Cannabinoid-induced actomyosin contractility shapes neuronal morphology and growth
- ESPCI-ParisTech, CNRS UMR8249, France
- Harvard University, United States
- Aix-Marseille Université, Parc Scientifique et Technologique de Luminy, France
Figures
Figure 1 with 1 supplement
CB1R activation induces retraction of actin-rich growth cones.
Cultured DIV8 hippocampal neurons co-expressing Flag-CB1R-eGFP and LifeAct-mCherry on (A–G) and LifeAct-mCherry only on (H and I). (A) Treatment with CB1R agonist WIN55,212-2 (WIN, 100 nM, added at …
Figure 1—figure supplement 1
mCherry-LifeAct label (red channel) from Figure 1A. Scale bar: 20 µm.
https://doi.org/10.7554/eLife.03159.004
Figure 2 with 3 supplements
CB1R-induced retraction is mediated by non-muscle myosin II dependent actomyosin contraction.
Cultured hippocampal neurons co-expressing Flag-CB1R-eCFP, LifeAct-mCherry, and EB3-eGFP at DIV6 were treated by WIN (100 nM) at 0 min. (A) Microtubules (MT) bend and form small loops (arrowhead on A…
Figure 2—figure supplement 1
Averaged F-actin relocalization in the distal 60 µm in growth cones in the first 4 min after WIN treatment in five randomly chosen neurons from Figure 1C.
Black, orange, and red curves represent the mean intensity of LifeAct-mCherry labeling at baseline, at 2 min and 4 min after addition of WIN (100 nM), respectively. Scale bar: 20 µm.
Figure 2—figure supplement 2
CB1R-induced periodic microtubule bends are not due to EB3-eGFP and LifeAct-mCherry expression.
Cultured hippocampal neurons were transfected at DIV6 with CB1R-eCFP and treated after 24 hr with 100 nM WIN55,212-2 for 10 min before fixation. Presence of periodic bends is shown by immunolabeling …
Figure 2—figure supplement 3
Concentration-response curve for the blebbistatin effect on the growth cone retraction essay after treatment with WIN (100 nM).
*p < 0.05; ***p < 0.001 calculated using Kruskal–Wallis one-way ANOVA followed by Dunn's post-tests on (I) and using one-way ANOVA followed by Newman–Keuls post-tests. Scale bar: 20 µm on A, B, and B…
Figure 3 with 2 supplements
CB1Rs activate non-muscle myosin II through heterotrimeric G12/G13 proteins, Rho GTPase, and ROCK.
Cultured hippocampal neurons at DIV6 co-expressing a combination of LifeAct-mCherry, Flag-CB1R-eCFP, and EB3-eGFP as indicated and treated by WIN (100 nM) at 0 min. (A–B) Representative …
Figure 3—figure supplement 1
Another representative LifeAct-mCherry expressing growth cone 2 min after treatment with WIN (100 nM), labeled with the phosphoMLC antibody, similarly to Figure 3B.
The region of interest used for the quantification of phosphoMLC labeling intensity, approximately 50–60 µm on each image, is marked with dotted lines. This region was delimited using the …
Figure 3—figure supplement 2
Amplitude of 100 nM WIN-induced growth cone retraction in neurons co-expressing LifeAct-mCherry, EB3-eGFP, and Flag-CB1R-eCFP with (C3T) or without (WIN) pre-treatement with 1 µg/ml C3T.
Results are pooled from two independent experiments and outliers were removed in accordance with Grubb's test. Results in are expressed as boxplots. ***p < 0.001 calculated using Student's t-test.
Figure 4
Activation of exogenous or endogenous CB1Rs modifies growth cone dynamics ex vivo.
Progression of dynamic, F-actin-rich corticofugal growth cones from organotypic slices cultured for 24 to 48 hr, prepared from P4-6 rat brains, previously electroporated in utero at E16 to express …
Figure 5 with 1 supplement
Actomyosin contractility is required for the correct targeting of CB1R expressing corticofugal axons.
