Blanc SVSE 5 - Blanc - SVSE 5 - Physique, chimie du vivant et innovations biotechnologiques

Caged Peptides to Block Endocytosis – CaPeBlE

To visualise the formation of endocytic vesicles, we use fluorescence microscopy and protocols developed to distinguish internalised receptors from those remaining on the plasma membrane. We synthesize candidate peptides which we validate with in vitro tests before introducing them throug a patch pipette to observe their effect on endocytosis.

We have adapted the method to detect the formation of endocytic vesicles to cultured neurons (manuscipt in preparation).
We are finishing the validation of the method to simultaneously visualise the formation of endocytic vesicles in live cells and dialyse the cells with the candidate peptides through a patch pipette.
In parallel, we have synthesized a library of simple and modified peptides (dimeric peptides, branched peptides), and we test them with biophysical tests (fluorescence anisotropy, surface plasmon resonance, peptide pull-down). These tests will permit the selection of the best candidate to test in live cells.

The project will help better understand the molecular and cellular mechanisms of endocytosis, essential step towards the quantitative understanding of this process and its role in cell biology. Moreover, photoactivatable blockers will permit the block of endocytosis at chosen times and locations. This will permit to understand the role of endocytosis in the establishment of cell polarity, chemotaxis or receptor trafficking, in particular in neurons. These tools will be protected with patents or licenced for their diffusion in the scientific community.

One mauscript in preparation on the visualisation of endocytic vesicles in cultured neurons.

Submission summary

The interaction of a cell with its environment occurs through the plasma membrane. To regulate the shape and composition of their plasma membranes, and to internalize nutrients and signaling factors, eukaryotic cells use endocytosis, the formation of vesicles from the plasma membrane. Clathrin mediated endocytosis (CME) is the most widely used form of endocytosis. The dynamics and molecular mechanisms of CME, and its role in a number of cellular functions, has been the subject of intense study. A vast number of proteins and interactions implicated in this process have been identified, but the dynamics of these interactions is largely unknown. Our interdisciplinary team of biologists and chemists will develop new tools to visualize and interfere with selected key interactions leading to clathrin coated vesicle (CCV) formation.
First, we will synthesize photoactivatable, or caged, peptides to block the interaction between the proline rich domain of dynamin, a protein essential for CME, and the SH3 (Src homology 3) domain of its partners, such as amphiphysin. This will block CME in live cells in a spatially and temporally controlled way, which we will test with live cell imaging of CCV formation. Second, we will construct pairs of endocytic proteins fused with fluorescent proteins to visualize their interaction by Förster Resonance Energy Transfer-Fluorescence LIfetime Microsopy (FRET-FLIM) imaging in live cells. We will connect the timing of this interaction to the stages of endocytic vesicle creation. This set of experiments will provide unprecedented details on the dynamics of molecular mechanisms of CME.
Moreover, the caged peptides developed in this project will be usable in a variety of cellular contexts. In particular, CME is essential for the expression of synaptic long term depression (LTD), a neuronal substrate of certain forms of learning and memory. We will perform electrophysiology patch clamp experiments to determine with the caged peptide where and when CME is required for LTD, and whether endocytosis contributes to the synapse specificity of LTD. We anticipate that these tools, once made available to the scientific community, will clarify the involvement of CME in a number of cellular functions.

Project coordination

David PERRAIS (Centre National de la Recherche Scientifique) – david.perrais@u-bordeaux.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CNRS Centre National de la Recherche Scientifique

Help of the ANR 300,000 euros
Beginning and duration of the scientific project: December 2012 - 36 Months

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