Cell Biology 2: Cellular Processes

The microscopes we use today are far more complex than those used in the 1600s by Antony van Leeuwenhoek, a Dutch shopkeeper who had great skill in crafting lenses. Despite the limitations of his now-ancient lenses, van Leeuwenhoek observed the movements of protista (a type of single-celled organism) and sperm, which he collectively termed “animalcules”.

In a 1665 publication called Micrographia, experimental scientist Robert Hooke coined the term “cell” for the box-like structures he observed when viewing cork tissue through a lens. In the 1670s, van Leeuwenhoek discovered bacteria and protozoa. Later advances in lenses, microscope construction, and staining techniques enabled other scientists to see some components inside cells.

By the late 1830s, botanist Matthias Schleiden and zoologist Theodor Schwann were studying tissues and proposed the unified cell theory. The unified cell theory states that: all living things are composed of one or more cells; the cell is the basic unit of life; and new cells arise from existing cells. Rudolf Virchow later made important contributions to this theory.

Schleiden and Schwann proposed spontaneous generation as the method for cell origination, but spontaneous generation (also called abiogenesis) was later disproven. Rudolf Virchow famously stated “Omnis cellula e cellula” (all cells only arise from pre-existing cells). The parts of the theory that did not have to do with the origin of cells, however, held up to scientific scrutiny and are widely agreed upon by the scientific community today. The generally accepted portions of the modern cell theory are as follows:

• The cell is the fundamental unit of structure and function in living things.
• All organisms are made up of one or more cells.
• Cells arise from other cells through cellular division.

The expanded version and modern interpretation of the cell theory can also include:

• Cells carry genetic material passed to daughter cells during cellular division
• All cells are essentially the same in chemical composition
• Energy flow (metabolism and biochemistry) occurs within cells

Movement into and out of cells

One of the most important functions of the plasma membrane (a semipermeable membrane) is to control the movement of molecules into and out of the cell, as internal conditions must be maintained within a certain range despite any changes in the external environment. The regulation of transport of substances across the plasma membrane allows cells to maintain the intracellular environment within homeostatic ranges.

Cells use various modes of transport across the plasma membrane. Some small molecules, like carbon dioxide or water, may cross the membrane bilayer directly by simple diffusion. Simple diffusion is the movement of molecules from an area of higher concentration of molecules to an area of lower concentration of molecules, driven solely by the random motion of particles (Figure 2.2). This process does not require energy (such as ATP) and occurs directly through the phospholipid bilayer of the cell membrane. Only small, nonpolar molecules can easily diffuse in this manner due to their ability to pass through the hydrophobic core of the membrane. Passive transport, on the other hand, encompasses all forms of transport across the membrane that do not require energy, including simple diffusion. It also includes facilitated diffusion, where molecules move down their concentration gradient with the help of specific membrane proteins, such as channel proteins or carrier proteins. These proteins assist in the movement of larger, polar, or charged molecules, like glucose or ions, which cannot pass through the lipid bilayer on their own. Both simple diffusion and facilitated diffusion are vital for processes such as nutrient uptake, waste removal, and gas exchange in cells. By relying on the natural movement of molecules along concentration gradients, passive transport allows cells to efficiently regulate their internal environments without expending energy. (Figure 2.2).

Molecules other than water, such as charged molecules and large molecules, need the help of carriers or channels in the membrane. These carriers or channels serve to ferry molecules across the membrane; a process known as facilitated diffusion and doesn’t require energy to work (Figure 2.3).

Osmosis, the movement of water, is a special type of passive transport process which protects the cell from changes in osmotic pressure (Figure 2.4). Osmotic pressure occurs because of differences in the concentration of solutes, such as salts, sugars, and other dissolved molecules, on opposing sides of a semipermeable membrane. While water can freely pass through a semipermeable membrane, but solutes (dissolved molecules like salts, sugars and other compounds) cannot. When one side of the membrane has a higher concentration of solutes, and therefore less water, compared to the other side, water will move across the membrane from the side with lower solute concentration (more water) to the side with higher solute concentration (less water). This diffusion, or movement, of water which is called osmosis continues until the concentrations of solutes on both sides of the membrane become equal. This can cause extreme osmotic pressure on a cell when its external environment changes relative to the concentration inside the cell.

Imagine a cell in a solution where the extracellular space (outside the cell) has a higher concentration of salt compared to the intracellular space (inside the cell). Since the cell membrane is semipermeable, water can move across it, but the salt cannot. Because there is more salt outside the cell, water will move from inside the cell (where there is more water and less salt) to the outside (where there is less water and more salt) in an attempt to balance the concentrations. If too much water leaves the cell, it can shrink, leading to potential stress or damage to the cell.

Now, imagine the opposite scenario, where the cell is in a solution where the extracellular space (outside the cell) has a lower concentration of salt compared to the intracellular space (inside the cell). Because there is more salt inside the cell, water will move from outside the cell into the cell to try to balance the concentrations. If too much water enters the cell, it can swell and, in extreme cases, may even burst, causing damage or death to the cell.

Tonicity

The degree to which a particular cell can withstand changes in osmotic pressure is called tonicity (the tone of the cell and how it changes shape). Cells that have a cell wall are better able to withstand subtle changes in osmotic pressure and maintain their shape. The external environment surrounding a cell can be described as an isotonic, hypertonic, or hypotonic medium depending on the relative concentration of solutes in the environment compared to those inside the cell. These terms are always defined in relation to the cell’s internal concentrations, as they determine how water will move across the cell membrane, affecting the cell’s shape, size, and function.

In an isotonic medium, the solute concentrations inside and outside the cell are approximately equal, so there is no net movement of water across the cell membrane and the osmotic pressure is balanced. As a result, the cell maintains its normal shape and volume, without swelling or shrinking, because water moves in and out of the cell at equal rates. In a hypertonic medium, the solute concentration outside the cell exceeds that inside the cell, so water diffuses out of the cell and into the external medium (dehydration of the cell). Cells that lack a cell wall can become dehydrated, causing crenation, or shrivelling of the cell; the plasma membrane contracts and appears scalloped or notched, leading to potential cell damage or dysfunction. By contrast, cells that possess a cell wall undergo plasmolysis rather than crenation. In plasmolysis, the plasma membrane contracts and detaches from the cell wall and there is a decrease in interior volume, but the cell wall remains intact, thus allowing the cell to maintain some shape and integrity for a period of time. In a hypotonic medium, the solute concentration inside the cell exceeds that outside of the cell, so water will move by osmosis into the cell. This influx of water can cause the cell to swell and potentially burst, a process called lysis in animal cells or turgor pressure in plant cells, which helps maintain plant cell rigidity. Cells that lack a cell wall are more prone to lysis in hypotonic environments. The presence of a cell wall allows the cell to maintain its shape and integrity for a longer time before lysing (Figure 2.5).

