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Posted by PLOS_CompBiol on 26 Feb 2008 at 14:21 GMT
Originally submitted as a Reader Response on 26th July 2007
It would have been nice for the authors to address some recent experimental data (Yang & Musser, 2006 JCB 174:951-961) that have a direct bearing on their theoretical results. As it is, it is unclear to me whether their proposed model is consistent with our experimental data. Specifically:
1) We observe shorter interaction times under conditions that yield increased transport efficiencies. This seems at odds with the main conclusion of Equation 4 that "an attractive potential inside the NPC increases the time the complex spends inside the NPC and thus increases the probability that it reaches the nuclear side, rather than returns to the cytoplasm." I.e., a shorter interaction time predicts a decreased transport efficiency.
2) One prediction of the authors' model is that "eventually, complexes spend so much time in the channel that they impede the passage of other complexes through the channel." Effectively, high numbers of molecules in the channel cause a traffic jam. Again, this is seems at odds with what we have seen experimentally. Namely, when there are many empty importin beta molecules bound within the NPC, transport is faster and more efficient.
3) A minor issue. We have estimated that the diffusion constant of the cargo in the channel is about 1 um2/s, approximately 10 times slower than in buffer with a viscosity of 3.5 cP. What happens if this is included in the analysis?
Finally, for the sake of accuracy, it is not at all clear that a 30 nm diameter cargo would be transported in 10 ms, as the authors seem to imply in their first paragraph. Further, the 10 ms interaction times that have been measured are for the cargo in the NPC (that is the only thing directly visualized) - it is not clear how much of that time the cargo is part of a transport complex.
Submitted by: Siegfried Musser
E-mail: smusser@tamu.edu
Occupation: Asst. Professor
Texas A&M Health Science Center
Response to Musser
PLOS_CompBiol replied to PLOS_CompBiol on 26 Feb 2008 at 14:48 GMT
Originally submitted as a Reader Response on 22nd October 2007
We are grateful to Dr. Musser for an interesting comment, as it provides an opportunity to discuss several important issues. However, it is important to note that the experiments of Yang and Musser do not directly test the predictions of our theory, since their experimental manipulations do not correspond to the variables we manipulate theoretically. Below we briefly comment on the issues raised by Dr. Musser.
1. The purpose of our theory is to explore the consequences of the binding strength combined with the limited space inside the pore. Yang and Musser, on the other hand, vary the concentration of importin-beta (that can potentially change many experimental variables).
Our theory predicts that as the binding strength increases, the transport efficiency increases at first, but at the expense of longer transport time. By contrast, the ‘conditions of increased transport efficiency’ of Yang and Musser are not due to increased binding strength but due to some other mechanism (presently unknown) mediated by high concentrations of importin-beta; much higher than we consider. Because we do not discuss this putative mechanism in our paper, we also do not predict how it could influence the transport times.
2. Our theory indeed predicts that as the concentration of kap-cargo complexes increases, the pore eventually becomes jammed. This superficially appears at variance with the results of Yang and Musser. However, as Figure 3 in our paper shows, this jamming manifests only above some critical value of the flux J through the pore – below this critical flux, jamming does not occur.
In this respect, it appears that the experiments of Yang and Musser are not in the jammed regime.
First, although Yang and Musser do not report how flux through the pore behaves as a function of the cargo concentration at different imp-beta concentrations, from their previous work it appears that the flux is linearly proportional to the concentration of cargo (Musser PNAS 2006). This indicates that the mutual occlusion between the complexes inside the pore is negligible. Second, when the flux through the pore is J, and the transport time is T, the average number of complexes in the pore is JT. At 12 μM concentration of importin-beta and the same concentration of cargo, the transport time is T ~ 2 msec. Thus, even assuming a very high flux of J = 1000 complexes per second, at each moment there are only about two complexes present inside the pore. This makes it very unlikely that the complexes interfere with each other’s passage.
3. It is not clear how Yang and Musser estimated the diffusion coefficient. It appears that it was done without taking into account that binding slows down the passage, which may result in a higher apparent diffusion constant. Just assuming that the diffusion coefficient inside the pore is different from the outside would not change our conclusion.
4. All we mean is that the NPC can transport up to 1000 cargoes per second. Some of these cargoes can be as large as 30 nm in diameter – however they are not necessarily transported at such high rates.
To summarize, the experiments of Yang and Musser appear to explore a different aspect of the transport through the NPC that that studied in our paper. Without further quantitative experiments it is not possible to determine whether our theory and Yang and Musser’s experiments indeed contradict each other.
Submitted by: Marcelo Magnasco
E-mail: magnasco@rockefeller.edu
Occupation: Professor
Rockefeller University
Additional authors: Anton Zilman and Michael Rout