How do you discover something extraordinarily fundamental that nobody has ever known or seen before? If you have a pretty good idea of what you’re seeking, you might take Walther Flemming’s approach. In Cell Division I: The Cell Cycle, we learned that Flemming observed how chromosomes became visible in patterns that repeated each time the cells of fire salamanders divided. This important discovery was made possible by using various dyes, a technique that Flemming pioneered (Figure 1). This is a good example of how a new instrument or technique can facilitate a discovery, provided that the researcher already knows more or less what he or she might find.
This was the case with Flemming. Scientists in the preceding years had already been seeing faint structures in cells, but their dyes were not good enough to reveal what any of these structures did. Throughout the 19th century, as microscopes developed, scientists had been seeing clues of structures in dividing cells of eukaryotes. Like Flemming, earlier scientists had been experimenting with dyes. These were not as good as the aniline dyes that would facilitate Flemming’s discovery, but they helped the scientists to see something. Unfortunately, the dyes killed the cells, and since the structures under the microscope were difficult to see as it was, Flemming’s forerunners weren’t sure they were seeing anything characteristic of a live, functional cell. Were they simply artifacts, something that formed only after the cells died? If so, that would not explain how a cell replicates in a living organism, or in vivo.
Knowing what he wanted to find, Flemming set out to do a better job of staining the internal details of cells. By doing so, he realized that he could also determine whether the structures were artifacts or part of cellular function. Using the fire salamander embryos, through a long, painstaking process, he cut his samples into very thin slices and treated them with his new dyes. This killed the cells, just as the earlier dyes had killed the cells of other laboratory animals. However, Flemming repeated this technique with many embryos, arresting their life process at different points in time. This protocol was as much a novel technique as his utilization of the aniline dyes. By stopping the life process at different points, he could investigate whether the structures looked any different at Time A compared with Time B or Time C and so forth.
It turned out that they did look different, and this proved that the structures were not artifacts. They were part of the life process of the cells. Coupled with the improving resolution of microscopes of the era, the aniline dyes could make the differing structures clearly visible. This led Flemming to discover the cell process that we call mitosis: division of the eukaryotic cell nucleus that occurs just prior to cytokinesis, which is the division of the cell itself. So revealing were the new dyes and so meticulous was his technique that Flemming was able to define the phases of mitosis that we still talk about today (Figure 2).
The phases of mitosis
Flemming coined the term chromatin to describe the material of which chromosomes are made. When he observed cell division in the fire salamander embryos, he saw the same pattern of events occur in each cell, beginning with the appearance of visible chromosomes. He described the events as four periods of time, which he named prophase, metaphase, anaphase, and telophase. Today, we speak of five phases, since we split up Flemming’s prophase, the longest phase, into prophase and prometaphase.
It’s important to remember that the process of cell division is cyclical, with one phase feeding into the next. For example, telophase overlaps with cytokinesis, the splitting of the rest of the cell that generates the two new daughter cells. Following cytokinesis, the two new cells then go through a long period called interphase, during which each new cell carries out normal life functions and replicates its chromatin, eventually leading to prophase and another cycle of mitosis. Thus, as mitosis begins, the nucleus already contains a double set of chromatin. Since chromatin contains the genes that give organisms their characteristics, this means that a cell entering prophase contains two copies of what is called the genetic sequence, or the genome, of an organism. What happens from this point forward is simply a matter of repackaging and relocating the chromatin.
Prophase is the time when we can first see the chromosomes under an optical microscope. As noted above, the cell’s genetic sequence replicates prior to prophase (during interphase). During interphase, the chromatin is relatively decondensed, bundled loosely, like spaghetti, and dispersed throughout the nucleus. With the onset of prophase, the chromatin folds up into a compact form that, when stained with a dye, can be seen as individual chromosomes, even with the primitive microscopes available in Flemming’s era. Each chromosome consists of a pair of sister chromatids, each containing the same genetic sequence that was duplicated during interphase, and these two chromatids are connected by a structure called a centromere. Also, during prophase, a prominent structure called a nucleolus disappears from the nucleus.
Prometaphase is marked by the breakdown of the membrane that surrounds the cell nucleus. Additionally, pairs of protein complexes called kinetochores bind to the centromere of each chromosome, one kinetochore for each chromatid. These two key events will allow for connections to form between the chromosomes and special structures located just outside of the nucleus.
Metaphase is characterized by a repositioning of the duplicated chromosomes so that they are ready to be pulled apart. During interphase, most animal cells contain a structure called a centrosome, located near the nucleus but outside of its membrane. Like the chromatin, the centrosome also replicates toward the end of interphase, and by the onset of metaphase each of the two daughter centrosomes has migrated to opposite ends of the nuclear membrane. Throughout the cell cycle, the centrosome acts as the control center for microtubules, a complex system of protein fibers that make up the part of the cytoskeleton. Just as bones give shape to your body on a large scale, the cytoskeleton provides each cell with a shape, while also helping to transport materials. With the nuclear membrane now dissolved and the two centrosomes positioned on opposite sides of the cell, the condensed chromosomes line up along an imaginary line in the center of the cell called the metaphase plate. Microtubule fibers then begin to extend from each centrosome toward the centromere that connects the two sister chromatids of each chromosome. This cage-like structure of microtubules is called the mitotic spindle. Specifically, the microtubule fibers attach to the kinetochores; as noted above, there are two kinetochores, one for each chromatid. This provides the setup for the chromatids to be pulled apart during the next phase.
Anaphase is characterized by the separation of the two identical chromatids of each chromosome. With the mitotic spindle complete, the two centrosomes start moving outward, pulling each chromatid away from its sister and toward opposite ends of the cell.
Telophase begins when the two sets of chromatids reach distinct regions of the cell and a new nuclear membrane starts to form around each set. Cytokinesis also begins during telophase, even before the new nuclear membranes are complete. Once formed, however, each new nuclear membrane encloses a full set of chromosomes. These then decondense into the ordinary chromatin of interphase, a nucleolus appears in each newly formed nucleus, and the cell cycle begins anew.
Interphase is not a part of mitosis, but is the cell's state between nuclear divisions when it is preparing for mitosis and cytokinesis. Interphase is discussed in more detail below.
What name did Flemming give to the material that forms chromosomes?
Structure of cell components important to mitosis
Structure of chromatin
Chromatin consists of DNA and special proteins called histones. DNA is a long molecule consisting of two strands of repeating chemical units called nucleotides. There are four types of nucleotides, and the genetic sequence is based on the order in which these four types of nucleotide are connected, one after the other, over the length of the molecule. It’s like a language built of words composed of only four possible letters, but it works well, because the DNA molecule allows each word to be very long (learn more in our series on DNA, specifically DNA II: The Structure of DNA). The density of chromatin changes throughout the cell cycle; this depends on how tightly the DNA strand is wrapped and tethered to histones and other associated proteins (Figure 4).
While chromosomes are a way of organizing the chromatin of eukaryotic organisms into individual packages, the number of chromosomes varies widely among eukaryotes. Humans have 46, cats and other felines have 38, dogs have 78, and wheat has 42, while the Jack Jumper ant has only 2, and a certain kind of protozoan is famous for having nearly 16,000.
It should be emphasized that mitosis occurs only in eukaryotic cells, since only eukaryotes have membrane-bound nuclei. Bacteria and Archaea, the other two domains of life, have chromosomes that are not separated from the rest of the cell; consequently, they can reproduce through a simpler process called binary fission (to learn more, see our module The Discovery and Structure of Cells).
All living organisms have the same number of chromosomes.
Microtubules and centrosomes
Just as DNA is a large molecule constructed of building blocks, microtubules are made of repeating units of protein called tubulin. In addition to playing a structural role akin to the skeleton of your body, large molecules built of tubulin subunits are vital to mitosis and several other dynamic cell functions. They actually move, which is why chromosomes can be pulled apart, and why the entire cell can be made to divide.
All of this takes a great deal of organization, and so eukaryotic cells depend on components known as microtubule organizing centers (MTOCs). In animal cells, the centrosome is one of the main types of MTOC. As we shall see in the next section, two centrosomes are needed during mitosis of an animal cell, each member of the pair using microtubules to pull a set of daughter chromosomes toward one end of the dividing cell. A centrosome consists of two centrioles that are made of tubulin. The two centrioles are arranged at right angles, or orthogonally, and are surrounded by other proteins that make the centrosome more than just a bent section of microtubule (Figure 5).
Interphase: normal life functions and preparation for mitosis
Although not part of mitosis, interphase is important to discuss because it places mitosis into context with respect to the cell cycle. For vertebrates (the subphylum of animals to which humans belong), the duration of the life cycle of each cell varies, depending on the cell type. Certain white blood cells may live and be replaced over a period lasting less than a day. Most other body cell types have life cycles ranging from days to months. Others, such as bone cells, typically are replaced in cycles measured in decades, while certain brain cells and muscle cells will endure for the entire lifespan of the organism. These cells are said to be in a permanent interphase; specifically, they are locked in a phase of interphase known as G1.
For cells that will be moving from interphase into a new round of mitosis, the G1 phase ends at what’s called the restriction point, when the cell commits to replication, and enters the phase of DNA synthesis, or S phase. Throughout G1, sections of the decondensed chromosomes are accessed as needed by enzymes using the DNA sequence to make proteins, but in the S phase the entire collection of genetic material is copied. Thus, by the end of the S phase, each decondensed chromosome exists in duplicate, the two copies destined to become the two sister chromatids when the chromosome condenses at prophase. Generally the S phase leads into a transitional phase known as G2, although the cells of some animal species proceed from the S phase directly into mitosis. During G2, proteins are synthesized that will support mitosis and cytokinesis. Additionally, many cell types undergo a kind of self-testing to make sure that everything is correct before mitosis begins, and certain cancers are thought to result from cells missing the G2 phase and thus avoiding the testing that would prevent mitosis in cases when all is not right. (You can learn about interphase in detail in our Cell Division I: The Cell Cycle module.) A representation of cell cycle phases is shown in Figure 6.
____________ remain in a permanent interphase.
Questions and answers from the periwinkle plant
Painstaking, systematic work like Flemming’s is one way to make a discovery. Indeed, in modern science, it’s the most common way. But it’s not the only way. One major discovery very relevant to mitosis came unexpectedly, and from a surprising source: tea leaves. Not conventional tea, but leaves of the Madagascar Periwinkle, a plant known for its beautiful flowers.
Vinca rosea and diabetes
In many parts of the world, people brew tea from leaves of the Periwinkle (Figure 7), previously called Vinca rosea and now designated as Catharanthus roseus (we'll use the older Vinca name here). This tea is used as a folk remedy for a plethora of ailments, but especially for diabetes when insulin and other conventional treatments are not available. It’s an ancient remedy whose potential in diabetes treatment science has only recently begun to uncover, but it first met the scrutiny of modern research back in the 1950s. Fascinated to hear of the tradition, a Canadian endocrinologist from Toronto, Clark Noble, accepted a sample of 25 Periwinkle leaves from a patient who had acquired them in Jamaica. Although recently retired from endocrinology research, Noble had been a key player in the discovery of insulin 30 years earlier, but the Nobel Prize for this milestone medical advance had eluded him. Others who had worked closely with Noble had received the award, but he was remembered as merely a sideline figure. If diabetics in Jamaica and elsewhere really were benefiting from the Vinca plant, Noble wanted to know how it worked. Lacking a lab of his own, he sent the envelope to the lab of his younger brother, Robert.
Also an endocrinology researcher, Robert Noble jumped at the opportunity to study the leaves. As noted above, insulin treatment had been available for only 30 years at this point. It was obtained from pigs, and supplies were not particularly abundant. Moreover, it didn’t work well for all diabetic patients. Today we know this has to do with the fact that there are two main types of diabetes, both of which manifest as an inability to absorb sugar from the blood into the body’s muscle cells, leading to a range of long-term complications in many body systems. Some diabetics are unable to produce insulin, so taking insulin works very well for them. In others, however, the problem is that their muscle cells do not respond well to insulin. They produce insulin, and yet their blood sugar levels are still high. Insulin may help them a little, but not completely, and for some it does not help at all. Today, we have drugs to make their muscle cells more sensitive to insulin, but the situation was very different back in the 1950s. And thus, Robert Noble happily set out to study the Vinca leaves that his older brother had sent him.
Noble started by formulating questions that could be answered through experiments on laboratory animals such as rabbits and mice. When injected, would an extract of the leaves lower an animal’s blood sugar? Would it prevent the development of diabetic symptoms like excessive urination? Would it prevent the development of blood circulation problems and blindness? Or, injected into an animal that already has full-blown diabetes, would it reverse the condition? Using laboratory animals that have a certain medical condition in order to test an agent that might affect that condition is known as an animal model. In this case, Noble was employing rabbit and mice models of diabetes.
After running a series of experiments, the younger Noble found that the Vinca rosea extract actually had no effect on diabetes whatsoever. In fact, at very high doses, it made the animals really sick. They were dying of infections, because their white blood cell counts were too low. Something from the Vinca leaves was preventing the bone marrow from producing new white blood cells, which form the basis of the immune system.
