Visualizing the Cell Cycle

You have got to see this video.

Sakaue-Sawano et al. may have created the coolest molecular biology video I have ever seen. They developed a system of reporters to watch the cells transition between the different stages of the cell cycle.

This is cool, but it is going to take a bit of explaining to understand why.

Background

All cells go through a cycle of steps in order to replicate their DNA and divide into two new cells. This cycle is called the cell cycle, and it is characterized by the presence of certain tightly coordinated proteins during each particular stage. I am referring to the cell cycle for mitosis here. Mitosis produces two cells with an equal number of chromosomes. The production of the majority of the cells in your body occurred through mitosis. Another process, called meiosis divides the number of chromosomes in half during cell division; it occurs during the production of germ line cells -- sperm and eggs. (I know that we are dusting off the cobwebby parts of your dull recollection of high school biology, so I am taking this as slow as I can.)

There are four stages to the mitotic cell cycle:

• G1 phase - G1 is a stage of highly variable duration for cells. During G1, the cell performs it's normal functions (provided it is not terminally differentiated -- read: not going to divide again -- in which case we say it is in G0). At the end of G1 stage when the cell is ready to divide, it begins to produce the proteins necessary for the replication of DNA which occurs in S phase.
• S phase - During S phase, the cell replicates it's DNA and chromosomes. At this point, the pairs of replicate chromosomes are still attached to one another and are called sister chromatids. The length of S phase tends not to vary in cells.
• G2 phase - During G2, the cell produces the proteins that are necessary for cell division. Among these proteins are microtubules which are structural proteins used to pull the sister chromatids apart during cell division.
• M phase - During M phase, the cell actually divides. The nuclear membrane dissolves. One of each of the sister chromatids is pulled into each daughter cell. Then the cells actually separate and close off the membrane between them. The nucleus reforms around the chromosomes, and now you have two new cells.

Everyone coming with me so far?

This whole process is very elaborate, and it is regulated at various points called checkpoints. At checkpoints, certain proteins confirm that specific events have taken place. Further, they prevent things which should not happen in a particular stage from happening.

One example of this -- which is critical for this paper -- is the initiation of DNA synthesis. DNA synthesis is not something that you want to do willy-nilly. You only want to replicate each of the chromosomes once during each cell cycle. This is why to begin DNA replication, the existing DNA must go through a process called licensing. In licensing, proteins called the Origin Recognition Complex (ORC) attached to a specific part on each chromosome called the Origin of Replication. The ORC then recruits other proteins called licensing factors to initiate DNA replication. An example of a licensing factor is a protein called Cdt1 -- which I only mention because it figures heavily in this paper.

Here is an important point: the proteins that initiate DNA replication are only present in the cell at the checkpoint between G1 and S phase. This is important because this is the only time you want to replicate the chromosomes.

How are these proteins prevented from accumulating during other periods in the cell cycle? How is DNA replication prevented? Well, there are two mechanisms to prevent unwanted DNA replication. First, the licensing factors are selectively degraded. (How is a subject way too long for me to discuss here.) Second, DNA replication is actively inhibited by a protein called geminin. Geminin inhibits the actions of licensing factors including Cdt1. Importantly, geminin is present in the cell during G2 and M phase.

These two proteins -- Cdt1 and geminin -- are involved in coordinating DNA replication in the cell cycle. They are also oscillate inversely. During G1 and S, Cdt1 is present. During G2 and M, geminin is present.

The authors use these inversely oscillating factors to label cells in different points in the cell cycle.

Sakaue-Sawano et al.

This is where Sakaue-Sawano et al. are particularly clever. In order to observe different parts of the cell cycle, they create specially labeled geminin and Cdt1 constructs and insert them into the cells that they want to observe. The label in this case is a protein that fluoresces a certain color under a light. (I explained how fluorescence works here.) What the construct does is create a protein in the cells that is identical to geminin and Cdt1 except that attached to it is this fluorescent label.

The label for Cdt1 fluoresces red, and the label for geminin fluoresces green. This is illustrated in the figure below (Figure 1F from the paper):

The first row shows cells in culture with the construct for Cdt1 with the red label (mKO2). The second row shows cells in culture with the construct for geminin with the green label (mAG). Note how they are expressed at different points in the cell cycle. (The rows below is a merged image of the two. The row below that is a blue label that is incorporated into replicating DNA. As expected it is present in S phase.)

Which brings us finally to our sweet video. By inserting both constructs into cultures of dividing cells and taking images over time, you can watch them go through the cell cycle.

Crazy right? There are many more pictures and videos, so I strongly suggest that you look at the paper.

What is the significance of this work?

Well, besides being just generally awesome, technical advances of this sort are really helpful to molecular biologists. Say you do something to a culture of cells, and you want to know what effect it had on the cell cycle. It is much easier to add these constructs than many of the other methods for measuring points in the cell cycle.

Furthermore -- and this is just amazing -- the authors create a transgenic mouse that expresses these constructs. That means that every cell in that mouses body is either red or green depending on what point it is in the cell cycle. The applications of this mouse strain are staggering. Say I want to find when neuronal precursors proliferate, for instance. Well, all I need to do is find the green cells.

This is one experiment that they do in a developing mouse as a proof of process, shown in the figure below (Figure 4 in the paper):

This figure depicts sections of a 13 day old mouse embryo that has these constructs. The cells that are undergoing division are green.

Frankly, I don't remember the last time I saw a paper this cool and this useful. Congratulations to Sakaue-Sawano et al. on this tremendous new advance!

SAKAUESAWANO, A. (2008). Visualizing Spatiotemporal Dynamics of Multicellular Cell-Cycle Progression. Cell, 132(3), 487-498. DOI: 10.1016/j.cell.2007.12.033