Active transport

Active transport does need energy to move molecules across the cell membrane against a concentration gradient (Figure 2.6). A major difference between passive and active transport is that active transport requires adenosine triphosphate (ATP) or other forms of energy to move molecules “uphill.” Therefore, active transport structures are often called “pumps.”

The sodium-potassium pump, which is also called Na+/K+ ATPase, transports sodium out of a cell while moving potassium into the cell. The Na+/K+ pump is an important ion pump found in the membranes of many types of cells. These pumps are particularly abundant in nerve cells, which are constantly pumping out sodium ions and pulling in potassium ions to maintain an electrical gradient across their cell membranes. An electrical gradient is a difference in electrical charge across a space. In the case of nerve cells, for example, the electrical gradient exists between the inside and outside of the cell, with the inside being negatively charged (at around -70 mV) relative to the outside. The negative electrical gradient is maintained because each Na+/K+ pump moves three Na+ ions out of the cell and two K+ ions into the cell for each ATP molecule that is used (Figure 2.6). This process is so important for nerve cells that it accounts for the majority of their ATP usage.

Active transport pumps can also work together with other active or passive transport systems to move substances across the membrane. For example, the sodium-potassium pump maintains a high concentration of sodium ions outside of the cell. Therefore, if the cell needs sodium ions, all it has to do is open a passive sodium channel, as the concentration gradient of the sodium ions will drive them to diffuse into the cell. In this way, the action of an active transport pump (the sodium-potassium pump) powers the passive transport of sodium ions by creating a concentration gradient. When active transport powers the transport of another substance in this way, it is called secondary active transport.

Symporters are secondary active transporters that move two substances in the same direction. For example, the sodium-glucose symporter uses sodium ions to “pull” glucose molecules into the cell. Because cells store glucose for energy, glucose is typically at a higher concentration inside of the cell than outside. However, due to the action of the sodium-potassium pump, sodium ions will easily diffuse into the cell when the symporter is opened. The flood of sodium ions through the symporter provides the energy that allows glucose to move through the symporter and into the cell, against its concentration gradient.

Conversely, antiporters are secondary active transport systems that transport substances in opposite directions. For example, the sodium-hydrogen ion antiporter uses the energy from the inward flood of sodium ions to move hydrogen ions (H+) out of the cell. The sodium-hydrogen antiporter is used to maintain the pH of the cell’s interior.

Endocytosis

The processes of simple diffusion, facilitated diffusion, and active transport are used in both eukaryotic and prokaryotic cells. However, eukaryotic cells also have the unique ability to perform diverse types of endocytosis, which is a type of vesicle transport that moves a substance into the cell through plasma membrane invagination and vacuole/vesicle formation (Figure 2.7). Vesicles and vacuoles are membrane-bound sacs that function in storage and transport. Vacuoles are somewhat larger than vesicles, and the membrane of a vacuole does not fuse with the membranes of other cellular components. Vesicles can fuse with other membranes within the cell system. Endocytosis is used by all cells of the body because most substances important to them are polar and consist of big molecules and thus cannot pass through the hydrophobic plasma membrane.

A type of endocytosis involving the engulfment of large particles through membrane invagination is called phagocytosis, which means “cell eating.” In phagocytosis, particles such as dead cells, cellular debris, pathogens or even other cells, are enclosed in a pocket within the membrane, which then pinches off from the membrane to form a vacuole that surrounds the particle. This vacuole is digested by the cell, effectively destroying and “cleaning up” the area.  Another type of endocytosis is called pinocytosis, which means “cell drinking.” Pinocytosis is a cellular process in which a cell engulfs extracellular fluid, along with dissolved nutrients and molecules, by forming small vesicles from its membrane, allowing the cell to absorb and utilise these essential substances.

Phagocytosis and pinocytosis take in large portions of extracellular material, and they are typically not highly selective in the substances they bring in. Cells regulate the endocytosis of specific substances via receptor-mediated endocytosis. Receptor-mediated endocytosis is a type of endocytosis that is initiated by ligands, molecules such as a hormones, neurotransmitters, or ions, that bind specifically to a receptor on the surface of or within a cell, initiating a series of events that trigger a biological response. Receptor-mediated endocytosis is the mechanism that peptide and amine-derived hormones use to enter cells and is also used by various viruses and bacteria for entry into host cells. Once the ligand-receptor complex forms, the cell membrane folds inward, forming a vesicle that encloses this complex and brings it into the cell. This process allows the cell to efficiently capture specific molecules from the extracellular environment. The internalised vesicle then typically fuses with endosomes or lysosomes, where the captured molecules are processed or degraded. If passage across the membrane of the target of receptor-mediated endocytosis is ineffective, it will not be removed from the tissue fluids or blood. Instead, it will stay in those fluids and increase in concentration. Some human diseases are caused by a failure of receptor-mediated endocytosis. For example, the carrier of cholesterol termed low-density lipoprotein or LDL (also commonly referred to as “bad” cholesterol) is removed from the blood by receptor-mediated endocytosis. In the human genetic disease familial hypercholesterolemia, the LDL receptors are defective or missing entirely. People with this condition have life-threatening levels of cholesterol in their blood because their cells cannot clear the chemical from their blood.

Exocytosis

The process by which secretory vesicles release their contents to the cell’s exterior is called exocytosis (Figure 2.8). Vesicles containing substances such as proteins, hormones, neurotransmitters, or waste products form within the endomembrane system and migrate towards the plasma membrane to fuse with it, ejecting their contents out of the cell. This process is tightly regulated and is essential for a variety of cellular functions, not just waste removal. For example, in nerve cells, exocytosis is crucial for the release of neurotransmitters that transmit signals between neurons. In secretory cells, such as those in the pancreas, exocytosis is responsible for releasing hormones like insulin into the bloodstream. Additionally, exocytosis plays a role in the insertion of membrane proteins and lipids into the cell membrane, contributing to its renewal and expansion.

