Neurons are complex cellular machines that utilize a dynamic cytoskeleton to elaborate long axonal processes. During embryonic development, these long processes eventually terminate and form a synapse with a prescribed target. Elongation is driven in part by a unique structure called the growth cone at the tip of the axon. A recently developed biophysical model for axonal elongation has proposed that forces cause the growth cone to translocate in bulk, while stretching the axon. This is followed by intercalated mass addition along the length of the axon to prevent thinning. As a result of axonal stretching, the cytoskeleton undergoes en masse translocation. While this has been observed in cultured neurons from a variety of different species, whether this occurs in vivo is unknown. In addition, the molecular force generating mechanisms in the axon that regulate axonal stretching and cytoskeleton translocation have not been characterized. Here, we use mitochondria docked to the cytoskeleton as fiduciary markers for bulk cytoskeletal movements. We use this technique in cultured Drosophila neurons to show that cytoskeleton translocation is conserved between vertebrates and invertebrates. Then we track the movement of docked mitochondria in the aCC motoneuron in stage 16 Drosophila embryos to show that the cytoskeleton translocates during axonal elongation. This suggests that axons grow by stretching in vivo .Non-muscle myosin II is a well-known force generating motor found in neurons. To characterize how myosin II contributes to axonal stretching and cytoskeleton translocation, we used the pharmacological agent blebbistatin to disrupt myosin II function in chick sensory neurons and genetic reduction of myosin II heavy chain in primary Drosophila neurons. We found an antagonistic relationship between myosin II in the growth cone and along the axon: myosin II in the growth cone promotes growth cone translocation, while myosin II along the axon restrains it by preventing axonal stretching. Cytoplasmic dynein is a microtubule motor previously implicated in axonal elongation. To test if dynein contributes to cytoskeleton translocation, we used microinjection of function-blocking antibodies and the pharmacological dynein inhibitor Ciliobrevin D to disrupt dynein function and found that elongation decreases due to a reduction in cytoskeleton translocation. We also found an increase in axonal tension upon dynein disruption, suggesting that dynein pushes microtubules embedded in the cytoskeletal meshwork forward. Altogether these results lead to a model for axonal elongation in which the cytoskeleton can be pulled forward by myosin II in the growth cone or pushed forward by dynein along the length of the axon, while myosin II along the length of the axon restrains growth cone advance by preventing axonal stretching. This offers the axon a convenient mechanism to regulate the rate of elongation and has the potential to illuminate new strategies for augmenting axonal regeneration following nerve damage.
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