The Journal of Neuroscience, March 22, 2006, ():
Constitutive Activation Drives Compartment-Selective Endocytosis and Axonal Targeting of Type 1 Cannabinoid Receptors
J. Neurosci. Leterrier et al.
26: 3141
Supplemental data
Files in this Data Supplement:
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Supplemental figure 1: Pattern of FCB1-eGFP expression
(A) and (B) Comparison of the distribution of endogenous CB1R (A, C-Ter antibody revealed in green) and transfected FCB1-eGFP (B, in green) in DIV 9 neurons. Top panel is a montage showing soma, dendrites and axonal arborization of a CB1R-positive or a FCB1-eGFP transfected neuron. CB1R as well as FCB1-eGFP show vesicular staining in the somatodendritic compartment (boxed) and homogenous staining on the axon (arrowheads). Bottom panel is a maximal projection of a deconvoluted Z-stack showing details of the somatodendritic labeling boxed on top panel. Vesicular staining is clearly detectable in the soma and in dendrites (see dendritic marker MAP2 in red). Scale bars, 20 µm (top panels) and 5 µm (bottom panels).
(C) Correlated light and electron microscopy of a FCB1-eGFP transfected neuron with revelation of FCB1-eGFP using an anti-eGFP antibody followed by immunoperoxidase staining. FCB1-eGFP is enriched on the surface of the axon (arrowheads). Note the passage of the stained axon (upper optical panel, arrowhead) along a non-transfected neuron (asterisk). The somatodendritic compartment exhibits numerous large CB1R-positive endosomes (boxed) detected on optical image (bottom brightfield panel) as well as on EM detail (bottom EM panel). Scale bars, 100 µm (top brightfield image), 10 µm (bottom brightfield image), 1 µm (top EM image) and 0.5 µm (bottom EM image).
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Supplemental figure 2: Endocytosed CB1Rs colocalize with clathrin
Neurons coexpressing FCB1-eGFP and a clathrin light chain-DsRed fusion protein at DIV 8 were incubated 60’ with anti-FLAG M1 antibody. After fixation and permeabilization, primary antibody was revealed using a far-red Alexa-647 secondary antibody. Left panel, image of a transfected neuron showing FCB1-eGFP in green, clathrin-DsRed in red, and M1 antibody (corresponding to CB1Rs that transited at the neuronal surface in the last 60’) in blue. The axon (arrowheads) is uniformly labeled both for total CB1R (green) and M1 antibody (blue), whereas clathrin appears as puncta in both the somatodendritic and axonal compartments. Scale bar, 50 µm. Image (middle panel) and zoom corresponding to the boxed area show clearly endocytic vesicles in the soma and proximal dendrites that are labeled for FCB1-eGFP, clathrin-DsRed and M1 antibody, thus appearing white on the overlay image (lower panel, arrows). Scale bar, 10 µm.
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Supplemental figure 3: MBCD is a potent inhibitor of transferrin uptake in hippocampal neurons
Neurons at DIV 9 were starved for 2h in Neurobasal medium without B27 or any other source of transferrin before being incubated with either vehicle (transferrin-free Neurobasal) or vehicle plus MBCD at 5 mM for 15’. Human transferrin conjugated to Alexa-568 was then added to the media and incubated for 15’ before fixation.
(A) Representative 20X (upper panel) and 100X (lower panel) images of neuronal cultures in the two conditions. Transferrin uptake (in green) is notably attenuated after pretreatment of neurons with 5 mM MBCD (right). Scale bar, 50 µm (upper panel) and 10 µm (lower panel).
(B) Quantification of transferrin uptake. Between 15 and 20 images of neuronal somas were taken using the 100X objective as in lower panel in (A) and pixel intensities in the transferrin channel were summed for pixels above a globally defined threshold. Results are plotted as mean ±SEM. MBCD significantly inhibits transferrin uptake, confirming its efficacy as inhibitor of clathrin-mediated endocytosis, in line with results of the literature (Subtil et al., 1999).
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Supplemental figure 4: Representative neurons for surface CB1R distribution classes in the BFA block experiment
After transfection with FCB1-eGFP (3h, green) and BFA block (overnight), BFA is washed out and trafficking of CB1R toward the plasma membrane resumes. 0, 4, 8 and 24h after washout, neurons are labeled for surface CB1Rs using the M1 antibody (red) and fixed. Transfected neurons on each coverslip are classified based on their surface CB1R distribution (see Materials & Methods) : “Not Yet on Surface” (A), “Uniform” (B), “Axonal” (C) and “Somatodendritic” (D). For each category, a 40X projection of a Z-stack showing axonal arborization (upper panel, scale bar, 20 µm) and a confocal slice inside the somatodendritic compartment (lower panel, scale bar, 5 µm) is shown.
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Supplemental figure 5: The constitutive activity-dependent axonal targeting cycle: a targeting mechanism for neuronal GPCRs
In hippocampal neurons, constitutive activation of CB1R drives a constitutive trafficking cycle between plasma membrane and endosomes. At the somatodendritic plasma membrane, CB1Rs are subject to efficient endocytosis (green arrow), followed by slow recycling (blue arrow). This cycle results in very low surface expression of CB1Rs on the soma and dendrites (red dots). In the axon, CB1Rs are maintained at the plasma membrane (red outline), due to less efficient endocytosis (little green arrow). A diffusion barrier between somatodendritic and axonal compartments (in black) prevents proteins to diffuse laterally outside of their respective compartment. In this model, accordant with our kinetic studies, most receptors appear first on the somatodendritic surface before being endocytosed and sent to the axon.
