8.1: Regulating the Cell Cycle- Checkpoint Control

Learning Goals

• Review the stages of the cell cycle, including the checkpoints, and identify the key features of each stage.
• Describe how specific protein modifications (e.g., phosphorylation and ubiquitination) result in activation/deactivation of cyclin-CDK complexes to regulate cell cycle checkpoints.
• Explain how the activation of the cyclin-CDK complexes results in the start of the next phase of the cell cycle. Use examples from both M- and S-cyclin-CDK complexes to explain this.
• Detail how fluorescence-activated cell sorting (FACS) can be used to identify the stage of the cell cycle for a population of cells.

Introduction to the Cell Cycle and Checkpoints

A discussion of the cell cycle and mitosis is a very good way to end this book, as it is a wonderful example of how the concepts we’ve covered in this book are interconnected. The progression of the cell from interphase to cell division is precisely regulated, and it involves every other cellular component in some way.

The cell cycle is defined as the events that enable cells to proceed from one cell division event to the next. Cell division itself consists of the overlapping processes of mitosis (nuclear division) and cytokinesis (division of the cytoplasm).

The cell cycle is divided up into four separate phases based on the primary event that is taking place in that stage:

• G₁ (gap or growth 1) phase: This is the “gap” between the end of cytokinesis and the start of DNA synthesis. A lot of the work of this phase involves cell growth so that it can support itself and also have the resources it needs for the next phase.
• S (synthesis) phase: This phase is defined by the initiation and termination of DNA synthesis.
• G₂ (gap or growth 2) phase: This second “gap” phase lasts from the end of DNA synthesis to the onset of mitosis. The cell continues to grow but also prepares for what’s to come in the next phase.
• M (mitosis) phase: This is the phase in which cell division occurs.

Figure 08-01 shows an overview of the stages of the cell cycle. Collectively, we consider G₁, S, and G₂ to be interphase (i.e., the phases “in between” M phase).

Figure 08-01: The four stages of the cell cycle. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

It is important to keep in mind that while cells do need a mechanism to control growth, that is not always their primary concern. There must also be provision for cells to “step away” from the cell cycle for a time and into other developmental pathways (mating, meiosis, differentiation). Some cell types will hit a point in their development where division isn’t a good option anymore for one reason or another, so they withdraw from the continuous cycle of growth and replication. Whether the cell leaves the cell cycle on a temporary or permanent basis is also highly controlled, and we will discuss how the cell makes these kinds of decisions later in this topic. (Hint: Signaling proteins are involved.)

Signaling and regulation are at the heart of the proper progression of the cell cycle. Using these tools, the cell ensures that

• discrete events, such as DNA synthesis and mitosis, do not occur before the cell is ready, and they occur in the right order
• the cell pauses DNA replication or mitosis when errors are identified and repairs are attempted
• only cells that should divide are allowed to do so
  • Some cells must undergo terminal differentiation, stall temporarily or permanently, or even go through programmed cell death (i.e., apoptosis). These are all cell cycle decisions.
• growth is coordinated with cell division so that the size of cells is maintained over the many cellular generations
  • Some cells are designed to get bigger or smaller over several cell cycles. Some even divide asymmetrically so that one large and one small daughter cell is produced. All of these will require precise cell cycle control.

One of the most important controls placed on the progression of the cell cycle is a series of checkpoints in which the cell is required to meet certain criteria before it is allowed to proceed. In this way, these checkpoints act as a form of quality control. The control of the checkpoints and the mechanism of when/how cell division takes place is a perfect example of how the cell uses signaling to understand its environment and to effect internal change. Cells do not pass through these checkpoints randomly. They are constantly receiving cues from the exterior (such as growth factors, for example) and from the interior that help them decide exactly when and how to divide.

Each of these checkpoints is controlled by one or more “gatekeeper proteins” that respond to cellular conditions and will only allow the cell to move forward into the next phase of the cell cycle if conditions are “right.” As a result, the checkpoints help ensure that specific criteria are met before the cell cycle is allowed to continue. There are a few different checkpoints, but the ones we are going to focus on are the following: the G₁/S checkpoint, which allows the cell to pass into S phase, and the G₂/M checkpoint, which controls when the cell enters mitosis. We will also look at the checkpoint that is in the middle of mitosis, which ensures that the mitotic spindle is set up correctly prior to chromosome separation. We call this the metaphase checkpoint, but we have seen many other names used as well (spindle assembly checkpoint, M phase checkpoint, M/G₁ checkpoint, etc.).