Information flow within the cell

A gene is a fundamental unit of heredity that carries the instructions necessary for building and maintaining an organism’s cells and for passing traits from one generation to the next. It encodes the information required to produce specific molecules which play crucial roles in the structure, function, and regulation of the body’s tissues and organs. Genes are organised on chromosomes and are responsible for passing genetic information from parents to offspring, influencing a wide array of characteristics, from physical traits like eye colour to complex behaviours and disease susceptibilities. A gene is composed of DNA that is “read” or transcribed to produce an RNA molecule during the process of transcription. One major type of RNA molecule, called messenger RNA (mRNA), provides the information for the ribosome to catalyse protein synthesis in a process called translation. The processes of transcription and translation are collectively referred to as gene expression. Gene expression is the synthesis of a specific protein with a sequence of amino acids that is encoded in the gene. The flow of genetic information from DNA to RNA to protein is described by the central dogma (Figure 2.9).

The DNA molecules never leave the nucleus and instead use an intermediary molecule to communicate with the rest of the cell. This is the messenger RNA (mRNA). When proteins need to be made, the mRNA enters the nucleus and attaches itself to one of the DNA strands. Being complementary, the sequence of nitrogen bases of the RNA is opposite that of the DNA. This is called transcription. For example, if the DNA strand reads TCCAAGTC, then the mRNA strand would read AGGUUCAG. The mRNA then carries the code out of the nucleus to organelles called ribosomes for the assembly of proteins.

Once the mRNA has reached the ribosomes, they do not read the instructions directly. Instead, another type of RNA called transfer RNA (tRNA) needs to translate the information from the mRNA into a usable form. The tRNA attaches to the mRNA, but with the opposite base pairings. It then reads the sequence in sets of three bases called codons. Each possible three letter arrangement of A,C,U,G (e.g., AAA, AAU, GGC, etc) is a specific instruction, and the correspondence of these instructions and the amino acids is known as the “genetic code”. Though exceptions to or variations on the code exist, the standard genetic code holds true in most organisms, and consists of 64 codons, with each codon specifying one of the 20 standard amino acids or a stop signal.

The ribosome acts like a giant clamp, holding all of the players in position, and facilitating both the pairing of bases between the messenger and transfer RNAs, and the chemical bonding between the amino acids. The ribosome has special subunits known as ribosomal RNAs (rRNA) because they function in the ribosome. These subunits do not carry instructions for making a specific protein (i.e., they are not messenger RNAs) but instead are an integral part of the ribosome machinery that is used to make proteins from mRNAs. The making of proteins by reading instructions in mRNA is generally known as translation (Figure 2.10).

Commonly, an mRNA transcription will be translated simultaneously by several adjacent ribosomes. This increases the efficiency of protein synthesis. A single ribosome might translate an mRNA molecule in approximately one minute; so multiple ribosomes aboard a single transcript could produce multiple times the number of the same protein in the same minute. A polyribosome is a string of ribosomes translating a single mRNA strand.

A human, as well as every sexually reproducing organism, begins life as a fertilised egg or zygote. Trillions of cell divisions subsequently occur in a controlled manner to produce a complex, multicellular human. In other words, that original single cell is the ancestor of every other cell in the body. Once a being is fully grown, cell reproduction is still necessary to repair or regenerate tissues. For example, new blood and skin cells are constantly being produced. All multicellular organisms use cell division for growth, maintenance, and repair of cells and tissues. Cell division is tightly regulated, and the occasional failure of regulation can have life-threatening consequences. Single-celled organisms use cell division as their method of reproduction.

Cells in the body replace themselves over the lifetime of a person. For example, the cells lining the gastrointestinal tract must be frequently replaced when constantly “worn off” by the movement of food through the gut. But what triggers a cell to divide, and how does it prepare for and complete cell division? The cell cycle is the sequence of events in the life of the cell from the moment it is created at the end of a previous cycle of cell division until it then divides itself, generating two new cells . The cell cycle and cell division are fundamental processes in biology that govern the growth, development, and reproduction of cells, ensuring the proper replication and distribution of genetic material to maintain the structure and function of living organisms. These processes are tightly regulated by a series of checkpoints and molecular signals that monitor the cell’s internal and external environment, ensuring that DNA is accurately replicated and that any errors or damage are corrected before the cell divides. This regulatory system involves complex interactions between proteins, enzymes, and signalling pathways that work together to maintain cellular integrity and prevent uncontrolled cell division, which can lead to conditions such as cancer.

Cell Cycle

Throughout the life of a cell, many processes are undertaken including cell proliferation (growth and division), differentiation, cellular movement and programmed cell death or apoptosis. There are many processes, checks and balances to ensure the cell cycles proceed as precisely and smoothly are possible and for apoptosis to occur when needed and appropriate.

The cell cycle is a highly ordered series of events involving cell growth and cell division that produces two new daughter cells. This mitotic replication happens to almost every cell in the body, except for sex cells or gametes. Gametes replicate through a slightly different process called meiosis. Mitotic cell division involves a series of precisely timed and carefully regulated stages of growth, DNA replication and division that produce two genetically identical cells. The cell cycle has two major phases: interphase and the mitotic phase (Figure 2.11). During interphase, the cell grows, completes normal metabolic activities, and the DNA is replicated. During the mitotic phase, the replicated DNA and cytoplasmic contents are separated and the cell divides. The cell cycle is completed in about 24 hours in most human cells however some cell types have slower or faster cell cycle times and there are a number of controls and checkpoints to ensure the cell cycle is completed correctly.

During interphase, the cell undergoes normal processes while also preparing for cell division. For a cell to move from interphase to the mitotic phase, many internal and external conditions must be met. The three stages of interphase are called the first gap (G1), phase of DNA synthesis (S phase), and the second gap (G2). During interphase, there are no visible changes to the cell’s morphology.

