Biochemical switches in the cell cycle

From Wikipedia, the free encyclopedia

 

A series of biochemical switches control transitions between and within the various phases of the cell cycle. The cell cycle is a series of complex, ordered, sequential events that control how a single cell divides into two cells, and involves several different phases. The phases include the G1 and G2 phases, DNA replication or S phase, and the actual process of cell division, mitosis or M phase. During the M phase, the chromosomes separate and cytokinesis occurs.

 

The switches maintain the orderly progression of the cell cycle and act as checkpoints to ensure that each phase has been properly completed before progression to the next phase. For example, Cdk, or cyclin dependent kinase, is a major control switch for the cell cycle and it allows the cell to move from G1 to S or G2 to M by adding phosphate to protein substrates. Such multi-component (involving multiple inter-linked proteins) switches have been shown to generate decisive, robust (and potentially irreversible) transitions and trigger stable oscillations. As a result, they are a subject of active research that tries to understand how such complex properties are wired into biological control systems.

Feedback loops

Many biological circuits produce complex outputs by exploiting one or more feedback loops. In a sequence of biochemical events, feedback would refer to a downstream element in the sequence (B in the adjacent image) affecting some upstream component (A in the adjacent image) to affect its own production or activation (output) in the future. If this element acts to enhance its own output, then it engages in positive feedback (blue arrow). A positive feedback loop is also known as a self-reinforcing loop, and it is possible that these loops can be part of a larger loop, as this is characteristic of regulatory circuits.

Conversely, if this element leads to its own inhibition through upstream elements, this is canonically negative feedback (red blunt arrow). A negative feedback loop is also known as a balancing loop, and it may be common to see oscillations in which a delayed negative feedback signal is used to maintain homeostatic balance in the system.

Feedback loops can be used for amplification (positive) or self-correction (negative). The right combination of positive and negative feedback loops can generate ultrasensitivity and bistability, which in turn can generate decisive transitions and oscillations.

Combination of positive and negative feedback loops

Positive and negative feedback loops do not always operate distinctly. In the mechanism of biochemical switches, they work together to create a flexible system. For example, according to Pfeuty & Kaneko (2009), to overcome a drawback in biochemical systems, positive feedback regulation loops may interact with negative regulation loops to facilitate escape from stable states. The coexistence of two stable states is known as bistability, which is often the result of positive feedback regulations. 

An example that reveals the interaction of the multiple negative and positive feedback loops is the activation of cyclin-dependent protein kinases, or Cdks14. Positive feedback loops play a role by switching cells from low to high Cdk-activity. The interaction between the two types of loops is evident in mitosis. While positive feedback initiates mitosis, a negative feedback loop promotes the inactivation of the cyclin-dependent kinases by the anaphase-promoting complex. This example clearly shows the combined effects that positive and negative feedback loops have on cell-cycle regulation.

Ultrasensitivity

An "all-or-none" response to a stimulus is termed ultrasensitivity. In other words, a very small change in stimulus causes a very large change in response, producing a sigmoidal dose-response curve. An ultrasensitive response is described by the general equation V = Sⁿ/(Sⁿ + K_m), known as the Hill equation, when n, the Hill coefficient, is more than 1. The steepness of the sigmoidal curve depends on the value of n. A value of n = 1 produces a hyperbolic or Michaelian response. Ultrasensitivity is achieved in a variety of systems; a notable example is the cooperative binding of the enzyme hemoglobin to its substrate. Since an ultrasensitive response is almost ‘digital’, it can be used to amplify a response to a stimulus or cause a decisive sharp transition (between ‘off’ and ‘on’ states).

Ultrasensitivity plays a large role in cell-cycle regulation. For example, Cdk1 and Wee1 are mitotic regulators, and they are able to inactivate each other through inhibitory phosphorylation. This represents a double negative feedback loop in which both regulators inactivate each other. According to Kim et al. (2007), there must be an ultrasensitive element to generate a bistable response. It turns out that Wee1 has an ultrasensitive response to Cdk1, and this likely arises because of substrate competition among the various phosphorylation sites on Wee1.

Bistability

Bistability implies hysteresis, and hysteresis implies multistability. Multistability indicates the presence of two or more stable states for a given input. Therefore, bistability is the ability of a system to exist in two steady states. In other words, there is a range of stimulus values for which the response can have two steady-state values. Bistability is accompanied by hysteresis, which means that the system approaches one of the two steady states preferentially depending on its history. Bistability requires feedback as well as an ultrasensitive circuit element. 

Under the proper circumstances, positive and negative feedback loops can provide the conditions for bistability; for example, by having positive feedback coupled to an ultrasensitive response element with the circuit. A hysteretic bistable system can act as a robust reversible switch because it is harder for the system to transition between ‘on’ and ‘off’ states (compared to the equivalent monostable ultransensitive response). The system could also be poised such that one of the transitions is physically unattainable; for example, no amount of reduction in the stimulus will return the system to the ‘off’-state once it is already in the ‘on’ state. This would form a robust irreversible switch.

There is no one-to-one correspondence between network topology, since many networks have a similar input and output relationship. A network topology does not imply input or output, and similarly input or output does not imply network topology. It is for this reason that parameterization is very important for circuit function. If the dynamics of the input are comparable or faster than the response of the system, the response may appear hysteretic. 

Three cell cycle switches are described below that achieve abrupt and/or irreversible transitions by exploiting some of the mechanisms described above.

The G1/S switch

The G1/S transition, more commonly known as the Start checkpoint in budding yeast (the restriction point in other organisms) regulates cell cycle commitment. At this checkpoint, cells either arrest before DNA replication (due to limiting nutrients or a pheromone signal), prolong G1 (size control), or begin replication and progress through the rest of the cell cycle. The G1/S regulatory network or regulon in budding yeast includes the G1 cyclins Cln1, Cln2 and Cln3, Cdc28 (Cdk1), the transcription factors SBF and MBF, and the transcriptional inhibitor Whi5. Cln3 interacts with Cdk1 to initiate the sequence of events by phosphorylating a large number of targets, including SBF, MBF and Whi5. Phosphorylation of Whi5 causes it to translocate out of the nucleus, preventing it from inhibiting SBF and MBF. Active SBF/MBF drive the G1/S transition by turning on the B-type cyclins and initiating DNA replication, bud formation and spindle body duplication. Moreover, SBF/MBF drives expression of Cln1 and Cln2, which can also interact with Cdk1 to promote phosphorylation of its targets.

This G1/S switch was initially thought to function as a linear sequence of events starting with Cln3 and ending in S phase. However, the observation that any one of the Clns was sufficient to activate the regulon indicated that Cln1 and Cln2 might be able to engage positive feedback to activate their own transcription. This would result in a continuously accelerating cycle that could act as an irreversible bistable trigger. Skotheim et al. used single-cell measurements in budding yeast to show that this positive feedback does indeed occur. A small amount of Cln3 induces Cln1/2 expression and then the feedback loop takes over, leading to rapid and abrupt exit of Whi5 from the nucleus and consequently coherent expression of G1/S regulon genes. In the absence of coherent gene expression, cells take longer to exit G1 and a significant fraction even arrest before S phase, highlighting the importance of positive feedback in sharpening the G1/S switch.

The G1/S cell cycle checkpoint controls the passage of eukaryotic cells from the first gap phase, G1, into the DNA synthesis phase, S. In this switch in mammalian cells, there are two cell cycle kinases that help to control the checkpoint: cell cycle kinases CDK4/6-cyclin D and CDK2-cyclin E. The transcription complex that includes Rb and E2F is important in controlling this checkpoint. In the first gap phase, the Rb-HDAC repressor complex binds to the E2F-DP1 transcription factors, therefore inhibiting the downstream transcription. The phosphorylation of Rb by CDK4/6 and CDK2 dissociates the Rb-repressor complex and serves as an on/off switch for the cell cycle. Once Rb is phosphorylated, the inhibition is released on the E2F transcriptional activity. This allows for the transcription of S phase genes encoding for proteins that amplify the G1 to S phase switch.

Many different stimuli apply checkpoint controls including TGFb, DNA damage, contact inhibition, replicative senescence, and growth factor withdrawal. The first four act by inducing members of the INK4 or Kip/Cip families of cell cycle kinase inhibitors. TGFb inhibits the transcription of Cdc25A, a phosphatase that activates the cell cycle kinases, and growth factor withdrawal activates GSK3b, which phosphorylates cyclin D. This leads to its rapid ubiquitination.

