See, the Grand Prix is indeed evolving. Perhaps less like a squid and more like a snail: You have to lean in close to see it happening. But a little maturation, judiciously applied, can produce helpful improvements. So it is with this Grand Prix makeover. More refinements, less Space Patrol in the dashboard design, and a manumatic shift that actually works quite well.
Better all-around responses and improved ride quality, even in this performance-oriented Comp G. More power too. With no torque steer. Pontiac has done a good job here. Now, just move the power delivery to the correct end of the car… —Tony Swan.
Back here in North Craterville, my enthusiasm has dampened just a skosh. More compliance is badly needed in the middle inch or two of shock travel. Th e-throttle also needs to be tamed. Who really wants to peel out at every intersection? Inside and out, the appearance has matured. The mechanicals still have some growing up to do. It was only the bright-red brake calipers that gave me a clue.
The supercharged 3. Inside, the Grand Prix is overloaded with plastic textures. The Nintendo-inspired TAPshift and the driver controls all function well but look low rent. New Cars. Buyer's Guide.
Type keyword s to search. Today's Top Stories. Could GTP tubulin exist in either the expanded or the compacted state according to a conformational equilibrium? It should be possible to obtain these data without generating a high resolution structure, perhaps by using TPX2 binding experiments or by a 2D cryo-EM analysis. Further, the conclusions about a conformational gradient are based on a limited set of data, and as, pointed out by the reviewers, alternative explanations of the obtained results are possible.
One could consider performing experiments with a monomeric EB3 protein, because they might help to rule out or provide support for the model proposed by reviewer 1. Another idea raised during the consultation between the reviewers was to use as a binding substrate microtubules grown from mixtures of EA and wild type tubulin at different ratios.
Two reviewers questioned the assignment of 'mono exponential' distributions in Figure 4, and this critique should be addressed. Finally, two reviewers found that the split comets deserve some analysis, and the faster growth rate displayed by ED microtubules requires some attention. Roostalu et al. They make the surprising observation that the dwell time of EB3 molecules changes as you go deeper in the cap. As expected from the Surrey lab, this is a high quality paper addressing a central issue in the microtubule field, namely the role of GTP hydrolysis.
The decrease in dwell times is hypothesized to be caused by a gradual "conformational gradient" that reduces EB3 affinity. The visual representation of this idea is the color gradient from red to yellow in the schematic in Figure 4J.
I have an alternative hypothesis for why the dwell times decrease. Consider that EB3 is an "obligatory dimer" Sen et al. At the very end of the microtubule, EB3 is likely to find two adjacent sites where all of the relevant tubulin dimers are in the GTP state. The site has 4 dimers, but we can leave aside for the moment the question of their relative relevance.
These states have different affinities for EB3. EB3 becomes functionally monomeric, hanging on to the microtubule with only one hand. In contrast, EB3 at the very end of the microtubule is holding on with both hands. Note: the EB3 construct they are using appears to be full-length when I trace back through their Materials and methods references. Can this alternative framework explain the decrease in dwell times with depth inside the cap Figure 4C?
More specifically: a gradual transition from dimeric to monomeric affinity conditions, driven by the probabilistic availability to two binding sites with different affinities? Are the measurements precise enough to rule out this hypothesis? The visual representation of this idea would NOT be a color gradient but rather an increased "speckling" of red and yellow blocks. They divide the microtubules into bins based on distance from the plus-end. The bin size is approximately 0. These bins are relatively small when you consider that: 1 the microtubule end-position is not determined with sub-pixel accuracy, but is determined rather using the "traced end position" from a manual-tracing of a kymograph, which they state has 0.
The first two points make me uncertain whether the molecules are being correctly placed into their bins. The 3rd point is more conceptual: what is the best way to treat a molecule whose bin changes beneath it? They appear to start off linear but then deviate from linearity at, e.
The central argument of the paper hinges on the fact that Figure 4B is not mono-exponential but Figure 4C is mono-exponential. Are the fits really strong enough to support this? The distributions are described as "strikingly mono-exponential". What does strikingly mean in terms of goodness of fit? How often are they observed? How bright are the split comets compared to a full comet, etc. That's fine; the Roll-Mecak lab uses similar constructs. The Materials and methods are clear about the retention of the internal His tag but the main text is not.
