Wednesday, 2 December 2009

Meet the chickens - Fluorescnet Microscopy and Centrosome Proteins

Hello Again in the Meet the Chickens section,

Today we will have a look at the DT40 cell line centrosomes but first I will give you a short background.
Centrosome is large cytoplasmic organelle. Its core is compsed of mother and doughter cetrioles which is surrounded by a pericentriolar matrix (which compose of dozens of proteins). The doughter centriole is always formed de novo and the other one comes from the previous cell cycle from the mother cell (mother centriole).
During the cell cycle, centrosome duplicates and just before the cell division there are two (and there should be only two) centrosomes in each cell. Higher number of centrosomes can be dangerous for the cell as division of the genetic material could be impaired (look at this animation McGraw Hill - Mitosis and Cytokinesis). Abnormal genetic material division can lead to gain or loss of chromosomes. Such situation may lead to pathogenic state (for example cancer).
Centrosomes are often called microtubule organizing centres. What it means is that microtubules nucleate at centrosome providing connection between chromosomes and centrosomes. When microtubules attachement to chromosomes is completed they can be pulled to opposite poles of the cell which are designated by position of the centrosomes (see picture below).

Centrosomes are involved not only in cell division. They also serve as a platform for different proteins and processes. Many proteins have been shown to localize to centrosomes and for some of them this localization is necessary for their activation or proper function.

We can visualize centrosomes in the cell using specific antibodies which recognize proteins associated with them. A list of the proteins which localize to centrosomes is still growing. The well known centrosomal proteins are: centrin, gamma-tubulin, pericentrin, Kizuna, Nedd1 and more. On the picture below you can see a example of such staining.

 Picture taken by Kliszczak M.

On the photos above you can cell in late stage of mitosis (anaphase). Each separate channel is represented in gray scale.
DNA was stained with DAPI and on the merge photo it appears as blue stained. You can see that DNA is already sepeareted to opposite poles of the cell.
On the Centrosome layer which appear green on the merge picture you can see a GFP fusion of centrosonal protein (Green Fluorescent Protein) which is known to localize to centrosomes (notice the two red circles). The cell has a lot of the green background which is common when fluorescent protein is expressed in the cell (GFP signal).
On the red channel (Microtubule) the protein which builds up the microtubules is stained. You can see the microtubules connecting opposite poles of the cell. Notice that at the poles microtubule staining is very bright. Those big blobs are actually centrosomes where microtubules nucleate. Notice also that those microtubules centres do colocalize with the green spots (GFP fusion of centrosomal protein).
Merge image represents all channel superimposed together. Microtubule staining is not very visibile at this photo, beside at the poles where you can also see a GFP fusion protein. On the merge picture you can clearly see that DNA is pulled to the opposite poles of the cell which are designated by centrosomes.

Scheme below represent another example of centrosomal protein staining. Where gamma-tubulin and another centrosomal protein are visualised.

Picture taken by Kliszczak M.

Again the DNA stained with DAPI appers blue on the merge photo. In this case there are three different cells. One which is not dividing (an interphase cell) and the other which just finished the division (two cells on the right hand side, you can tell it because DNA is sitll compacted).
On the green and red channels you can see centrosomal proteins which appear as bright spots.
Merge channel talks for himself:)

As you probably noticed the Flurorecent Microscopy is a powerful tool which allows to visualize a specific protein, protein complex or even an organelle. With this technique we can monitor protein behaviour. For example if the protein X is a cytpolasmic protein but during mitosis or after, let say stress response (like DNA damage, or heat shock response) it is targeted to nucleus or chromatin (DNA) we can easily detect that shift.
The ultimate technique now for visualizing protein of interest is live imaging (which will appear in on this blog soon:). Using a specially designed microscopes we can follow live cell which express a fluorescent fusion (our protein + fluorescent tag) of our protein of interest and in real time its localization.

I hope you enjoy todays post:)

Have a nice day.


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