Interphase: G1 phase

The G1 phase, or the first gap phase, is a crucial period in the cell cycle that occurs immediately after the cell has completed mitosis and cytokinesis, and before the S phase, where DNA replication begins. One of the primary roles of the G1 phase is cell growth. During this phase, the cell increases in size, creating a variety of proteins and producing new organelles. The cell membrane expands, and the cytoplasm becomes more substantial as the cell accumulates the resources it will need for later stages of the cell cycle. The cell also synthesises RNA, which is essential for the production of proteins. These proteins include enzymes required for DNA replication and other molecules necessary for cellular functions. In addition, the G1 phase is characterised by its role in monitoring the internal and external environment to determine whether conditions are favourable for the cell to proceed with division. The cell undergoes a series of checkpoints during G1, most notably the G1/S checkpoint, also known as the restriction point in some organisms. At this checkpoint, the cell evaluates several critical factors such as nutrient availability, DNA integrity, growth signals, and size of the cell. The G1 phase is also a time when the cell can differentiate into a specific cell type if it receives the appropriate signals. In some cases, if a cell is not ready to proceed to the S phase, it can enter the G0 phase, a quiescent state where it can remain for an extended period. In G0, the cell carries out its specialised functions without preparing for division. This phase is common in cells that do not divide frequently, such as neurons and muscle cells.

Interphase: S phase

The S phase, or synthesis phase, is a critical stage of the cell cycle during which DNA replication occurs, ensuring that each daughter cell will receive an identical set of genetic material after cell division. This phase follows the G1 phase and represents one of the most vital processes in cell biology, as it ensures the accurate duplication of the cell’s genome. During the S phase, the entire DNA content of the cell’s nucleus is duplicated. Each chromosome, which originally consists of a single DNA molecule, is replicated to produce two identical DNA molecules. These identical copies, known as sister chromatids, remain connected at a region called the centromere. The replication process is highly regulated and involves multiple steps and key enzymes to ensure that the DNA is copied with high fidelity. The DNA replication begins at specific locations on the DNA molecule called origins of replication. These origins are recognised by proteins that bind to the DNA and initiate the unwinding of the double helix, creating replication forks where the actual synthesis of new DNA strands occurs. Helicase enzymes unwind the DNA helix, separating the two strands to allow each strand to serve as a template for the synthesis of a new complementary strand. DNA polymerase is the primary enzyme responsible for adding nucleotides to the growing DNA strand. It reads the template strand and incorporates the complementary nucleotides (A pairs with T, and C pairs with G) to form a new strand. The synthesis of new DNA is semi-conservative, meaning that each of the two resulting DNA molecules consists of one original strand and one newly synthesised strand. This mechanism ensures that the genetic information is preserved in each daughter cell. DNA replication occurs in a bidirectional manner, with two replication forks moving away from the origin in opposite directions. On one strand, known as the leading strand, DNA is synthesised continuously in the same direction as the replication fork. The other strand, known as the lagging strand, DNA is synthesised in short fragments called Okazaki fragments because the synthesis occurs in the opposite direction to the replication fork. These fragments are later joined together by the enzyme DNA ligase to form a continuous strand. DNA polymerase has a proofreading ability, which allows it to detect and correct errors in nucleotide incorporation. If an incorrect nucleotide is added, the enzyme can remove it and replace it with the correct one, significantly reducing the error rate during replicaDNA ligasetion. Additional repair mechanisms operate during and after the S phase to identify and fix any remaining errors or DNA damage, ensuring the integrity of the genome.

In addition to DNA replication, the S phase is also when the cell duplicates its centrioles, which are essential for the formation of the mitotic spindle during cell division. Centrioles are not present in the centrosomes of many eukaryotic species, such as plants and most fungi. The mitotic spindle is responsible for separating the sister chromatids into the two daughter cells during mitosis. By the end of the S phase, the cell has successfully duplicated its entire genome, resulting in two complete sets of chromosomes. Each chromosome now consists of two sister chromatids that are held together at the centromere. This ensures that during mitosis, each daughter cell will receive an exact copy of the genetic material, maintaining genetic continuity across generations of cells. The completion of the S phase is critical for the cell’s progression to the G2 phase, where further preparations for mitosis occur. Any errors or issues that arise during the S phase, such as incomplete replication or DNA damage, must be resolved before the cell can proceed to ensure that cell division occurs accurately and without introducing mutations or genomic instability.

Interphase: G2 phase

The G2 phase, or second gap phase, is a crucial stage in the cell cycle that occurs after the completion of DNA replication in the S phase and before the onset of mitosis. This phase is dedicated to ensuring that the cell is fully prepared for the complex process of mitosis, during which the cell will divide to produce two genetically identical daughter cells. Additionally, the G2 phase features critical checkpoints that help regulate the cell’s progression to mitosis, ensuring that division occurs accurately and efficiently. Throughout the G2 phase, the cell continues to grow, increasing in size and accumulating the resources necessary for mitosis. The cell generates a wide range of proteins that are essential for the upcoming mitotic events, including those that will form the mitotic spindle, a structure crucial for the segregation of chromosomes during cell division. The cell also expands its cytoplasmic volume and produces additional organelles, ensuring that each daughter cell will have sufficient cellular machinery and resources to function properly after division.

One of the primary functions of the G2 phase is to verify that the cell’s DNA has been accurately replicated and that there are no errors or damage that could compromise genomic integrity. The cell’s DNA repair mechanisms are highly active during this phase, identifying and correcting any replication errors or damage that may have occurred during the S phase. The G2 phase features a crucial regulatory point known as the G2/M checkpoint. At this checkpoint, the cell rigorously evaluates the integrity of its DNA to ensure that all chromosomes have been fully and correctly replicated. The cell checks for any DNA damage, incomplete replication, or structural abnormalities. If any issues are detected, the cell may pause its progression through the cell cycle, giving it time to repair the damage before moving on to mitosis. If the damage is beyond repair, the cell may initiate apoptosis, or programmed cell death, to prevent the propagation of defective genetic material.

During the G2 phase, the cell begins to assemble the mitotic spindle apparatus, which is responsible for separating the sister chromatids during mitosis. The centrioles, which were duplicated in the S phase, migrate to opposite poles of the cell, where they will serve as the organising centres for the spindle fibres. These will attach to the chromosomes and facilitate their movement during mitosis. The cell also reorganises its cytoskeleton in preparation for cytokinesis, the process of physically dividing the cell into two daughter cells. This ensures that the cell’s contents, including organelles and cytoplasm, are evenly distributed between the two new cells. The G2 phase ensures that all critical organelles, such as mitochondria, endoplasmic reticulum, and Golgi apparatus, are duplicated and appropriately positioned within the cell. This duplication is essential for maintaining the metabolic functions of the daughter cells after division. Proper distribution of these organelles is necessary to ensure that each daughter cell is equipped with the necessary components to survive and function effectively. Although the actual condensation of chromosomes into visible structures occurs during the early stages of mitosis (specifically during prophase), the G2 phase involves initial steps that prepare the chromatin for condensation. This preparation ensures that chromosomes will condense correctly and align properly on the metaphase plate during mitosis, facilitating accurate segregation of genetic material.

