Cell Division I: _{The cell cycle}

The discovery of cell division

Walther Flemming (Figure 2), a 19^th century professor at the Institute for Anatomy in Kiel, Germany, was the first to document the details of cellular division. The use of microscopes to study biological tissues was an emerging technology in Flemming's day, and he was highly regarded as an innovator in the field.

As a professor at Kiel, Flemming experimented with a technique for using dyes to color the specimens he wanted to examine under a microscope. Microscopes in the 1870s were not equipped with electric light sources as they are today, so dying the specimens allowed him to see them in greater detail. He found aniline dyes particularly useful because different types of tissues absorbed the dyes at varying intensities depending on their chemistry. The effect was that different parts of a cell would absorb more dye, in effect "highlighting" them, as in Figure 3, to reveal structures and processes that were invisible before.

Flemming used these dyes to study cells. In particular, he was interested in the process of cell division. He began a series of live observations under the microscope using dyed samples of animal tissues and found that a particular mass of material inside the nucleus of cells absorbed the dye quite well. He didn't have a name for it at the time, but later came to call the material "chromatin," from chroma, the Greek word for color (Zacharias, 2013). Flemming drew pictures of what he saw under his microscope to illustrate various publications he produced in his research (Figure 4).

Flemming did many of his experiments with tissue samples from Fire salamanders, a common species in Northern European forests, because the chromatin in their nuclei was large in comparison to other available study organisms. After many hours of observation, Flemming began to see a pattern whereby cells would periodically transition from a resting stage to a period of frenzied activity that turned one nucleus into two, and then pulled the entire cell apart creating two identical cells – each with its own complement of chromatin enveloped within its nucleus.

Today we call the process of the nucleus splitting into two nuclei mitosis, and the cell split itself, cytokinesis. The terms came into use years after Flemming's discovery, but he described the process fully in his book Zur Kenntniss der Zelle und ihrer Theilungs-Erscheinungen (To the knowledge of the cell and its phenomena of division) (Flemming, 1878).

Cell life cycles

The alternating patterns of activity and inactivity that Flemming saw in his samples are now commonly referred to as a cell's life cycle, or often just called the cell cycle. Different types of animal cells – like bone, skin, heart, or nerve cells – all have different life cycles. Life cycles vary between types of cells, but all eukaryotic cell cycles can be broken down into four distinct phases: the G₁ phase, when the cell grows in preparation for an eventual split; the S phase, where DNA inside the nucleus makes a complete copy of itself; the G₂ phase, when the cell checks and corrects any errors that may have occurred during DNA duplication; and an M phase (for mitosis), when the cell’s nucleus splits into two identical nuclei, immediately followed by cytokinesis – cell division. The length and frequency of these phases are different for different types of cells.

At this point, it is necessary to point out that, while all living cells are remarkably similar, cell division is one of those areas where eukaryotic cells (plants, animals, fungi, and protists) are very different than bacteria and other prokaryotes. This is because bacteria and other simple cells do not have a nucleus, so the process can be much simpler. In effect, bacteria simply grow and divide continuously with no distinguishable phases between one division and the next. The process by which prokaryotes divide is called binary fission, and the term “mitosis” never applies to them.

Another difference between prokaryotes and eukaryotes is that prokaryotes have one main circular chromosome, while eukaryotes typically have many linear chromosomes. When a prokaryote divides, it must copy its genetic material and separate the two copies between the two new cells that result from the division, just like eukaryotes (Figure 5). However, the process is different. In prokaryotes, the circular chromosome is physically attached to a certain point of the inside of the plasma membrane of the cell. As the cell copies the chromosome in preparation for cell division, it attaches the new copy in a separate place. This way, the two copies of the chromosome are attached away from each other. Then, when the cell splits into two, the bacterium is careful to ensure that each of the two new cells will have one copy of the chromosome.

In the more complex eukaryotic cells, the G₁, S, and G₂ phases are collectively referred to as interphase, as these phases cannot be distinguished by just looking at the cells under the microscope. Even cells that are growing and dividing very quickly in our bodies spend approximately 78% of their lives in interphase. During interphase, eukaryotic cells double in size, synthesize new strands of DNA, and prepare for mitosis and cytokinesis.

Some cells, like human skin cells, will enter the mitotic phase and divide frequently throughout life in order to accommodate changes in size as an organism grows or to generate new cells to repair tissues damaged by illness or injuries. Other cells, like muscle, nerve, and red blood cells, will remain in a permanent G₀ phase without ever re-entering the mitotic phase. Even cells that are busy reproducing constantly throughout their lives spend very little time in the actual mitotic phase (M phase) as compared to the other phases of their life cycle (Alberts, et al., 2002). Figure 6 illustrates how the various phases compare in length.

Experimenting with the cell cycle

So what causes one cell to linger in G₀ instead of launching into the phases of G₁ to S-phase, G₂ and on to mitosis? Arthur Pardee, an American biochemist working at Princeton University, was one of the first to examine that question. He experimented with live cultures of hamster cells to find what he called the "restriction point." Pardee hypothesized that there must be a single decision point in a cell's life cycle where a cell commits to one of two paths: one path that leads toward cell division and another that keeps the cell in a quiescent, or inactive, G₀ state (Figure 7).

Pardee began by restricting the amount of nutrients and hormones available to the experimental cultures to see if he could stop the cells' progress toward cell division. He did this by removing the cell growth signals at different time intervals. After the cycles were stopped, he attempted to restart the cycle by adding back the growth signals. Throughout these experiments, Pardee was careful to time each culture to see how long it took to reenter S-phase and mitosis.

