By Carla M. Quiñones
The main objective of the research described in this article by Rotello and coworkers was to use a synthetic non-peptide targeting motif that accesses the nucleus of the cell through an active transport mechanism. Active and passive transport take place in the cellular membrane as well as in the nuclear membrane. Intracellular targeting is as important as cellular targeting due to their relevance in drug delivery and potential therapies. The two challenges faced by the research group was to deliver the nuclear-targeted protein into the cytosol and to comprehend the mode of nuclear entry. They delivered the protein successfully to the cytosol with the help of a previously synthesised nanoparticle stabilized particle, which encapsulated the proteins of interest and liberated them into the cytosol via membrane fusion. The most significant finding was the efficiency of targeting the nucleus when modifying each protein of interest with benzyl boronate tags (BB tags).
They performed experiments with different proteins modified with BB tags resulting in a successful high-efficiency delivery to the nucleus. To assess the role of boronic acid in the BB tags, they modified GFP with the benzyl tag alone and saw less fluorescence inside the nucleus. This suggests that the boronic acid is necessary in the BB tags for a successful nuclear targeting. Furthermore, they determined if the mechanism of transport into the nucleus was either active or passive. To accomplish this goal, they added Ivermectin, an inhibitor for the α/β importin pathway (active transport), to the cells and also depleted ATP (required for all active transport pathways) in another set of experiments. They delivered GFP with 3 BB tags in both cases and saw a lower nuclear efficiency. They concluded that these modified proteins targeted the nucleus effectively through the importin α/β pathway (active transport) rather than passive transport.
Through the last part of the paper, I was curious of how they acknowledged that this happened the way the proposed since they didn’t explain the mechanism behind the α/β importin pathway, nor the chemistry of how the BB tag contributes to the nuclear targeting. Intrigued by this, I searched more about this phenomenon, but found no relevant studies, probably because this is a novel research field. In general, this was a good article and useful to our lab since we work with supramolecular systems which, in the future, we could adapt this strategy to achieve nuclear targeting.
Rating (synopsis): 4/5
Rating (figure): 5/5
Carla’s summary was clear and direct. I was able to understand everything she explained and it was interesting to me as they could go through this mechanism of the addition of the BB label and how they managed to reach the core efficiently. I think that this type of mechanism could lead to useful applications in the future.
The image is creative and original. In the image, the main goal of the experiment and the experimental part of the article are well presented.
Rating Synopsis: 4/5
Rating Image: 5/5
This summary clarified was very consist and straight forward on the goals of the research and the things done to get to that goal. As Carla said, I also expected to see the mechanism behind the α/β importing pathway with which they work with. Nevertheless, it was still really interesting how they could effectively target the nucleus with the modified proteins through active transport.
As for the figure, I really liked how it got to again summarized the synopsis already done.
Rating Synopsis 5/5
Rating Image 5/5
This summary was helpful to understand more clearly the objectives of this article. I understand the effectiveness of labeled the proteins with BB tags for targeting the nucleus. Also it was interesting how intensity of the fluorescence change because of different modifications when they include the boronic acid in the BB tag. This article I think is really useful for the lab, and more for Carla’s team because they work directly with boronic ester. Maybe they could use this article as a reference of modification, behavior or conditions for their experiments with their probes.
As for the figure I think it has the perfect amount of images to understand the main idea of this article, also it helps to understand more the article.
Rating (synopsis): 4/5
Rating (figure): 4/5
I can see that she worked hard on this summary and it shows. Focusing first on context, experimentation rational and comment. It was concise and analytical calling the paper out on its issues which we should be doing also on our summaries. Congrats. By organizing the legends of the image somewhat better, to each end maybe the image could be a bit clearer. But still is good work.
Rating (synopsis): 5/5
Rating (figure): 4/5
After reading the synopsis, I can see that it is an excellent summary of the paper. I like how Carla highlights the main idea, the importance and challenges of the research. Then, she explains very well the approach of the researchers by using benzyl boronate tags (BB tags) and the benzyl tag alone, both with GFP. She makes a good job in the conclusion when she talks about how the modified proteins with importin α/β pathway achieved active transport rather than passive transport (the purpose of the study). In the figure, the message is clear, however the Rnase A-(BB)7 and Chymotrypsin – BB are not presented in the synopsis, it can be confusing at first to see them, but they appear in the paper and the importance of them in the confocal microscopy studies is shown.
Rating (Synopsis): 4/5
Rating (Figure): 4/5
I enjoyed reading this synopsis, as it had a very nice flow that was akin to how the results were presented in the article. She managed to state the authors’ rationale and the challenges they were trying to address. The results obtained by the researchers very strongly suggest that their BB-tagged protein was being internalized in the nucleus by active transport, but I wonder if they did any experiments where passive transport mechanisms were inhibited and the results that would be obtained from that.
Carla’s TOC figure is a good attempt at summarizing the article’s main idea successfully, and it does a better job at that than the one published by the authors. However, if the legend was organized more efficiently, the minor misspellings were fixed and the graph (which is hard to read because it is too small) was deleted, the figure could be improved. Also, like Maria mentioned, chymotrypsin and RNAse A were not discussed in the synopsis; I don’t think they necessarily need to be deleted from the figure but instead should have been included in the synopsis if they were considered important.