The G2/M checkpoint is one of the most critical regulatory points in the cell cycle. It serves as a final quality control step before the cell commits to mitosis. This checkpoint ensures that the cell is fully prepared for division by assessing several key factors. The checkpoint verifies that the DNA has been completely and accurately replicated during the S phase. It checks for any DNA damage or errors that need to be repaired. If the checkpoint detects damage or incomplete replication, it halts the cell cycle, allowing time for repair mechanisms to address the issues. This ensures that only cells with intact and complete genomes proceed to mitosis, reducing the risk of mutations and genomic instability in the daughter cells. The checkpoint also ensures that the cell has grown sufficiently and has accumulated the necessary resources, such as proteins and organelles, to support the division process. A cell that has not reached an adequate size or lacks sufficient resources may be delayed at this checkpoint until it is ready to proceed. The checkpoint assesses the readiness of the cell’s mitotic machinery, including the assembly of the mitotic spindle and the organisation of the cytoskeleton. This ensures that the cell is fully equipped to carry out the complex processes of chromosome segregation and cytokinesis. If the cell successfully passes the G2/M checkpoint, it transitions into the mitotic phase (M phase), where the process of mitosis begins.

Mitotic phase

The mitotic phase of the  cell typically takes between 1 and 2 hours. During this phase, a cell undergoes two major processes. First, it completes mitosis, during which the contents of the nucleus are equitably pulled apart and distributed between its two halves. Cytokinesis then occurs, dividing the cytoplasm and cell body into two new cells. Mitosis is divided into four major stages that take place after interphase (Figure 2.12) and in the following order: prophase, metaphase, anaphase, and telophase. The process is then followed by cytokinesis.

Prophase

Prophase is the first phase of mitosis, during which the loosely packed chromatin coils and condenses into visible chromosomes. During prophase, each chromosome becomes visible with its identical partner attached, forming the familiar X-shape of sister chromatids. The nucleolus disappears early during this phase, and the nuclear envelope also disintegrates.

A major occurrence during prophase concerns a very important structure that contains the origin site for microtubule growth. Recall the cellular structures called centrioles that serve as origin points from which microtubules extend. These tiny structures also play a very important role during mitosis. A centrosome is a pair of centrioles together. The cell contains two centrosomes side-by-side, which begin to move apart during prophase. As the centrosomes migrate to two different sides of the cell, microtubules begin to extend from each like long fingers from two hands extending toward each other. The mitotic spindle is the structure composed of the centrosomes and their emerging microtubules.

Near the end of prophase there is an invasion of the nuclear area by microtubules from the mitotic spindle. The nuclear membrane has disintegrated, and the microtubules attach themselves to the centromeres that adjoin pairs of sister chromatids. The kinetochore is a protein structure on the centromere that is the point of attachment between the mitotic spindle and the sister chromatids. This stage is referred to as late prophase or “prometaphase” to indicate the transition between prophase and metaphase.

Metaphase

Metaphase is a critical stage of mitotic division as it plays a pivotal role in ensuring that chromosomes are accurately and evenly distributed to the two daughter cells. During metaphase, the chromosomes, which have already condensed and attached to the mitotic spindle, align precisely along the metaphase plate, also known as the equatorial plane, an imaginary plane between the two spindle poles. This alignment is essential for the subsequent segregation of chromosomes and is tightly regulated by several cellular mechanisms. Each chromosome, consisting of two sister chromatids joined at the centromere, aligns in such a way that the kinetochores of the sister chromatids are oriented toward opposite poles. This ensures that when the chromatids are separated during the next stage, each daughter cell will receive one identical copy of each chromosome. The mitotic spindle plays a crucial role in metaphase by maintaining the alignment of chromosomes at the metaphase plate. Spindle fibres, specifically kinetochore microtubules, attach to the kinetochores on each chromosome.

Proper tension is generated between the opposing spindle fibres as they pull on the kinetochores of each chromosome. This tension is critical for ensuring that chromosomes are correctly aligned and that each sister chromatid is attached to microtubules from opposite spindle poles. The balanced tension serves as a signal that the chromosomes are properly attached and ready for separation. Metaphase is under the control of a crucial regulatory mechanism known as the spindle assembly checkpoint (SAC). This checkpoint monitors the attachment of spindle fibres to the kinetochores and ensures that all chromosomes are correctly aligned at the metaphase plate before the cell proceeds to anaphase. The SAC prevents the onset of anaphase until all chromosomes are properly attached to the spindle apparatus and under appropriate tension. If any chromosome is not correctly aligned or attached, the checkpoint activates inhibitory signals that delay the progression to anaphase, allowing time for the cell to correct any errors. This checkpoint is vital for preventing chromosome misalignment, which could lead to aneuploidy (an abnormal number of chromosomes) in the daughter cells.

During metaphase, the sister chromatids remain tightly connected at their centromeres due to the action of cohesion proteins. These proteins form a complex that holds the chromatids together, ensuring they do not prematurely separate. This cohesion is maintained until the cell is ready to transition to anaphase, at which point the cohesion proteins are cleaved, allowing the chromatids to be pulled apart. Metaphase concludes with the cell receiving the “all-clear” signal from the spindle assembly checkpoint, indicating that all chromosomes are correctly aligned and attached to the spindle fibres. Once this checkpoint is satisfied, the cell transitions into anaphase, where the cohesion between sister chromatids is broken, and the chromatids are pulled toward opposite poles of the cell. This transition marks the beginning of chromosome segregation and the distribution of genetic material to the daughter cells.

