGN.
The majority of the data on raft-association of GPI-APs rely on the study of protein insolubility in non-ionic detergents and flotation to lighter fractions of sucrose density gradients together with DRMs (Brown and Rose, 1992). Although DRMs have been a good tool for defining properties of raft- and non raft-associated molecules they do not provide any information about the spatial organization of proteins in membranes (Zurzolo et al., 2003, Paladino et al 2004, Zurzolo and Paladino, 2009). One of our aims is therefore to better assess raft associations of apically and basolaterally sorted GPI-APs in FRT and MDCK cells at both plasma membrane and TGN levels. This will allow us to understand whether raft association is a prerequisite for apical sorting of GPI proteins and will tell us what is the spatial organization of these proteins in the Golgi and at the plasma membrane. To this aim we are using different imaging approaches, FRET and FRAP (Lebreton et al 2008) and super resolution microscopy (STORM and PALM). By using these combined approaches in control cells and different conditions perturbing the lipid metabolism and/or the cytoskeletal organization, we will be able to define both the spatial organization of different membrane proteins at the plasma membrane and at the Golgi and define the requirements for polarized sorting in living conditions.
Project 2: Prions Trafficking and Conversion
Intracellular and intercellular trafficking of the prion protein
Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative disorders affecting humans and animals. They can be of infectious, genetic or sporadic origin. They result from a post-translational alteration in the conformation of a host-encoded GPI-AP called PrPC into 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 still a mystery how PrPSc invades the CNS from the periphery and which mechanisms allow prions to spread from cell to cell.
We are currently analysing both the mechanisms of prion conversion and of prions spreading based on our two recent and important findings: 1) that prion conversion occur/involves the endosomal recycling compartment (Marjianovic, 2009), and 2) that tunnelling nanotubes are able to transfer PrPSc between different cells in culture (Gousset et al 2009).
Our first objective is to study the intracellular site and the factors involved in prion conversion.
The cell biology of the conversion PrPC → PrPSc is poorly understood. Using combined pharmacological and genetic approaches and biochemical and imaging techniques we have recently identified the perinuclear endosomal recycling compartment (ERC) as the site of prion conversion. We demonstrated that in infected neuronal GT1 cells PrPSc preferentially accumulates in the ERC and modulation of PrP trafficking through the ERC influenced PrPSc levels. We propose that PrP retention in the ERC is necessary to concentrate prion proteins in a specific membrane domain for enough time to promote conversion.
We are currently setting a siRNA interference screening in order to identify factors involved in prion conversion in permanently prion-infected neuronal cells. To simplify the approach we plan to restrict our screen to the genes encoding proteins that pass through the ERC. To this aim we will use siRNA Mouse Genome Libraries purchased from Dharmacon. Similarly, pre-designed library containing siRNA directed towards genes involved in lipid metabolism will be used to identify lipid components important for scrapie production. The scrapie levels will be monitored by immunofluorescence, and automated detection, 4-6 days post-transfection. The best candidates will be further analyzed for their particular role in PrPSc production.
Another major objective is to characterize the mechanism of prion uptake and intercellular spreading from periphery to CNS.
To visualize PrPSc by immunofluorescence, cells must be subjected to a harsh treatment with guanidine hydrochloride (Gnd), which prevents studies in living cells. Therefore we created fluorescently labeled PrPSc by coupling prions-enriched brain homogenate with Alexafluor. To dissect the endocytic pathway involved in Alexa-PrPSc uptake, we follow its internalization together with Alexa-labeled transferrin, dextran or cholera toxin in living neurons. Furthermore, once Alexa-PrPSc is internalized we will analyse its subsequent intracellular trafficking using markers for different subcellular compartments in living cells and on fixed samples.
The mechanisms involved in spreading of infectious prions from one cell to another are largely unknown. A central question in the prion field that remains unanswered is how infection is transmitted from the periphery to the CNS. We have recently shown that PrP and PrPSc are found in tunnelling nanotubes (TNTs) in between neuronal cells (Gousset K et al, 2009). These are very fine membrane channels between distant cells which were shown to transfer proteins and vesicles (Gerdes H.H. et al., 2007, FEBS Lett., Davis DD and Sowinski S, Nature Reviews, 2008). Recent data showing TNT-mediated HIV transfer from infected to uninfected T-cells (Sowinski S. et al., 2008) implicates TNTs as a general way of pathogen spreading. Thus understanding TNT formation and subsequent transfer of proteins and entire organelles have became very important issues in cell biology. We are currently studying the role of TNTs in neuronal cells and in primary neurons, as well as their biological significance and possible role in transferring PrPSc in different neuronal cell cultures, between dendritic cells and neurons and in organotypic cultures and live tissue.
Based on co-culture experiments between Dendridic Cells (DCs) loaded with prions and primary neurons we have proposed that DCs are mediating the spreading of PrPSc from the intestine to the CNS. We are currently following the movement of Alexa-PrPSc in live cells in real time using spinning disk confocal microscopy. Following the transfer of Alexa scrapie we also monitor whether infection can be spread by DCs by analyzing scrapie production in neurons. Finally, in order to identify the organelle which mediates Alexa-PrPSc passage through TNTs we analyse colocalization of Alexa-PrPSc with various markers.
Fig. 2. TNTs between neuronal cells and transfer of lysotraker vesicles (top panels). Transfer of Alexa-scrapie from DC to a neurite of CAD cells (white arrow) via a TNT (yellow arrow lower panel).
Role of TNTs in vivo
To unequivocally demonstrate that TNTs represent a mechanisms for prions spreading in vivo we need to morphologically characterize these structures in cell culture, to understand how they are formed and to identify specific markers that in turn will be necessary to recognize TNTs in live tissues. To this aim we are in the process of setting up a high content siRNA screen in order to 1) identify key molecules critical for TNT formation and 2) identify TNT specific molecular markers that can be use in culture experiments to identify TNTs, in vitro and in vivo. In collaboration with JC Olivo-Marin we are setting an image analysis program for automated detection of TNTs in a siRNA interference screening. Once a few potentially important candidates are identified, we will study their individual roles in TNT formation, protein transfer, and as TNT specific markers.