What is the importance of the cell cycle to multicellular organisms with respect to tissue repair?

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Within multicellular organisms, tissues are organized communities of cells that work together to carry out a specific function. The exact role of a tissue in an organism depends on what types of cells it contains. For example, the endothelial tissue that lines the human gastrointestinal tract consists of several cell types. Some of these cells absorb nutrients from the digestive contents, whereas others (called goblet cells) secrete a lubricating mucus that helps the contents travel smoothly.

However, the multiple cell types within a tissue don't just have different functions. They also have different transcriptional programs and may well divide at different rates. Proper regulation of these rates is essential to tissue maintenance and repair. The spatial organization of the cells that form a tissue is also central to the tissue's function and survival. This organization depends in part on polarity, or the orientation of particular cells in their place. Of course, external signals from neighboring cells or from the extracellular matrix are also important influences on the arrangement of cells in a tissue.

What Is the Source of New Cells for Tissues?

Without cell division, long-term tissue survival would be impossible. Inside every tissue, cells are constantly replenishing themselves through the process of division, although the rate of turnover may vary widely between different cell types in the same tissue. For example, in adult mammal brains, neurons rarely divide. However, glial cells in the brain continue to divide throughout a mammal's adult life. Mammalian epithelial cells also turn over regularly, typically every few days.

Neurons are not the only cells that lose their ability to divide as they mature. In fact, many differentiated cells lose this ability. To help counteract this loss, tissues maintain stem cells to serve as a reservoir of undifferentiated cells. Stem cells typically have the capacity to mature into many different cell types. Transcription factors — proteins that regulate which genes are transcribed in a cell — appear to be essential to determining the pathway particular stem cells take as they differentiate. For example, both intestinal absorptive cells and goblet cells arise from the same stem cell population, but divergent transcriptional programs cause them to mature into dramatically different cells (Figure 1).

Whenever stem cells are called upon to generate a particular type of cell, they undergo an asymmetric cell division. With asymmetric division, each of the two resulting daughter cells has its own unique life course. In this case, one of the daughter cells has a finite capacity for cell division and begins to differentiate, whereas the other daughter cell remains a stem cell with unlimited proliferative ability.

How Do Non-Growing Tissues Maintain Themselves?

Although most of the tissues in adult organisms maintain a constant size, the cells that make up these tissues are constantly turning over. Therefore, in order for a particular tissue to stay the same size, its rates of cell death and cell division must remain in balance.

A variety of factors can trigger cell death in a tissue. For example, the process of apoptosis, or programmed cell death, selectively removes damaged cells — including those with DNA damage or defective mitochondria. During apoptosis, cellular proteases and nucleases are activated, and cells self-destruct. Cells also monitor the survival factors and negative signals they receive from other cells before initiating programmed cell death. Once apoptosis begins, it proceeds quickly, leaving behind small fragments with recognizable bits of the nuclear material. Specialized cells then rapidly ingest and degrade these fragments, making evidence of apoptosis difficult to detect.

What Cellular Components Support Tissue Structure?

Tissue function depends on more than cell type and proper rates of death and division: It is also a function of cellular arrangement. Both cell junctions and cytoskeletal networks help stabilize tissue architecture. For instance, the cells that make up human epithelial tissue attach to one another through several types of adhesive junctions. Characteristic transmembrane proteins provide the basis for each of the different types of junctions. At these junctions, transmembrane proteins on one cell interact with similar transmembrane proteins on adjacent cells. Special adaptor proteins then connect the resulting assembly to the cytoskeleton of each cell. The many connections formed between junctions and cytoskeletal proteins effectively produces a network that extends over many cells, providing mechanical strength to the epithelium.

The gut endothelium — actually an epithelium that lines the inner surface of the digestive tract — is an excellent example of these structures at work. Here, tight junctions between cells form a seal that prevents even small molecules and ions from moving across the endothelium. As a result, the endothelial cells themselves are responsible for determining which molecules pass from the gut lumen into the surrounding tissues. Meanwhile, adherens junctions based on transmembrane cadherin proteins provide mechanical support to the endothelium. These junctions are reinforced by attachment to an extensive array of actin filaments that underlie the apical — or lumen-facing — membrane. These organized collections of actin filaments also extend into the microvilli, which are the tiny fingerlike projections that protrude from the apical membrane into the gut lumen and increase the surface area available for nutrient absorption. Additional mechanical support comes from desmosomes, which appear as plaque-like structures under the cell membrane, attached to intermediate filaments. In fact, desmosome-intermediate filament networks extend across multiple cells, giving the endothelium sheetlike properties. In addition, within the gut there are stem cells that guarantee a steady supply of new cells that contribute to the multiple cell types necessary for this complex structure to function properly (Figure 2).