Anaphase

Anaphase is a critical stage of mitotic division that directly follows metaphase and is described by the separation and movement of sister chromatids toward opposite poles of the cell. This stage marks the point at which the duplicated genetic material is evenly distributed between the two daughter cells, ensuring that each new cell inherits a complete set of chromosomes. Anaphase is a highly dynamic and tightly regulated process that plays a pivotal role in maintaining genomic integrity. The most defining event of anaphase is the separation of sister chromatids, which were previously held together at the centromeres by cohesion proteins. This separation is triggered by the activation of a protein complex called the anaphase-promoting complex/cyclosome (APC/C). The APC/C targets the cohesion proteins for degradation by tagging them with ubiquitin, leading to their cleavage and the release of the sister chromatids. Once the cohesion is removed, each chromatid, now considered an individual chromosome, is free to move toward opposite poles of the cell. This separation ensures that each daughter cell will receive an identical set of chromosomes, crucial for maintaining genetic continuity.

The movement of chromatids toward the poles is driven by the shortening of kinetochore microtubules, which are attached to the kinetochores of the chromosomes. As these microtubules depolymerise, break down into their tubulin subunits, they pull the chromatids toward the centrosomes located at opposite poles of the cell. At the same time, the polar microtubules, which extend from one pole to the other, lengthen and push against each other, helping to elongate the cell and increase the distance between the two sets of separating chromosomes. This coordinated action of microtubule dynamics ensures efficient and directed movement of chromosomes. During anaphase, the spindle poles themselves move further apart from each other, a process facilitated by the elongation of the polar microtubules and the action of motor proteins, such as kinesins and dyneins. This separation of spindle poles contributes to the physical separation of the two sets of chromosomes and helps prepare the cell for the final stages of division. The increase in distance between the poles ensures that the chromosomes are well-separated by the time they reach the poles, reducing the risk of chromosome entanglement or misalignment.

Anaphase can be divided into two distinct sub-phases: Anaphase A and Anaphase B. During Anaphase A, the primary focus is on the movement of the chromatids toward the spindle poles, while Anaphase B, on the other hand, involves the further separation of the spindle poles themselves. Both sub-phases are critical for ensuring that the chromosomes are correctly and efficiently divided between the two daughter cells. Anaphase concludes as the chromatids reach the poles of the cell, completing their segregation. The cell then transitions into telophase, the final stage of mitosis.

Telophase and Cytokinesis

One of the primary events in telophase is the reformation of the nuclear envelope around each set of separated chromosomes. After the chromosomes reach the opposite poles of the cell, vesicles derived from fragments of the original nuclear envelope and the endoplasmic reticulum begin to assemble around the chromosomes. These vesicles fuse to form a complete nuclear envelope, effectively encapsulating each set of chromosomes within a newly formed nucleus. This re-establishment of the nuclear boundary is essential for segregating the genetic material and protecting it from the cytoplasmic environment. As the nuclear envelope re-forms, the chromosomes, which had been highly condensed and visible during the earlier stages of mitosis, begin to decondense. This process involves the uncoiling of the chromatin fibres, making the chromosomes less compact and returning them to a more extended, thread-like state. Decondensation is important because it allows the chromatin to resume its normal function, including the transcription of DNA into RNA, which is essential for the cell’s activities and functions during interphase.

During telophase, the nucleolus, which had disappeared during prophase, reappears within each newly formed nucleus. The nucleolus is responsible for synthesising ribosomal RNA (rRNA) and assembling ribosomes, which are critical for protein synthesis. The reappearance of the nucleolus marks the resumption of ribosome production, which is essential for the cell’s metabolic activities as it prepares to enter the next interphase. As telophase progresses, the mitotic spindle, which played a key role in the segregation of chromosomes during anaphase, begins to disassemble. The spindle fibres, made of microtubules, depolymerise and are broken down into tubulin subunits that can be recycled for use in the cytoskeleton or other cellular structures. The disassembly of the mitotic spindle is necessary to complete the transition from mitosis to cytokinesis and to allow the cell to resume its normal structural organisation.

In animal cells, telophase is closely followed by the formation of the cleavage furrow, which is the beginning of cytokinesis. The cleavage furrow is an indentation of the cell membrane that forms around the equator of the cell, marking the site where the cell will eventually split into two daughter cells. The cleavage furrow is formed by the contraction of a ring of actin filaments, which tightens like a drawstring to progressively pinch the cell membrane inward. This process will continue through cytokinesis, eventually leading to the complete separation of the two daughter cells. Telophase sets the stage for cytokinesis, the final step in cell division, where the cytoplasm is divided between the two daughter cells. While cytokinesis overlaps with telophase, it is a distinct process that finalises the physical separation of the daughter cells. In plant cells, instead of a cleavage furrow, a cell plate forms, eventually leading to the construction of a new cell wall that separates the two daughter cells.

Telophase concludes with the complete reformation of the nuclei and the disassembly of the mitotic machinery, marking the end of mitosis. The cell now enters cytokinesis, where the cytoplasm is fully divided, resulting in two separate daughter cells. After cytokinesis is complete, the daughter cells enter interphase, where they begin the process of growth and preparation for the next cycle of cell division. Telophase is essential for ensuring that the genetic material segregated during anaphase is properly enclosed within two distinct nuclei, each containing a complete set of chromosomes. The accurate reformation and re-establishment are critical for restoring the cell’s normal functions after division. Any errors during telophase could lead to issues with gene expression or cell cycle regulation in the daughter cells.

Imagine a cell that completed mitosis but never underwent cytokinesis. In some cases, a cell may divide its genetic material and grow in size but fail to undergo cytokinesis. This results in larger cells with more than one nucleus. Usually this is an unwanted aberration and can be a sign of cancerous cells.

Size regulation by surface to volume ratio

Both the initiation and inhibition of cell division are triggered by events external to the cell when it is about to begin the replication process. An event may be as simple as the death of nearby cells or as far-reaching and impactful as the release of growth-promoting hormones, such as human growth hormone (HGH or hGH). A lack of HGH can inhibit cell division, resulting in dwarfism, whereas too much HGH can result in gigantism. Crowding of cells can also inhibit cell division. In contrast, a factor that can initiate cell division is the size of the cell. As a cell grows, its volume increases at a faster rate than its surface area, leading to a decreased surface-to-volume ratio. This reduction can limit the efficiency of nutrient uptake, waste removal, and gas exchange across the cell membrane, as the surface area becomes insufficient to support the metabolic demands of the enlarged cell volume. To restore balance, cells often undergo division when they reach a certain size, ensuring that the resulting daughter cells have a more favourable surface-to-volume ratio. This regulation prevents cells from becoming too large to function efficiently and allows for the proper distribution of cellular components during division. Additionally, certain signalling pathways, such as those involving growth factors and cell cycle checkpoints, are sensitive to changes in cell size and surface-to-volume ratio, further coordinating the timing of cell division with the cell’s physical and metabolic needs. This dynamic interplay ensures that cells divide at the appropriate time to maintain optimal size and functionality within tissues and organs.

