UPDATE: I feel as though I should add a bit of an update to this. These are very preliminary opinions. It may be somewhat unfair of me to even have posted them, but they’re what first came to mind. As I think more about what La Clair said, the more it makes a bit of sense, but it’s still nothing that’s hitting me as great. Take my opinions or leave them, you won’t hurt my feelings any.
Jesus, that was a whirlwind lecture. J.J. went at fifty brazillion miles an hour and presented more images of glowing gels then an organic chemist should EVER SEE in his or her lifetime. I don’t even know where to begin. If I’m not mistaken, he started off with something along the lines of ‘you make enormous molecules synthetically and then put a label on them and that’s dumb when you can just use natural products.’ He then shows these enormous natural products that glow and says you can append them to proteins and they can travel into the cell and stain different components. At least I think that’s what he said – he said it so quickly and his PowerPoint slides moved so fast it wasn’t trivial for me to follow along.
Of course, he passed over the controversial hexacyclinol in the beginning, suggesting some people don’t believe him and that he is currently remaking it to prove us all wrong – stuff we all already know. And if you’re curious, there was no clashing with Rychnovsky’s student at all. Her talk was in no way related to hexacyclinol.
I think the biggest problem with his idea is that, not only is it done, but it’s done ridiculously inexpensively with commercially available things. Cell trackers are available from a very large number of companies and you can buy them which selectively stain the ER, the nucleus, the cell membrane, the ribosome membrane, etc. etc. So, even if there was a point, I missed it. That could also have been my bad as well… I had to pee the entire time. There was time for one question at the end of his talk but no one bothered. I wanted to state the above, but the room was PACKED and HOT. There wasn’t even enough room to move around. The doors were jammed with people. It was crazy. His last slide had an image of that t-shirt with ‘the proof is in the product.’ At least he has a sense of humor. When it was all over, the moderator got up at then end and, clearly confused, said something like “I know I’m supposed to ask a question here, but I’m just as much at a loss as you guys.” And with that, a mass exodus took place out of the room.
Update II La Clair had an interesting point with a peptide based macrocycle that would ring open and move from one organelle to another. I dunno if I’ve read anything that does that before.
Update III There is a second take on it at Totally Synthetic right now.
Update La Fin: (that’s French) There is a third take on it at Tenderbutton right now. Also, Dr. La Clair has graced us with his presence to answer questions via the internets for those of us that were too chicken shit (or baffled) to do it in the meeting. Please see the comments. (are there any accents on any letters of “La Fin”? My French is, how you say, total crap.)



I didn’t see a SINGLE control costain in that entire talk… did anyone else?
you can’t prove a molecule is in a cellular compartment without demonstating colocalization. Cells don’t look like the cartoon pictures from your general biology class in highschool.The ER looks like the mitochondria that looks like endocytotic vessicular transport system. one giant blob of colors
Holy shit, you’re right. I didn’t see one either. I have no idea why I wasn’t paying attention. Though, I have to admit, nothing he was saying was outside the realm of possibility. But, yeah. You’re right.
“outside the realm of possibility” does seem to be his speciality…
He’s a fraud. Just so you all know.
Then why didn’t you point that out to him at the question session?
You have to hand it to La Clair, he is an excellent talker. He was cool and petty collected the entire time. He’s one hell of a salesman… so… it isn’t any wonder that he has so many collaboratins.
Speaking of which, did he actually collaborate with Scott Rychnovsky?
I think they exchanged an e-mail or two. Does that count?
Sure, why not? I emailed Sharpless once, can I say he’s my co advisor now?
Snake-oil salesmen and magicians are interesting to watch and can talk your arm off; just be careful what you believe. They’ll also have an answer for any challenge you throw at them.
There are many ways to identify the cellular localization co-localization is one. It is done all the time and I did present those images using a series of natural product analogs. All images can be compared to a series of commercial multicolored organelle labels and this was done. In addition I usually extract the cell components as well check the uptake in those extracts. I find this provides a second level of data. I presented this data as well.
Lets get over the name calling and focus on science. If questions were not asked then please feel free to bring them online…
Jim La Clair
Jim La Clair,
I never said I doubted the data presented here, it’s really not that off the cuff. I do have a few questions for you; however.
1. I’m not sure why you want to use huge natural products? Culture and extraction is a pain in the ass compared to the synthesis of simple dyes like congo red. Hell, a cy7 or cy5 dye can be made faster than a fungus can be grown and blended.
2. What other cell lines did you use? It didn’t seem like the ones you presented were ideal, being the clumpy sort. Did you use CHO lines or anything else?
3. What’s the advantage of this ring opening macrocycle that moves from one organelle to the other? What applications do you foresee it having?
thx!
