Hi Anthony,

Can I ask, is the GFP attached to the N- or C-terminal?

It may be that the GFP (~230 amino acids) is too bulky to enable tubulin incorporation into the tightly packed microtubule polymer lattice. Similarly, it may prevent correct folding prior to alpha/beta dimerisation.

I have previously tried both N- and C-terminally tagged alpha-tubulins in HEK293 with little luck. I have only been able to visualise polymerised tubulin using a C-terminal FLAG- tag (8AA's) and anti-FLAG antibodies. Annoyingly, this requires fixation so cells aren't live.

Hope this is of some help!

Cheers

Tom

In plant communities there are TUA6-GFP lines that work very well (and other like MAP4-GFP). Maybe try to find the plasmid(s) that was (were) used there to create the lines and have a look?

Thanks both.

Tom, the GFP is at the N-terminus on alpha-Tubulin and others seem to use it ok (to varying degrees of success with other cell lines). I totally accept the steric argument, but others seemed to get it to work! I had assumed that the monomeric/polymeric ratio was too high, but wasn't sure how to make that more favourable.

Depressing that you couldn't get it to work either in HEKs. Oh well... perhaps this is a non-starter.

Thanks,

Ant

Hela MTs are not very pretty in interphase. Still, when you have the right conditions this will work. We typically use stable lines, but you should see at least some MT also by transient transfection.

Some comments:

1) Try to find mitotic cells (round), it might be easier to see the very intense signal from mitotic spindles.

2) Expression level might be an issue in transient transfection, try transfecting 10:1 ratio empty plasmid/GFP-tubulin to reduce expression level, or wait several days before imaging for expression levels to go down (multiple rounds of division will reduce the signal).

3) If still no luck try other cells or make a stable line and chose clones with appropriate expression levels.

Thank you Jens, that is very helpful and comprehensive advice.

Unfortunately, small round cells will not be too helpful as we require large flat cells for optimal imaging, but your point is well taken. Perhaps another cell line will be the way forward if dropping the expression levels also fails.

Many thanks,

A

I hope your microtubules are not depolymerized by the time you image in the microscope. If they are, you won't see the tubules but GFP-tubulin labelling will appear as cytosolic dots. If your transfection is ok, use the media strictly at 37oC and incubate at 37oC stage for sometime before imaging. I agree that HeLa cells are not the best ones to image microtubules but many people have done.

Thanks Suparna, that is also very useful. Perhaps a stupid question, but why would the microtubules be depolymerized by the time I image? Because of an excess of monomer?

Because by the time you prepare for imaging, the cells are out of 37o incubator and microtubules in live cells are very sensitive to temperature drop induced depolymerization.

I agree with Suparna, but I was assuming you do the live imaging at 37C?

Unless the temperature is quite low, my guess is the expression level is the problem.

Another thing you can try to see what is wrong is to fix in ice-cold methanol for >5 min and stain cells with tubulin antibody (DM1A, Sigma). Compare untransfected (you should see nice MTs) and transfected cells. This will give you an idea whether the problem is caused by temperature, overexpression, etc. Methanol fixation washes out some soluble protein and you might still be able to see MTs in some cells, e.g. in those with lower expression levels. Then you know you are on the right track.

Both of you have been super helpful, thanks so much.

I wasn't aware of the temp-sensitivity and I am actually imaging at room temp (~22˚C) which may be a major issue then.

I like the suggestion of fixing and performing IF to see the status of the MTs. Thanks for the antibody suggestion, Jens.

As an MT novice, I really appreciate your advice.

Kind regards,

Ant

Your construct isn't working.

re-prep your plasmid, I'm assuming it is a pEGFP backbone?

I would transfect helas with lipofectamine 2000.

tube a) 125ul optimem tube + 2-3ug DNA

tube b)125 ul optimem + 5 ul lipo 2000

(Add in that order)

leave at RT for 5 min then combine tubes, flick to mix, leave for 20 min at RT

replace the media on your 3cm dish with 2ml fresh media.

add complexes dropwise and rock dish back and forth, side to side. Don't swirl.

If you do this in the afternoon, your cells will be expressing the next day.

Change the media the next morning.

microtubules require 4C to depol, not 22C.

Always image at 37C and add hepes to your imaging buffer.

If you are using wide field, use phenol free media.

If you are trying to look at microtubule dynamics, you should use EB1...... tubulin photobleaches too fast and can't be used for most tracking software.

Hi Samantha, thanks for taking the time, this is most helpful.

To put your mind at rest, I routinely and successfully transfect HeLas with other GFP-chimaeric constructs (and the JetPEI uses a similar cationic approach to Lipo 2000), this was the odd one out. Perhaps I should get it sequenced, even though it was from Addgene.

Thanks for the reassurance about the temp, I was alarmed that MTs could be *so* sensitive that RT would be catastrophic.

I have seen some beautiful EB1 images so thanks for highlighting that for me.

Thanks,

A

Hi Anthony,

We usually work in transiently transfected cells with fluorescent tubulin and have been puzzled by this question before. It is not a matter of transfection protocol. All you have to do is wait for your transgene to repress the expression of endogenous alpha-tubulin gene(s), then you'll see your microtubules fluorescent. Usually this is done within 48 to 72h. Unfortunately, HeLa is not the best model to visualize microtubule dynamics and many collinear microtubules make them difficult to distinguish but the principle is still the same with other cell lines…

Best wishes

Christian Poüs

Thanks very much, Christian. Perhaps I'm not leaving them long enough, what you say makes sense. I will have to move cells too, I suspect.

Best, Ant

Hi Anthony,

we and our collegues tried a lot to get these very HeLa expressing clontech GFP tubulin plasmid. We got rather nice result when put the plasmid under the control of low effective promoter.There is a lot of tubulin in the cells, and when you additionally express (or even overexpress) a lot of labeled one, MT stabilise a bit, and you get bright background from depolymerized tubulin plus a dense bright network of MT. So try to get lower expression level.

Good Luck,

Elena Kornilova

Elena, many thanks indeed, this is very helpful indeed. I will indeed try what you suggest and hope for some good luck!

Kind regards,

Anthony

Nitin, it seems I was using too high a DNA concentration (see Elena's point above). For most plasmids I use 1-2ug, but when I dropped to 0.4ug (or less) it started to look much better.

Since Elena's helpful comment, I have noticed some people use 10-fold lower DNA for tagged tubulins (http://www.ncbi.nlm.nih.gov/pubmed/17925382) compared to other constructs.

Perhaps this might help in your cells?

Best,

A

Not a protocol as such, Nitin. Just a regular transfection using JetPEI (but I guess any cationic lipid-based system will work).

Cells ~ 60% confluent on 25mm coverslips in a 6-well plate.

Mixed DNA:JetPEI (1:2 ratio) in 200µl NaCl > allow complex formation 15-20 min > then added this drop-wise to the well.

Incubate 4-5 hrs 37˚C.

Exchange for fresh medium, leave overnight, image next day.

Hope this helps.