Cell Cycle Control

A very elaborate and precise system of regulation controls direct the way cells proceed from one phase to the next in the cell cycle and begin mitosis. The control system involves molecules within the cell as well as external triggers. These internal and external control triggers provide “stop” and “advance” signals for the cell. Precise regulation of the cell cycle is critical for maintaining the health of an organism, and loss of cell cycle control can lead to cancer.

Mechanisms of Cell Cycle Control

As the cell proceeds through its cycle, each phase involves certain processes that must be completed before the cell should advance to the next phase. A checkpoint is a point in the cell cycle at which the cycle can be signalled to move forward or stopped. At each of these checkpoints, different varieties of molecules provide the stop or go signals, depending on certain conditions within the cell. A cyclin is one of the primary classes of cell cycle control molecules (Figure 2.13). A cyclin-dependent kinase (CDK) is one of a group of molecules that work together with cyclins to determine progression past cell checkpoints. By interacting with many additional molecules, these triggers push the cell cycle forward (unless prevented from doing so by “stop” signals, if for some reason the cell is not ready). At the G1 checkpoint, the cell must be ready for DNA synthesis to occur. At the G2 checkpoint the cell must be fully prepared for mitosis. Even during mitosis, a crucial stop and go checkpoint in metaphase ensures that the cell is fully prepared to complete cell division. The metaphase checkpoint ensures that all sister chromatids are properly attached to their respective microtubules and lined up at the metaphase plate before the signal is given to separate them during anaphase.

Homeostatic Imbalances

Cancer is an extremely complex condition, capable of arising from a wide variety of genetic and environmental causes. Typically, mutations or aberrations in a cell’s DNA that compromise normal cell cycle control systems lead to cancerous tumours. Cell cycle control is an example of a homeostatic mechanism that maintains proper cell function and health. While progressing through the phases of the cell cycle, a large variety of intracellular molecules provide stop and go signals to regulate movement forward to the next phase. These signals are maintained in an intricate balance so that the cell only proceeds to the next phase when it is ready. This homeostatic control of the cell cycle can be thought of like a car’s cruise control. Cruise control will continually apply just the right amount of acceleration to maintain a desired speed, unless the driver hits the brakes, in which case the car will slow down. Similarly, the cell includes molecular messengers, such as cyclins, that push the cell forward in its cycle.

In addition to cyclins, a class of proteins that are encoded by genes called proto-oncogenes provide important signals that regulate the cell cycle and move it forward. Examples of proto-oncogene products include cell-surface receptors for growth factors, or cell-signalling molecules, two classes of molecules that can promote DNA replication and cell division. In contrast, a second class of genes known as tumours suppressor genes sends stop signals during a cell cycle. For example, certain protein products of tumours suppressor genes signal potential problems with the DNA and thus stop the cell from dividing, while other proteins signal the cell to die if it is damaged beyond repair. Some tumours suppressor proteins also signal a sufficient surrounding cellular density, which indicates that the cell need not presently divide. The latter function is uniquely important in preventing tumours growth: normal cells exhibit a phenomenon called “contact inhibition;” thus, extensive cellular contact with neighbouring cells causes a signal that stops further cell division.

These two contrasting classes of genes, proto-oncogenes and tumour suppressor genes are like the accelerator and brake pedal of the cell’s own “cruise control system,” respectively. Under normal conditions, these stop and go signals are maintained in a homeostatic balance. Generally speaking, there are two ways that the cell’s cruise control can lose control: a malfunctioning (overactive) accelerator, or a malfunctioning (underactive) brake. When compromised through a mutation, or otherwise altered, proto-oncogenes can be converted to oncogenes, which produce oncoproteins that push a cell forward in its cycle and stimulate cell division even when it is undesirable to do so. For example, a cell that should be programmed to self-destruct (a process called apoptosis) due to extensive DNA damage might instead be triggered to proliferate by an oncoprotein. On the other hand, a dysfunctional tumours suppressor gene may fail to provide the cell with a necessary stop signal, also resulting in unwanted cell division and proliferation.

A delicate homeostatic balance between the many proto-oncogenes and tumours suppressor genes delicately controls the cell cycle and ensures that only healthy cells replicate. Therefore, a disruption of this homeostatic balance can cause aberrant cell division and cancerous growths.

Cell differentiation

The transformation of a single fertilised egg into the diverse and complex array of cell types that make up a fully developed human organism is a process that highlights the remarkable capabilities of cellular differentiation. This journey begins with a zygote, a single cell formed by the union of sperm and egg, which contains all the genetic information necessary to form every cell type in the body. As the zygote undergoes repeated cycles of division, it gives rise to a population of unspecialised cells known as stem cells. A stem cell is an unspecialised cell that can divide without limit as needed and can, under specific conditions, differentiate into specialised cells. Stem cells are divided into several categories according to their potential to differentiate. The first embryonic cells that arise from the division of the zygote are the ultimate stem cells; these stems cells are described as totipotent. Totipotent stem cells are the most versatile and powerful type of stem cells, possessing the unique ability to develop into any cell type within an organism, including both embryonic and extraembryonic tissues such as the placenta. Because of their extraordinary potential, totipotent stem cells are essential in the initial stages of embryonic development, laying the foundation for all the tissues and organs that will form the complete organism. The totipotency of these cells is limited to the very early stages of development; as the embryo progresses, these cells undergo further divisions and begin to specialise, losing their totipotency and transitioning into pluripotent stem cells, which are still highly versatile but no longer capable of forming an entire organism.

The embryonic cells that develop from totipotent stem cells and are precursors to the fundamental tissue layers of the embryo are classified as pluripotent. Pluripotent stem cells are a type of stem cell that have the remarkable ability to differentiate into nearly any cell type within the three primary germ layers—ectoderm, mesoderm, and endoderm—that form all the tissues and organs in a body. Unlike totipotent stem cells, which can give rise to an entire organism including extraembryonic tissues like the placenta, pluripotent stem cells are limited to forming only the embryonic cells that will develop into the body’s various systems. These cells are derived from the inner cell mass of the blastocyst during early embryonic development and are crucial for the formation of all the specialised tissues that make up the organism, such as neurons, muscle cells, and skin cells.