Cells can only move through the checkpoint and into the next stage of the cell cycle when they have met the required conditions. For example, if biotin, a vitamin, is missing from the growth medium, yeast cells will not pass the G₁/S checkpoint even if all other conditions are perfect. Low nutrient levels reduce the growth rate, which, if severe enough, can make it so a newly divided cell will not survive. Thus, having the ability to confirm that everything is in place before dividing is key to survival for the cell.

G₁/S Checkpoint

The first checkpoint a new cell will encounter is the G₁/S checkpoint (also sometimes known as the “restriction” point or “Start”). As its name implies, this checkpoint marks the transition from G₁ to S phase. Since S phase involves the replication of DNA, it is important that the cell and the environment are both ready before replication starts. Not only is this an energy- and nutrient-intensive process, but replication is when any preexisting errors in DNA become permanent mutations, so the DNA must be in good shape before the cell starts this process. Once again, making an error with the timing of replication could result in the death of the cell, so the checkpoint plays a key role here.

Cell signaling is important for ensuring that conditions are ideal for cell division. The cell responds to internal and external cues in order to “decide” when to divide. Some of the conditions that must be met include the following:

• Proper nutrients (carbon source, energy source, inorganic phosphate, nitrogen, vitamins, etc.) must be present at specific concentrations.
• Sister chromatid separation (from the previous mitosis) must be complete.
• There must be no detectable DNA damage.
• The cell must have reached a critical threshold size.

Additionally, external factors must also be appropriate. For example, in yeast, if the appropriate mating factor is present in the environment, the cells cannot proceed to S phase and are switched instead into an alternative pathway (called the sexual pathway). Similarly, in mammalian cells, appropriate growth factors must be present to allow cells to pass this checkpoint. If not, the cell remains in G₁.

In some cases, cells are stalled for extended periods of time…maybe indefinitely. We say that these cells have removed themselves from the cell cycle and that they are in G₀ phase. This could be due to a long-term deprivation of nutrients or other resources required for cell division. More commonly, however, this is a normal part of the development of certain cell types. For example,

• Stem cells for specific tissues will enter G₀ for short periods of time until replacement cells are required. This allows tissues to grow to a certain size and then stop growing and maintain a relatively stable size and distribution. Your blood cell system (called the hematopoietic system) does this. New blood cells are grown only when specific cell types are needed or cells are lost through injury.
• Some cell types undergo terminal differentiation, which means that when these cells reach maturity, they do not need to go through mitosis anymore. Good examples of this are muscle cells (multiple muscle cells fuse together at maturity to produce multinucleate muscle fibers), neurons (with their extremely long axons), and osteocytes (bone cells, which are intricately embedded in the calcified matrix of the bone).

G₂/M Checkpoint

The second is the G₂/M checkpoint, which stops the cell from entering mitosis before its ready. It is also sometimes called “CD” or “Commitment to Division,” as the cell cannot stop the process of mitosis once it has passed this point.

As its name suggests, this checkpoint controls the transition from G₂ to M phase. Some conditions that must be met here are the following:

• DNA replication must be complete and accurate. No DNA damage can be detected (through a robust biochemical surveillance system) or this checkpoint cannot be passed. This is the most important factor for passing the G₂/M checkpoint.
• The cell must also have reached a certain minimum size so that it is big enough that, when split in two, the two daughter cells will also be large enough to survive.

It is considered exceedingly rare that cells would stall and enter G₀ from G₂. One would think that there is not really much point in doing all of the work of replication unless there is an intention for the cell to complete mitosis. However, since this is biology, there are examples of chromosomes called polytene chromosomes that can have thousands of sister chromatids, instead of simply two. These cells undergo repeated rounds of replication without moving forward to mitosis. It is thought to help increase the number of copies of genes in the cell, which can significantly impact gene expression. While this is thought to be relatively common in some cell types, such as the salivary glands of certain insects (like flies), and examples can be found throughout eukaryotes, it is still considered to be a relatively rare occurrence overall.

Metaphase Checkpoint

This checkpoint marks the halfway point of mitosis, but it’s also the point right before the actual division of the genetic material, so it makes sense that this would be a point that the cell would verify before proceeding. To separate the sister chromatids of the chromosome, the mitotic spindle, composed of microtubules and associated motor proteins, must be assembled, and the chromosomes must be properly attached. If the spindle is not assembled correctly, an entire chromosome could get destroyed or mislocalized. Considering how important the genetic material is to the cell, you can imagine how bad this kind of “mitotic misfire” would be; both daughter cells would likely die, if they could even complete mitosis. Interestingly, you can see this checkpoint if you watch closely as cells divide under the microscope (Video 08-01). The chromosomes line up at the metaphase plate and then wait at the checkpoint for a little while until suddenly the sister chromatids split and move to opposite ends of the spindle.