How Does the Extracellular Matrix Support Tissue Structure?

The extracellular matrix (ECM) is also critical to tissue structure, because it provides attachment sites for cells and relays information about the spatial position of a cell. The ECM consists of a mixture of proteins and polysaccharides produced by the endoplasmic reticula and Golgi apparatuses of nearby cells. Once synthesized, these molecules move to the appropriate side of the cell — such as the basal or apical face — where they are secreted. Final organization of the ECM then takes place outside the cell.

To understand how the ECM works, consider the two very different sides of the gut endothelium. One side of this tissue faces the lumen, where it comes in contact with digested food. The other side attaches to a specialized ECM support structure called the basal lamina. The basal lamina is composed of collagen and laminin proteins, as well as various other macromolecules. On this side of the endothelium, adhesive junctions attach cells to the ECM. Transmembrane integrin proteins in the junctions bind components of the ECM and recruit signaling proteins to their cytoplasmic sides. From there, the signals travel to the nucleus of each cell.

Conclusion

Tissues are communities of cells that have functions beyond what any single cell type could accomplish. Healthy tissues require the proper mix of cells, and the cells within them must be oriented correctly and dividing at an appropriate rate. In order to coordinate their function, organization, and rates of death and division, the cells in a tissue are constantly processing and responding to signals from one another and from the ECM around them.

The most basic function of the cell cycle is to duplicate accurately the vast amount of DNA in the chromosomes and then segregate the copies precisely into two genetically identical daughter cells. These processes define the two major phases of the cell cycle. DNA duplication occurs during S phase (S for synthesis), which requires 10–12 hours and occupies about half of the cell-cycle time in a typical mammalian cell. After S phase, chromosome segregation and cell division occur in M phase (M for mitosis), which requires much less time (less than an hour in a mammalian cell). M phase involves a series of dramatic events that begin with nuclear division, or mitosis. As discussed in detail in Chapter 18, mitosis begins with chromosome condensation: the duplicated DNA strands, packaged into elongated chromosomes, condense into the much more compact chromosomes required for their segregation. The nuclear envelope then breaks down, and the replicated chromosomes, each consisting of a pair of sister chromatids, become attached to the microtubules of the mitotic spindle. As mitosis proceeds, the cell pauses briefly in a state called metaphase, when the chromosomes are aligned at the equator of the mitotic spindle, poised for segregation. The sudden separation of sister chromatids marks the beginning of anaphase, during which the chromosomes move to opposite poles of the spindle, where they decondense and reform intact nuclei. The cell is then pinched in two by cytoplasmic division, or cytokinesis, and cell division is complete (Figure 17-2).

Most cells require much more time to grow and double their mass of proteins and organelles than they require to replicate their DNA and divide. Partly to allow more time for growth, extra gap phases are inserted in most cell cycles—a G1 phase between M phase and S phase and a G2 phase between S phase and mitosis. Thus, the eucaryotic cell cycle is traditionally divided into four sequential phases: G1, S, G2, and M (Figure 17-3). G1, S, and G2 together are called interphase. In a typical human cell proliferating in culture, interphase might occupy 23 hours of a 24 hour cycle, with 1 hour for M phase.

The two gap phases serve as more than simple time delays to allow cell growth. They also provide time for the cell to monitor the internal and external environment to ensure that conditions are suitable and preparations are complete before the cell commits itself to the major upheavals of S phase and mitosis. The G1 phase is especially important in this respect. Its length can vary greatly depending on external conditions and extracellular signals from other cells. If extracellular conditions are unfavorable, for example, cells delay progress through G1 and may even enter a specialized resting state known as G0 (G zero), in which they can remain for days, weeks, or even years before resuming proliferation. Indeed, many cells remain permanently in G0 until they or the organism dies. If extracellular conditions are favorable and signals to grow and divide are present, cells in early G1 or G0 progress through a commitment point near the end of G1 known as Start (in yeasts) or the restriction point (in mammalian cells). After passing this point, cells are committed to DNA replication, even if the extracellular signals that stimulate cell growth and division are removed.

