Meet the chickens - sister chromatid labelling

Yo all,

welcome again in Meet the chickens section. Today a specific labelling of chromosomes (actually sister chromatids) will be presented.

As you probably remember from previous post (Meet the chickens - chook chromosomes at hand), there is a way to examine karyotype (set of chromosomes) of chicken DT40 cell line (or any other cell line:). In this post we will combine this technique with a labelling procedure. This protocol will give us oportunity to see differential staining of sister chromatids.

Ok lets go.

In this protocol a nucleotide derivative, called BrdU (5-bromo deoxyuridine) is used to labell DNA for two cell cycles (see the picture below).

Simply saying, cells are treated with BrdU, which is incorporated to DNA during S-phase (DNA synthesis phase of cell cycle). It is important that labelling is performed for exactly two cell cycles. This is because after two cell cycles (two subsequent S-phases) one of sister chromatids is complately substituted with BrdU where other one is half substituted half not. This feature of labelling can be now used to distinguish between sister chromatids (they simply have different physical and chemical properties).

First cells are stopped in metaphase (cell division phase) using a specific drug (usually colcemid a microtubule depolymerysing agent) that prevents mitotic spindle formation. After that, cells are swollen (to make them fragile) and fixed (to fix their state:). Further, cells are droped onto slide to open them and release chromosome spreads. Next few steps leads to differential staining of sister chromatids (see picture below).

In the first step, cells are treated with a DNA intercalating dye called Hoechst 33258 (this is a fluorophore that adsorbs UVC light at 258nm). Intercalating means that it gets in between the DNA base pairs stacks (see picture below)

Red bars represent an intercalating agent bound to DNA/

Picture taken from http://en.wikipedia.org/wiki/File:DNA_intercalation.jpeg

As you remember one of the chromatids is complately BrdU substituted. BrdU contains bromide and occupy more space than a regular nucleotide (remember that bromide atom is way more bigger than carbon, hydrogen or nitrogen). Additionally BrdU nucleotide is light sensitive. Hoeachst intercalation occurs only in monosubstituted chromatid but not in disubstituted one (there is not enough space for Hoechst intercalation, because of two bromide atoms).

Further, slides are exposed to UVC light. Hoechst 33258 adsorbs the UVC light protecitng monosubstituted chromatid from UV light degradation. Subsequently DNA is stained with another dye called Giemsa stain. Degraded DNA do not stain with Giemsa dye revealing a differential effect as on picture below. Additionally to UVC light effect it is believed that different set of proteins are bound to BrdU disubstituted chromatid comparing to monosubstituted (what leads to differential staining). 

Picture taken by Kliszczak M.

As you can see on the picture above mono- and disubstituted chromatid are indicated with blue and red arrowheads, respectively. Monosubstituted chromatid appears darker (as a result of Giemsa staining) than disubstituted (pale colour, no staining).

This technique is being used to assay chromosomal stability. Any DNA damage that occurs in the cell and is repaired by recombination (exchange of DNA information between the sister chromatids), will be visualized by this technique. You can see such events on the picture above. Two of them are indicated with black arrowheads. Some gene mutations (for example Rad54 or Rad51 - these genes code for proteins involved in DNA recombination pathways) leads to decreased number of Sister Chromatid Exchanges (SCE). Usually a specific drug (in this case Mitomycin C) is used to induced SCE's. After induction SCE's frequencies between Wild Type and mutant strains are comapred.

I hope you enjoy it.

Maciek GGSTEAM

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.

Maciek GGSTEAM

Meet the chickens - taste of fluorescent microscopy

Welcome again. This time in the Meet the Chickens theme we will look at the DT40 chicken cells under the microscope.

Picture taken by Kliszczak M.(NUIG)

What you can see here is just a bunch of DT40 cells where DNA is stained with DAPI (blue dye). This is a grayscale format and normally under microscope DNA would appear as bright blue. Let's take a closer look to cells indicated with the red frames.
Cell with number 2 is a regular interphase cell (interphase cell is a cell which is not in the division phase). As you see there is a lot of cells looking similar to cell number 2. Mmajor population of the DT40 cells is "sitting" in the interphase (either G1, S or G2 phase of the cell cycle).

Cell number 5 is a cell which just started to condense the DNA. Final stage of the DNA condensation are chromosomes which you could see in the previous post on the Meet the chickens section (see the post rom 16th October). What you can observe here is that shape of the DNA is no longer round like but becomes clumpy and more dense.

Cell number 3 is a cell which is in the metaphase of the mitosis where already condensed chromosomes align in the center of the cell (metaphase plate). In the next step chromosomes will be pulled to opposite poles of the cell. Final stage of that event is being captured in the cells number 4.

Cell number 4, actually cells number 4 are just finising the division. You can see that each cell line got its set of the chromosomes. In the next step chromosomes will decondensed and each of the cells will enter next cell cycle.

If you look at the cell number 1 what you will see is a abnormal mitosis where condensed chromosomes are pulled to three (instead of two) different poles. This is abnormal as the cell before the division duplicates the DNA (cell do not triplicate it) so there is not enough of the genetic material to divide it between three new cells.

Usually this kind of the abnormal cells are eliminated because they simply are not healthy cells (they contain wrong amount of the DNA).

I hope you enjoy it and start to like the chickens.

GGS

Meet the chickens - chook chromosomes at hand

Ladies and Gentelman,

please welcome the chicken DT40 cell line chromosomes.

Picture taken by Kliszczak M.(NUIG)

What you can see here is a methaphase spread where macrochromosomes are visible (chromosome pairs 1, 3, 4, 5, signle sex chromosome Z and trisomic chromosome 2). Rest of the chromosomes appear as a blurry spots.

Metaphase spreads of this kind are investigated in search form any abnormalities like: chromosome breaks, chromosome loss or gain and many more.

I hope you enjoy:)

Till Next Time NUIG:)

GGS