Video 08-01: Several examples of mitosis observed with live fluorescence imaging. In all cases, DNA is labeled in red and often looks like small worms in the center. Microtubules are labeled in green and look like long filaments. These videos are sped up and do not show mitosis in real time.

Cell Cycle Checkpoint Control: Cyclins and CDKs

Like all things in cell biology, there was a time when the details of the cell cycle were not known to scientists. Mitosis was observed extremely early (as early as the 1600s), as even the most rudimentary light microscopes could be used to observe it. The fact that cells arose from other cells was identified in the 1800s, but it wasn’t until the 1950s that the rest of the cell cycle was suggested. By the late 1960s, scientists believed that “something” in the cytoplasm was controlling the cell cycle, but they had no proof. Dr. Yoshio Masui, a Japanese Canadian researcher at the University of Toronto, ran some experiments while working as a postdoctoral fellow at Yale University that provided the evidence needed. In these experiments, Dr. Masui extracted cytosol from frog’s eggs that were in mitosis and then injected it into a cell that was stalled at the end of interphase. (Aside: This is a normal part of frog oocyte maturation.) As a control, they compared this with oocytes that were injected with cytosol from another oocyte also in interphase. They found that when the cytosol from a mitotic oocyte was injected into the interphase oocyte, a mitotic spindle would begin to form. This did not happen in the control, in which interphase cytosol was injected into an interphase oocyte.

The results of these experiments provided evidence of the following:

1. The active agent that promotes the next stage of the cell cycle is in the cytosol.
2. Control of DNA synthesis and mitosis is positive—that is, the active agent promotes mitosis in the recipient cell.
3. Cells can be advanced into the next stage before they planned it by adding the appropriate factors to their cytosol.

After this initial discovery, it was replicated using cytosol from mitotic cells from many different species and by taking cytosol from one species and using it to induce mitosis in another species. This meant that the “factor” in the cytosol was universal and helped promote the “maturation” of the cell. As a result, they called this molecule the maturation promoting factor (MPF). It was called a factor, and not a protein, because at this point, no one knew what it was. About 20 more years of research were required to figure out that the “factor” in question was actually a protein.

MPF = Activated Cyclin-CDK Complex

We now know that MPF is actually a set of proteins that work together to control the checkpoints and thus the entire cell cycle. The active agent is a protein called cyclin-dependent kinase (CDK). As you remember from Chapter 7, a kinase is an enzyme that specifically adds phosphate groups to other proteins. This particular kinase is only active in the presence of a second protein known as cyclin. Cyclin binds to CDK (Figure 08-02), and acts as a regulatory unit; CDK can only perform its function as a kinase when cyclin is bound. If cyclin is removed from CDK, then CDK is inactivated. This is also the source of its name…cyclin-dependent kinase. These two proteins combine to produce a single enzyme called the cyclin-CDK complex. The cyclin-CDK system is somewhat unique due to the use of cyclins to regulate CDK activity. The cell controls the activity of CDKs by controlling the synthesis and destruction of cyclins.

Figure 08-02: Cyclin and cyclin-dependent kinase (CDK) work together to control the cell cycle checkpoints. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

CDK concentrations in the cell remain constant throughout the cell cycle, but they are not always active. On the other hand, cyclin concentrations show a cyclical pattern. They increase as the cell moves through the cell cycle, which coincides with increasing enzymatic activity of CDK, peaking at the appropriate point in the cell cycle (usually a checkpoint). After the relevant checkpoint is passed, cyclin concentrations crash down to almost nothing. Once again, this change in cyclin concentration coincides with the end of the CDK enzymatic activity. It is this “cycling” of the cyclin concentrations that regulates CDK activity, allowing the cell to progress through checkpoints of the cell cycle.

Since the initial studies last century, we have learned that there are actually several classes of cyclins and CDKs, and each of these classes is responsible for controlling a specific part of the cell cycle. The four major classes of cyclins are listed in Table 08-01.

Table 08-01: The different classes of mitotic cyclins

Cyclin class	Function
G1 cyclins	Unique cyclins that are thought to help the cell respond to external signals to leave G0 and initiate cell division. We won’t discuss these any further in this book.
G1/S cyclins	Cyclins that control the G1/S checkpoint and control the transition from G1 to S phase.
S cyclins	Cyclins that activate at the start of S phase (by the G1/S cyclins) and directly induce replication of DNA. The concentration of these cyclins remains high right through to M phase.
M cyclins	Cyclins that control the G2/M checkpoint. They remain active in the first half of mitosis until their destruction is signaled by the anaphase-promoting complex (APC).