The G2/M switch

G2 is commenced by E2F-mediated transcription of cyclin A, which forms the cyclin A-Cdk2 complex. In order to proceed into mitosis, the cyclin B-Cdk1 complex (first discovered as MPF or M-phase promoting factor; Cdk1 is also known as Cdc2 in fission yeast and Cdc28 in budding yeast) is activated by Cdc25, a protein phosphatase. As mitosis starts, the nuclear envelope disintegrates, chromosomes condense and become visible, and the cell prepares for division. Cyclin B-Cdk1 activation results in nuclear envelope breakdown, which is a characteristic of the initiation of mitosis.

The cyclin B-Cdk1 complex participates in a regulatory circuit in which Cdk1 can phosphorylate and activate its activator, Cdc25 (positive feedback), and phosphorylate and inactivate its inactivator, the kinase Wee1 (double-negative feedback). This circuit could act as a bistable trigger with one stable steady state in G2 (Cdk1 and Cdc25 off, Wee1 on) and a second stable steady state in M phase (Cdk1 and Cdc25 active, Wee1 off). However, Wee1 is itself regulated by other factors, such as Cdr2. 

It was suggested and defended by Jin et al. in their series of experiments with the human HeLa cell line in 1998 that it is the spatial location of cyclin B within the cell that initiates mitosis. Known from previous experiments in both human cells and starfish oocytes, Jin et al. summarize that cyclin B1 is abundant in the cytoplasm during non-dividing phases of mitosis, but is identified in the nucleus, in complex with Cdk1, immediately before the cell enters mitosis. Other experimenters showed that cells would not divide if cyclin B remains in the cytoplasm. In order to further investigate the effect of spatial location of cyclin B on cell division and cycle control, Jin et al. tagged cyclin B with a nuclear localization signal (NLS) that would keep the cyclin within the nucleus. Initially, this NLS cyclin B did not induce the expected effect of accelerated mitotic entry. This result is due to the inhibition detailed in the figure below. Wee1, an inhibitor on the cyclin B-Cdk1 complex, is localized in the nucleus, and likely phosphorylating the NLS cyclin B, rendering it unable to perform as predicted. This postulation was confirmed when Jin et al. employed Cdc2AF, an unphosphorylatable mutant of Cdk1, and saw accelerated entry to cell division due to the nuclear localization of the cyclin B. Therefore, nuclear localization of cyclin B is necessary but not sufficient to trigger cell division.

In investigation of cell cycle regulation, Jin et al. manipulated cells in order to evaluate the localization of cyclin B in cells with DNA damage. Through combination of DNA damage and nuclear localization of exogenous cyclin B, they were able to determine that cells would divide even with DNA damage if the cyclin B were forced to be expressed in the nucleus. This suggests that spatial localization of cyclin B may play a role as a checkpoint of mitosis. If the cells, under normal circumstances, don’t divide when their genetic information is damaged, but will enter mitosis if endogenous cyclin B is expressed in the nucleus, it is likely that the translocation of the cyclin B to the cytoplasm is a mechanism that prevents immature mitotic entry. This hypothesis was further supported by Jin et al.’s analysis of cells arrested in G2 due to DNA damage. In these cells, Jin et al. observed high levels of cyclin B-Cdc2 complex activity in the cytoplasm. This is supporting evidence for the previously mentioned theory because it shows that the Cdc2 can activate the cyclin without immediate translocation to the nucleus. Additionally, the accumulation of cyclin B-Cdk1 complexes in the cytoplasm of cells that are not dividing due to DNA damage supports the theory that it is nuclear localization of cyclin B that initiates mitotic entry.

To conclude, spatial localization of cyclin B plays a role in mitotic entry. Translocation of cyclin B from the cytoplasm to the nucleus is necessary for cell division, but not sufficient, as its inhibitors do not allow the cell to enter mitosis prematurely. In addition to the back up inhibition of the cyclin B-Cdk1 complex, premature cellular division is prevented by the translocation of the cyclin B itself. The cyclin B-Cdk1 complex will remain in the cytoplasm in cells with DNA damage, rather than translocate to the nucleus, keeping the cell inhibiting the cell from entering mitosis. The next question addressed by researchers in this field is by which specific mechanism is this translocation regulated. 

Santos et al. hypothesized that the translocation of cyclin B is regulated by a mechanism of positive feedback, similar to that which regulates the activation of the cyclin B-Cdk1 complex. They believed that the positive feedback loop involves the phosphorylation of the cyclin B and its translocation to the nucleus. To begin to investigate this, they first reconfirmed some of the results of the Jin et al. experiments, utilizing immunofluorescence to show cyclin B in the cytoplasm prior to division, and translocation to the nucleus to initiate mitosis, which they operationalized by comparing relative to nuclear envelope breakdown (NEB). Using nuclear cyclin that cannot be inactivated by Wee1 or Myt1, Santos et al. observed that active nuclear cyclin recruits more cyclin from the cytoplasm to be translocated to the nucleus. They confirmed this observation by employing a rapamycin treatment, iRap. iRap induces translocation of tagged cyclin B from the cytoplasm to the nucleus. Remarkably, Santos et al. saw that untagged cyclin B migrated with the cyclin B influenced by iRap. The untagged cyclin is insentive to the treatment, and moves independently from the treated cyclin. This supports the first part of the positive feedback loop, that nuclear localization of cyclin B, which leads to mitotic entry, promotes increased translocation of cytoplasmic cyclin B to the nucleus, further promoting the remaining cytoplasmic cyclin B to migrate to the nucleus, etc. 

Santos et al. further hypothesize that phosphorylation of the cyclin B is another component of the positive feedback loop. They observed that the cyclin B naturally enters the nucleus before NEB. In contrast, mutated, unphosphorylatable cyclin B enters the nucleus during NEB. This is unexpected because it is characteristic of the cell cycle for the cyclin to translocate to the nucleus prior to NEB in order to induce cell cycle progression into mitotic division. Therefore, Santos et al. conclude that the phosphorylation of the cyclin B promotes the translocation to the nucleus. However, in addition, translocation to the nucleus promotes phosphorylation of the cyclin. It is noted by the authors that phosphorylation of cyclin B is nineteen times more favorable in the nucleus than in the cytoplasm, due to the smaller overall volume of the nucleus, allowing a faster phosphorylation rate. The increased translocation due to phosphorylation and increased phosphorylation due to translocation exemplify the positive feedback loop that resembles that previously discovered, which activates the cyclin B-Cdk1 complex.

In conclusion, nuclear localization of cyclin B is necessary for cellular entry into mitosis. The translocation of the cyclin from the cytoplasm to the nucleus, which allows for cellular division, is regulated by a positive feedback loop. Active cyclin B translocates to the nucleus and promotes activation and translocation of additional units of cyclin residing in the nucleus. This phenomenon is enhanced when considering phosphorylation. Phosphorylation of cyclin B promotes translocation to the nucleus, and cyclin B in the nucleus is much more likely to be phosphorylated, so nuclear localization promotes cyclin B phosphorylation in return. 

Once cells are in mitosis, cyclin B-Cdk1 activates the anaphase-promoting complex (APC), which in turn inactivates cyclin B-Cdk1 by degrading cyclin B, eventually leading to exit from mitosis. Coupling the bistable Cdk1 response function to the negative feedback from the APC could generate what is known as a relaxation oscillator, with sharp spikes of Cdk1 activity triggering robust mitotic cycles. However, in a relaxation oscillator, the control parameter moves slowly relative to the system’s response dynamics which may be an accurate representation of mitotic entry, but not necessarily mitotic exit. 

It is necessary to inactivate the cyclin B-Cdk1 complex in order to exit the mitotic stage of the cell cycle. The cells can then return to the first gap phase G1 and wait until the cycle proceeds yet again.

In 2003 Pomerening et al. provided strong evidence for this hypothesis by demonstrating hysteresis and bistability in the activation of Cdk1 in the cytoplasmic extracts of Xenopus oocytes. They first demonstrated a discontinuous sharp response of Cdk1 to changing concentrations of non-destructible Cyclin B (to decouple the Cdk1 response network from APC-mediated negative feedback). However, such a response would be consistent with both a monostable, ultrasensitive transition and a bistable transition. To distinguish between these two possibilities, they measured the steady-state levels of active Cdk1 in response to changing cyclin levels, but in two separate experiments, one starting with an interphase extract and one starting with an extract already in mitosis. At intermediate concentrations of cyclin they found two steady-state concentrations of active Cdk1. Which of the two steady states was occupied depended on the history of the system, i.e.whether they started with interphase or mitotic extract, effectively demonstrating hysteresis and bistability.

