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Membrane diffusion of glycine receptors

par Pierre-Louis Porté - 7 mai 2007

Glycine is one of the two main inhibitory neurotransmiters. Synaptic plasticity (temporel modification of the synaptic efficiency) depends directly on the number of receptors available in the postsynaptic area. Here we want to understand the role of the membranous diffusion in the temporel regulation of the number of glycine receptors.

The use of fluorescent semi-conductor nanocristals allows to follow the movement of individual receptors during several dickers of minutes. Thus it enables us not only to caracterize the diffusif behavior of individual receptors (so that to distinguish different types of population) but also to see changes of comportement within a same trajectory.

Thus, we have caraterized their diffusive movement in the neuronal membrane. The fluorescent approach has been completed by electron microscopy imaging of nanocristal-tagged receptors allowing a localization with a nanometrical resolution.

1. The glycine receptor (RGly) : generality and questions raised

1.1. Structure of the glycine receptor

Glycine is one of the two main aminoacids that mediates inhibition by activating chloride channels. The correspondant ionotropic receptor functions as a channel which allows to hyperpolarized the neuronal membrane. The glycine receptor is composed of 5 subunits (3 alpha subunits and 2 beta subunits), each containing 4 transmembrane spanning domains. Note that cellular membranes are fluid lipidic bi-layers inside which the membranous protein can diffuse.

In the post-synaptic membrane, the receptors are generally accumulated in front of presynaptic release sites containing the correpondant neurotransmitter. The RGLy are concentrated in functionnal microdomains in front of active presynaptic area, the formation of such microdomains is done thanks to a high density of proteins constituing the subsynaptic scaffold. Indeed, without it, the receptors freely diffuse within the membrane. The stabilisation of RGly is mediated by a cytoplasmic anchor protein : the gephyrin who interacts both with the beta subunit of the receptor and with molecules of the cytosqueleton, the microtubules.

1.2. Why study the lateral diffusion of RGly ?

The synaptogenesis mechanismes, when the synapses are being formed, and those of the synaptic plasicity, that is to say how the efficiency of a synaptic junction is modified, are current and significant issues in neurobiology field. Note that the cellular plasticity is likely to play a key role in the memory and learning processes.

We already know that the synaptic transmission efficiency depends on the number of receptors present in front of synaptic release sites. The reciprocal regulation is one of the current hypothesis. Thus, a modification of the synaptic efficiency at the long time scale could result from a modification of the equilibrium between freely diffusive receptors, receptors anchored by gephyrin and those recycled by endocytosis. Then, to go further in the understanding of all the phenomenons at stake, it seems very usefull to study the lateral dynamics of the receptors, that is to say how the receptors, free or clustered, diffuse within the membrane and how they are stabilised at the synapses.

1.3. Diffusion-capture mechanism

The analysis of the RGLy membranous diffusion, in videomicroscopy, on living cells, has revealed that they toggle between phasis of free diffusion, slow and fast, and confined ones due to the interaction with gephyrin. Thus the diffusion-capture mechanism must play a key role in the membranous organisation of the glycine receptor.

2. Technical aspects

2.1. Cell cultures

All cultures (cultures of neurons or line cells) are done in the Laboratoire de Biologie Cellulaire de la Synapse, ENS Biology. Primary cultures of spinal cord neurons from embryonic rats day 14.

2.2. RGLy labelling

To ensure a specific linkage between RGLy and a single nanocristal, we use one or two intermediate antibodies.


In neurons, the alpha 1 subunit of RGLy is labeled	In fibroblasts, the myc tag of transfected RGly is labeled

2.3. Epifluorescent set up

Optical resolution : around 250 nm The set up also contains an air system in order to regulate the working temperature

2.4. Image processing

See the project concerning the "Images processing for Single Particle Tracking" in order to fully understand the technics that enable us to obtain the nanocrital trajectories from the films aquired.

Let’s just note that, after the image processing, we can localize a single nanocristal with an accurancy of around 10 nm (which depends on the nanocristal emission wavelength and on the intensity of the incident ligth).

2.5. Diffusion coefficient calculation

By making the realistic assumption that the membranous proteins have a brownien diffusive movement (free, directed or confined), we obtain their diffusion coefficient by the calculation of the Mean Square Displacement (MSD) function.

