Subcellular Structure and cellular Dynamics

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The Traffic, Signalling and Delivery group is focused on several aspects of how cells communicate with their environment through endocytosis (the process whereby cells internalise proteins, solutes etc. from the extracellular space by engulfing them within plasma membrane vesicles). We are also investigating how endocytic pathways may be exploited to deliver therapeutic and diagnostic compounds to the interior of the cell. We study novel trafficking pathways and mechanisms, especially also that of the interferon receptor, which we use to address the relationship between traffic and signalling.

The group is composed of two teams whose projects are tightly intertwined sharing many molecular tools (siRNAs, antibodies, etc.) as well as our expertise on trafficking and signalling assays:

• The Traffic and Delivery team, directed by Ludger Johannes,
• The Traffic and Signalling team, directed by Christophe Lamaze.

Traffic and Delivery

Fig. 1Intracellular transport pathways of protein toxins.
Shiga toxin enters cells, at least in part, by a non-classical clathrin-independent endocytic pathway (red arrow), as well as by clathrin-dependent endocytosis (black arrow). The blue circle represents the interface between early endosomes, recycling endosomes and the trans-Golgi network. By making use of Shiga toxin, we have identified the first molecular players at this interface. Shiga toxin, cholera toxin and some other toxins are then transported to the Golgi apparatus and on to the endoplasmic reticulum by the retrograde transport route (red arrows).  

This team studies two poorly characterised intracellular transport pathways: clathrin-independent endocytosis and retrograde transport from early/recycling endosomes to the endoplasmic reticulum via the Golgi apparatus (Fig. 1).

First, we are identifying the molecular mechanisms of clathrin-independent endocytosis and retrograde transport. These studies concern the role of membrane microdomains in retrograde sorting and membrane translocation, the structural requirements of retrograde sorting, and the role of cytosolic machinery in retrograde trafficking to the trans-Golgi network. We are using many different techniques including in vitro reconstitution of transport in cell-free assays and secondary ion mass spectroscopy (SIMS) for high-resolution imaging of lipids. We have a close collaboration with the Chemistry Department at the Institut Curie to obtain the modified lipid species that are required for these latter studies.

Secondly, we have developed a vectorial proteomics technique to screen for cellular proteins that use the retrograde transport route. We study how and to what extent the functions of these proteins depend on their trafficking via the retrograde transport route.

Fig. 2A new route for delivery of tumour and viral antigens into antigen-presenting cells
To stimulate a patient's immune response against tumour cells or against virally infected cells, the B-subunit of Shiga toxin (red symbols) is coupled to an antigenic peptide from a tumour or viral antigen (green ovals). The toxin-peptide complex enters the cell by binding to the cellular receptor for the toxin. After translocation into the cytosol, the antigenic peptide is released from the complex by the proteolytic activity of the proteasome. It is then processed like any other antigenic peptide for presentation on the cell surface where it can stimulate cytotoxic T lymphocytes to kill cells bearing the same antigen.

Finally, we are exploiting these two pathways in the development of anti-tumour and anti-viral immunotherapies (Fig. 2) as well as to image cancer cells and to target therapies to them. To this end, we have developed a well-established retrograde transport cargo - the B-subunit of Shiga toxin (STxB) - as a novel ‘delivery tool'. Ludger Johannes is cofounder of a biotech company, ShigaMediX (a spin-off from the Institut Curie), the goal of which is to bring the STxB technology into clinics.

Traffic and Signalling

This team is working mainly on the role of membrane trafficking in the signalling functions of cytokine and growth factor receptors - an emerging field of study. Our research is aimed at understanding the roles that the different endocytic pathways (i.e. clathrin-independent and clathrin-dependent endocytosis) play in the trafficking, signalling and biological activities of interferon receptors. Our ultimate goal is to understand how endocytosis controls type I- and type II-interferon signalling and to identify key factors that regulate both processes. These factors, at the interface between signalling, receptor transport and cell proliferation, will be potential targets for treatment of proliferative diseases.

More specifically, we have set up novel assays and characterised in molecular detail the endocytic pathways followed by the receptors for interferon-γ and interferon-α. We are currently analysing the molecular mechanisms that control the endocytosis and intracellular transport of these receptors. Also, we are developing new tools to visualise the receptors and their signalling partners using innovative cell imaging techniques.

Fig. 3Differential membrane sorting and signalling of interferon receptors.
The ligand-bound receptors for interferon-γ (IFNGR) and interferon-α (IFNAR) are segregated into different membrane domains due to their differential interaction with lipid microdomains. Once inside clathrin-coated pits, interferon-γ and its receptor are simply endocytosed, whereas interferon-α and its receptor are both endocytosed and signal to the inside of the cell. Thus, interferon-γ and interferon-α differentially control intracellular signalling pathways.

We are also analysing the roles of endocytosis and intracellular trafficking in the signalling functions and the immune, antiviral and antitumoural activities of interferons. We have shown recently that clathrin-dependent endocytosis controls the antitumoral activity of interferon-α but not that of interferon-γ, even though both receptors are endocytosed through the clathrin-dependent pathway (Fig. 3). We are currently investigating the molecular mechanisms behind this differential effect in terms of trafficking and signalling.

As we first showed for interleukin-2 and its receptor, we have found that treatment of cells with interferon-γ stimulates the association of its receptors with microdomains in the plasma membrane. We are currently investigating the role of this association in signalling and trafficking of the interferon-γ receptor.