Dynamic balance between vesicle transport and microtubule growth enables neurite outgrowth
Fig 9
Imbalanced SCP dynamics disturb neurite outgrowth.
(A-B) We predicted four sets of kinetic parameters that allow coordinated NOG at the velocities 5, 10, 15 and 20 μm/h. To simulate the effect of uncoordinated vesicle SCP dynamics we decreased the count and production rate of v-SNARE V to 25%, 50% and 75% of the original prediction, without modifying any other parameters. Numerical simulations predict that reduced exocytosis causes an increase in the count of vesicles in the growth cone cytoplasm (A) without reducing NOG velocity (B). (C) We validated our prediction by analyzing neurite outgrowth after knockdown of the v-SNARE VAMP7. P1 rat cortical neurons transfected with siRNA against VAMP7 or scrambled siRNAs were stained for β-III tubulin to document neurite growth across the microgroove of the chamber. In agreement with our prediction, VAMP7 knock down did not decrease total outgrowth length. It caused a significant number of physiological growth cones to turn into dystrophic bulbs. (D) Analysis of quantified neurite lengths confirmed that there was no significant difference in outgrowth length. Shown are average values and standard deviations (three independent experiments). (E) Neurites growing from neurons after VAMP7 knock down showed a significant increase in the number of dystrophic bulbs, indicative for impaired NOG. (F) To simulate the effect of reduced MT stabilization, we reduced the stabilization rate that turns dynamic into stable MTs to 25%, 50% and 75% of the original prediction, without changing the predicted values for all other kinetic parameters. Numerical simulations propose that decreasing stabilization leads to a significant decrease in NOG outgrowth over the investigated time. (G) We validated our predictions by knock down of the microtubule crosslinking protein MTCL1. MTCL1 knock down caused reduced NOG outgrowth and dystrophic bulb formation. (H) Outgrowth quantification confirms significant decrease in NOG (3 independent experiments). (I) Mtcl1 also significantly increased the number of dystrophic bulbs. *p<0.05, **p<0.01, ***p<0.001based on unpaired t-test for all panels.