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Home > TEAMS > TEAM CHOQUET > RESEARCH AXES > Membrane Trafficking

Membrane Trafficking


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Contact: David PERRAIS

Tel. (+33)5 05 33 51 47 33







Scientific Context


The Membrane Trafficking axis is integrated into the Dynamics of Synapse Organization and Function group .

This activity is headed by David Perrais (DR2, senior researcher) and currently involves, Magalie Martineau (post-doctoral fellow: Marie Curie fellowship) and Léa Claverie (PhD student). The main objective is to study the molecular and cellular mechanisms of endocytosis, exocytosis and recycling and their relevance for cellular function. We are specifically interested in these processes in neuronal dendrites, and how it organizes neuronal function, in particular synaptic transmission and plasticity. We focus on the imaging of single exo-and endocytic events in living cells, which permit us to:
  • record the precise location and timing of these events, and thus map them in cells
  • determine the recruitment of associated proteins, hence their role in these process
  • accurately measure the changes occurring during acute perturbation or physiological processes


  • Technical approaches


    We use essentially fluorescence imaging on three inverted microscopes: two with total internal reflection fluorescence (TIRF) microscopy, and one with spinning disk confocal microscopy. These imaging modalities can be combined with local fast solution exchange for the use of the pulsed pH protocol, and patch clamp electrophysiology, to record currents in neurons but also to manipulate the intracellular environment of cells. This imaging data is complemented by semi-automated, quantitative analysis performed with in house routines programmed with Matlab®.




    Detection of single exocytosis and endocytosis events in living cells


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    Methods to detect single exo- and endocytosis events with fluorescence microscopy:
    Cells are transfected with the transferrin receptor (TfR) fused to supereclipticphluorin (SEP, a mutant of GFP which is not fluorescent at pH 5.5).
    (A) Exocytosis event detected in a living neuron with TfR-SEP (see Jullié et al. 2014).
    (B) Principle of the pulsed pH protocol to detect the formation of single endocytic events. A vesicle is created at pH 7.4 (blue arrow) and detected 2s later at pH 5.5 (green arrow).
    (C) Detection of an endocytic event in a living 3T3 cell with TfR-SEP and the pulsed pH protocol. Dynamin1-mCherry is recruited maximally at the time of vesicle formation (see Taylor et al. 2011).


    Display event during pH oscillations, recorded at 10 Hz with a wide field epifluorescence microscope. Opening (sudden increase in fluorescence which oscillates with pH, green arrow) and closing (the fluorescence of the structure stops oscillating, orange arrow) are clearly visible. Movie is slowed down 2 times, scale bar is 1µm.





    A display event is followed 24s later by a burst event corresponding to the same organelle. Movie is accelerated 5 times, scale bar is 1µm.









    Exocytosis events recorded at 10 Hz with spinning disk confocal microscopy in the soma and dendrites of a neuron (14 DIV) transfected with TfR-SEP. Movie is displayed in real time, scale bar is 5µm.








    Funding


    This axis is funded by the ANR (CaPeBLE 2013) the Conseil Regional d’Aquitaine (2015) and the European Union (Marie Sklodowska-Curie fellowship 2015 to MM).




    Selected publications

    Cauvin C, Rosendale M, Gupta-Rossi N, Rocancourt M, Larraufie P, Salomon R, Perrais D*, Echard A* (2016) Current Biology,26(1):120-8.
    Rab35 GTPase Triggers Switch-like Recruitment of the Lowe Syndrome Lipid Phosphatase OCRL on Newborn Endosomes.

    Shen Y*, Rosendale M*, Campbell RE, Perrais D (2014) J Cell Biol 207:419–432.
    pHuji, a pH-sensitive red fluorescent protein for imaging of exo- and endocytosis.

    Jullié D, Choquet D, Perrais D (2014) J Neurosci 34:11106–11118.
    Recycling Endosomes Undergo Rapid Closure of a Fusion Pore on Exocytosis in Neuronal Dendrites.

    Taylor MJ, Perrais D & Merrifield CJ (2011). PloS Biology 9(3):e1000604.
    A high precision survey of the molecular dynamics of mammalian clathrin-mediated endocytosis.




    Former Members

    Julien Veran (PhD student and post-doctoral fellow)
    Damien Jullié (PhD student and post-doctoral fellow)
    Morgane Rosendale (PhD student and post-doctoral fellow)
    Thi Nhu Ngoc Van (post-doctoral fellow)
    Julia Krapivkina (PhD student)