Increasing cyclin concentrations occur directly prior to a cell passing a cell cycle checkpoint. This is because the cyclin-CDK activity must hit a certain level before the checkpoint can be passed, and cyclins are required components of CDK activity. In some cases, such as the M cyclin shown in Figure 08-03, the activity of the CDK decreases rapidly not long after the checkpoint has been passed, but in others, like the G₁-cyclin shown here, the activity of the CDK is activated at one checkpoint and then stays high throughout the rest of the cycle.

Figure 08-03: The concentration curves of the different cyclins. The three checkpoints are indicated by the vertical dashed lines. “Control of the cell cycle: Figure 2” by OpenStax College, Biology, shared under a CC BY 3.0 license, was modified by WikiMaMa.

Phosphorylation Controls Cyclin-CDK Enzymatic Activity

The activity of the CDKs must be very tightly controlled. The consequences of a cell moving to the next stage of the cell cycle before its ready could be disastrous. As such, cyclin-CDKs are at the heart of a complex signaling pathway involving potentially hundreds of enzymes that are fighting with each other to either activate or deactivate the cyclin-CDK complex. Figure 08-04 shows an extremely simplified version of a regulatory pathway that includes two different cyclin-CDKs (highlighted by yellow arrows). In particular, take note of how the pathways labeled cell growth (labeled as cell proliferation in the figure) and programmed cell death (also known as apoptosis) are connected to each other. This gives a strong hint about how important it is to get this right. If it goes wrong, the cell has the option to initiate apoptosis and die if necessary.

Figure 08-04: CDK2 (and its cyclin, CyclE) and CDK4 (+CyclD) within the signaling cascade that controls growth proliferation and/or apoptosis. “Signal Transduction pathways” by BohHog2 modified by Dr. Robin Young. This image is in the public domain.

Because CDK activation must be tightly controlled, there are multiple layers of regulation. Note that while the binding of cyclin to CDK is necessary for CDK activity, it is not sufficient for activation of the CDK on its own. (Remember our discussion of necessary and sufficient from Chapter 3.) This means that other factors are also needed to activate the CDK complex. In this case, the CDK-cyclin complex itself must also be phosphorylated (by other kinases). Interestingly, phosphorylation of CDK can also be used to keep the CDK inactive as well, depending on the location of the phosphate addition on the protein.

The activation of CDKs follows a set pattern, which requires both the presence of cyclin and proper phosphorylation of the CDK. We’ll use the activation of the M-CDK/cyclin complex as an example, which is illustrated by Figure 08-05 and Video 08-02. In a nutshell, the binding of cyclin initiates the activation process. The signaling proteins upstream activate two kinases in particular: Wee1 and CAK. Both phosphorylate the CDK, but for different purposes. The phosphate group that Wee1 adds is considered to be an inhibitory phosphate, which limits the activity of the CDK. On the other hand, CAK (which stands for cyclin-activating kinase) adds a phosphate that is necessary for the CDK to become active. Once everything else is ready and it’s time for the checkpoint to be passed, a third protein called cdc25 comes in and removes the inhibitory phosphate (thus cdc25 is a phosphatase), and the CDK becomes fully active.

Figure 08-05: The activation of the cyclin-CDK complex by both the addition of phosphate at an activating site and the loss of a phosphate at an inhibitory site. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

You can think about this a little bit like a race car at the starting line of a race. By the time you get to the starting line, the car is all gassed up (cyclin) and running (activating phosphate). The drivers might even be revving the engines a bit, but with the break on (inhibitory phosphate) so that the car can’t go anywhere until the signal is received that the race has started. By being ready to go before the race starts, they can leave the starting line as quickly as possible once the signal to start is received.

Video 08-02: The molecular mechanisms required to initiate M phase.

In yeast, Wee1 and Cdc25 are key regulators of M-CDK. The antagonistic (i.e., opposing) relationship of Wee1 and Cdc25 was discovered using genetic experiments in which the “dosage” of the genes was experimentally manipulated. This simply means that the amount of protein present in the cell was increased or decreased depending on the mutation. Increasing the gene dosage (i.e., a gain-of-function mutation) increases the concentration of enzyme in the cell, whereas decreasing gene dosage (i.e., a loss-of-function mutation) leads to a decrease in enzyme concentration. The effects of these mutations on cell division are summarized in Table 08-02.