In the same year, Sha et al. independently reached the same conclusion revealing the hysteretic loop also using Xenopus laevis egg extracts. In this article, three predictions of the Novak-Tyson model were tested in an effort to conclude that hysteresis is the driving force for "cell-cycle transitions into and out of mitosis". The predictions of the Novak-Tyson model are generic to all saddle-node bifurcations. Saddle-node bifurcations are extremely useful bifurcations in an imperfect world because they help describe biological systems which are not perfect. The first prediction was that the threshold concentration of cyclin to enter mitosis is higher than the threshold concentration of cyclin to exit mitosis, and this was confirmed by supplementing cycling egg extracts with non-degradable cyclin B and measuring the activation and inactivation threshold after the addition of cycloheximide (CHX), which is a protein synthesis inhibitor. Furthermore, the second prediction of the Novak-Tyson model was also validated: unreplicated deoxyribonucleic acid, or DNA, increases the threshold concentration of cyclin that is required to enter mitosis. In order to arrive at this conclusion, cytostatic factor released extracts were supplemented with CHX, APH (a DNA polymerase inhibitor), or both, and non-degradable cyclin B was added. The third and last prediction that was tested and proven true in this article was that the rate of Cdc2 activation slows down near the activation threshold concentration of cyclin. These predictions and experiments demonstrate the toggle-like switching behavior that can be described by hysteresis in a dynamical system.

Metaphase-anaphase switch

In the transition from metaphase to anaphase, it is crucial that sister chromatids are properly and simultaneously separated to opposite ends of the cell. Separation of sister-chromatids is initially strongly inhibited to prevent premature separation in late mitosis, but this inhibition is relieved through destruction of the inhibitory elements by the anaphase-promoting complex (APC) once sister-chromatid bi-orientation is achieved. One of these inhibitory elements is securin, which prevents the destruction of cohesin, the complex that holds the sister-chromatids together, by binding the protease separase which targets Scc1, a subunit of the cohesin complex, for destruction. In this system, the phosphatase Cdc14 can remove an inhibitory phosphate from securin, thereby facilitating the destruction of securin by the APC, releasing separase. As shown by Uhlmann et al., during the attachment of chromosomes to the mitotic spindle the chromatids remain paired because cohesion between the sisters prevents separation. Cohesion is established during DNA replication and depends on cohesin, which is a multisubunit complex composed of Scc1, Scc3, Smc2, and Smc3. In yeast at the metaphase-to-anaphase transition, Scc1 dissociates from the chromosomes and the sister chromatids separate. This action is controlled by the Esp1 protein, which is tightly bound by the anaphase inhibitor Pds1 that is destroyed by the anaphase-promoting complex. In order to verify that Esp1 does play a role in regulating Scc1 chromosome association, cell strains were arrested in G1 with an alpha factor. These cells stayed in arrest during the development. Esp1-1 mutant cells were used and the experiment was repeated, and Scc1 successfully bound to the chromosomes and remained associated even after the synthesis was terminated. This was crucial in showing that with Esp1, Scc1 is hindered in its ability to become stably associated with chromosomes during G1, and Esp1 can in fact directly remove Scc1 from chromosomes. 

It has been shown by Holt et al. that separase activates Cdc14, which in turn acts on securin, thus creating a positive feedback loop that increases the sharpness of the metaphase to anaphase transition and coordination of sister-chromatid separation. Holt et al. probed the basis for the effect of positive feedback in securin phosphorylation by using mutant 'securin' strains of yeast, and testing how changes in the phosphoregulation of securin affects the synchrony of sister chromatid separation. Their results indicate that interfering with this positive securin-separase-cdc14 loop decreases sister chromatid separation synchrony. This positive feedback can hypothetically generate bistability in the transition to anaphase, causing the cell to make the irreversible decision to separate sister-chromatids.

Mitotic exit

Mitotic exit is an important transition point that signifies the end of mitosis and the onset of new G1 phase for a cell, and the cell needs to rely on specific control mechanisms to ensure that once it exits mitosis, it never returns to mitosis until it has gone through G1, S, and G2 phases and passed all the necessary checkpoints. Many factors including cyclins, cyclin-dependent kinases (CDKs), ubiquitin ligases, inhibitors of cyclin-dependent kinases, and reversible phosphorylations regulate mitotic exit to ensure that cell cycle events occur in correct order with the fewest errors. The end of mitosis is characterized by spindle breakdown, shortened kinetochore microtubules, and pronounced outgrowth of astral (non-kinetochore) microtubules. For a normal eukaryotic cell, mitotic exit is irreversible.

Proteolytic degradation

Fig. 1 Immunofluorescence patterns of cyclin B and phosphorylated cyclin-dependent kinase1 (Cdk1) in HeLa cells change as they go from G2 to anaphase.

 

Many speculations were made with regard to the control mechanisms employed by a cell to promote the irreversibility of mitotic exit in a eukaryotic model organism, the budding yeast Saccharomyces cerevisiae. Proteolytic degradation of cell cycle regulators and corresponding effects on the levels of cyclin-dependent kinases were proposed as a mechanism that promotes eukaryotic cell cycle and metaphase-to-anaphase transition in particular. In this theory, anaphase promoting complex (APC), a class of ubiquitin ligase, facilitates degradation of mitotic cyclins (Clb2) and anaphase-inhibiting factors (PDS1, CUT2) to promote mitotic exit. APC ubiquitinates nine-amino acid motif known as the destruction box (D box) in the NH2-terminal domain of mitotic cyclins for degradation by proteasome. APC in association with Cdc20 (APC-Cdc20) ubiquitinates and targets mitotic cyclins (Clb2) for degradation at initial phase. Simultaneously, APC-Cdc20 mediates degradation of securins, which inhibit separases through binding, at anaphase onset. Released and active separase cleaves cohesin that held sister chromatids together, facilitating separation of sister chromatids and initiates mitotic exit by promoting release of Cdc14 from nucleolus. At later phase, downregulation of Cdk1 and activation of Cdc14, a Cdh1-activating phosphatase, promotes formation of APC in association with Cdh1 (APC-Cdh1) to degrade Clb2s. Cdc20 and Cdh1, which are the activators of APC, recruit substrates such as securin and B-type cyclins(Clb) for ubiquitination. Without Cdk1-Clb2 complexes to phosphorylate proteins that are involved in spindle dynamics such as Sli15, Ase1, and Ask1, spindle elongation and chromosomal segregation are promoted, facilitating mitotic exit. The importance of proteolytic degradation in eukaryotic cell cycle changed the view of cell division as a simple kinase cascade to a more complex process in which interactions among phosphorylation, ubiquitination, and proteolysis are necessary. However, experiments using budding yeast cells with cdc28-as1, a INM-PP1 (ATP analog)-sensitive Cdk allele, proved that destruction of B-type cyclins (Clb) is not necessary for triggering irreversible mitotic exit. Clb2 degradation did shorten the Cdk1-inhibition period required for triggering irreversible mitotic exit indicating that cyclin proteolysis contributes to the dynamic nature of the eukaryotic cell cycle due to slower timescale of its action but is unlikely to be the major determining factor in triggering irreversible cell cycle transitions.

Sic1 levels

Discoveries were made which indicated the importance of the level of the inhibitors of cyclin-dependent kinases in regulating eukaryotic cell cycle. In particular, the level of Sic1, a stoichiometric inhibitor of Clb-CDK complexes in budding yeast, was shown to be particularly important in irreversible G1-S transition by irreversibly activating S phase kinases. Sic1 level was shown to play a major role in triggering irreversible mitotic exit (M-G1 transition) as well as in G1-S transition. During mitosis, decreasing levels of Cdk1 leads to the activation of Cdc14, a phosphatase that counteracts Cdk1 via activation of Cdh1 and Swi5, a transcriptional activator of Sic1 proteins. While degradation of Sic1 to a certain low level triggered the onset of S phase, accumulation of Sic1 to a certain high level was required to trigger irreversible mitotic exit. Cdk1-inhibitors could induce mitotic exit even when degradation of B-type cyclins was blocked by expression of non-degradable Clbs or proteasome inhibitors. However, sister chromatids failed to segregate, and cells reverted to mitosis once the inhibitors were washed away, indicating that a threshold level of the inhibitors needs to be achieved to trigger irreversible mitotic exit independently of cyclin degradations. Despite different thresholds of Sic1 level that are required to trigger mitotic exit compared to G1-S transition, the level of Sic1 was shown to play a key role in regulating eukaryotic cell cycle by inhibiting the activity of CDKs.

Dynamical systems approach

Fig. 2 Irreversible and bistable switch in mitotic exit with control parameter being Sic1 level and order parameter being cell cycle phases.

