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 arrowheads: neurite tip at 0 min. (B) Blebbistatin (25 µM) significantly reduces 100 nM WIN-induced cell rounding. Results are expressed as mean ± SEM. (C) Phase-contrast image of a Neuro2A cell and the AFM cantilever. Stiffness response to 100 nM WIN at different cell locations (crosses). Subsequent measurements were focused on the cell bodies, corresponding to positions 2 and 3. (D) Blebbistatin (25 µM) significantly reduces 100 nM WIN-induced increase of cell stiffness. Results are pooled from at least three independent experiments and are expressed as mean stiffness between 2 and 8 min after stimulation ±SEM. (E) 3D reconstruction shows neurite retraction, cell rounding, and transitory blebbing (arrows) following WIN treatment (100 nM). n.s p > 0.05; ***p < 0.001, calculated using Student's t-test on B and using one-way ANOVA followed by Newman–Keuls post-tests on (D). Scale bars: 10 µm on (A) and (E), 15 µm on (C).