Table 08-02: The effects of mutations in WEE1 and CDC25 on yeast cell growth and division

The Effects of Mutations in WEE1 and CDC25 on Yeast Cell Growth and Division
Gene	Gain-of-Function Mutation^*	Loss-of-Function Mutation^**
WEE1—inhibitory kinase	Cells divide later at a larger-than-normal size	Cells divide early at a smaller-than-normal size (i.e., they’re “wee”!)
CDC25—activating phosphatase	Cells divide early at a smaller-than-normal size	Cells divide later at a larger-than-normal size
*i.e., too much protein produced or produced all of the time without regulation **i.e., no protein produced

Specific Cyclins and Their Role in the Cell Cycle

As mentioned earlier in this topic, we are focusing on three specific checkpoints in the cell cycle. Of those, two are controlled directly by cyclin-CDKs: the G₁/S checkpoint and the G₂/M checkpoint. Each of these checkpoints is controlled by its own cyclin-CDK complexes. Cyclins and CDKs involved in the start of mitosis are called M cyclins. The progression into S phase is more complicated, and multiple cyclin/CDK complexes are needed (see Figure 08-03). Interestingly, the metaphase checkpoint is indirectly controlled by cyclin/CDK complexes. The M cyclin-CDK complex also activates the process by which the metaphase checkpoint is set up and then passed. We’ll look at how the different checkpoints work here.

Control of the G₁/S Checkpoint and S Phase Progression

The transition from G₁ to S phase is controlled by multiple cyclin-CDK complexes, including a G₁ cyclin-, an S-cyclin-, and a G₁/S cyclin-CDK complex (Figure 08-03). The G₁/S cyclin-CDK complex will be de-activated once the checkpoint is passed, but the others will continue to function. Once the G₁/S checkpoint is passed, DNA replication will begin.

One of the most important criteria for the passing of the G₁/S checkpoint is that there can be no detectable DNA damage. The protein that manages checking for DNA damage is a transcription regulator called p53. p53 is sometimes called “the guardian of the genome” because of the vital role it plays in ensuring that DNA remains intact and undamaged. It also functions in a way that is somewhat counterintuitive—in its inactive state, p53 is constantly translated and then immediately degraded (Figure 08-06). When DNA damage is detected, p53 gets phosphorylated, which stops it from being degraded. It then binds to the promoter sequence for a CDK inhibitor called p21, thereby activating its transcription and subsequent translation. p21 blocks the activity of the G₁/S cyclin-CDK complex, which will stop the cell from passing the G₁/S checkpoint.

Figure 08-06: The protein p53 inhibits entry into S phase when DNA damage is detected. Presence of p53 upregulates the production of p21, an inhibitor of the G₁/S cyclin-CDK complex. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

It is also interesting to note that p53 is mutated in as many as 50% of all cancers, which is a clear indication of just how important this protein is in the proper function of the cell cycle. If you lose p53 function in a cell, you lose your ability to delay the cell cycle so that there’s time to repair DNA damage. At that point, damage is less likely to get fixed before replication, and mutations accumulate at a much faster rate, with each new round of replication.

Once the G₁/S checkpoint has been passed successfully, the active cyclin-CDK complexes help initiate S phase via phosphorylation of the replication machinery, which, in turn, helps it assemble on the DNA. DNA helicase—responsible for DNA unwinding—is also activated via phosphorylation, allowing replication to begin.

While there is much focus on how S phase is initiated, it is equally important to consider how it is terminated. DNA replication machinery must be deactivated at the end of replication as well. This ensures that the entire genome is replicated once, and only once, during S phase. Again, the cyclin-CDKs control this by phosphorylating key enzymes, which will lead to the eventual shutdown of replication.

Prior to the start of S phase, each chromosome in the genome was made of a single helix of DNA that is in a complex with histones to form a chromatin fiber. (Review Chapter 3 if needed.) It has its own telomeres at each end of the chromosome and a centromere near the middle of the DNA. During DNA replication, the DNA double helix is duplicated, and the chromosome now includes two sister chromatids. This requires that a whole new set of histones is synthesized and imported into the nucleus to form the new chromatin fiber. Also, the two sister chromatids must be held together until the point at which they separate, during mitosis. This is done using a protein we’ve seen before (in Chapter 3) known as cohesin (Figure 08-07). Cohesin attaches the two chromatids together along their entire length at specific sites on the DNA called cohesin attachment regions (or CARs).

