hi
I observed such a-tubulin distribution changes in control and flattened(compressed) MDA-MB-231 cells. do let me know if you disagree or find other differences.
1) Upon compression, the orderliness or tidiness of a-tubulin decreased. They are all over the place.
2) Upon compression, there seems to be greater concentration of tubulin around nucleus compared to the other parts.
for 1), can you suggest a procedure where I can quantify this randomness of microtubules?
for 2) do you agree of this observation. Is looking at ratio of intensity of microtubule around nucleus vs the other parts of cell a good way of quantifying this? using image J etc.
first two images are compressed cells, last two are uncompressed cells
Resolution of all figures are not same; therefore it is difficult to comment on that just by looking at one or two panels. Some of the cells microtubule networks are looking a little depolymerized in figure 3. But Just by looking one figure it is difficult to judge.
Dear Iwan, What you see is quite normal and expected. Nothing strange or exceptional/remarkable. Microtubule orientation/distribution is always affected by forces applied on cells. Only if you over-press, they may depolymerize.
Hi Emanuel. Thanks for the feedback. But what I would like to know next is whether there are indeed changes to cell morphology and microtubules arrangements? I actually see changed but it will be good to have an expert agree on this.
How would you describe the tubulin changes?
Dear Iwan,
as Diptiman already mentioned, we cannot judge by just two images. It is you, who have the whole set of sections, that can understand and monitor any change in cell shape (BTW, what is the the axis of cell compression?) and microtubule orientation. Just a kind correction: please, when you refer to microtubules do not call them just "tubulin". In fact, there might be some degree of depolymerization, with subsequent relesase of free tubulin and increase of background fluorescence. But you have to measure this by some relevant application on the original images.
Sorry for not being very helpful, if I have to judge by the 4 images that you attach, I would say that nothing really significant can be observed.
Good luck with your experiments!
Thanks emanuel. i still have a long way more to go and am still learning the ropes.
i have constructed this compression device and i was hoping to show that indeed this compression device work and the evidence is the cytoskeleton changes. what i did was grew the cancer cells on transwell insert, then place a 1mm thick agarose cushion over it, followed by a weight over it. Thus it is like compression (flattening) kind of axis.
i get what you mean by no significant observation can be made. I have attached a zoomed out version of the images. first 2 images refer to compressed, last 2 images is uncompressed. Please do comment on it, thank you!
Now I see what you intend to show! My suggestion is to try F-actin or even better some kind of intermediate filaments, instead of microtubules. Their response to compression should be more prominent.
I cannot see anything very prominent but I just saw two images. You have to substantiate any change by statistic analysis in a large sample (ca. 100 cells).
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology