19.3: Cell Division in Eukaryotes

Depending on cell type, typical eukaryotic cells have a cell cycle of sixteen to twenty-four hours, which is divided into phases. One of these phases, mitosis, was discovered in the late 1800s with light microscopy (you may have seen mitosing onion root-tip cells in a biology class laboratory). In any given cluster of cells, some were seen to lose their nuclei and to form chromosomes (from chroma, colored; soma, bodies). In mitosis, paired chromosomes (chromatids) are attached at their centromeres. The chromatids were seen to separate and to be pulled apart by spindle fibers. Once separated and on their way to opposite poles of dividing cells, they are once again called chromosomes. Thus, homologous chromosomes were equally partioned to the daughter cells at the end of cell division. Because the same chromosomal behavior was observed in mitosis in diverse organisms, chromosomes were soon recognized as the stuff of inheritance, the carrier of genes! The events of mitosis, lasting about an hour, were seen as occurring in four phases over that short time (Figure 19.2).

Figure 19.2: Mitosis and cytokinesis in eukaryotic cells are separated in time, and mitosis is further divisible into five phases.

To the early microscopists, this short period of intense mitotic activity was in stark contrast to a much longer “quiet” time in the life of the cell. For lack of a more descriptive term, they called this period interphase. Also, depending on whom you ask, cytokinesis (the cell movements that divide a cell in two) might not be considered a part of mitosis. In that sense, we can think of three stages in the life of a cell: interphase, mitosis, and cytokinesis. Of course, it turned out that interphase is not cellular “quiet time” at all!

19.3.1 Defining the Phases of the Eukaryotic Cell Cycle

Correlation of the inheritance of specific traits with the inheritance of chromosomes was shown early in the twentieth century, most elegantly in genetic studies of the fruit fly, Drosophila melanogaster. At that time, chromosomes were assumed to contain the genetic material, and it was assumed that both were duplicated during mitosis. The first clue that this was not so came only after the discovery that DNA was in fact the chemical stuff of genes. The experiment outlined in Figure 19.3 distinguished the time of chromosome formation from the time of DNA duplication.

Figure 19.3: This experiment asked how many cells in a random culture are in mitosis and how many were actively replicating their DNA. The results showed that mitosing cells were not synthesizing DNA.

Here are the details of the experiment:

1. Cultured cells were incubated with \({}^3\)H-thymine, the radioactive base that cells will incorporate into deoxythymidine triphosphate (dTTP, one of the four nucleotide precursors to DNA synthesis).
2. After a short period of culture, unincorporated \({}^3\)H-thymine was washed away, and the cells were fixed and spread on a glass slide.
3. Slides were dipped into a light-sensitive emulsion containing chemicals like the lightsensitive chemical coat on the emulsion-side of film. (Do you remember photographic film?)
4. After sufficient time to allow the radioactivity on the slide to “expose” the emulsion, the slides were developed (in much the same way as developing photographic film).
5. The resulting autoradiographs in the microscope revealed images in the form of dark spots created by exposure to hot (i.e., radioactive) DNA.

If DNA replicates in chromosomes undergoing mitosis, then when the developed film is placed back over the slide, any dark spots should lie over the cells in mitosis, and not over cells that are not actively dividing. But observation of the autoradiographs showed that none of the cells in mitosis were radioactively labeled. On the other hand, some of the cells in interphase were! Therefore, DNA synthesis must take place sometime in interphase, before mitosis and cytokinesis, as illustrated in Figure 19.4.

Figure 19.4: The autoradiographic data from the experiment outlined in Figure 19.3 demonstrates that DNA synthesis begins and ends some time before the beginning of mitosis.

340-2 Experiments Revealing Replication in Cell-Cycle Interphase

As we will see, the synthesis of nuclear DNA in eukaryotic cells (replication) occurs many hours before the start of mitosis, and in fact it lasts a good chunk of the time of interphase.

Next a series of pulse-chase experiments were done to determine when in the cell cycle DNA synthesis takes place. Cultured cells given a short pulse (exposure) to \({}^3\)H-thymine and then allowed to grow in a nonradioactive medium for different times (the chase). At the end of each chase time, cells were spread on a glass slide and again prepared for autoradiography. Analysis of the autoradiographs identified distinct periods of activity in interphase: \(\rm G_1\) (Gap 1), S (a time of DNA synthesis), and \(\rm G_2\) (Gap 2). Described here are the details of these very creative experiments, performed before it became possible to synchronize cells in culture so that they would all be growing and dividing at the same time:

1. Cells were exposed to \({}^3\)H-thymine for just five minutes (the pulse) and then centrifuged. The radioactive supernatant was then discarded.
2. The cells were rinsed and spun again to remove as much labeled precursor as possible.
3. The cells were resuspended in fresh medium containing unlabeled (i.e., nonradioactive) thymine and were further incubated for different times (the chase periods).
4. At each chase time, cells were washed free of radioactive precursor, then spread on glass slides.

After dipping the slides in light-sensitive emulsion and exposing and developing the film, the autoradiographs were examined, with the results shown in Figure 19.5.

Figure 19.5: These autoradiographs from a pulse-chase labeling experiment led to the identification of the phases of the eukaryotic cell cycle.

Here is a description of the results:

• After a three-hour (or shorter) chase period, the slides looked just as they would immediately after the pulse: that is, while 7% of the cells were in mitosis, none of those were radioactively labeled. In contrast, many interphase cells showed labeled nuclei.
• After four hours of chase, a few of the 7% of the cells that were in mitosis were labeled, along with others in interphase.
• After a five-hour chase, most cells in mitosis (still about 7% of cells on the slide) were labeled; many fewer cells in interphase were labeled.
• After a twenty-hour chase, none of the 7% of cells that were in mitosis were labeled. Instead, all the labeled cells were in interphase.

CHALLENGE

Here is a question to ponder: in these illustrations, the way the radioactive labeling is depicted is not exactly correct. Can you see the “error” and explain what you might really see?

The graph in Figure 19.6 plots a count of radiolabeled mitotic cells against chase times.

Figure 19.6: A graph plotting the number of radioactive cells in mitosis over the time of chase in the pulse chase experiment described in Figure 19.5 and in the text.

The plot defines the duration of events (phases) of the cell cycle as follows:

1. The first phase (interval #1 on the graph) must be the time between the end of DNA synthesis and the start of mitosis, defined as Gap 2 (\(\rm G_2\)).
2. Cell doubling times are easily measured. Assume that the cells in this experiment doubled every twenty hours. This would be consistent with the time interval of twenty hours between successive peaks in the number of radiolabeled mitotic cells after the pulse (interval #2).
3. Interval #3 is easy enough to define. It is the time when DNA is synthesized, from start to finish; this is the synthesis, or S phase.
4. One period of the cell cycle remains to be defined, but it is not on the graph! That would be the time between the end cell division (i.e., mitosis and cytokinesis) and the beginning of DNA synthesis (replication). That interval can be calculated from the graph as the time of the cell cycle (about twenty hours) minus the sum of the other defined periods of the cycle. This phase is defined as the Gap 1 (\(\rm G_1\)) phase of the cycle.

CHALLENGE

So, what then is interval #4 on the graph? Think about it and try to explain this roughly nine-to-ten-hour period.

Events in each phase of a typical eukaryotic cell cycle are summarized in Figure 19.7.

Figure 19.7: In a typical eukaryotic cell cycle, phases \(\rm G_1\), S, and \(\rm G_2\) follow cytokinesis and \(\rm G_2\) immediately precedes prophase of mitosis. The length of time for each phase differs for different cell types.

During interphase (\(\rm G_1\), S, \(\rm G_2\)) cells grows in size, preparing for the next cell division. As you might guess, \(\rm G_1\) includes synthesis of enzymes and other proteins needed next for replication.

CHALLENGE

Review and name some of these proteins!

DNA replicates in the S phase, along with the synthesis of new histone and other proteins that will be needed to assemble new chromatin. \(\rm G_2\) is the shortest time of interphase and is largely devoted to preparing the cell for the next round of mitosis and cytokinesis. Among the proteins whose synthesis increases in this time are the tubulins and proteins responsible for condensing chromatin into paired chromatids that represent the duplicated chromosomes. Cohesion is a more-recently discovered protein made in the run-up to mitosis. It holds centromeres of chromatids together until they are ready to separate.

341-2 Events in the Phases of the Cell Cycle

Recall that generation times of dividing cells range from sixteen to twenty-four hours. Atypical cells, like newly fertilized eggs, might divide every hour or so! In these cells, events that normally take many hours must be completed in just fractions of an hour.

CHALLENGE

How might a shortened cell cycle look? How would events that normally take a long time occur in a much shorter time?

19.3.3 When Cells Stop Dividing…

Terminally differentiated cells are those that spend the rest of their lives performing a specific function. These cells no longer cycle. Instead, shortly after entering \(\rm G_1\), they are diverted into a phase called \(\rm G_0\), as shown in Figure 19.8.

Figure 19.8: Terminally differentiated cells no longer divide but instead enter the \(\rm G_0\) state. While they can sometimes resume cycling, such cells more typically experience cell death and replacement by stem cells.

Referred to as terminally differentiated, these cells normally never divide again. With a few exceptions (e.g., many neurons), most terminally differentiated cells have a finite lifespan, and must be replaced by stem cells. A well-known example is the erythrocyte, or red blood cell. With a half-life of about sixty days, these cells are regularly replaced by precursor reticulocytes produced in bone marrow.