I think it's important to be clear throughout. Alternatively, in the concluding paragraph, the authors say that high-resolution structures are on the way. But one doesn't necessarily need a 3. What motivates this hypothesis? Are there data, structures, kinetic measurements, conceptual arguments, etc, that would motivate this idea? Are there measurements of the GTPase activity with non-human proteins that would provide support for this idea?
This is an interesting and well-executed paper that addresses interesting questions about the microtubule's stabilizing cap, how it relates to nucleotide state, and what is the preferred state that EB proteins recognize.
The experiments are performed rigorously and for the most part described clearly, and the work has been done to a high technical standard. The main findings of the paper are: i that abolishing or at least substantially slowing; EA GTPase results in very stable microtubules akin to GMPCPP microtubules, ii that moderately slowing ED GTPase results in microtubules with longer EB comets that are also more stable, and iii that in these longer comets and also wild-type comets , there appears to be a gradient of EB binding affinity, with the highest affinity being closest to the growing end.
Although it has not been shown directly before using a mutant, I did not find it all surprising that reducing GTPase rate increased microtubule stability, but it is nice to see this in the way that it is shown here.
The findings involving EB binding are more interesting: they provide some of the most direct support for the idea of an adaptable microtubule lattice, and they raise questions about what states commonly used GTP analogs are giving.
I think the site-specific EB analysis is probably the most interesting and mechanistic part of the manuscript, and the authors might want to place a little more emphasis there and place less emphasis on some of the obvious-sounding claims. I don't think new experiments are required, but they may want to go deeper into some of the analysis of the EB binding.
Could the authors commend on whether they think the switch in protofilament number or the different conformation of tubulin or both account for the lack of EB3 binding?
I think this should be made more clear. This is unexpected, and the authors really don't say much about it. This is what they measured, of course. Since they know the growth rates, is there anything interesting to learn from plotting the decay against time or just transforming to get the time dependence? A few more sentences about this might be useful. In particular, while the authors ascribe various deficiencies to nucleotide analogs, they do not seem to consider the possibility that the mutation s they made might also perturb the structure.
This criticism applies to the main text also. Some version of the discussion of induced fit should probably be incorporated into the main text. The study by Roostalu et al. The present study examines the binding of the major plus end tip tracking protein, EB3, and its ability to specifically recognize the GTP form of tubulin.
Here, it is shown, using tubulin mutants that either fail to hydrolyze or hydrolyze slowly, that EB3 specifically recognizes the GTP form of tubulin. This is an important finding for the field, one that is achieved through elegant experimental studies of mutant tubulin and careful quantitative analysis, for which the authors are to be commended.
However, in the final analysis, the authors invoke a GDP-Pi intermediate state, without strong evidence to justify it. Rather, it seems possible that a simpler alternative explanation that only assumes GTP and GDP states, as suggested by their data, is not ruled out. Thus, I am concerned that the study, while making an important contribution to the field, may be misleading in its final interpretation.
The spatially resolved dwell time is interesting, but the two positions that are farther away from the tip appear that they may be bi-exponential. It seems the non-exponential distribution might be expected as the k off jumps when the hydrolysis occurs, which would give spatially varying dwell times and nonexponential distributions as the hydrolysis can occur at random Poisson process during the observation time.
To rule this out it will be necessary to do model-convolution to make it convincing that the analysis method is not yielding spurious results. Even then, the EB binding could be dependent on the local neighborhood of nucleotide state see Seetapun et al. Overall, model-convolution on the microtubule addition-loss-hydrolysis and EB binding-unbinding to assess the model is needed to rule out the simpler GTP-GDP model. Even then the argument for a GDP-Pi state is not compelling.
Why is this, and are the in vitro results informative of the tip tracking in living cells? Note: "cap size" is in the title, but it is not estimated, despite a lot of nice quantitation. These papers should be cited, as previous estimates of cap size. Note: need to account for tubulin concentration, e.
