Visualisation and Perturbation of the Spatio-Temporal Dynamics of Endocytosis
Defended on June 18th, 2015
Clathrin mediated endocytosis (CME) is a fundamental process of all eukaryotic cells. At the level of the plasma membrane, it is characterised by the formation of deep invaginations resulting in the creation of small vesicles after membrane scission by the large GTPase dynamin. In the central nervous system, it is involved in the expression of synaptic long term depression, a proposed cellular correlate of learning and memory. The complex morphology of neurons and the precise timing of neuronal firing suggest that endocytosis may be spatially and temporally regulated in those cells.
The aim of my PhD was to develop new tools to visualise and perturb CME in order to study such regulation. The first tool to be characterised was pHuji, a genetically encoded red pH-sensor. I used it in combination with an existing green pH-sensor to demonstrate that in NIH-3T3 cells, the β2-adrenergic receptor was internalised upon agonist binding in a subset of vesicles containing the constitutively endocytosed transferrin receptor. This work was published in the Journal of Cell Biology in November 2014 (Shen and Rosendale et al., 2014).
The second tool is a new imaging method that allowed me to monitor the endocytic activity of optically stable clathrin coated structures in hippocampal neurons. By adapting a protocol developped in cell lines to monitor endocytosis with a temporal resolution of 2 s. (Merrifield et al., 2005), I was able to visualise for the first time the kinetics of internalisation of AMPA-type glutamate receptors under plasticity inducing conditions.
Finally, I set up an assay combining imaging and cell dialysis in order to develop a specific peptide-based inhibitor of CME. In collaboration with chemists of our institute, we found by using dimeric peptides, that the interplay between dynamin and its binding partners relies on multimeric interactions.
Altogether, this work provides a toolbox to decipher the mechanisms of vesicle formation with high spatial and temporal resolution.”
pHuji, a pH-sensitive red fluorescent protein for imaging of exo- and endocytosis.
Shen Y, Rosendale M, Campbell RE, Perrais D.
J Cell Biol. 2014 Nov 10;207(3):419-32. doi: 10.1083/jcb.201404107.
- Nathalie Sans (Neurocentre Magendie, Bordeaux, France) – Présidente du jury
- Volker Haucke (Leibnitz Institut Berlin, Germany) – Rapporteur
- Aurélien Roux (Geneva University, Switzerland) – Rapporteur
- Britta Qualmann (Jena University, Germany) – Examinateur
- David Perrais (Institut Interdisciplinaire de Neurosciences, Bordeaux, France) – Directeur de thèse
Team : Dynamics of synapse organization and function