Figure 08-07: Cohesins holding the two sister chromatids together by binding the DNA (light blue) from two adjoining sister chromatids. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

One interesting final point: S-CDK has been shown to remain active right until the start of mitosis (Figure 08-03) despite the fact that S phase is over long before that. The reasons for this are not entirely clear, but there is some evidence that S-cyclin-CDK helps with the activation of M-CDKs. This provides further continuity in the cell cycle and shows that the different cyclins are influenced by each other.

M-CDK Controls the G₂/M Checkpoint and Reentry into G₁

The transition from G₂ phase to M phase is complex. It requires an almost complete rearrangement of the cytoplasm, including shutting down all transcription and translation, preparing all of the organelles for separation (which often means deconstructing them), building a second microtubule organizing center (MTOC) for the mitotic spindle, and completely rearranging the cytoskeleton to allow for division. M-CDK-cyclin becomes enzymatically active at the end of the G₂ phase and is the primary control for this transition. M cyclin concentrations peak in metaphase, before crashing, which deactivates the M-CDK and marks the beginning of the transition back to G₁.

As a kinase, the role of M-CDK is to phosphorylate other proteins, which, in turn, will activate or deactivate them depending on the protein. Some of the targets of the activated M cyclin-CDK complex include the following:

• Histone H1—Phosphorylating H1 leads to changes in chromatin configuration and, in conjunction with other proteins, leads to the tighter packing of chromatin required for mitosis.
• Condensins—These are a class of DNA-binding proteins that bind to chromatin to help with higher-order chromosome condensation. They work to loop up the chromatin fiber into the tightly coiled mitotic chromosome.
• Nuclear lamins—Phosphorylated lamins have a lower affinity for each other, and as such, the nuclear lamina falls apart. Disassembly of nuclear lamina results in the breakup of the nuclear envelope. (We explored this in detail in Chapter 3.)
• Structural proteins of the nucleolus—Since DNA from multiple chromosomes is used to form the nucleolus, it must be taken apart prior to mitosis. Phosphorylation of the structural proteins results in dispersion of the nucleolar proteins and disintegration of the nucleolus.
• A variety of protein kinases that regulate the cytoskeleton—In order for mitosis to happen, the cytoskeleton needs to be completely taken apart and rebuilt. A number of proteins are involved in this, including microtubule-associated proteins (MAPs) and even some actin-binding proteins (ABPs), and must be activated directly or indirectly by the M-CDK.
• cdc25, the M-CDK-activating phosphatase—This creates a positive feedback loop, resulting in further activation of M-CDK-cyclin. As a result, M-CDK activity rises increasingly rapidly as more M-CDK becomes active (Figure 08-08).
• Anaphase-promoting complex (APC)—This protein is key to passing the metaphase checkpoint. At the beginning of anaphase, APC degrades the cohesin proteins that bind sister chromatids together, releasing the daughter chromosomes. Interestingly, APC also activates enzymes that tag M cyclin for degradation. This ensures that M-CDK will be properly deactivated when its task is done. The cell cannot complete mitosis and return the cytoplasm to its interphase state unless M-CDK is inactive.

We will be breaking down the events of mitosis and how CDKs and other proteins drive that process in the next topic of this chapter.

Figure 08-08: Positive feedback loop of Cdc25. Active M-CDK helps activate Cdc25, and this activation helps activate more M-CDK. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

The Metaphase Checkpoint Ensures Proper Mitotic Spindle Formation

Unlike the other two checkpoints, this checkpoint is not directly controlled by a cyclin-CDK complex. However, that doesn’t mean that the CDKs and cyclins have no role to play. This checkpoint is in the middle of M phase and ensures that the mitotic spindle is formed properly before mitosis is allowed to proceed. The M cyclins and their associated CDKs are involved in this checkpoint, as they activate the proteins required to create the mitotic spindle. Passing the metaphase checkpoint not only allows mitosis to proceed but also sets in motion a series of events that will eventually shut down M phase, which we will talk about in the next section.

Proper spindle assembly is essential to the metaphase checkpoint, so it stands to reason that the proteins involved in regulating this checkpoint would be interacting directly with the spindle itself. At some point during G₂ phase, a large protein complex known as a kinetochore assembles at the centromere of each sister chromatid in the chromosome. This complex will help capture microtubules during the assembly of the mitotic spindle so that the sister chromatids can be separated. Several proteins are also assembled at the kinetochore, the most important of which is one called anaphase-promoting complex, or APC, and they will remain there until microtubules are properly attached to the kinetochores of both sister chromatids. Once that happens, the checkpoint proteins assembled at the kinetochore, including APC, are released and activated. Once that happens, the cohesins holding the sister chromatids together are degraded and anaphase begins.