Seetapun et al. We thank the reviewers for their overall very positive evaluation of the quality, novelty and importance of our work. We however respectfully disagree with the view that the demonstration of EBs binding GTP microtubules is less significant, because it is perceived as not surprising. The question whether EBs bind to the GTP conformation of microtubules could not be answered with certainty in the past.
Previous studies have used various nucleotide analogs to address questions of the nucleotide state. In our Introduction, we now provide more background and explain these different interpretations that arose from partly contradictory observations. We believe that this helps to provide a better context and highlights the value of our experiments with GTP microtubules. In our view, we present here for the first time evidence that EBs indeed bind the GTP state of microtubules with high affinity, providing clarity concerning a central question about microtubule biochemistry that has remained unsolved and has been a matter of speculation.
We have performed additional analysis going beyond what the reviewers asked which helped us to improve the clarity of the description of the measured affinity gradient. We describe this in detail in our response to the individual points raised by the reviewers and provide an improved description and discussion in the manuscript. EBs sense the conformation of their binding site. And the conformation of this binding site is affected by the nucleotide state.
We make this clear in the revised Discussion. Fluorescence microscopy experiments cannot visualise the degree of lattice 'compaction' or 'expansion' that can be observed by cryo-electron microscopy.
Therefore, we do not make statements about such lattice characteristics. Following a suggestion of a reviewer, we added experiments with a fragment of TPX2 to the manuscript but consider electron microscopy experiments beyond the scope of this already extensive study. We respectfully disagree with the view that our data set is too limited.
Not many labs are currently able to make high quality recombinant tubulin. Although it cannot be produced in amounts as large as for animal brain tubulin, our single molecule imaging data sets here are larger than previous data sets, even when compared to experiments made with brain tubulin.
Otherwise our new spatially resolved dwell time analysis at growing microtubule ends would not have been possible. We consider experiments with monomeric EB and mixed microtubule lattices beyond scope, because they have their own challenges and become easily studies in their own right.
We have addressed the issue of the 'mono-exponential distributions' by additional analysis. This is an interesting point and it turns out that it is important to consider whether EBs diffuse on the microtubule lattice while they are bound:.
If they do not diffuse, the reviewer's model is not supported by the data. This would then result in complicated, clearly non-mono-exponential local dwell time distributions, which we do not observe. However, if EBs can diffuse on the lattice, as reported earlier Lopez and Valentine, , they are expected to have a "mixed" affinity resulting from the various affinities of the binding sites they visit during lattice diffusion, leading again to mono-exponential local dwell time distributions.
In our experiments, we observe much less diffusion than reported in Lopez and Valentine possibly due to our lower ionic strength buffer. Therefore, our data do not allow to distinguish between the reviewer's model of a speckled nucleotide state lattice in the cap of growing microtubule ends and a model in which tubulins hypothetically change their conformation in a concerted manner. Nevertheless, we clearly observe an affinity gradient for EB binding at growing microtubule ends, as we state in our manuscript.
We explain now in our Discussion that the measured affinities are 'average' affinities integrating information of the conformation of several EB binding sites visited by EBs during lattice diffusion.
We also clarify that our colour gradient in the Legend of the schematic figure is intended to illustrate affinity gradient for EB binding. Option Pack No. Always a class act, retired General Motors Corp. This year was no different. It was after the meeting that Stempel said GM may have to close more plants and lay off some more workers to meet its committment to percent plant capacity utilization by The topic was not mentioned at the meeting.
We also spotted retired GM Chairman Roger Smith, now carrying a noticeable paunch, before the start of the meeting. Smith kept a distance from the press in a room reserved for directors. While shareholders applauded, the directors sat mute. The proposals all were defeated soundly.
The meeting also provided a laugh when shareholder gadflys John Gilbert and Evelyn Y. The reason Saturn will bring out a station wagon before a convertible, said Saturn President Richard ''Skip'' LeFauve, is that the wagon requires only a stretch of the four-door sedan, and the ragtop means surgical removal of the top from the coupe and then the addition of structural reinforcement.
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