Thursday, December 10, 2009

A PLoS ONE Success Story--Taxol Crystals Masquerading as Microtubules


ResearchBlogging.org
Andy Maloney, a Ph.D. student in our lab, recently read and summarized a very interesting paper in his open lab notebook. The paper, "Taxol Crystals Can Masquerade as Stabilized Microtubules," was published in PLoS ONE in January of 2008 by Margit Foss, Buck W. L. Wilcox, G. Bradley Alsop, and Dahong Zhang1. Since our lab is now heavily involved in experiments involving kinesin and microtubules, and because it addresses something that had been a mystery to us, the paper really caught my interest. I'll explain more about that below. But before doing that, I wanted to talk about something probably of more general interest: a success story for publishing in PLoS.

Andy noticed that in their methods they defined BRB80 as having 4% glycerol. Glycerol is used to promote tubulin polymerization, and I've never seen it included in the BRB80 (aka PEM) definition. It could also affect solubility of Taxol, so it's an important detail whether or not a substantial amount of glycerol was in their standard BRB80 buffer. I strongly suspected that this was just an oversight by the authors...and I could easily have assumed this and moved on. But what about future readers of the article? Was there anyway to correct that article? For most journals today, even in the year 2009, the answer would have been, "no." However, this is no ordinary journal, this is PLoS ONE! All I had to do was select the text in question, and then click to add a note. After adding my note, an icon appeared in the article, allowing any future reader to see the question.
PLoS Comment Image

I don't know whether authors are notified when their article is commented on. (If not, it would be an important feature for PLoS to add.) So, I sent an email to the corresponding author of the paper (D. Zhang) pointing out the question. In less than a day, D. Zhang wrote back saying that he'd asked M. Foss to look into the issue. And then again in less than a day, Margit wrote me back to say that she'd looked at the original lab notes and indeed they'd made a bit of a typo in how they described BRB80 in their report. She added a very clear response to my note. She also went out of her way to point me to two subsequent papers that have extended their taxol microcrystal research2,3. These authors deserve a lot of praise for responding to this question so quickly! A few months ago received a similarly rapid response from authors of another PLoS article...only two data points, but I wonder if PLoS authors are indeed more likely to respond quickly to questions from readers?

Now, why am I so happy and why do I think this is a success story for PLoS? It's because now, for the rest of time, when readers of this excellent paper do look into the methods, they will be able to see the corrected definition of the buffer used. Given how many times I've been burned by incomplete or incorrect methods, I do believe this will save substantial amount of time for at least a couple people down the road. (Will the PDF version of the article ever incorporate this note? As it stands now, I don't think it does...it would be very valuable if technology could be worked out to include links to these comments in future PDF downloads.) One more thing: I just noticed that Margit Foss today also posted a new comment on her article. She links to the two papers she'd told me about in her email, as "Relevant references on Taxol crystals." This is a great service to readers, especially since the newer reports2,3 support a different mechanism for Taxol microcrystal / fluorescent tubulin binding. In summary, many thanks to PLoS for this wonderful journal and to these authors for their dedication to excellent science!

Now, if you're still reading, I'd like to also comment on the very interesting science in their report. Taxol (generic name is paclitaxel, I think) is a drug used in cancer chemotherapy. It's proposed mechanism of action is to inhibit mitosis by stabilizing microtubules in the spindle apparatus. In vitro, Taxol dramatically reduces the rate of microtubule depolymerization. Many people, including kinesin researchers in our lab, leverage this microtubule-stabilizing effect by adding Taxol to microtubule-containing solutions. What I learned from the Foss et al. paper is that the concentration of Taxol typically used in microtubule gliding assays (10-20 micromolar) is far above the solubility limit of Taxol (somewhere around 0.8 micromolar in aqueous solutions). Furthermore, they show that Taxol forms microcrystals above this solubility limit (even at 0.92 micromolar) and that often these microcrystals form a striking resemblence to microtubule bundles and asters! DIC images of these microcrystals (formed in absence of tublin) are shown in these images from Foss et al.1:

(scale bar 10 microns)


The final piece of crucial information provided by this article is: these Taxol microcrystals rapidly bind fluorescently-labeled tubulin! (Later reports indicate that it's the fluorophore, not the tubulin that is binding to Taxol2,3.) This means that many kinesin researchers (including me) likely have Taxol microcrystals in their samples, and because they become coated with fluorescent tubulin, there is a huge risk of misidentifying these structures as microtubule structures. Indeed, here is a recent fluorescence microscopy video that Andy took of something that at the time was a mystery but which we now know is likely a Taxol microcrystal decorated with rhodamine-labeled tubulin!

Likely Taxol microcrystal in kinesin / microtubule gliding motility assay (using rhodamine-labeled tubulin). Andy Maloney data.

In my past, I've also often seen these structures which I attributed to "clumpy" or "weird" microtubule structures. For example, I often noticed very bright, thick, and stick-like structures that I called "microtubule logs." It never occurred to me that they were Taxol crystals! (Also I remember that these structures were much less prone to photobleaching. I wonder if that's because (a) there are buried fluorophores inside the crystals, protected from oxygen, or (b) even on the surface of the crystals, Taxol somehow protects fluorophores from photobleaching?)