Vinca rosea and white blood cells
Noble didn’t know why the Vinca extract killed the white blood cells of mice, but he wondered if this property could be useful for people who have too many white blood cells. In other words, he wondered if the Vinca extract could be used to treat leukemia, a type of cancer characterized by excessively high numbers of white blood cells? To find out, Noble joined forces with chemist C.T. Beer to isolate the specific chemical compound from the Vinca extract that caused the effect.
They found the compound that belongs to a class of chemicals known as alkaloids, and they named it vinblastine. Switching from an animal model of diabetes to one of leukemia, Robert Noble began a new series of experiments looking at the effects of vinblastine on leukemia and some other diseases that are caused by uncontrolled replication of cells.
Following success with the animal experiments, vinblastine proved to be very effective in clinical trials of cancer patients in Toronto. Soon, a related compound called vincristine was isolated by another investigator. A whole range of additional Vinca compounds followed, and each proved useful against various types of cancer, though vinblastine and vincristine are the most famous.
How well did they work? To give you an idea, Vinca drugs are still used today, often in combinations with other chemotherapy drugs, and they have led to dramatic increases in cancer survival. Vincristine, for instance, is part of the combination cocktail against the most common childhood leukemia known as acute lymphoblastic leukemia (ALL). In 1950, an ALL diagnosis was a virtual death sentence for a child, with a survival rate of 5 percent. Today, the survival rate of ALL is up to 95 percent. Similarly, Hodgkin disease – a type of cancer of the lymph nodes that often affects young adults – had a pitiful survival rate in the 1950s, but by 1980 the death rate from Hodgkin disease had decreased by 75 percent, thanks in large part to vinblastine, the drug that Noble discovered in the Vinca leaves.
All of this came from two brothers who had not even set out to do cancer research. Unlike Walther Flemming, who had a plan and knew precisely what he was looking for, the discovery of vinblastine is a story of serendipity, or a fortunate accident.
The Vinca extract was effective in treating
Cancer and mitosis
How could a chemical drawn from a plant be so effective against leukemia? What does vinblastine do to the cells of rabbits, mice, and people with cancer? Today, when pathologists look at suspected cancer under a microscope, they pay a lot of attention to mitosis. Each time mitosis occurs, it leads to the parent cell splitting into two new daughter cells. While that formula is always the same, the rate at which mitosis occurs varies substantially. Just like other cells in a body, the life cycle of different cancer cells can vary. Some have a very short life cycle, with mitosis occurring frequently, while in other cancer cells mitosis is infrequent. When cancer is suspected, the pathologist looks at how fast and how often mitosis occurs. Cancer cells that undergo more mitosis tend to be more aggressive than cancer cells in which mitosis is more relaxed. This means that if you slow down mitosis, you might then be able to slow down, or even reverse, the progression of cancer.
It turns out that this is exactly how the Vinca alkaloids work. When Robert Noble gave the Periwinkle tea to laboratory animals, and later when he gave the isolated vinblastine compound to human patients, cell division slowed down in the white blood cells. It was later discovered that the compound interferes with mitosis. In addition, the various Vinca compounds that were eventually discovered each interfere with mitosis at different phases and for different reasons. It turns out that the compounds disrupt the assembly of microtubules – the special fibers that provide structure in the cell. Vinblastine binds to the tubulin subunits, preventing them from coming together.
The questions that Robert Noble and the generations of cancer researchers who stood on his shoulders were inspired to ask ultimately were investigated in a very systematic way. Having an idea of what they were looking for, researchers isolated new drugs and honed in more closely on the workings of the microtubule system. So while it may start with a lucky find, ultimately scientific advancement requires a clear plan, and long lasting, painstaking work.
Beginning with the discovery of mitosis, the module details each phase of this cell process. It provides an overview of the structure of cell components that are critical to mitosis. The module describes Clark Noble’s experiments with the Madagascar Periwinkle, which led to the discovery of an effective cancer treatment drug. The relationship between mitosis and cancer is explored as is the mechanism by which anti-cancer drugs work to slow down or prevent cell division.
The term mitosis refers specifically to the process whereby the nucleus of a eukaryotic cell splits into two identical daughter nuclei prior to cell division.
Mitosis is a cyclical process consisting of five phases that feed into one another: prophase; prometaphase; metaphase; anaphase; telophase.
The rate at which mitosis occurs depends on the cell type. Some cells replicate faster and others slower, and the entire process can be interrupted.
Chromosomes are made of a material called chromatin, which is dispersed throughout the cell nucleus during interphase. During mitosis, however, the chromatin condenses making individual chromosomes visible under an ordinary light microscope.