DYNamics Of The Endocytic Proteome : a systemic perspective of trafficking and maturation – DYNOTEP

TThe DYNOTEP project involves multidisciplinary approaches including cell biology, bio-informatics, quantitative proteomics and original mathematical tools. Magnetic purification of the endocytic compartments at different stages of maturation will provide us with the pertinent biological material. Analysis of their composition at the protein level will involve advanced quantitative proteomics strategies based on mass spectrometry analysis. Exploitation of the data such as the establishment of time-resolved abundance profiles for each protagonist will require mathematical tools dedicated to complex time-series. The data will be explored by biologists to further dissect the cell endocytic dynamics.

The results derived from the DYNOTEP project include:

- data collection in the field of biology, and more specifically on the endocytic process. The project is structured around 4 interconnected tasks aimed at the identification of the proteins present on the endocytic pathway, the quantification of their relative abundance on the compartments and the analysis of their relationships.

- Methodological developments applicable to others biological processes or model organisms.

The DYNOTEP project is at the crossroad of fundamental research on endocytosis and experimental developments on proteome dynamics. It will generate completely novel data as to the dynamics of the endocytic process and underlying molecular mechanisms that should help integrate our current knowledge of the system in a more detailed and global view of the process. It should open new perspectives to investigate how pathological disorders originate from endocytic dysfunctions. Methodological spinoffs are expected in the field of mass spectrometry by establishing the basis for future systematic studies.

Habourdin C, Klein G, Tsuyoshi A, Williams J and Aubry L. (2013) The arrestin-domain containing protein AdcA is a response element to stress. Cell Comm. Signaling 11 :91-115

This paper is focused on the functional characterization of an actor of the endocytic pathway, the arrestin-related protein AdcA.

Endocytosis is a process whereby cells internalize fluid and solutes, macromolecules and particles, as well as membrane components by invagination of the plasma membrane and subsequent formation of intracellular vesicles. Besides its role in nutrition, endocytosis acts also as an integral element of signal transduction.
Implication of endocytosis in various pathological situations has largely stimulated research efforts regarding that process. Endocytosis dysfunctions are implicated in cancerogenesis and neurodegenerative disorders. The endocytic pathway also constitutes a weak link in cases of intracellular pathogen infections, as various viruses and bacteria have evolved strategies to subvert the endocytic process and thereby achieve replication and propagation. Furthermore, endocytosis has great potential as a cell entry mechanism for therapeutic biologicals such as membrane impermeant drugs, proteins or nucleic acids. But, despite considerable efforts devoted to the dissection of the molecular processes and pathways governing the endocytic activity, our current knowledge of endocytosis remains fragmentary. Most of the available data is limited to specific trafficking steps, components or sub-compartments; and the organization and multi-functionality of the endocytic network is clearly too complex to be decoded by the mere addition of information derived from these targeted approaches. Deep understanding of endocytosis therefore requires an integrative map combining molecular level information, dynamic aspects of trafficking, and crosstalks between the endocytic pathway and the rest of the cell.
The objective of the DYNOTEP project is to provide a systemic perspective on the endocytic process from the angle of protein dynamics. The project is at the crossroad of fundamental research on endocytosis and experimental development on proteome dynamics. Modern large-scale mass spectrometry-based quantitative proteomics methods now offer the possibility to establish the repertoire of proteins and to gain insight into their temporal distribution profile in dynamic biological systems. While the technology to achieve the throughput and data quality required for studying proteome dynamics is available, the concepts for multidimensional temporal profile analysis are emerging, and the underlying mathematical tools still require refinement.
The DYNOTEP project will use the Dictyostelium macropinocytic pathway as a model system. It will exploit cell biology, bioinformatics, advanced MS-based quantitative proteomics approaches and original mathematical tools for temporal data processing through 4 interconnected tasks designed to 1) provide a deep curated annotation of the Dictyostelium endocytic proteome we obtained previously, 2) enrich it with a spatio-temporal dimension by the establishment of time-resolved abundance profiles for all the protagonists, 3) supplement the proteome with functional information regarding interaction networks and 4) elaborate an integrated dynamic map of the endocytic process. This map will substantially complement targeted cell biology approaches and contribute to a systems-level knowledge of the endocytosis function(s).
The multidisciplinarity requirements of the project will be fulfilled by the association of three research teams that are recognized experts in their respective fields.