Because eukaryotic cell cycle involves a variety of proteins and regulatory interactions, dynamical systems approach can be taken to simplify a complex biological circuit into a general framework for better analysis. Among the four possible input/output relationships, the relationship between Sic1 level and mitotic exit seems to show the characteristics of an irreversible bistable switch, driven by feedback between APC-Cdh1, Sic1, and Clb2-Cdk1. Bistability is known to control biological functions such as cell cycle control and cellular differentiation and play a key role in many cellular regulatory networks. Bistable input/output relationship is characterized by two stable states with two bifurcation points. Multiple outputs are possible for one specific input in the region of bistability, marked by two bifurcation points. In addition, the bistable relationship displays hysteresis: the final state/output depends on the history of the input as well as the current value of input because the system has a memory. One bifurcation point has a negative control parameter value (the bifurcation point is on the other side of the axis), resulting in disconnection between the two stable states and irreversibility of the transition from one state to the other. With regard to mitotic exit, the two stable states are defined by mitosis and G1 phase. Once Sic1 level (input) accumulates beyond the threshold, irreversible transition occurs from mitosis (stable state I) to G1 phase (stable state II). In the imperfect environment, the only bifurcation that remains intact is saddle-node bifurcation. Saddle-node bifurcation does not break down (saddle-node is the expected generic behavior), while transcritical and pitchfork bifurcations break down in the presence of imperfections. Thus, the only one-dimensional bifurcation that can exist in imperfect biological world is the saddle-node bifurcation. The bistable relation between M-G1 transition and Sic1 level can be represented as a diagram of two saddle-node bifurcations in which the system’s behavior changes qualitatively with a small change in control parameter, the amount of Sic1.

Systems-level feedback

Fig. 3 Simplified network involving Cdk1-Clb2, APC-Cdh1, Sic1, and Cdc14. Double negative feedback loop, mediated by APC-Cdh1 and Sic1, is required to suppress Cdk1-Clb2 and trigger mitotic exit.

 

Because the behavior of cell cycle critically depends on the amount of Sic1 at the M-G1 transition state, the amount of Sic1 is tightly regulated by systems-level feedbacks. Because Cdk1-Clb2 inhibits Sic1 by phosphorylating Sic1 and making Sic1 available for degradation via ubiquitylation, APC-Cdh1-dependent degradation of Cdk1-Clb2 not only decreases the level of available Cdk1-Clb2 complexes but also increases the level of Sic1 which in turn further inhibits the function of Cdk1-Clb2. This activation of the double negative feedback loop is initiated from APC-Cdc20-dependent degradation of Cdk1-Clb2 and release of Cdc14 from nucleolar protein Net1/Cfi1. FEAR (Cdc14 early anaphase release) pathway facilitates Clb2-Cdk1-dependent phosphorylation of Net1 which transiently releases Cdc14 from Net1. The released Cdc14 and Clb2-Cdk1 complexes go onto form spindles that activates mitotic exit network (MEN). MEN allows sustained release of Cdc14 from the nucleolus, and Cdc14 counters the activity of Clb2-Cdk1 by activating Cdh1 and stabilizing Sic1 through activation of Sic1-transcriptional activator Swi5. Sic1 positively regulates itself by inhibiting Cdk1-Clb2 to release inhibition of Swi5, and Cdh1 also positively regulates itself by inhibiting Clb2-Cdk1 to release inhibition of MEN which can activate Cdc14 and subsequently Cdh1 itself. The double-negative feedback loop, formed by APC-Cdh1 and Sic1, is required to maintain low Clb2-Cdk1 activity because Clb2 auto-activates its synthesis by activating transcriptional factors, Fkh2–Mcm1 Ndd1 complex.

Implications

Eukaryotic cell cycle consists of various checkpoints and feedback loops to ensure faithful and successful cell division. During mitosis for example, when duplicated chromosomes are improperly attached to mitotic spindle, spindle assembly checkpoint (SAC) proteins including Mad and Bub inhibit APC-Cdc20 to delay entry into anaphase and B-type cyclin degradations. In addition, when mitotic spindles are misaligned, MEN and subsequently Cdc14 are inhibited in a Bub2 and Bfa1-dependent manner to prevent degradation of mitotic cyclins and anaphase entry. Sic1 is a nice example demonstrating how systems-level feedbacks interact to sense the environmental conditions and trigger cell cycle transitions. Even though actual M-G1 transition is vastly complex with numerous proteins and regulations involved, dynamical systems approach allows simplification of this complex system into bistable input/output relation with two saddle-node bifurcations in which the output (mitotic exit) depends on critical concentration of Sic1. Using one-dimensional analysis, it might be possible to explain many of the irreversible transition points in the eukaryotic cell cycle that are governed by systems-level control and feedback. Other examples of irreversible transition points include Start (irreversible commitment to a new cell division cycle) that can be explained by irreversible bistable switch whose control parameter is tightly regulated by the systemic feedbacks involving Cln2, Whi5, and SBF.

Cell cycle

From Wikipedia, the free encyclopedia

Life cycle of the cell

 

Onion (Allium) cells in different phases of the cell cycle. Growth in an 'organism' is carefully controlled by regulating the cell cycle.

 

The cell cycle, or cell-division cycle, is the series of events that take place in a cell leading to its division and duplication of its DNA (DNA replication) to produce two daughter cells. In bacteria, which lack a cell nucleus, the cell cycle is divided into the B, C, and D periods. The B period extends from the end of cell division to the beginning of DNA replication. DNA replication occurs during the C period. The D period refers to the stage between the end of DNA replication and the splitting of the bacterial cell into two daughter cells. In cells with a nucleus, as in eukaryotes, the cell cycle is also divided into three periods: interphase, the mitotic (M) phase, and cytokinesis. During interphase, the cell grows, accumulating nutrients needed for mitosis, preparing it for cell division and duplicating its DNA. During the mitotic phase, the chromosomes separate. During the final stage, cytokinesis, the chromosomes and cytoplasm separate into two new daughter cells. To ensure the proper division of the cell, there are control mechanisms known as cell cycle checkpoints. 

The cell-division cycle is a vital process by which a single-celled fertilized egg develops into a mature organism, as well as the process by which hair, skin, blood cells, and some internal organs are renewed. After cell division, each of the daughter cells begin the interphase of a new cycle. Although the various stages of interphase are not usually morphologically distinguishable, each phase of the cell cycle has a distinct set of specialized biochemical processes that prepare the cell for initiation of cell division

Phases

The cell cycle consists of four distinct phases: G₁ phase, S phase (synthesis), G₂ phase (collectively known as interphase) and M phase (mitosis or meiosis). M phase is itself composed of two tightly coupled processes: karyokinesis, in which the cell's chromosomes are divided, and cytokinesis, in which the cell's cytoplasm divides forming two daughter cells. Activation of each phase is dependent on the proper progression and completion of the previous one. Cells that have temporarily or reversibly stopped dividing are said to have entered a state of quiescence called G₀ phase. 

Schematic of the cell cycle. Outer ring: I = Interphase, M = Mitosis; inner ring: M = Mitosis, G₁ = Gap 1, G₂ = Gap 2, S = Synthesis; not in ring: G₀ = Gap 0/Resting

 

State	Phase	Abbreviation	Description
Resting	Gap 0	G0	A phase where the cell has left the cycle and has stopped dividing.
Interphase	Gap 1	G1	Cells increase in size in Gap 1. The G1 checkpoint control mechanism ensures that everything is ready for DNA synthesis.
	Synthesis	S	DNA replication occurs during this phase.
	Gap 2	G2	During the gap between DNA synthesis and mitosis, the cell will continue to grow. The G2 checkpoint control mechanism ensures that everything is ready to enter the M (mitosis) phase and divide.
Cell division	Mitosis	M	Cell growth stops at this stage and cellular energy is focused on the orderly division into two daughter cells. A checkpoint in the middle of mitosis (Metaphase Checkpoint) ensures that the cell is ready to complete cell division.

After cell division, each of the daughter cells begin the interphase of a new cycle. Although the various stages of interphase are not usually morphologically distinguishable, each phase of the cell cycle has a distinct set of specialized biochemical processes that prepare the cell for initiation of cell division.

G₀ phase (quiescence)

Plant cell cycle

 

Animal cell cycle

 

G₀ is a resting phase where the cell has left the cycle and has stopped dividing. The cell cycle starts with this phase. The word "post-mitotic" is sometimes used to refer to both quiescent and senescent cells. Non-proliferative (non-dividing) cells in multicellular eukaryotes generally enter the quiescent G₀ state from G₁ and may remain quiescent for long periods of time, possibly indefinitely (as is often the case for neurons). This is very common for cells that are fully differentiated. Cellular senescence occurs in response to DNA damage and external stress and usually constitutes an arrest in G₁. Some cells enter the G₀ phase semi-permanently and are considered post-mitotic, e.g., some liver, kidney, and stomach cells. Many cells do not enter G₀ and continue to divide throughout an organism's life, e.g., epithelial cells. 

