PREreview of Dynein and dynactin move long-range but are delivered separately to the axon tip

This review reflects comments and contributions from Kamaldeep Singh, Prithviraj Rajebhosale, Luciana Gallo, Ryan Cubero & Femi Arogundade. Review synthesized by Kamaldeep Singh.

In this study, Fellows et al. investigated the motility of endogenously-tagged dynein motors and its regulators along the length of the axons using live imaging of neuron-inducible human-stem cell lines (iNeurons) as a model system. Using highly inclined and laminated optical sheet (HILO) imaging of iNeurons, they show that dynein and dynactin are transported at different speeds to the distal tip of the axon. Further, use of SNAP and Halo-tag conjugated with highly stable fluorophores also allowed them to show that single molecules of dynein and dynactin can traverse the entire length of the axon (>500 um). In summary, this study has contributed in advancing the cell biological understanding of dynein and its regulators in mammalian axons.

We believe that the following positive aspects make the findings of the study strong and convincing:

1. Use of neuron-inducible human stem cell-derived iNeurons as a model system is a significant advantage as it provides a better understanding of mammalian cell biology for studying axonal transport compared to traditional cancer cell lines.

2. Use of microfluidic devices to separate axons from the somatodendritic compartments allows a convincing and clear demonstration of retrograde and anterograde transport in the axons and dynamics thereof.

3. Use of CRISPR to endogenously tag the dynein heavy chain and the ARP11 subunit of dynactin with a SNAP and HaloTag allowed the authors to selectively label the motos with high spatio-temporal resolution. This not only aided in imaging dynein/dynactin at a near single-molecule level but also allowed them to figure that single molecules of dynein and dynactin can traverse the entire length of the axon (>500um).

4. The photobleaching analysis on dynein spots in iNeurons provides valuable insights into the possible number of dynein molecules per cargo (which also agrees well with recent measurements of dynein number on endosomes in literature) and supports the claim of detecting single molecules under the given experimental conditions.

We also noted several points regarding the study and the manuscript (as major or minor comments) which if addressed, could possibly make the study better:

Major comments:

• The authors observed mostly 1 and 2 step bleaching for dynein (Figure 2). Here, while they commented on the two step events being predominant and representative of the cytoplasmic dynein dimer, an explanation for the single bleaching events is lacking. Is it possible that these could be Halo-tagged dynein molecules dimerized with endogenous, untagged dynein from monoallelic targeting by CRISPR? It would therefore be nice to clarify and show the data that validates their CRISPR knock-in efficiency.  

• The authors mention: “We saw many distinct dynein spots in the axonal compartment, most of which were diffusing, often along microtubules (Fig S2A, Video 4).” How did the authors determine that these events were diffusive and that they occured along microtubules?

Minor comments: 

• Figure 3 is incorrectly labeled as Figure 2 in the legend. Kindly correct this.

• “Thought” is misspelled as “thught” in the last paragraph of Introudction section.

• Fig 3C is incorrectly addressed as “Fig 3B” under the results section “Dynein moves long range”.

• In the videos attached along with the manuscript: It would be nice if there could be additional markers - for instance arrows tracking the particles (just like box on the spot where photobleaching was performed) -  to help readers focus on the main point the authors are trying to make with respect to a given video.

• While the microfluidic devices utilized in the study might be standard in the trafficking field, it would be nice if the authors could provide a detailed description such as the devices' exact dimensions and manufacturer/supplier details.

Authors have already hinted towards many unanswered questions, possible experiments and also listed anticipated outcomes for many such questions. Here are a few suggestions for lines of investigation that one could undertake in the future research:

• As it has already been emphasized by authors, LIS1 is an important regulator of dynein activation. Therefore, simultaneous imaging of LIS1 and dynein might allow authors to identify whether the pausing of dynein is due to exchange of LIS1 within the complexes. Similar studies conducted with dynein together with other dynein cofactors, activators, or even kinesins will be useful to address many unanswered questions in the field.

• Given that the authors can compartmentalize the somatodendrites and axons, it would also be interesting to know whether supply of dynein at the distal tip is regulated in response to different stimuli for e.g. in response to perturbations in neuronal activity.

We wish the authors best of luck for all of their future research endeavours!

Competing interests

The author declares that they have no competing interests.