Some features of the cell cycle, including the time required to complete certain events, vary greatly from one cell type to another, even in the same organism. The basic organization of the cycle and its control system, however, are essentially the same in all eucaryotic cells. The proteins of the control system first appeared over a billion years ago. Remarkably, they have been so well conserved over the course of evolution that many of them function perfectly when transferred from a human cell to a yeast cell. We can therefore study the cell cycle and its regulation in a variety of organisms and use the findings from all of them to assemble a unified picture of how eucaryotic cells divide. In the following section, we briefly review the three eucaryotic systems in which cell-cycle control is commonly studied—yeasts, frog embryos, and cultured mammalian cells.

Yeasts are tiny, single-celled fungi whose mechanisms of cell-cycle control are remarkably similar to our own. Two species are generally used in studies of the cell cycle. The fission yeast Schizosaccharomyces pombe is named after the African beer it is used to produce. It is a rod-shaped cell that grows by elongation at its ends. Division occurs by the formation of a septum, or cell plate, in the center of the rod (Figure 17-4A). The budding yeast Saccharomyces cerevisiae is used by brewers, as well as by bakers. It is an oval cell that divides by forming a bud, which first appears during G1 and grows steadily until it separates from the mother cell after mitosis (Figure 17-4B).

Despite their outward differences, the two yeast species share a number of features that are extremely useful for genetic studies. They reproduce almost as rapidly as bacteria and have a genome size less than 1% that of a mammal. They are amenable to rapid molecular genetic manipulation, whereby genes can be deleted, replaced, or altered. Most importantly, they have the unusual ability to proliferate in a haploid state, in which only a single copy of each gene is present in the cell. When cells are haploid, it is easy to isolate and study mutations that inactivate a gene, as one avoids the complication of having a second copy of the gene in the cell.

Many important discoveries about cell-cycle control have come from systematic searches for mutations in yeasts that inactivate genes encoding essential components of the cell-cycle control system. The genes affected by these mutations are known as cell-division-cycle genes, or cdc genes. Many of these mutations cause cells to arrest at a specific point in the cell cycle, suggesting that the normal gene product is required to get the cell past this point.

A mutant that cannot complete the cell cycle cannot be propagated. Thus, cdc mutants can be selected and maintained only if their phenotype is conditional—that is, if the gene product fails to function only in certain specific conditions. Most conditional cell-cycle mutations are temperature-sensitive mutations, in which the mutant protein fails to function at high temperatures but functions well enough to allow cell division at low temperatures. A temperature-sensitive cdc mutant can be propagated at a low temperature (the permissive condition) and then raised to a higher temperature (the restrictive condition) to switch off the function of the mutant gene. At the higher temperature, the cells continue through the cell cycle until they reach the point where the function of the mutant gene is required for further progress, and at this point they halt (Figure 17-5). In budding yeasts, a uniform cell-cycle arrest of this type can be detected by just looking at the cells: the presence or absence of a bud, and bud size, indicate the point in the cycle at which the mutant is arrested (Figure 17-6).

While yeasts are ideal for studying the genetics of the cell cycle, the biochemistry of the cycle is most easily analyzed in the giant fertilized eggs of many animals, which carry large stockpiles of the proteins needed for cell division. The egg of the frog Xenopus, for example, is over 1 mm in diameter and carries 100,000 times more cytoplasm than an average cell in the human body (Figure 17-7). Fertilization of the Xenopus egg triggers an astonishingly rapid sequence of cell divisions, called cleavage divisions, in which the single giant cell divides, without growing, to generate an embryo containing thousands of smaller cells (Figure 17-8). In this process, almost the only macromolecules synthesized are DNA—required to produce the thousands of new nuclei—and a small amount of protein. After a first division that takes about 90 minutes, the next 11 divisions occur, more or less synchronously, at 30-minute intervals, producing about 4096 (212) cells within 7 hours. Each cycle is divided into S and M phases of about 15 minutes each, without detectable G1 or G2 phases.