You can find below a real trajectory following a free brownian movement and next, the MSD corresponding with the linear fit done on the short time scale which gives us the diffusion coefficient (1/4 of the slope).

3. Study of RGly dynamics

3.1. In neurons

In a first step, we have managed to caracterize, in rat spinal cord neurons, the lateral dynamics of the RGly which depends on its location (synaptic, perisynaptic or extrasynaptic).

We are now interested in understanding how its dynamics is changed by applying drugs (like TTX, strychnine, gabazine, APV, cnqx) in order to better know its ways of regulation. These studies are ongoing.

One film for exemple :

Transmission image	FM image	Nanocristals

On the right, you can see the trajectories calculated by the Single Particle Tracking program plus the transmission image and the thresholded image of the labeled synapses (FM 4-64, in red) in the background.

Finally, we obtain the following trajectories (in bleu the extrasynaptic receptors, in green the synaptic ones and in yellow the perisynaptic ones). From these trajectories, we are able to calculate the diffusion coefficients and the times spent at the synapses.

A few results :

You can find below the empirical cumulative distribution function of the diffusion coefficients obtained for the extrasynaptic receptors in controle conditions (without adding any drug, the closest condition to the culture medium) and with the add of TTX.

Collaborations

This is a collaborative work with Sabine Lévi and Antoine Triller (Laboratoire de Biologie Cellulaire de la Synapse, ENS Biologie) and with Stéphane Bonneau and Laurent Cohen CEREMADE, Université de Paris-Dauphine) for the image processing part.

3.2. In Hela cells

3.2.1. Why the study of these model cells is interesting ?

To study the RGly dynamics in neuron culture raises a few difficulties :
on a technical point of view, it is quite hard to localize precisely the synapses because the FM 4-64 staining is diffuse and bleach rapidly hence the measurement of dwell times and the precise localization of synapses is fastidious.
concerning the interpretation of the results obtained, neurons form a very complicated sytem so that it is quite hard to find the causes of an effect observed.

So, before coming back to the neuron culture (which are whatever the system we want to understand), we will look at simpler system, fibroblasts, in which we can selectively express the protein we want to study. Thus, once we have some interesting figures in these model cells, we will dispose of reference values that we can compare with the values obtained (or that will be otained) in neurons.

3.2.2. Caracterization of the RGLy-gephyrine interaction

In order to reduce the number of actors acting in RGLy dynamics regulation and to concentrate on the RGly-gephyrin interaction, the use of model cells like Hela cells (or COS cells or HEK cells) is a good tool. Indeed, in fibroblasts, it is possible to choose (with some limitation of course) which proteins we want the cell to express.

So by transfecting the cells, or not, with gephyrin:YFP and with RGly a1 or a1-bgb (containing or not the interaction loop with gephyrin), we will be able to precisely caracterize the RGly dynamics with and without the associated anchored protein. In a first step, we will compare the values obtained here with nanocristals with those already available obtained with beads.

The localization of the gephyrin (labeled with a very brigth YFP) should be easier and more accurate than the synapses staining (with the FM staining, moving vesicules in the presynaptic zones were labeled). Eventuelly, it is likely that we will be able to caraterize the RGly-gephyrin interaction.

3.2.3. A few images obtained with Hela cells

The transfection functions on Hela cells :

In a view field of 110*110 microns, you can see below 2 cotransfected cells (transfected with gephyrin and with RGly containing the interaction loop with gephyrin) and 1 cell not transfected. Here the cells are fixed.


Transmission image	Gephyrin (YFP)	RGly (labeled with an antibody linked with an organic fluorophore)

The antibody-nanocristal probe well functions :

Below, on living cells, you can see that our probe functions : the non specific labeling is very rare and coincide in most cases with casts.


Transmission image	Gephyrin (YFP)	RGLy labeled by nanocristal in the single particle state

One film for exemple :

This film contains 512 images taken at a frequency of 13Hz (one image every 75 ms), and on a view field of around 28*28 microns. Note the nanocristal blinking, it ensures us that it is a single probe.


Transmission image	Gephyrin (YFP)	Film : RGLy

3.2.4. Collaborations

This is a collaborative work with Cyril Hanus and Antoine Triller (Laboratoire de Biologie Cellulaire de la Synapse, ENS Biologie) and with Stéphane Bonneau and Laurent Cohen CEREMADE, Université de Paris-Dauphine) for the image processing part.

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