This is not the end of APC’s role in mitosis, however.

Deactivation of the Cyclin-CDK Complex

Just like in S phase, the cell must eventually end M phase, put everything back where it was, and allow the new daughter cells to reenter G₁. To do this, the M cyclin-CDK must be deactivated so that it stops phosphorylating its target proteins and the cell can exit M phase. APC is directly involved in the degradation of M cyclins, which deactivates the M-CDK.

Incidentally, that means that M cyclin-CDK not only creates a positive feedback loop to activate itself, through cdc25 (Figure 08-08), but it also creates a negative feedback loop, using APC, which will result in its eventual deactivation. This is a fascinating case study on the complexity of cell signaling and how it regulates cellular function.

Like most signaling events, the cyclin-CDK complex must be deactivated once its job is complete. This is done by a combination of

1. shutting down the transcription and translation of new cyclin and
2. degrading the cyclin proteins that already exist.

Negative feedback loops are often built into signaling pathways, and CDKs are no different. The mechanism is illustrated in Figure 08-09. APC is phosphorylated by M-CDK so that it is active and controls the metaphase checkpoint. Unlike many of the regulatory proteins we’ve seen, APC is not a kinase or a phosphatase. APC is a protein that transfers a small protein tag called ubiquitin to other proteins. (Review Chapter 4 if needed.) APC tags the cyclin with ubiquitin, which results in it being sent to the proteasome for degradation. Once the cyclin is degraded, the CDK is no longer active, and all of the phosphorylation targets of the CDK can then be dephosphorylated and returned to their original state. The result is that the concentration of cyclin in the cell drops rapidly, and the CDK is also deactivated, and the cell can start the process of putting the cytosolic contents back together again in the new daughter cells, complete cytokinesis, and return to G₁. For its part, APC is deactivated in G₁. It is one of the phosphorylation targets of the activated G₁/S-cyclin-CDK complex.

Figure 08-09: Ubiquitination and degradation of cyclin, which deactivates CDK. The anaphase-promoting complex (APC) is activated by M-CDK. Activated APC adds ubiquitin to target proteins including m-cyclin. Ubiquitination targets proteins for degradation in the proteasome. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

Studying Cells: Experimental Techniques to Identify the Cell Cycle

Initially, we knew two things about the cell cycle: there was mitosis, and there was the time in between mitosis (i.e., interphase). We could observe this in the most rudimentary light microscopes roughly 200 years before we knew that DNA was the molecule that stored genetic information. As we now know, there are four major stages to the life cycle of a cell, and only one of them is mitosis. When researchers study the cell cycle, it is important that they can differentiate among the four stages. DNA content is a useful cue, since the amount of DNA changes in predictable ways throughout the cycle. In addition, sometimes researchers will need to work with a population of cells that are all in the same stage of development. In this section, we look at two ways to identify, and possibly synchronize, a population of cells based on cell cycle.

We will explore two different techniques and discuss each in turn:

1. Chemically synchronizing cells in a population: This technique is the starting point for many experiments to ensure all cells are starting at the same stage in the cell cycle.
2. Fluorescence-activated cell sorting (FACS): This is a subtype of a more commonly known procedure called flow cytometry, where cells are monitored for certain properties and grouped based on these properties.

Chemically Synchronizing Cells in a Population

When studying cells as they progress through the cell cycle, it’s common to want to take measurements based on a particular parameter. We can watch cells go through mitosis using a simple light microscope, but some of the other phases are more difficult to detect. As such, we may want to work with a population of cells that we know are all at the same phase. However, that generally does not happen naturally in a population of cells. Just like your classmates in elementary and high school hit their growth spurts at slightly different times even though you were all roughly the same age, cells will not naturally align their cell cycles.

In order to synchronize a population of cells, the scientist must apply some kind of block that stalls the cell cycle at a known step in the cell cycle. The two most common blocks are based on biological concepts we’ve seen before in this book.

• In Chapter 4, we discovered the existence of temperature-sensitive mutations, and how they could be used to study the essential proteins of the secretory pathway. Most cell cycle proteins are equally essential, as a cell that cannot progress through its cell cycle cannot divide. So temperature-sensitive mutants are useful in this context as well.
• In Chapter 6, we also saw that chemical inhibitors could be used to disrupt cytoskeletal function temporarily. This, too, is a strategy that can be applied to the cell cycle. There are a few known chemical inhibitors that block further progression in the cell cycle. DNA replication is often targeted by these compounds, and the cell stalls at the start of S phase as a result of the chemical inhibition.

