In many eukaryotic cells, directed molecular transport occurs along microtubules. Within neuronal axons, transport over vast distances particularly relies on uniformly oriented microtubules, whose plus-ends point towards the distal axon tip (anterogradely polymerizing, or plus-end-out). However, axonal microtubules initially have mixed orientations, and how they orient during development is not yet fully understood. Using live imaging of primary Drosophila melanogaster neurons, we found that, in the distal part of the axon, catastrophe rates of plus-end-out microtubules were significantly reduced compared to those of minus-end-out microtubules. Physical modelling revealed that plus-end-out microtubules should therefore exhibit persistent long-term growth, while growth of minus-end-out microtubules should be limited, leading to a bias in overall axonal microtubule orientation. Using chemical and physical perturbations of microtubule growth and genetic perturbations of the anti -catastrophe factor p150, which was enriched in the distal axon tip, we confirmed that the enhanced growth of plus-end-out microtubules is critical for achieving uniform microtubule orientation. Computer simulations of axon development integrating the enhanced plus-end-out microtubule growth identified here with previously suggested mechanisms, that is, dynein-based microtubule sliding and augmin-mediated templating, correctly predicted the long-term evolution of axonal microtubule orientation as found in our experiments. Our study thus leads to a holistic explanation of how axonal microtubules orient uniformly, a prerequisite for efficient long-range transport essential for neuronal functioning.
Keywords: D. melanogaster; axons; microtubule orientation; microtubule polarity; neurons; physics of living systems.
For humans to be able to wiggle their toes, messages need to travel from the brain to the foot, a distance well over a meter in many adults. This is made possible by neurons, the cells that form the nervous system, which transmit electrical signals along long extensions called ‘axons’. Axons can only transmit signals if all the required molecules, which are produced in a part of the neuron known as the cell body, are ferried to the ends of the axons. This ferrying around of molecules is carried out by long, filamentous molecules called microtubules, which act as a directed carrier system, shuttling molecules along the axon, either towards or away from the cell body. Microtubules can be thought of as asymmetrical rods. One end – known as the plus end – is dynamic and can undergo growth or shrinkage, while the other end – called the minus end – is stable. For transport along the axon to happen efficiently, microtubules in the neuron need to be oriented with their plus end pointing towards the ends of the axon. Microtubules in growing neurons develop this orientation, but how that is achieved is not fully understood. To understand the basis of this cellular phenomenon, Jakobs, Zemel and Franze examined the behaviour of microtubules in developing neurons from fruit fly larvae. A fluorescent protein, which emits light when the microtubules are growing, helped the researchers visualise the plus end of microtubules, the microtubule orientation, and their growth in developing axons. This experiment showed that microtubules that had their plus end pointing towards the axon end shrank more slowly than those with the opposite orientation, leading them to grow longer. This resulted in a higher proportion of the correctly-oriented microtubules in the axon. Treating the neurons with Nocodazole, a chemical that disrupts microtubule growth, or with sodium chloride, which changes the osmotic pressure, caused the microtubules that were oriented with their plus end towards the axon to grow less, and disrupted the uniform orientation of the microtubules in the axon. The next step was to determine whether specific axonal proteins such as p150 – a protein that is enriched at the tip of the axon and decreases microtubule shrinkage rates – are involved in this process. Reducing the levels of p150 in fruit flies using molecular and genetic methods resulted in microtubules with their plus end pointing towards the axon tip shrinking faster, reducing the proportion of microtubules with this orientation in the axon. This role of proteins enriched in the axonal tip, along with previously discovered mechanisms, explains how microtubules align unidirectionally in axons. These findings open new avenues of research into neurodegenerative diseases like Alzheimer’s and Parkinson’s, which might manifest due to a breakdown of transport along microtubules in neurons.
© 2022, Jakobs et al.