Cellular senescence is also a state that occurs in response to DNA damage or degradation that would make a cell's progeny nonviable; it is often a biochemical alternative to the self-destruction of such a damaged cell by apoptosis.

Interphase (intermitosis)

Before a cell can enter cell division, it needs to take in nutrients. All of the preparations are done during interphase. Interphase is a series of changes that takes place in a newly formed cell and its nucleus, before it becomes capable of division again. It is also called preparatory phase or intermitosis. Previously it was called resting stage because there is no apparent activity related to cell division.Typically interphase lasts for at least 91% of the total time required for the cell cycle. 

Interphase proceeds in three stages, G₁, S, and G₂, followed by the cycle of mitosis and cytokinesis. The cell's nuclear DNA contents are duplicated during S phase but may continue till G₂ in case of heterochromatin.

G₁ phase (First growth phase or Post mitotic gap phase)

The first phase within interphase, from the end of the previous M phase until the beginning of DNA synthesis, is called G₁ (G indicating gap). It is also called the growth phase. During this phase, the biosynthetic activities of the cell, which are considerably slowed down during M phase, resume at a high rate. The duration of G₁ is highly variable, even among different cells of the same species. In this phase, the cell increases its supply of proteins, increases the number of organelles (such as mitochondria, ribosomes), and grows in size. In G₁ phase, a cell has three options. (1) To continue cell cycle and enter S phase (2) Stop cell cycle and enter G₀ phase for undergoing differentiation. (3) Get arrested in G₁ phase hence it may enter G₀ phase or re-enter cell cycle. The deciding factor is availability of Nitrogens and storage of energy rich compounds at the deciding point called check point (Restriction point). This check point is called the restriction point or START and is regulated by G₁/S cyclins, which cause transition from G₁ to S phase. Passage through the G₁ check point commits the cell to division.

S phase (DNA replication)

The ensuing S phase starts when DNA synthesis commences; when it is complete, all of the chromosomes have been replicated, i.e., each chromosome has two (sister) chromatids. Thus, during this phase, the amount of DNA in the cell has effectively doubled, though the ploidy of the cell remains the same. Rates of RNA transcription and protein synthesis are very low during this phase. An exception to this is histone production, most of which occurs during the S phase.

G₂ phase (growth)

G2 phase occurs after DNA replication and is a period of protein synthesis and rapid cell growth to prepare the cell for mitosis. During this phase microtubules begin to reorganize to form a spindle.

Mitotic phase (chromosome separation)

The relatively brief M phase consists of nuclear division (karyokinesis). It is a relatively short period of the cell cycle. M phase is complex and highly regulated. The sequence of events is divided into phases, corresponding to the completion of one set of activities and the start of the next. These phases are sequentially known as:

• prophase,
• prometaphase,
• metaphase,
• anaphase,
• telophase

Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets in two nuclei. During the process of mitosis the pairs of chromosomes condense and attach to fibers that pull the sister chromatids to opposite sides of the cell.

Mitosis occurs exclusively in eukaryotic cells, but occurs in different ways in different species. For example, animals undergo an "open" mitosis, where the nuclear envelope breaks down before the chromosomes separate, while fungi such as Aspergillus nidulans and Saccharomyces cerevisiae (yeast) undergo a "closed" mitosis, where chromosomes divide within an intact cell nucleus. Prokaryotic cells, which lack a nucleus, divide by a process called binary fission.

Cytokinesis phase (separation of all cell components)

Mitosis is immediately followed by cytokinesis, which divides the nuclei, cytoplasm, organelles and cell membrane into two cells containing roughly equal shares of these cellular components. Mitosis and cytokinesis together define the division of the mother cell into two daughter cells, genetically identical to each other and to their parent cell. This accounts for approximately 10% of the cell cycle.

Because cytokinesis usually occurs in conjunction with mitosis, "mitosis" is often used interchangeably with "M phase". However, there are many cells where mitosis and cytokinesis occur separately, forming single cells with multiple nuclei in a process called endoreplication. This occurs most notably among the fungi and slime moulds, but is found in various groups. Even in animals, cytokinesis and mitosis may occur independently, for instance during certain stages of fruit fly embryonic development. Errors in mitosis can either kill a cell through apoptosis or cause mutations that may lead to cancer. 

The process of mitosis is complex and highly regulated. The sequence of events is divided into phases, corresponding to the completion of one set of activities and the start of the next. These stages are prophase, prometaphase, metaphase, anaphase and telophase. During the process of mitosis the pairs of chromosomes condense and attach to fibers that pull the sister chromatids to opposite sides of the cell. The cell then divides in cytokinesis, to produce two identical daughter cells.

Regulation of eukaryotic cell cycle

Regulation of the cell cycle involves processes crucial to the survival of a cell, including the detection and repair of genetic damage as well as the prevention of uncontrolled cell division. The molecular events that control the cell cycle are ordered and directional; that is, each process occurs in a sequential fashion and it is impossible to "reverse" the cycle.

Role of cyclins and CDKs

Nobel Laureate Paul Nurse

Nobel Laureate Tim Hunt

Two key classes of regulatory molecules, cyclins and cyclin-dependent kinases (CDKs), determine a cell's progress through the cell cycle. Leland H. Hartwell, R. Timothy Hunt, and Paul M. Nurse won the 2001 Nobel Prize in Physiology or Medicine for their discovery of these central molecules. Many of the genes encoding cyclins and CDKs are conserved among all eukaryotes, but in general more complex organisms have more elaborate cell cycle control systems that incorporate more individual components. Many of the relevant genes were first identified by studying yeast, especially Saccharomyces cerevisiae; genetic nomenclature in yeast dubs many of these genes cdc (for "cell division cycle") followed by an identifying number, e.g. cdc25 or cdc20. 

Cyclins form the regulatory subunits and CDKs the catalytic subunits of an activated heterodimer; cyclins have no catalytic activity and CDKs are inactive in the absence of a partner cyclin. When activated by a bound cyclin, CDKs perform a common biochemical reaction called phosphorylation that activates or inactivates target proteins to orchestrate coordinated entry into the next phase of the cell cycle. Different cyclin-CDK combinations determine the downstream proteins targeted. CDKs are constitutively expressed in cells whereas cyclins are synthesised at specific stages of the cell cycle, in response to various molecular signals.

General mechanism of cyclin-CDK interaction

Upon receiving a pro-mitotic extracellular signal, G₁ cyclin-CDK complexes become active to prepare the cell for S phase, promoting the expression of transcription factors that in turn promote the expression of S cyclins and of enzymes required for DNA replication. The G₁ cyclin-CDK complexes also promote the degradation of molecules that function as S phase inhibitors by targeting them for ubiquitination. Once a protein has been ubiquitinated, it is targeted for proteolytic degradation by the proteasome. However, results from a recent study of E2F transcriptional dynamics at the single-cell level argue that the role of G1 cyclin-CDK activities, in particular cyclin D-CDK4/6, is to tune the timing rather than the commitment of cell cycle entry.

Active S cyclin-CDK complexes phosphorylate proteins that make up the pre-replication complexes assembled during G₁ phase on DNA replication origins. The phosphorylation serves two purposes: to activate each already-assembled pre-replication complex, and to prevent new complexes from forming. This ensures that every portion of the cell's genome will be replicated once and only once. The reason for prevention of gaps in replication is fairly clear, because daughter cells that are missing all or part of crucial genes will die. However, for reasons related to gene copy number effects, possession of extra copies of certain genes is also deleterious to the daughter cells.

Mitotic cyclin-CDK complexes, which are synthesized but inactivated during S and G₂ phases, promote the initiation of mitosis by stimulating downstream proteins involved in chromosome condensation and mitotic spindle assembly. A critical complex activated during this process is a ubiquitin ligase known as the anaphase-promoting complex (APC), which promotes degradation of structural proteins associated with the chromosomal kinetochore. APC also targets the mitotic cyclins for degradation, ensuring that telophase and cytokinesis can proceed.