Pardee found that it made no difference at all as to when in the cycle he removed the growth signals. All of the samples took the same amount of time to re-enter mitosis. This result led Pardee to conclude that all of the cells must have ended up at the same point, regardless of where they were in their cycle when he first removed the growth factor.

Pardee called the point where the cells halted the "restriction point," and he hypothesized that it functioned as a “point of no return.” In other words, if growth signals are present, cells will proceed forward, and once they pass the restriction point, they will complete their current cycle – even if you remove the growth signals. Some people still refer to this restriction point found in the G₁ phase of all mammalian cells as the "Pardee point." It is the point in the life cycle at which a cell either commits to a path toward division, or stops proliferation and enters the G₀ phase. Scientists later found another checkpoint at G₂ that halts cell division if DNA was not synthesized properly during S-phase.

Pardee published his results in 1974 (Pardee, 1973). At that same time, scientists at the University of Colorado Medical Center began experimenting with a special line of human cancer cells, called HeLa cells, to see if they could get cells to go backward in the cell cycle or jump from one stage to another out of order. They used HeLa cells because they proliferated quickly and could be kept alive indefinitely in a laboratory setting. In their experiments, the team fused different HeLa cells together that were at different phases of the cell cycle. They wanted to see if they could “trick” a nucleus in one phase of the cell cycle to enter another phase by fusing it with the cytoplasm of a cell in a different phase.

What they found was very interesting. They found that when they fused a G₁ cell together with an S-phase cell, the nucleus of the G₁ cell quickly entered S-phase. They predicted that something in the cytoplasm of the S phase cell caused the G₁ nucleus to begin DNA synthesis and enter S-phase. However, when they fused a G₂ cell with an S-phase cell, the G₂ nucleus would not enter S-phase. Because the G₂ nucleus had already duplicated its DNA, it would not enter another S-phase and re-duplicate its DNA.

Because the nucleus could be tricked into moving forward in the cycle, but not backward, this clever experiment revealed that cells can proceed through the cell cycle in only one direction. In addition, their results confirmed what many scientists had suspected – there are factors in the cytoplasm of cells that control the progression through the phases of the cell cycle (Rao & Johnson, 1970). The hunt was on to find them.

Controlling the cell cycle

Several years after the experiments in Colorado, Tim Hunt, an English biochemist, began to look for the cellular factors that control cell division and other life cycle activities. He found his answers while conducting research as a visiting professor at Woods Hole Oceanographic Institute in Massachusetts.

Hunt began by looking for a protein that might be responsible for triggering the various stages of cell division. He got the idea from research that showed cells would not enter the mitotic phase if treated with drugs that inhibit protein synthesis. This meant that the cells had to make some new proteins in order to begin mitosis. The question became, “What are these mitosis-causing proteins?” Proteins, however, cannot be seen under a microscope in the bustling environment of living cells. So Hunt, like Flemming, had to be an innovator and adapt a tool from biochemistry, called radioactive tagging, for use in his experiments.

Hunt injected radioactive amino acids into sea urchin eggs (Figure 8) to help him “see” proteins in much the same way that Flemming had used his dyes to highlight the chromatin he wanted to see. As eggs used the radioactive amino acids to synthesize new proteins, the newly generated proteins would be tagged with radioactivity and visible when viewed with x-ray imaging devices.

Using the bio-tagging technique, Hunt tracked the new proteins as they developed in the sea urchin eggs over time and found that levels of one protein in particular would rise and fall at regular intervals as the cell entered the mitotic phase. The levels would build dramatically just prior to mitosis and then fall suddenly just prior to cell division. It seems that Hunt had found his mystery protein (Evans, et al., 1983).

Hunt called the protein "cyclin" – one that we now know to be an integral part of the cell cycle control system. Cyclins work in tandem with a family of enzymes called kinases to control the cell cycle. These kinases are found in a cell's cytoplasm; but unlike cyclins, kinases do not build up and disappear over time. The cell cycle kinases exist at relatively constant levels in a dormant state in a cell's cytoplasm until they are activated by cyclins. When activated, these cyclin-dependent kinases, or CDKs, trigger the chain reactions that initiate DNA replication, mitosis, and other events in the life cycle of a cell.

Checkpoints in the cell cycle

Although it is the cyclins and CDKs that manage when eukaryotic cells enter each phase, the system relies on checkpoints like the one discovered by Pardee to ensure that all systems are ready before launching into the most critical phases of the cycle – DNA synthesis, and following that, mitosis. The cell cycle control system keeps the life cycle moving forward in an orderly manner, sort of like the mechanical timer on a washing machine ensures that clothes are washed, rinsed, and spun dry in the correct order. The cell cycle control system, like a washing machine timer, is automatic, unidirectional, and dependent on signal inputs at certain checkpoints to keep the process moving forward (Figure 9).

Tim Hunt, who discovered the cyclins, won the Nobel Prize in medicine 2001, together with Paul Nurse, who discovered the cyclin-dependent kinases (CDKs). They also shared the prize with Leland Hartwell, who pioneered the research into the checkpoints of the cell cycle.

The network of proteins that make up the cell cycle control system manage an extremely complex series of operations that allow the cells in our bodies – and those in all the plants and animals around us – to grow and sustain life. From the careful replication of DNA that becomes the blueprint of life for new daughter cells to the final cleave that pinches one cell into two during cytokinesis, every phase must go off without a hitch – millions and millions of times during the life of an organism. Most the time the process goes smoothly. However, occasionally errors occur or the cell cycle control systems get damaged. When this happens, the result can be disastrous for the cell and can even lead to cancer. In fact, because the main feature of a cancer cell is constant unrestrained growth, cancer is often referred to as a disease of the cell cycle.