PREreview of Cytoplasmic ribosomes hitchhike on mitochondria to dendrites

The work by Renken and colleagues presents a significant new discovery about ribosome transport in neurons, addressing a gap in our understanding of local protein synthesis. The study uncovers the mechanism regulating the localization of ribosomes to dendrites, using C. elegans chemosensory neurons as a model. The data presented are consistent with a role for mitochondria and mitochondrial transport in directing the localization of ribosomes,  highlighting the crucial role of mitochondria in local protein synthesis.

Positive aspects of the study: 

• The study is written in a clear language and is concise. 

• The thorough study reveals the fascinating result that ribosomes are transported down dendrites by hitchhiking on mitochondria. The authors demonstrated this result in multiple ways, and the study is ultimately convincing. 

• The novel mechanism provides valuable insights into the upstream mechanisms that enable local protein synthesis in neurons and contributes to our understanding of the importance of mitochondria in neuronal processes beyond energy production. The findings may be utilized in neurodegenerative disease research.

• The authors reuse previously published  EM data set to strengthen their conclusions and good scientific practices

While the data presented are consistent, reviewers have some suggestions that will help to strengthen and confirm the authors conclusions and may increase even more the overall quality of the manuscript.

Major comments: 

• For ribosome localization, the authors rely primarily on a gfp-tagged small ribosomal protein, RPS-2::GFP, as a marker for ribosomes. The reviewers consider that there are some important controls that should be performed to ensure that it is being incorporated into functional ribosomes, and that the transgene itself is not disrupting ribosome function. The authors partially address this by citing a yeast study that examines these points, however it should really be tested in the system under study, given the different organism, context, and potentially different level of expression of their construct. While we are aware that some additional work would be involved to confirm that RPS-2::GFP is properly incorporated into ribosomes without disrupting their function, it is very important to establish this given that the RPS-2::GFP readout forms the basis of the majority of their experiments. We would like to suggest different alternatives:

  • Addressing the incorporation into intact ribosomes by polysome profiling. A nice example of a set of experiments that can be done in C. elegans to test for proper incorporation of their RPS-2::GFP construct into translating ribosomes, without disrupting ribosome function, is found in the study here: https://doi.org/10.1016/j.crmeth.2023.100433. 

  • Immunostaining with antibodies against other ribosomal proteins to confirm colocalization with RPS-2::GFP would be also interesting.

  • Use puromycin labeling to confirm that RPS-2::GFP-positive structures are translationally active.

  • The authors could perform a similar approach labelling the ribosomal 60S large subunit and then perform colocalization analysis of 60S large subunit and 40S small subunit.

All these suggestions would strengthen the use of RPS-2::GFP to label assembled and functional ribosomes.

• Similarly to drp-1, the authors should also perform cell-specific trak-1 expression in AWB and AWC to rescue RPS-2::GFP distribution in dendrites, especially as the impact of trak-1 is further studied in Figure 3.

• Similar to establishing that RPS-2::GFP properly marks functional ribosomes, the authors should confirm independently, using alternative mitochondrial markers, that their mitoRFP marker does indeed label intact, functional mitochondria, without disrupting mitochondrial function or localization. This is important to establish, because again, many of their experiments rely on this marker.

Minor comments:

Introduction

• The first time that C.elegans is mentioned it is advised to use the full word Caenorhabditis elegans in italics.

• Regarding the paragraph introducing the filamentous fungus U. maydis, reviewers appreciate the author's attempt to bring an evolutionary perspective on the problem. Nevertheless we would recommend to do so only after they have resolved outstanding questions about the C. elegans model system. The paragraph might be rewritten giving a more conclusive idea of the meaning of the evolutionary comparison

• One important piece of information that might be missing is if ribosome localization in dendrites requires the presence of the endoplasmic reticulum or not.

• Reviewers considered that more relevant literature should be included. For example: https://www.ncbi.nlm.nih.gov/books/NBK6352/#top

Results

First results section

• We would recommend that in the sub-title the specification that AWB and AWC are chemosensory neurons are included. For example: "Ribosomes are stereotypically positioned in the dendrites of chemosensory neurons AWB and AWC"

• The authors should specify the rationale for selecting odr-1p promoter to express RPS-2GFP.

• When describing the localization of RPS-2::GFP  around the nucleus, we believe the authors are referring to Figure 1C instead of Figure 1B.

• In figure 1E and 1SB, in the stacks of representative images, it would be recommended to add an anterior/posterior reference for precision. It is difficult to understand if the "first 10 microns of the dendrite" considered in the fluorescence quantification correspond to the proximal segment of the dendrite (proximal to cell body) or beginning from the base of the cilium.

Second results section

• The screen of 5,000 haploid genomes may not provide full coverage of the C. elegans genome. The study should address whether this sample size is sufficient.

• The reviewers would appreciate a little more detail about the forward genetic screen, especially in the main text, but also could be in the methods.

• We consider that the results for drp-1(shy54) mutant worms are great and deserve a place in the very first figure instead of  in a supplementary figure. Especially because both mutants showed different phenotypes and different disruptions of ribosome distribution along the dendrite.