The cells in early embryos of Xenopus, as well as those of the clam Spisula and the fruit fly Drosophila, are thus capable of exceedingly rapid division in the absence of either growth or many of the control mechanisms that operate in more complex cell cycles. These early embryonic cell cycles therefore reveal the workings of the cell-cycle control system stripped down and simplified to the minimum needed to achieve the most fundamental requirements—the duplication of the genome and its segregation into two daughter cells. Another advantage of these early embryos for cell-cycle analysis is their large size. It is relatively easy to inject test substances into an egg to determine their effect on cell-cycle progression. It is also possible to prepare almost pure cytoplasm from Xenopus eggs and reconstitute many events of the cell cycle in a test tube (Figure 17-9). In such cell extracts, one can observe and manipulate cell-cycle events under highly simplified and controllable conditions.

It is not easy to observe individual cells in an intact mammal. Most studies on mammalian cell-cycle control therefore use cells that have been isolated from normal tissues or tumors and grown in plastic culture dishes in the presence of essential nutrients and other factors (Figure 17-10). There is a complication, however. When cells from normal mammalian tissues are cultured in standard conditions, they often stop dividing after a limited number of division cycles. Human fibroblasts, for example, permanently cease dividing after 25–40 divisions, a process called replicative cell senescence, which we discuss later.

Mammalian cells occasionally undergo mutations that allow them to proliferate readily and indefinitely in culture as “immortalized” cell lines. Although they are not normal, such cell lines are used widely for cell-cycle studies—and for cell biology generally—because they provide an unlimited source of genetically homogeneous cells. In addition, these cells are sufficiently large to allow detailed cytological observations of cell-cycle events, and they are amenable to biochemical analysis of the proteins involved in cell-cycle control.

Studies of cultured mammalian cells have been especially useful for examining the molecular mechanisms governing the control of cell proliferation in multicellular organisms. Such studies are important not only for understanding the normal controls of cell numbers in tissues but also for understanding the loss of these controls in cancer (discussed in Chapter 23).

How can one tell at what stage an animal cell is in the cell cycle? One way is to simply look at living cells with a microscope. A glance at a population of mammalian cells proliferating in culture reveals that a fraction of the cells have rounded up and are in mitosis. Others can be observed in the process of cytokinesis. The S-phase cells, however, cannot be detected by simple observation. They can be recognized, however, by supplying them with visualizable molecules that are incorporated into newly synthesized DNA, such as 3H-thymidine or the artificial thymidine analog bromo-deoxyuridine (BrdU). Cell nuclei that have incorporated 3H-thymidine are visualized by autoradiography (Figure 17-11A), whereas those that have incorporated BrdU are visualized by staining with anti-BrdU antibodies (Figure 17-11B).

Typically, in a population of cells that are all proliferating rapidly but asynchronously, about 30–40% will be in S phase at any instant and become labeled by a brief pulse of 3H-thymidine or BrdU. From the proportion of cells in such a population that are labeled (the labeling index), one can estimate the duration of S phase as a fraction of the whole cell cycle duration. Similarly, from the proportion of these cells in mitosis (the mitotic index), one can estimate the duration of M phase. In addition, by giving a pulse of 3H-thymidine or BrdU and allowing the cells to continue around the cycle for measured lengths of time, one can determine how long it takes for an S-phase cell to progress through G2 into M phase, through M phase into G1, and finally through G1 back into S phase.

Another way to assess the stage that a cell has reached in the cell cycle is by measuring its DNA content, which doubles during S phase. This approach is greatly facilitated by the use of DNA-binding fluorescent dyes and a flow cytometer, which allows large numbers of cells to be analyzed rapidly and automatically (Figure 17-12). One can also use flow cytometry to determine the lengths of G1, S, and G2 + M phases, by following over time a population of cells that have been preselected to be in one particular phase of the cell cycle: DNA content measurements on such a synchronized population of cells reveal how the cells progress through the cycle.

Cell reproduction begins with duplication of the cell's contents, followed by distribution of those contents into two daughter cells. Chromosome duplication occurs during S phase of the cell cycle, whereas most other cell components are duplicated continuously throughout the cycle. During M phase, the replicated chromosomes are segregated into individual nuclei (mitosis), and the cell then splits in two (cytokinesis). S phase and M phase are usually separated by gap phases called G1 and G2, when cell-cycle progression can be regulated by various intracellular and extracellular signals. Cell-cycle organization and control have been highly conserved during evolution, and studies in a wide range of systems—including yeasts, frog embryos, and mammalian cells in culture—have led to a unified view of eucaryotic cell-cycle control.