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Proteases and immunity
Group leader: Ana-Maria Lennon-Duménil
My project, at the junction between immunology, cell biology and biophysics aims to identify the molecular mechanisms involved in the space and time-related control of antigen-presenting cell (APC) function and to assess their impact in vivo on the immune response, in particular in the context of tumor immunity. More specifically, we analyzes the rules and players governing the protein trafficking events associated to antigen (Ag) recognition, processing and presentation as well as to Ag presenting cell (APC) activation and migration.
The capture and processing of antigens (Ag) for their presentation onto MHC molecules to T lymphocytes is a key step in the establishment of the specific immune response. It is largely mediated by receptors expressed at the surface of APCs. The transport of vesicles containing internalised Ag-Receptor complexes and of intracellular vesicles containing the MHC molecules must therefore be finely coordinated to ensure that they merge into a compartment where the MHC-peptide complexes are formed.
Our team is attempting to understand the molecular mechanisms enabling APCs to organise their intracellular trafficking network in response to Ag internalisation in order to allow its processing.
For this, we use techniques from the fields of immunofluorescence (confocal microscopy), time-lapse imaging, micro-fabrication, electronic microscopy, molecular biology and biochemistry (FPLC, immunoprecipitation, radioactive labelling).
We recently demonstrated the existence of a network of interactions between cytoskeleton filaments and endocytic vesicles, which is finely regulated by antigenic stimulation in APCs. We have shown that these interactions serve to coordinate Ag processing for presentation by class II MHC molecules, along with APC migration for their encounter with T lymphocytes.
From a molecular standpoint, our results show that antigen-mediated stimulation induces APCs activation by phosphorylation of Myosin II (protein that drives binding to actin). Activated Myosin II in turn interacts with the vesicles carrying MHCII/Invariant chain
(li) complexes, thus enabling them to merge with the vesicles carrying the internalised Ag for preparation of MHCII/peptide complexes. The use of siRNA and Myosin II inhibitors has demonstrated that its activity is indeed essential for Ag processing. Myosin II is the first molecular motor identified as playing a key role in Ag presentation.*
Based on previous results, we are currently developing the following lines of research:
· How does Myosin II regulate MHC II trafficking?
Our results suggest that, rather than directly conveying the MHC II/li+ vesicles to the actin filaments, Myosin II indirectly regulates their transport along microtubules (MTs). Thus, Myosin II would appear to facilitate connections between MHCII/li+ vesicles, associated with MTs and those transporting the internalised Ag, associated with the actin cortex.
· What signals control APC polarization?
We have shown that tyrosine kinase Syk and small GTPase CDC42 are essential for polarization of the MT organisation centre and for the convergence of MHCII/li+ vesicles with Ag. We now wish to identify the CDC42 effectors involved. We are particularly interested in WASP, an actin polymerization regulator, and in the PAR polarity complex.
· Co-regulation of Ag preparation and APC migration
We postulate that the use of common proteins to regulate Ag processing and migration enable APCs to integrate and coordinate theses processes in time and space according to extracellular signals. We currently want to precisely define the mechanisms involved in this regulation.