Third results section:

• Regarding the colocalization of RPS-2::GFP and mitoRFP in the dendrite, it would be helpful to include another fluorescent marker. We would suggest something generally cytoplasmic or membrane-bound to show that the morphology of the dendrites is normal, and that the accumulation of ribosomes and mitochondria at the observed puncta doesn't correspond to some physical deformation of the dendrite that might be caused by the expression of these constructs.

• We would also recommend labeling another dendritic structure/organelle that is not supposed to interact with ribosomes to strengthen that the interaction is specific and not only because of overcrowding of structures in a limited space such as a dendrite. 

• Regarding the statement that RPS-2::GFP and mitoRFP do not colocalize in the soma,  we understand what the authors want to declared here, however, the mean Pearson's Correlation Coefficient for the soma is around 0.5, meaning that the colocalization of both signals is moderate. It would be recommended changing "but not in the soma" to "while the colocalization in the soma is moderate".

• Regarding the analysis of the proximity of mitochondria and ribosomes in EM images (fig 2G), the reviewers find that this is a promising result that aligns well with the colocalization data. However, they have few considerations: 

  • Since this analysis was conducted in a single animal (n=1), it’s important to include a statement acknowledging that the result does not account for potential biological variability. 

• The data presented in Fig. 2G appears to deviate from a normal distribution. If this has not been checked, we highly recommend doing so. If the distribution is indeed non-normal, the median value should be reported instead of the mean. Based on the current visualization, the median seems to be below 20 nm, which further supports the authors' hypothesis.

Fourth results section

• The reviewers would like to ask the authors to comment on why dli-1 has such a distinctive localization pattern compared to the other mitochondrial transport mutants examined in Figure 3?

• Why null alleles of trak-1(wy50182), miro-1(wp88) and mtx-2(wy50266) have a  stronger phenotype than loss of dynein light intermediate chain?

• The reviewers find it difficult to justify the need to analyze the effect of the double mutant miro-1; mtx-2 double mutant when both single mutants have already almost a complete deleterious effect. In case the authors have a strong supporting argument, we would recommend that if they want to claim that it exhibits a phenotype similar to both single mutants, they could show the statistical comparison between single and double mutants to support this affirmation.

Fifth results section 

• In the sub-title, we  recommend using caution with the word 'sufficient'. The authors express the concept more precisely in the conclusion of this section: 'mitochondria are both necessary and instructive for dendritic ribosome positioning in this system.' While they demonstrated that mitochondria are essential and play an instructive role in this process, proving sufficiency required the experiments that manipulate alternative regulatory pathways that authors performed later.

Sixth results section

• The authors report that from all detectable RPS-2 movements, 74% have associated mitochondria. The reviewers wonder if  this percentage is consistent across proximal vs distal dendrites

Eighth results section

• In figure S3A (in orange), we believe the authors are referring to RAB-5 and not RAB-6-

• As RPS-2 puncta colocalize extensively with RAB-7, it would be important to decipher how much of the motile RPS-2 are co-transported with RAB-7, in a similar analysis as the authors did for mitochondria and RAB-7 co-transport.bTherefore, we would recommend the authors to perform time-lapse imaging of RAB-7 and RPS-2 and analysis as in Figure S3 C.

Last results section

• We would like to encourage the authors to include time-lapse experiments showing that ribosomes and mitochondria are co-transported in the absence of rab7-positive endosomes to strengthen the conclusion even more. Moreover, are the numbers of ribosome localization in distal dendrites decreased?

Discussion

• Reviewers would appreciate if authors could expand on the alternative mechanisms involving mRNP granules or endolysosomal organelles on the transport of ribosomes along the dendrites

Methods

• Regarding the generation of transgenic nematode strains, the authors explain that F2 progeny were visualized on a microscope to identify strains with desired expression levels. The reviewers express concern about how they decided what expression levels were desirable? Expression level could have an important impact on the functionality and localization of the transgenes. 

Comments on reporting:

• In figure 2E, the datasets do not seem to have a clear normal distribution when looking at the points in the graph, especially regarding dendrite one. Did the authors analyze normality in this case before running the parametric test declared? How many animals did they use? It would be recommended to further clarify what this n is about.

• In Fig 4C, the n listed is 36 worms, but there are many more data points displayed on the graph. It is not statistically appropriate to calculate p-values based on multiple measurements within each sample. The t-test and ANOVA assume that each measurement is independent, and multiple mitochondria within the same sample are not independent. Either do not report p-values or you can average together the values from each sample and calculate the p-value using those sample-level means. For more information, see  https://doi.org/10.1371/journal.pbio.2005282 and https://doi.org/10.1083/jcb.202001064

• Regarding the EM Data from Witvliet et al. (2021),  On a data reproduction note, the authors should declare to have permission to reproduce other authors' images.

Suggestions for future studies:

• Explore the potential implications of impaired dendritic ribosome transport or mitochondrial dysfunction in neurodegenerative diseases or disorders associated with impaired local protein synthesis. Investigate whether targeting these processes could be a therapeutic strategy.

• Investigate which protein complexes and/or RNA-binding proteins (RBPs) may be facilitating the attachment of ribosomes to mitochondria.

• Explore the effect of mitochondrial dynamics on the translation rate in dendrites

• Study whether these observations change in other neuron types or under stress conditions

• Explore the mechanism of mitochondria-independent transport

Conflicts of interest of reviewers 

• None declared.

Competing interests

The authors declare that they have no competing interests.