Specific action of cyclin-CDK complexes

Cyclin D is the first cyclin produced in the cell cycle, in response to extracellular signals (e.g. growth factors). Cyclin D binds to existing CDK4, forming the active cyclin D-CDK4 complex. Cyclin D-CDK4 complex in turn phosphorylates the retinoblastoma susceptibility protein (Rb). The hyperphosphorylated Rb dissociates from the E2F/DP1/Rb complex (which was bound to the E2F responsive genes, effectively "blocking" them from transcription), activating E2F. Activation of E2F results in transcription of various genes like cyclin E, cyclin A, DNA polymerase, thymidine kinase, etc. Cyclin E thus produced binds to CDK2, forming the cyclin E-CDK2 complex, which pushes the cell from G₁ to S phase (G₁/S, which initiates the G₂/M transition). Cyclin B-cdk1 complex activation causes breakdown of nuclear envelope and initiation of prophase, and subsequently, its deactivation causes the cell to exit mitosis. A quantitative study of E2F transcriptional dynamics at the single-cell level by using engineered fluorescent reporter cells provided a quantitative framework for understanding the control logic of cell cycle entry, challenging the canonical textbook model. Genes that regulate the amplitude of E2F accumulation, such as Myc, determine the commitment in cell cycle and S phase entry. G1 cyclin-CDK activities are not the driver of cell cycle entry. Instead, they primarily tune the timing of E2F increase, thereby modulating the pace of cell cycle progression.

Inhibitors

Overview of signal transduction pathways involved in apoptosis, also known as "programmed cell death"

 

Two families of genes, the cip/kip (CDK interacting protein/Kinase inhibitory protein) family and the INK4a/ARF (Inhibitor of Kinase 4/Alternative Reading Frame) family, prevent the progression of the cell cycle. Because these genes are instrumental in prevention of tumor formation, they are known as tumor suppressors. 

The cip/kip family includes the genes p21, p27 and p57. They halt cell cycle in G₁ phase, by binding to, and inactivating, cyclin-CDK complexes. p21 is activated by p53 (which, in turn, is triggered by DNA damage e.g. due to radiation). p27 is activated by Transforming Growth Factor of β (TGF β), a growth inhibitor. 

The INK4a/ARF family includes p16^INK4a, which binds to CDK4 and arrests the cell cycle in G₁ phase, and p14^ARF which prevents p53 degradation. 

Synthetic inhibitors of Cdc25 could also be useful for the arrest of cell cycle and therefore be useful as antineoplastic and anticancer agents.

Transcriptional regulatory network

Current evidence suggests that a semi-autonomous transcriptional network acts in concert with the CDK-cyclin machinery to regulate the cell cycle. Several gene expression studies in Saccharomyces cerevisiae have identified 800–1200 genes that change expression over the course of the cell cycle. They are transcribed at high levels at specific points in the cell cycle, and remain at lower levels throughout the rest of the cycle. While the set of identified genes differs between studies due to the computational methods and criteria used to identify them, each study indicates that a large portion of yeast genes are temporally regulated.

Many periodically expressed genes are driven by transcription factors that are also periodically expressed. One screen of single-gene knockouts identified 48 transcription factors (about 20% of all non-essential transcription factors) that show cell cycle progression defects. Genome-wide studies using high throughput technologies have identified the transcription factors that bind to the promoters of yeast genes, and correlating these findings with temporal expression patterns have allowed the identification of transcription factors that drive phase-specific gene expression. The expression profiles of these transcription factors are driven by the transcription factors that peak in the prior phase, and computational models have shown that a CDK-autonomous network of these transcription factors is sufficient to produce steady-state oscillations in gene expression).

Experimental evidence also suggests that gene expression can oscillate with the period seen in dividing wild-type cells independently of the CDK machinery. Orlando et al. used microarrays to measure the expression of a set of 1,271 genes that they identified as periodic in both wild type cells and cells lacking all S-phase and mitotic cyclins (clb1,2,3,4,5,6). Of the 1,271 genes assayed, 882 continued to be expressed in the cyclin-deficient cells at the same time as in the wild type cells, despite the fact that the cyclin-deficient cells arrest at the border between G₁ and S phase. However, 833 of the genes assayed changed behavior between the wild type and mutant cells, indicating that these genes are likely directly or indirectly regulated by the CDK-cyclin machinery. Some genes that continued to be expressed on time in the mutant cells were also expressed at different levels in the mutant and wild type cells. These findings suggest that while the transcriptional network may oscillate independently of the CDK-cyclin oscillator, they are coupled in a manner that requires both to ensure the proper timing of cell cycle events. Other work indicates that phosphorylation, a post-translational modification, of cell cycle transcription factors by Cdk1 may alter the localization or activity of the transcription factors in order to tightly control timing of target genes.

While oscillatory transcription plays a key role in the progression of the yeast cell cycle, the CDK-cyclin machinery operates independently in the early embryonic cell cycle. Before the midblastula transition, zygotic transcription does not occur and all needed proteins, such as the B-type cyclins, are translated from maternally loaded mRNA.

DNA replication and DNA replication origin activity

Analyses of synchronized cultures of Saccharomyces cerevisiae under conditions that prevent DNA replication initiation without delaying cell cycle progression showed that origin licensing decreases the expression of genes with origins near their 3' ends, revealing that downstream origins can regulate the expression of upstream genes. This confirms previous predictions from mathematical modeling of a global causal coordination between DNA replication origin activity and mRNA expression, and shows that mathematical modeling of DNA microarray data can be used to correctly predict previously unknown biological modes of regulation.

Checkpoints

Cell cycle checkpoints are used by the cell to monitor and regulate the progress of the cell cycle. Checkpoints prevent cell cycle progression at specific points, allowing verification of necessary phase processes and repair of DNA damage. The cell cannot proceed to the next phase until checkpoint requirements have been met. Checkpoints typically consist of a network of regulatory proteins that monitor and dictate the progression of the cell through the different stages of the cell cycle. 

There are several checkpoints to ensure that damaged or incomplete DNA is not passed on to daughter cells. Three main checkpoints exist: the G₁/S checkpoint, the G₂/M checkpoint and the metaphase (mitotic) checkpoint.

G₁/S transition is a rate-limiting step in the cell cycle and is also known as restriction point. This is where the cell checks whether it has enough raw materials to fully replicate its DNA (nucleotide bases, DNA synthase, chromatin, etc.). An unhealthy or malnourished cell will get stuck at this checkpoint.

The G₂/M checkpoint is where the cell ensures that it has enough cytoplasm and phospholipids for two daughter cells. But sometimes more importantly, it checks to see if it is the right time to replicate. There are some situations where many cells need to all replicate simultaneously (for example, a growing embryo should have a symmetric cell distribution until it reaches the mid-blastula transition). This is done by controlling the G₂/M checkpoint. 

 

The metaphase checkpoint is a fairly minor checkpoint, in that once a cell is in metaphase, it has committed to undergoing mitosis. However that's not to say it isn't important. In this checkpoint, the cell checks to ensure that the spindle has formed and that all of the chromosomes are aligned at the spindle equator before anaphase begins.

While these are the three "main" checkpoints, not all cells have to pass through each of these checkpoints in this order to replicate. Many types of cancer are caused by mutations that allow the cells to speed through the various checkpoints or even skip them altogether. Going from S to M to S phase almost consecutively. Because these cells have lost their checkpoints, any DNA mutations that may have occurred are disregarded and passed on to the daughter cells. This is one reason why cancer cells have a tendency to exponentially accrue mutations. Aside from cancer cells, many fully differentiated cell types no longer replicate so they leave the cell cycle and stay in G₀ until their death. Thus removing the need for cellular checkpoints. An alternative model of the cell cycle response to DNA damage has also been proposed, known as the postreplication checkpoint.

Checkpoint regulation plays an important role in an organism's development. In sexual reproduction, when egg fertilization occurs, when the sperm binds to the egg, it releases signalling factors that notify the egg that it has been fertilized. Among other things, this induces the now fertilized oocyte to return from its previously dormant, G₀, state back into the cell cycle and on to mitotic replication and division.

p53 plays an important role in triggering the control mechanisms at both G₁/S and G₂/M checkpoints. In addition to p53, checkpoint regulators are being heavily researched for their roles in cancer growth and proliferation.

Fluorescence imaging of the cell cycle

Fluorescent proteins visualize the cell cycle progression. IFP2.0-hGem(1/110) fluorescence is shown in green and highlights the S/G₂/M phases. smURFP-hCdtI(30/120) fluorescence is shown in red and highlights the G₀/G₁ phases.

 

Pioneering work by Atsushi Miyawaki and coworkers developed the fluorescent ubiquitination-based cell cycle indicator (FUCCI), which enables fluorescence imaging of the cell cycle. Originally, a green fluorescent protein, mAG, was fused to hGem(1/110) and an orange fluorescent protein (mKO₂) was fused to hCdt1(30/120). Note, these fusions are fragments that contain a nuclear localization signal and ubiquitination sites for degradation, but are not functional proteins. The green fluorescent protein is made during the S, G₂, or M phase and degraded during the G₀ or G₁ phase, while the orange fluorescent protein is made during the G₀ or G₁ phase and destroyed during the S, G₂, or M phase. A far-red and near-infrared FUCCI was developed using a cyanobacteria-derived fluorescent protein (smURFP) and a bacteriophytochrome-derived fluorescent protein (movie found at this link).