(A) Experimental design. Left: in utero intracerebroventricular injection of E15 rat embryos. Right: analysis of axons in the lateral sub-ventricular zone (SVZ, red). (B) E15 corticofugal axons …
Figure 5—figure supplement 1
Cortifugal origin of Tuj1-expressing axons in the SVZ.
(A) Experimental design. Left: in utero transfection of cortical progenitors of E16 rat embryos with GFP. Right: analysis of GFP-expressing corticofugal axons of transfected and radially migrated …
Figure 6
CB1R-induced actomyosin contraction results in neurite retraction and transiently increased cell stiffness in Neuro2A cells.
(A and B) Cells expressing Flag-CB1R-eGFP and LifeAct-mCherry. F-actin accumulates in the extremity and shaft of neurites (arrowheads). Agonist WIN (100 nM) induces retraction of neurites. Open …
Figure 7
Acute and chronic effects of CB1R-mediated actomyosin contraction on somatodendritic morphology.
(A) Cultured hippocampal neurons expressing CB1R-eCFP, LifeAct-mCherry, and EB3-eGFP at DIV8. Application of 100 nM WIN results in rapid and significant reorganization of somatodendritic morphology, …
Videos
Video 1
CB1R activation induces retraction of actin-rich growth cones.
Dynamic, F-actin-rich growth cone of a cultured hippocampal neuron co-expressing CB1R-eCFP, LifeAct-mCherry, and EB3-eGFP at DIV6 treated with 100 nM WIN at 10 min. Scale bar: 20 μm.
Video 2
Effect of microtubule depolymerization on CB1R-induced growth cone retraction.
Dynamic, F-actin-rich growth cone of a cultured hippocampal neuron co-expressing Flag-CB1R-eGFP and LifeAct-mCherry at DIV6, pre-treated with 10 µM Nocodazole at 20 min before treatment with 100 nM …
Video 3
Effect of actin depolymerization on CB1R-induced growth cone retraction.
Dynamic, F-actin-rich growth cone of a cultured hippocampal neuron co-expressing CB1R-eCFP, LifeAct-mCherry, and EB3-eGFP at DIV6 pre-treated with 1 µM cytochalasin D at 20 min before treatment with …
Video 4
Effect of NM II inhibition on CB1R-induced growth cone retraction.
Dynamic, F-actin-rich growth cone of a cultured hippocampal neuron co-expressing Flag-CB1R-eGFP and LifeAct-mCherry at DIV6 pre-treated with 25 µM blebbistatin at 20 min before treatment with 100 nM …
Video 5
CB1R activation induces retraction of actin-rich growth cones in organotypic slices.
Dynamic, F-actin-rich corticofugal growth cones from organotypic slices were cultured for 24 to 48 hr, prepared from P4-6 rat brains, previously electroporated with EB3-eGFP, LifeAct-mCherry, and …
Video 6
CB1R activation induces neurite retraction and cell rounding in Neuro 2A cells.
Neuro2A cell expressing Flag-CB1R-eGFP and LifeAct-mCherry. Treatment with 100 nM WIN at 30 min induces neurite retraction and cell rounding. Scale bar: 20 μm.
Video 7
CB1R activation induces neurite retraction, cell rounding, and temporary blebbing in Neuro 2A cells.
3D reconstruction of a Neuro2A cell expressing Flag-CB1R-eGFP and DsRed2. Treatment with 100 nM WIN at 7 min (420 s) induces neurite retraction, cell rounding, and transitory blebbing. Scale bar: 10 …
Video 8
CB1R activation induces rapid remodeling of the somatodendritic region in cultured hippocampal neurons.
Somatodendritic region of a cultured hippocampal neuron co-expressing CB1R-eCFP, LifeAct-mCherry, and EB3-eGFP at DIV8. The axon, whose initial segment is typically strongly labeled with EB3-GFP, …