Foss et al., go further and speculate on whether this has important implications in vivo (i.e. in cancer chemotherapy). I can't really comment on that, but it's interesting to think about. What's most important for us is that we now know we have a problem with our buffers (too much Taxol!) and we may be able to fix it. The concentration of tubulin that we typically use is about 0.4 micromolar of tubulin dimers. Thus, for a 1:1 ratio of Taxol to tubulin dimers, we'd need 0.4 micromolar starting concentration of Taxol, which is below the solubility limit. There's at least two things I don't know: (a) What is the binding affinity of Taxol for microtubules? and (b) Do we need a 1:1 ratio to get significant stabilization? If the answer to (a) is something like a few nanomolar, then we may be OK with something around 0.5 micromolar (500 nanomolar) Taxol. If not, then we may have to hope the answer to (b) is "no."

A quick search just now yielded a paper from 1994 that says the binding constant for taxol to microtubules in 10 nM. That'd be good, except that they also seem to say that they only get stabilizing effects when the concentration is in the micromolar range4. Dang! Well, it shouldn't be too hard to try out 500 nM Taxol and to see whether MTs are reasonably stable. It's possible our MTs may be more stable than those used in the Caplow et al. study. It's also possible that the Taxol microcrystals are not affecting the kinesin activity in our system, and that we can do our studies at high Taxol concentration. Even if so, it's great to know about this issue so we can keep on the lookout for Taxol problems.

References

1. Foss M, Wilcox BWL, Alsop GB, Zhang D (2008) Taxol Crystals Can Masquerade as Stabilized Microtubules. PLoS ONE 3(1):e1476. doi:10.1371/journal.pone.0001476

2. Castro, J. S., Deymier, P. a., Trzaskowski, B., & Bucay, J. (2009). Heterogeneous and homogeneous nucleation of Taxol crystals in aqueous solutions and gels: Effect of tubulin proteins. Colloids and surfaces. B, Biointerfaces. doi: 10.1016/j.colsurfb.2009.10.033.

3.
Castro, J. S., Trzaskowski, B., Deymier, P. a., Bucay, J., Adamowicz, L., Hoying, J. B., et al. (2009). Binding affinity of fluorochromes and fluorescent proteins to Taxol™ crystals. Materials Science and Engineering: C, 29(5), 1609-1615. doi: 10.1016/j.msec.2008.12.026

4. Caplow, M., Shanks, J., & Ruhlen, R. (1994). How taxol modulates microtubule disassembly. The Journal of biological chemistry, 269(38), 23399-402. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/7916343.

Foss M, Wilcox BW, Alsop GB, & Zhang D (2008). Taxol crystals can masquerade as stabilized microtubules. PloS one, 3 (1) PMID: 18213384

Link to FriendFeed discussion thread.

3 comments:

  1. Thanks for blogging about our paper, and for advocating for open access. Clearly there's still a lot to be learned about Taxol/tubulin interactions. Since this is not the focus of Dahong's lab, we're no longer working on this project. But I'm very pleased that other people are taking an interest.

    I see you’ve referenced the 1994 Caplow paper for the 10 nM binding constant. I’m curious as to whether their experiments may also have been affected by the presence of Taxol crystals.

    For example, in Table I, footnote c, the authors write, "Microtubules assembled with 1 mM GTP and 50 uM tubulin were made 50 uM in taxol and incubated for 10 min. This mixture was diluted 10-fold into 5 uM taxol and incubated an additional 10 min. Aliquots were then added to [3H]taxol contained in either a 5% volume or a 9-fold greater volume; after 90 s microtubules were isolated with an Airfuge. Radioactivity and protein were measured in the resultant pellet."

    My feeling is that some Taxol crystals would likely have been present in the 50 uM Taxol before the 50 uM tubulin mixture was added. It's unclear to me whether all such crystals would have remained intact after the addition of tubulin or whether there would have been some dissociation, freeing up additional Taxol to stabilize the MTs. Since Taxol is so insoluble in water, I suspect at least some crystals would remain even after the 10-fold dilution. If so, it's possible that some tritiated Taxol could get incorporated into the Taxol crystals rather than stabilizing the MTs. I also suspect that any good-sized Taxol crystals would pellet along with the stabilized MTs. So when the authors measure the tritium vs protein in the pellet, I'm not certain that these numbers reflect only Taxol that's MT-associated. And that's ignoring the complication that free tubulin heterodimers could nucleate Taxol crystals, according to the Castro 2009 reference. In addition, some tritiated Taxol may be associated with small Taxol crystals that are too light to pellet, perhaps accounting for the 15% of tritated Taxol that didn't pellet in fig. 2.

    Anyway, I've made a lot of assumptions above, and they may not all be true, or the effects may not be significant enough to affect the outcome. And I haven't gone through the whole paper to look for other possible discrepancies. But it may be worthwhile for you to consider these issues before fully accepting the 10 nM figure.

    Again, good luck with your experiments!

    -Margit

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  2. Fantastic blog. I completely agree that the power of journals to allow comments post publication, has given the meaning of publication and discussion a whole new twist.
    Indeed we also struggled to understand what was going on in our negative controls with taxol-microtubule mixtures with BRB80, till we saw the report by Foss et al. I have worked with microtubules before but in Xenopus extracts, so there was no question of adding taxol. Now in an in vitro system it isn't that simple.
    Thanks.
    -Chaitanya

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  3. I am glad if this blog helped you out at all! I am not in the field anymore, but I am still suspicious that a lot of people haven't picked up on Foss et al.'s excellent work. I'm glad you did, and wish you the best in your research!

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