When these blocks are applied, the cells will continue to progress through the cell cycle until they hit the blockage, and then they can go no farther. You can think of this like construction on a major bridge or roadway. The cars are able to move freely elsewhere as they work toward their destination, but once they arrive at the construction, they stop and stay there until the construction block is lifted so that the stopped traffic can again begin to move forward.

Fluorescence-Activated Cell Sorting (FACS)

This final technique is, in some ways, explained by its name. When we do FACS, we fluorescently label cells and then use that fluorescence to differentiate between cells that are in different states (Figure 08-10). The machine (called a flow cytometer) that measures the fluorescence is also able to separate a mixed population of cells based on this fluorescence (i.e., the cells are “sorted” into different groups based on the measured fluorescence).

Figure 08-10: A schematic representation of fluorescence-activated cell sorting (FACS). The cells (blue circles) are first labeled with fluorescent dye that binds quantitatively to DNA. The cells are sent through the machine, and a laser detects the fluorescence and redirects the cells into different test tubes based on the fluorescence intensity. The machine also produces a readout of the cells that were measured and their fluorescence intensity. This image was created by Dr. Robin Young and is shared under a CC BY-SA 4.0 license.

When using FACS to learn about the stage of the cell cycle that each cell in a population is in, a live fluorescent DNA stain like DAPI is commonly used. Since the amount of DNA changes as the cell cycle progresses, this is a very simple way to separate the cells in a sample. The result of this technique is twofold:

1. You now have synchronized populations of cells without using chemical inhibitors or temperature-sensitive mutations. These can be used for further experiments.
   • Cells in G₁ will have one “full set” of their DNA, while cells in G₂ and M phase will have duplicated their DNA (i.e., two “full sets”).
   • Cells in S phase will have more than one set but less than two sets, as replication has started in S phase but is not yet complete.
   • Additional visual separation may be required to differentiate between G₂ and M phase, but this can be done relatively easily, as a cell undergoing mitosis can be identified using a standard light microscope.
2. The flow cytometer also produces a graphical readout (Figure 08-10) that summarizes how many cells were found with each “amount” of fluorescent material (DNA in this case). These readouts can be used to learn important information about your population of cells.

Interpreting FACS Readouts

A simple FACS readout is shown in Figure 08-10. It is very much like a histogram, in which the y-axis represents the number of cells counted at each point on the x-axis (often listed as a percentage of the total population), and the x-axis measures the amount of DNA. (Note: This is usually done indirectly, by measuring the fluorescence intensity.) The amount of fluorescence intensity correlates to the amount of DNA in a given cell.

Since the amount of DNA in a cell correlates with the phase of the cell cycle, this readout can help us determine how many of the cells in our sample are in each stage of the cell cycle at the moment at which they were measured. We can compare cells at different time points, which might tell us whether our cells are able to progress through the cell cycle or whether they have stalled.

The following are some things to remember about FACS and the readout in Figure 08-10:

• The readout indicates “replicated” and “unreplicated” DNA, not whether it’s in G₁, S, G₂, or M phase. This is an important limitation of this technique. There are two phases in which the DNA has been replicated and as such are lumped together in the second peak (G₂ and M phase). FACS cannot differentiate between these, since the DNA content is the same for both. Depending on the question you’re trying to answer, this might be key to correctly interpreting your results. To separate G₂ from M phase in your sample, you would need to do something additional, like look at the cells in a microscope, for example.
• The part in between the peaks is more important than it might seem. In this area, we have more than one “full set” of DNA but less than two. There’s only one stage of the cell cycle where the amount of DNA goes from one set to two: S phase. Cells that are in the process of undergoing S phase will be found between the peaks that represent G₁ cells and G₂/M cells.

Like many of the techniques we’ve seen so far (FRAP, SDS-PAGE, etc.), FACS is a way to quantify what’s going on in your samples. It’s not actually the experiment itself. The experiment would be done before you put your samples through the FACS machine. If, for example, you wanted to explore the impact of a newly discovered mutation, you could compare the FACS readout from a wild-type (i.e., unmutated) and a mutated sample of cells and see how similar or different their FACS readouts are.

Changes in the FACS readout, even really strange and unexpected changes that don’t make a lot of sense at first glance, can tell us very important things about the samples and treatments we choose to study. Like all of the experiments we’ve covered in this book, understanding what the technique can, and cannot, reveal is essential to being able to accurately interpret the data you receive from your experiment.