Mutation of the ALS/FTD-associated RNA-binding protein FUS affects axonal development.

Aberrant condensation and localisation of the RNA-binding protein (RBP) fused in sarcoma (FUS) occur in variants of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Changes in RBP function are commonly associated with changes in axonal cytoskeletal organisation and branching in neurodevelopmental disorders. Here, we asked whether branching defects also occur in vivo in a model of FUS-associated disease. We use two reported Xenopus models of ALS/FTD (of either sex), the ALS-associated mutant FUS(P525L) and a mimic of hypomethylated FUS, FUS(16R). Both mutants strongly reduced axonal complexity in vivo. We also observed an axon looping defect for FUS(P525L) in the target area, which presumably arises due to errors in stop cue signalling. To assess whether loss of axon complexity also had a cue-independent component, we assessed axonal cytoskeletal integrity in vitro Using a novel combination of fluorescence and atomic force microscopy, we found that mutant FUS reduced actin density in the growth cone, altering its mechanical properties. Therefore, FUS mutants may induce defects during early axonal development.Significance statement This study demonstrates that mutation of the ALS/FTD (amyotrophic lateral sclerosis/frontotemporal dementia)-associated RNA-binding protein Fused in Sarcoma (FUS) can result in changes in axonal development. These changes occur both axon-autonomously in cytoskeletal organisation during axon extension and context-dependently during axonal branching. This indicates pre-symptomatic, developmental changes in axonal organisation may occur in familial disease variants.

Unrestrained growth of correctly oriented microtubules instructs axonal microtubule orientation.

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.

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.

Mechanical Regulation of Neurite Polarization and Growth: A Computational Study.

The densely packed microtubule (MT) array found in neuronal cell projections (neurites) serves two fundamental functions simultaneously: it provides a mechanically stable track for molecular motor-based transport and produces forces that drive neurite growth. The local pattern of MT polarity along the neurite shaft has been found to differ between axons and dendrites. In axons, the neurons' dominating long projections, roughly 90% of the MTs orient with their rapidly growing plus end away from the cell body, whereas in vertebrate dendrites, their orientations are locally mixed. Molecular motors are known to be responsible for cytoskeletal ordering and force generation, but their collective function in the dense MT cytoskeleton of neurites remains elusive. We here hypothesized that both the polarity pattern of MTs along the neurite shaft and the shaft's global extension are simultaneously driven by molecular motor forces and should thus be regulated by the mechanical load acting on the MT array as a whole. To investigate this, we simulated cylindrical bundles of MTs that are cross-linked and powered by molecular motors by iteratively solving a set of force-balance equations. The bundles were subjected to a fixed load arising from actively generated tension in the actomyosin cortex enveloping the MTs. The magnitude of the load and the level of motor-induced connectivity between the MTs have been varied systematically. With an increasing load and decreasing motor-induced connectivity between MTs, the bundles became wider in cross section and extended more slowly, and the local MT orientational order was reduced. These results reveal two, to our knowledge, novel mechanical factors that may underlie the distinctive development of the MT cytoskeleton in axons and dendrites: the cross-linking level of MTs by motors and the load acting on this cytoskeleton during growth.

KymoButler, a deep learning software for automated kymograph analysis.

Kymographs are graphical representations of spatial position over time, which are often used in biology to visualise the motion of fluorescent particles, molecules, vesicles, or organelles moving along a predictable path. Although in kymographs tracks of individual particles are qualitatively easily distinguished, their automated quantitative analysis is much more challenging. Kymographs often exhibit low signal-to-noise-ratios (SNRs), and available tools that automate their analysis usually require manual supervision. Here we developed KymoButler, a Deep Learning-based software to automatically track dynamic processes in kymographs. We demonstrate that KymoButler performs as well as expert manual data analysis on kymographs with complex particle trajectories from a variety of different biological systems. The software was packaged in a web-based 'one-click' application for use by the wider scientific community (http://kymobutler.deepmirror.ai). Our approach significantly speeds up data analysis, avoids unconscious bias, and represents another step towards the widespread adaptation of Machine Learning techniques in biological data analysis.

Many molecules and structures within cells have to move about to do their job. Studying these movements is important to understand many biological processes, including the development of the brain or the spread of viruses. Kymographs are images that represent the movement of particles in time and space. Unfortunately, tracing the lines that represent movement in kymographs of biological particles is hard to do automatically, so currently this analysis is done by hand. Manually annotating kymographs is tedious, time-consuming and prone to the researcher's unconscious bias. In an effort to simplify the analysis of kymographs, Jakobs et al. have developed KymoButler, a software tool that can do it automatically. KymoButler uses artificial intelligence to trace the lines in a kymograph and extract the information about particle movement. It speeds up analysis of kymographs by between 50 and 250 times, and comparisons show that it is as reliable as manual analysis. KymoButler is also significantly more effective than any previously existing automatic kymograph analysis programme. To make KymoButler accessible, Jakobs et al. have also created a website with a drag-and-drop facility that allows researchers to easily use the tool. KymoButler has been tested in many areas of biological research, from quantifying the movement of molecules in neurons to analysing the dynamics of the scaffolds that help cells keep their shape. This variety of applications showcases KymoButler's versatility, and its potential applications. Jakobs et al. are further contributing to the field of machine learning in biology with 'deepmirror.ai', an online hub with the goal of accelerating the adoption of artificial intelligence in biology.

Late Endosomes Act as mRNA Translation Platforms and Sustain Mitochondria in Axons.

Local translation regulates the axonal proteome, playing an important role in neuronal wiring and axon maintenance. How axonal mRNAs are localized to specific subcellular sites for translation, however, is not understood. Here we report that RNA granules associate with endosomes along the axons of retinal ganglion cells. RNA-bearing Rab7a late endosomes also associate with ribosomes, and real-time translation imaging reveals that they are sites of local protein synthesis. We show that RNA-bearing late endosomes often pause on mitochondria and that mRNAs encoding proteins for mitochondrial function are translated on Rab7a endosomes. Disruption of Rab7a function with Rab7a mutants, including those associated with Charcot-Marie-Tooth type 2B neuropathy, markedly decreases axonal protein synthesis, impairs mitochondrial function, and compromises axonal viability. Our findings thus reveal that late endosomes interact with RNA granules, translation machinery, and mitochondria and suggest that they serve as sites for regulating the supply of nascent pro-survival proteins in axons.