Role in tumor formation

A disregulation of the cell cycle components may lead to tumor formation. As mentioned above, when some genes like the cell cycle inhibitors, RB, p53 etc. mutate, they may cause the cell to multiply uncontrollably, forming a tumor. Although the duration of cell cycle in tumor cells is equal to or longer than that of normal cell cycle, the proportion of cells that are in active cell division (versus quiescent cells in G₀ phase) in tumors is much higher than that in normal tissue. Thus there is a net increase in cell number as the number of cells that die by apoptosis or senescence remains the same.

The cells which are actively undergoing cell cycle are targeted in cancer therapy as the DNA is relatively exposed during cell division and hence susceptible to damage by drugs or radiation. This fact is made use of in cancer treatment; by a process known as debulking, a significant mass of the tumor is removed which pushes a significant number of the remaining tumor cells from G₀ to G₁ phase (due to increased availability of nutrients, oxygen, growth factors etc.). Radiation or chemotherapy following the debulking procedure kills these cells which have newly entered the cell cycle.

The fastest cycling mammalian cells in culture, crypt cells in the intestinal epithelium, have a cycle time as short as 9 to 10 hours. Stem cells in resting mouse skin may have a cycle time of more than 200 hours. Most of this difference is due to the varying length of G₁, the most variable phase of the cycle. M and S do not vary much.

In general, cells are most radiosensitive in late M and G₂ phases and most resistant in late S phase.

For cells with a longer cell cycle time and a significantly long G₁ phase, there is a second peak of resistance late in G₁. 

The pattern of resistance and sensitivity correlates with the level of sulfhydryl compounds in the cell. Sulfhydryls are natural substances that protect cells from radiation damage and tend to be at their highest levels in S and at their lowest near mitosis.

Cell growth

From Wikipedia, the free encyclopedia

The term cell growth is used in the contexts of biological cell development and cell division (reproduction). When used in the context of cell development, the term refers to increase in cytoplasmic and organelle volume (G1 phase), as well as increase in genetic material (G2 phase) following the replication during S phase. This is not to be confused with growth in the context of cell division, referred to as proliferation, where a cell, known as the "mother cell", grows and divides to produce two "daughter cells" (M phase).

Cell populations

Cell populations go through a particular type of exponential growth called doubling. Thus, each generation of cells should be twice as numerous as the previous generation. However, the number of generations only gives a maximum figure as not all cells survive in each generation.

Cell size

Cell size is highly variable among organisms, with some algae such as Caulerpa taxifolia being a single cell several meters in length. Plant cells are much larger than animal cells, and protists such as Paramecium can be 330 μm long, while a typical human cell might be 10 μm. How these cells "decide" how big they should be before dividing is an open question. Chemical gradients are known to be partly responsible, and it is hypothesized that mechanical stress detection by cytoskeletal structures is involved. Work on the topic generally requires an organism whose cell cycle is well-characterized.

Yeast cell size regulation

The relationship between cell size and cell division has been extensively studied in yeast. For some cells, there is a mechanism by which cell division is not initiated until a cell has reached a certain size. If the nutrient supply is restricted (after time t = 2 in the diagram, below), and the rate of increase in cell size is slowed, the time period between cell divisions is increased. Yeast cell-size mutants were isolated that begin cell division before reaching a normal/regular size (wee mutants).

Figure 1:Cell cycle and growth

 

Wee1 protein is a tyrosine kinase that normally phosphorylates the Cdc2 cell cycle regulatory protein (the homolog of CDK1 in humans), a cyclin-dependent kinase, on a tyrosine residue. Cdc2 drives entry into mitosis by phosphorylating a wide range of targets. This covalent modification of the molecular structure of Cdc2 inhibits the enzymatic activity of Cdc2 and prevents cell division. Wee1 acts to keep Cdc2 inactive during early G2 when cells are still small. When cells have reached sufficient size during G2, the phosphatase Cdc25 removes the inhibitory phosphorylation, and thus activates Cdc2 to allow mitotic entry. A balance of Wee1 and Cdc25 activity with changes in cell size is coordinated by the mitotic entry control system. It has been shown in Wee1 mutants, cells with weakened Wee1 activity, that Cdc2 becomes active when the cell is smaller. Thus, mitosis occurs before the yeast reach their normal size. This suggests that cell division may be regulated in part by dilution of Wee1 protein in cells as they grow larger.

Linking Cdr2 to Wee1

The protein kinase Cdr2 (which negatively regulates Wee1) and the Cdr2-related kinase Cdr1 (which directly phosphorylates and inhibits Wee1 in vitro) are localized to a band of cortical nodes in the middle of interphase cells. After entry into mitosis, cytokinesis factors such as myosin II are recruited to similar nodes; these nodes eventually condense to form the cytokinetic ring. A previously un-characterized protein, Blt1, was found to co-localize with Cdr2 in the medial interphase nodes. Blt1 knockout cells had increased length at division, which is consistent with a delay in mitotic entry. This finding connects a physical location, a band of cortical nodes, with factors that have been shown to directly regulate mitotic entry, namely Cdr1, Cdr2, and Blt1. 

Further experimentation with GFP-tagged proteins and mutant proteins indicates that the medial cortical nodes are formed by the ordered, Cdr2-dependent assembly of multiple interacting proteins during interphase. Cdr2 is at the top of this hierarchy and works upstream of Cdr1 and Blt1. Mitosis is promoted by the negative regulation of Wee1 by Cdr2. It has also been shown that Cdr2 recruits Wee1 to the medial cortical node. The mechanism of this recruitment has yet to be discovered. A Cdr2 kinase mutant, which is able to localize properly despite a loss of function in phosphorylation, disrupts the recruitment of Wee1 to the medial cortex and delays entry into mitosis. Thus, Wee1 localizes with its inhibitory network, which demonstrates that mitosis is controlled through Cdr2-dependent negative regulation of Wee1 at the medial cortical nodes.

Cell polarity factors

Cell polarity factors positioned at the cell tips provide spatial cues to limit Cdr2 distribution to the cell middle. In fission yeast Schizosaccharomyces pombe (S. Pombe), cells divide at a defined, reproducible size during mitosis because of the regulated activity of Cdk1. The cell polarity protein kinase Pom1, a member of the dual-specificity tyrosine-phosphorylation regulated kinase (DYRK) family of kinases, localizes to cell ends. In Pom1 knockout cells, Cdr2 was no longer restricted to the cell middle, but was seen diffusely through half of the cell. From this data it becomes apparent that Pom1 provides inhibitory signals that confine Cdr2 to the middle of the cell. It has been further shown that Pom1-dependent signals lead to the phosphorylation of Cdr2. Pom1 knockout cells were also shown to divide at a smaller size than wild-type, which indicates a premature entry into mitosis.

Pom1 forms polar gradients that peak at cell ends, which shows a direct link between size control factors and a specific physical location in the cell. As a cell grows in size, a gradient in Pom1 grows. When cells are small, Pom1 is spread diffusely throughout the cell body. As the cell increases in size, Pom1 concentration decreases in the middle and becomes concentrated at cell ends. Small cells in early G2 which contain sufficient levels of Pom1 in the entirety of the cell have inactive Cdr2 and cannot enter mitosis. It is not until the cells grow into late G2, when Pom1 is confined to the cell ends that Cdr2 in the medial cortical nodes is activated and able to start the inhibition of Wee1. This finding shows how cell size plays a direct role in regulating the start of mitosis. In this model, Pom1 acts as a molecular link between cell growth and mitotic entry through a Cdr2-Cdr1-Wee1-Cdk1 pathway. The Pom1 polar gradient successfully relays information about cell size and geometry to the Cdk1 regulatory system. Through this gradient, the cell ensures it has reached a defined, sufficient size to enter mitosis.

Cell cycle regulation in mammals

Many different types of eukaryotic cells undergo size-dependent transitions during the cell cycle. These transitions are controlled by the cyclin-dependent kinase Cdk1. Though the proteins that control Cdk1 are well understood, their connection to mechanisms monitoring cell size remains elusive. A postulated model for mammalian size control situates mass as the driving force of the cell cycle. A cell is unable to grow to an abnormally large size because at a certain cell size or cell mass, the S phase is initiated. The S phase starts the sequence of events leading to mitosis and cytokinesis. A cell is unable to get too small because the later cell cycle events, such as S, G2, and M, are delayed until mass increases sufficiently to begin S phase.