Kyle
Jim…. are you going to answer these questions? We anticipate your responce!
La Clair, you did very well under all the pressure; If the roles were reversed I would not of faired as well. My knowledge of Biology is limited so I didn’t understand all you were doing, but the talk seemed fine to me. In the end, the real man of the day was the Red-Ticket Man, and we would all do well to pay our respects to him.
Mitch
I totally made the toilet in my hotel room a hat that night thanks to that guy (you helped there, buddy).
Well, look who’s decided to dodge your questions, kyle? Uh huh…
Hey JJ,
1. Did the government really take your hexacyclinol product away?
2. If you made 3 grams of said product, why are you re-making it?
3. Why did you use coumarin (a blue dye) for your peptide work when the cells autofluoresce blue? Wouldn’t another dye be more appropriate? And why did you once use dimethyl amino coumarin and once diethyl amino coumarin?
Dear Kyle,
1) Sure u can use stains to determine the regions of the cell however the reason to make a natural product fluorescent is quite a different query. It is one method that one can use to determine the biological role of the natural product. By determining the localization of where a material goes in a cell allows one to determine what potential role a natural product has? For instance it is not likely that a DNA binding natural product will appear in the cell membrane rather one would see it in the nucleus.
2) There are a number of quite easy to use human cell lines. I usually end up using what the laboratory I am collaborating with has grown a use to using. When I do work myself and I am in the mood for exhaustive efforts I will use between 20-60 cell lines but for target work this is quite exhaustive. It’s best to start with a small set of cell lines that you are comfortable with culturing.
3) The idea of that presentation was to create thought not close it. If I gave u the answer you would not be able to make the question. There are a number of programs in the cell that movement would around the cell would be interesting to investigate for instance Golgi genesis.
Dear Mitch,
Thanks man…
Dear Anonymous,
1+2 I hope someday that we learn that rumors are not good for science.
3) Good question. Actually over time I screened a large set of dyes for their lack of biological activity and easy in synthetic manipulation. The DMC (coumarin) label is easy to make (at least 20+ labs have done it at the gram scale) and it can be manipulated easily. It is uncharged enters cells well and can be washed out of them.
Human cells have only modest blue fluorescence and backgrounds can always be eliminated with the proper blue filter sets (Semrock makes excellent ones). Also there are several controls (with other colors one can do when concerned). However the key to moving the cell to a fluorescent probe in my hands has been to stay with the same dye for as many studies as possible as each natural-product dye conjugate serves as a control for the next study.
Good on ya for answering the questions.
I’ll admit that my first question was a rumor, but as I heard it directly from my advisor, I thought I would ask.
As for my question #2, I think it is valid. You reported synthesizing the molecule on the gram scale. Now you have publically said you are re-making it. These seem incompatible.
I’d like nothing more than for you to come out correct (having made the compound you claimed — it would be a good lesson for the gosspimongers, me included), but the question as to why it needs to be re-made is still valid.
But back to your talk… Your cyclic/linear peptide localization work was interesting for sure. Could the peptides be used as localization tags (for macromolecules)? What is the end goal for this work (meaning: it looks very cool, but what are the applications)?
Jim,
Hmmm… I may have missed the point of the first part of your talk. I understand the coumarin dye derivatives, that’s all fine. The mobile dye is neat, that’s fine, but I just couldn’t figure out what you were talking about with the Gambogic acid. I’m probably just missing the point so, please, what did you mean by that?
I agree with you, Kyle. I also didn’t understand the gambrogic acid beginning. Well, I understand that it is important to consider where to put the label (as JJLC said and I agree with), but I didn’t understand how that fit into the rest of the talk (which was cool, but still..).
(the previous two anonymous posters were me. i’ll just go by cheese from now on.)
JJ,
I’ll preface my question by noting that I am strictly a synthetic organic chemist with limited knowledge of cell work. I did attend your talk at ACS.
Have you considered that attaching a large fluorescent label to a natural product before treating cells with it can significantly impact its lipophilicity and membrane permeability, thereby changing dramatically its localization within a cell? It seemed from your talk that you meant to design the labelling into a natural product synthesis (thereby making it the synthesis of a natural product analog, and not a natural product synthesis). I can only guess that this would mean attaching the label earlier than the final step (following a global deprotection that, as you deftly pointed out, leaves a number of synthetic handles with which to attach said label).
Secondly, regarding your point about “design(ing) a natural product de novo” in order to effect a desired biological response: isn’t that what medicinal chemists have been doing for years using HTS and SAR? Drug discovery has been around a long time. I don’t see how locating a molecule within a cell is more useful than the battery of assays against specific proteins that are run in high throughput screening. It may be a nice complement to those assays.
Philip