These cells then become slightly more specialised and are referred to as multipotent cells. Multipotent stem cells are a more specialised type of stem cell with the ability to differentiate into a limited range of cell types within a specific lineage or tissue. They are restricted to forming cells related to a particular family or group of tissues, for example haematopoietic stem cells, which are a type of multipotent stem cell found in the bone marrow, can differentiate into various types of blood cells, such as red blood cells (erythrocytes), white blood cells (leukocytes), and platelets, but cannot develop into cells outside of the blood lineage. Similarly, mesenchymal stem cells, another type of multipotent stem cell, can give rise to bone, cartilage, fat, and muscle cells. These cells play a crucial role in the ongoing maintenance, repair, and regeneration of tissues throughout an individual’s life, as they are responsible for replenishing cells that are lost due to normal wear and tear, injury, or disease.

Finally, multipotent cells can become further specialised oligopotent cells. An oligopotent stem cell is limited to becoming one of a few different cell types. While they share some similarities with multipotent stem cells in that they are limited to a particular family of cells, oligopotent stem cells have an even narrower differentiation potential. For example, lymphoid or myeloid progenitor cells, which are oligopotent, can differentiate into specific types of immune cells such as T cells, B cells, or natural killer cells within the lymphoid lineage, or into various types of myeloid cells like macrophages, neutrophils, or platelets within the myeloid lineage. These cells are crucial for the body’s ongoing ability to maintain and replenish the cells within these specific systems, ensuring a continuous supply of functionally specialised cells.

In contrast, a unipotent cell is fully specialised and can only reproduce to generate more of its own specific cell type. Despite this limitation, unipotent cells are crucial for the body’s maintenance and regeneration processes. They are responsible for the continuous renewal of certain tissues that require frequent replacement due to wear and tear, such as the skin, where unipotent basal cells in the epidermis continuously divide to produce new keratinocytes, the cells that make up the outer layer of the skin. Similarly, in the liver, unipotent cells contribute to the regeneration of hepatocytes, the main functional cells of the liver. The highly specialised nature of unipotent cells makes them essential for the upkeep of tissue homeostasis, ensuring that specific cell types are replenished as needed to maintain proper tissue function.

Stem cells are unique in that they can also continually divide and regenerate new stem cells instead of further specialising. Different stem cells are present at different stages of a human’s life. They include the embryonic stem cells of the embryo, foetal stem cells of the foetus, and adult stem cells in the adult. One type of adult stem cell is the epithelial stem cell, which gives rise to the keratinocytes in the multiple layers of epithelial cells in the epidermis of skin. Adult bone marrow has three distinct types of stem cells: hematopoietic stem cells (which give rise to red blood cells, white blood cells, and platelets), endothelial stem cells (which give rise to the endothelial cell types that line blood and lymph vessels), and mesenchymal stem cells (which give rise to the different types of muscle cells). The process of haematopoiesis involves the differentiation of multipotent cells into blood and immune cells. The multipotent hematopoietic stem cells give rise to many different cell types, including the cells of the immune system and red blood cells.

When a cell differentiates (becomes more specialised), it may undertake major changes in its size, shape, metabolic activity, and overall function. Since all cells in the body, beginning with the fertilised egg, contain the same DNA, how do the different cell types come to be so different? The answer is analogous to a movie script. The different actors in a movie all read from the same script, however, they are each only reading their own part of the script. Similarly, all cells contain the same full complement of DNA, but each type of cell only “reads” the portions of DNA that are relevant to its own function. In biology, this is referred to as the unique genetic expression of each cell.

In order for a cell to differentiate into its specialised form and function, it need only manipulate those genes (and thus those proteins) that will be expressed, and not those that will remain silent. The primary mechanism by which genes are turned “on” or “off” is through transcription factors.

While each body cell contains the organism’s entire genome, different cells regulate gene expression with the use of various transcription factors. Transcription factors are proteins that affect the binding of RNA polymerase to a particular gene on the DNA molecule.

Theory in Practice – Stem Cell Research

Stem cell research aims to find ways to use stem cells to regenerate and repair cellular damage. Over time, most adult cells undergo the wear and tear of aging and lose their ability to divide and repair themselves. Stem cells do not display a particular morphology or function. Adult stem cells, which exist as a small subset of cells in most tissues, keep dividing and can differentiate into a number of specialised cells generally formed by that tissue. These cells enable the body to renew and repair body tissues.

The mechanisms that induce a non-differentiated cell to become a specialised cell are poorly understood. In a laboratory setting, it is possible to induce stem cells to differentiate into specialised cells by changing the physical and chemical conditions of growth. Several sources of stem cells are used experimentally and are classified according to their origin and potential for differentiation. Human embryonic stem cells (hESCs) are extracted from embryos and are pluripotent. The adult stem cells that are present in many organs and differentiated tissues, such as bone marrow and skin, are multipotent, being limited in differentiation to the types of cells found in those tissues. The stem cells isolated from umbilical cord blood are also multipotent, as are cells from deciduous teeth (baby teeth). Researchers have recently developed induced pluripotent stem cells (iPSCs) from mouse and human adult stem cells. These cells are genetically reprogrammed multipotent adult cells that function like embryonic stem cells; they are capable of generating cells characteristic of all three germ layers.

Because of their capacity to divide and differentiate into specialised cells, stem cells offer a potential treatment for diseases such as diabetes and heart disease (Figure 2.14). Cell-based therapy refers to treatment in which stem cells induced to differentiate in a growth dish are injected into a patient to repair damaged or destroyed cells or tissues. Many obstacles must be overcome for the application of cell-based therapy. Although embryonic stem cells have a nearly unlimited range of differentiation potential, they are seen as foreign by the patient’s immune system and may trigger rejection. Also, the destruction of embryos to isolate embryonic stem cells raises considerable ethical and legal questions.

In contrast, adult stem cells isolated from a patient are not seen as foreign by the body, but they have a limited range of differentiation. Some individuals bank the cord blood or deciduous teeth of their child, storing away those sources of stem cells for future use, should their child need it. Induced pluripotent stem cells are considered a promising advance in the field because using them avoids the legal, ethical, and immunological pitfalls of embryonic stem cells.