Many of the signal molecules that convey information to cells during the control of cellular differentiation or growth are called growth factors. The protein mTOR is a serine/threonine kinase that regulates translation and cell division. Nutrient availability influences mTOR so that when cells are not able to grow to normal size they will not undergo cell division. The details of the molecular mechanisms of mammalian cell size control are currently being investigated. The size of post-mitotic neurons depends on the size of the cell body, axon and dendrites. In vertebrates, neuron size is often a reflection of the number of synaptic contacts onto the neuron or from a neuron onto other cells. For example, the size of motoneurons usually reflects the size of the motor unit that is controlled by the motoneuron. Invertebrates often have giant neurons and axons that provide special functions such as rapid action potential propagation. Mammals also use this trick for increasing the speed of signals in the nervous system, but they can also use myelin to accomplish this, so most human neurons are relatively small cells.

Other experimental systems for the study of cell size regulation

One common means to produce very large cells is by cell fusion to form syncytia. For example, very long (several inches) skeletal muscle cells are formed by fusion of thousands of myocytes. Genetic studies of the fruit fly Drosophila have revealed several genes that are required for the formation of multinucleated muscle cells by fusion of myoblasts. Some of the key proteins are important for cell adhesion between myocytes and some are involved in adhesion-dependent cell-to-cell signal transduction that allows for a cascade of cell fusion events. 

Oocytes can be unusually large cells in species for which embryonic development takes place away from the mother's body. Their large size can be achieved either by pumping in cytosolic components from adjacent cells through cytoplasmic bridges (Drosophila) or by internalization of nutrient storage granules (yolk granules) by endocytosis (frogs). 

Increases in the size of plant cells are complicated by the fact that almost all plant cells are inside of a solid cell wall. Under the influence of certain plant hormones the cell wall can be remodeled, allowing for increases in cell size that are important for the growth of some plant tissues.

Most unicellular organisms are microscopic in size, but there are some giant bacteria and protozoa that are visible to the naked eye. See: Table of cell sizes —Dense populations of a giant sulfur bacterium in Namibian shelf sediments — Large protists of the genus Chaos, closely related to the genus Amoeba.

 

In the rod-shaped bacteria E. coli, Caulobacter crescentus and B. subtilis cell size is controlled by a simple mechanisms in which cell division occurs after a constant volume has been added since the previous division. By always growing by the same amount, cells born smaller or larger than average naturally converge to an average size equivalent to the amount added during each generation.

Cell division

Cell reproduction is asexual. For most of the constituents of the cell, growth is a steady, continuous process, interrupted only briefly at M phase when the nucleus and then the cell divide in two.

The process of cell division, called cell cycle, has four major parts called phases. The first part, called G₁ phase is marked by synthesis of various enzymes that are required for DNA replication. The second part of the cell cycle is the S phase, where DNA replication produces two identical sets of chromosomes. The third part is the G₂ phase in which a significant protein synthesis occurs, mainly involving the production of microtubules that are required during the process of division, called mitosis. The fourth phase, M phase, consists of nuclear division (karyokinesis) and cytoplasmic division (cytokinesis), accompanied by the formation of a new cell membrane. This is the physical division of "mother" and "daughter" cells. The M phase has been broken down into several distinct phases, sequentially known as prophase, prometaphase, metaphase, anaphase and telophase leading to cytokinesis. 

Cell division is more complex in eukaryotes than in other organisms. Prokaryotic cells such as bacterial cells reproduce by binary fission, a process that includes DNA replication, chromosome segregation, and cytokinesis. Eukaryotic cell division either involves mitosis or a more complex process called meiosis. Mitosis and meiosis are sometimes called the two "nuclear division" processes. Binary fission is similar to eukaryote cell reproduction that involves mitosis. Both lead to the production of two daughter cells with the same number of chromosomes as the parental cell. Meiosis is used for a special cell reproduction process of diploid organisms. It produces four special daughter cells (gametes) which have half the normal cellular amount of DNA. A male and a female gamete can then combine to produce a zygote, a cell which again has the normal amount of chromosomes. 

The rest of this article is a comparison of the main features of the three types of cell reproduction that either involve binary fission, mitosis, or meiosis. The diagram below depicts the similarities and differences of these three types of cell reproduction. 

Cell growth

Comparison of the three types of cell division

The DNA content of a cell is duplicated at the start of the cell reproduction process. Prior to DNA replication, the DNA content of a cell can be represented as the amount Z (the cell has Z chromosomes). After the DNA replication process, the amount of DNA in the cell is 2Z (multiplication: 2 x Z = 2Z). During Binary fission and mitosis the duplicated DNA content of the reproducing parental cell is separated into two equal halves that are destined to end up in the two daughter cells. The final part of the cell reproduction process is cell division, when daughter cells physically split apart from a parental cell. During meiosis, there are two cell division steps that together produce the four daughter cells. 

After the completion of binary fission or cell reproduction involving mitosis, each daughter cell has the same amount of DNA (Z) as what the parental cell had before it replicated its DNA. These two types of cell reproduction produced two daughter cells that have the same number of chromosomes as the parental cell. Chromosomes duplicate prior to cell division when forming new skin cells for reproduction. After meiotic cell reproduction the four daughter cells have half the number of chromosomes that the parental cell originally had. This is the haploid amount of DNA, often symbolized as N. Meiosis is used by diploid organisms to produce haploid gametes. In a diploid organism such as the human organism, most cells of the body have the diploid amount of DNA, 2N. Using this notation for counting chromosomes we say that human somatic cells have 46 chromosomes (2N = 46) while human sperm and eggs have 23 chromosomes (N = 23). Humans have 23 distinct types of chromosomes, the 22 autosomes and the special category of sex chromosomes. There are two distinct sex chromosomes, the X chromosome and the Y chromosome. A diploid human cell has 23 chromosomes from that person's father and 23 from the mother. That is, your body has two copies of human chromosome number 2, one from each of your parents.

Chromosomes

Immediately after DNA replication a human cell will have 46 "double chromosomes". In each double chromosome there are two copies of that chromosome's DNA molecule. During mitosis the double chromosomes are split to produce 92 "single chromosomes", half of which go into each daughter cell. During meiosis, there are two chromosome separation steps which assure that each of the four daughter cells gets one copy of each of the 23 types of chromosome.

Sexual reproduction

Though cell reproduction that uses mitosis can reproduce eukaryotic cells, eukaryotes bother with the more complicated process of meiosis because sexual reproduction such as meiosis confers a selective advantage. Notice that when meiosis starts, the two copies of sister chromatids number 2 are adjacent to each other. During this time, there can be genetic recombination events. Parts of the chromosome 2 DNA gained from one parent (red) will swap over to the chromosome 2 DNA molecule that received from the other parent (green). Notice that in mitosis the two copies of chromosome number 2 do not interact. It is these new combinations of parts of chromosomes that provide the major advantage for sexually reproducing organisms by allowing for new combinations of genes and more efficient evolution. However, in organisms with more than one set of chromosomes at the main life cycle stage, sex may also provide an advantage because, under random mating, it produces homozygotes and heterozygotes according to the Hardy-Weinberg ratio.

Disorders

A series of growth disorders can occur at the cellular level and these consequently underpin much of the subsequent course in cancer, in which a group of cells display uncontrolled growth and division beyond the normal limits, invasion (intrusion on and destruction of adjacent tissues), and sometimes metastasis (spread to other locations in the body via lymph or blood).

Measurement methods

The cell growth can be detected by a variety of methods. The cell size growth can be visualized by microscopy, using suitable stains. But the increase of cells number is usually more significant. It can be measured by manual counting of cells under microscopy observation, using the dye exclusion method (i.e. trypan blue) to count only viable cells. Less fastidious, scalable, methods include the use of cytometers, while flow cytometry allows combining cell counts ('events') with other specific parameters: fluorescent probes for membranes, cytoplasm or nuclei allow distinguishing dead/viable cells, cell types, cell differentiation, expression of a biomarker such as Ki67. 

Beside the increasing number of cells, one can be assessed regarding the metabolic activity growth, that is, the CFDA and calcein-AM measure (fluorimetrically) not only the membrane functionality (dye retention), but also the functionality of cytoplasmic enzymes (esterases). The MTT assays (colorimetric) and the resazurin assay (fluorimetric) dose the mitochondrial redox potential. 

All these assays may correlate well, or not, depending on cell growth conditions and desired aspects (activity, proliferation). The task is even more complicated with populations of different cells, furthermore when combining cell growth interferences or toxicity.