Servicio de Información Comunitario sobre Investigación y Desarrollo - CORDIS

Final Activity Report Summary - MVB BIOGEN. MOT. (Dynamics of Multivesicular and Late Endocytic Membranes: Invagination versus Tubulation)

The inside of eukaryotic cells, or cytoplasm, and, more specifically, of mammal cells, is separated from the extracellular medium by the plasma membrane which allows the selective entry and exit, called endocytosis and exocytosis respectively, of biological compounds as proteins and lipids. The cytoplasm contains the nucleus, in which the genetic material, i.e. Deoxyribonucleic acid (DNA) is packed, as well as many other intracellular compartments, or organelles, which are themselves also delimited by their own membrane and have precise functions to fulfil. To allow for cell survival, these organelles communicate between themselves using different pre-determined and highly regulated intracellular pathways, which can be compared to a motorway with many traffic lights. One of the main challenging questions of cell biology is to understand the mechanisms of sorting and transport of a specific molecule and how it is selectively sorted into one intracellular pathway and not in another.

The host laboratory was interested in the endocytosis and degradation pathways which allowed the entry of molecules into the cell by way of the plasma membrane to early endosomes, where they could be recycled back to the plasma membrane or directed to late endosomes and lysosomes to be degraded. This degradation process is necessary to maintain a tight equilibrium of the number of molecules per cell and regulate the complex cellular signalling machineries. Inside these endosomes, in a location which is called the lumen, we could observe some internal vesicles, or intralumenal vesicles, resulting from the invagination process of the limiting membrane of these intracellular compartments. Moreover, it was shown that endosomes could form tubules, which suggested that they were able to deform their limiting membrane in two topologically opposite directions, i.e. invagination into the endosomal lumen or tubulation towards the cytoplasm. Finally, it seemed that these two processes were linked and could be regulated by the same cellular components, since interfering with the membrane dynamics of intralumenal vesicles also inhibited the tubulation activity of late endosomes.

The aim of my project was to better investigate the invagination of the limiting membrane of late endosomes and to identify the key players in this cellular process. In order to do this, I developed an in vitro assay allowing for the reconstitution and precise measurement of the invagination process of endosomes. The implemented principle was that isolated endosomes were incubated in the presence of a small fluorescent compound which had a fluorescent intensity directly proportional to the efficiency of the invagination process.

Several control experiments were performed to confirm the validity and specificity of this new approach. Its strength and reproducibility opened the way to the second part of the project, i.e. the study of the regulation of this endosomal invagination process. Amongst many candidates, I identified Alix and Tsg101, two different proteins that could physically interact as important regulators of this invagination process. Furthermore, they had opposite effects on this mechanism. Alix was a negative regulator while Tsg101 seemed to enhance it. Finally, the interaction of each of these two proteins with late endosomal membranes was directly regulated by the presence of the other one. These observations altogether suggested that:
1. the limiting membrane of late endosomes was able to invaginate, thus leading to the formation of intralumenal vesicles; and
2. the tandem Alix/Tsg101 allows for tight regulation of the invagination of the limiting membrane of late endosomes.

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