The Cell Nucleus

The Cell Nucleus The nucleus is a highly specialized organelle that serves as the information processing and administrative center of the cell. This organelle has two major functions: it stores the cell's hereditary material, or DNA, and it coordinates the cell's activities, which include growth, intermediary metabolism, protein synthesis, and reproduction (cell division). Only the cells of advanced organisms, known as eukaryotes, have a nucleus. Generally there is only one nucleus per cell, but there are exceptions, such as the cells of slime molds and the Siphonales group of algae. Simpler one-celled organisms (prokaryotes), like the bacteria and cyanobacteria, don't have a nucleus. In these organisms, all of the cell's information and administrative functions are dispersed throughout the cytoplasm. The spherical nucleus typically occupies about 10 percent of a eukaryotic cell's volume, making it one of the cell's most prominent features. A double-layered membrane, the nuclear envelope, separates the contents of the nucleus from the cellular cytoplasm. The envelope is riddled with holes called nuclear pores that allow specific types and sizes of molecules to pass back and forth between the nucleus and the cytoplasm. It is also attached to a network of tubules and sacs, called the endoplasmic reticulum, where protein synthesis occurs, and is usually studded with ribosomes (see Figure 1). The semifluid matrix found inside the nucleus is called nucleoplasm. Within the nucleoplasm, most of the nuclear material consists of chromatin, the less condensed form of the cell's DNA that organizes to form chromosomes during mitosis or cell division. The nucleus also contains one or more nucleoli, organelles that synthesize protein-producing macromolecular assemblies called ribosomes, and a variety of other smaller components, such as Cajal bodies, GEMS (Gemini of coiled bodies), and interchromatin granule clusters. Chromatin and Chromosomes - Packed inside the nucleus of every human cell is nearly 6 feet of DNA, which is divided into 46 individual molecules, one for each chromosome and each about 1.5 inches long. Packing all this material into a microscopic cell nucleus is an extraordinary feat of packaging. For DNA to function, it can't be crammed into the nucleus like a ball of string. Instead, it is combined with proteins and organized into a precise, compact structure, a dense string-like fiber called chromatin. The Nucleolus - The nucleolus is a membrane-less organelle within the nucleus that manufactures ribosomes, the cell's protein-producing structures. Through the microscope, the nucleolus looks like a large dark spot within the nucleus. A nucleus may contain up to four nucleoli, but within each species the number of nucleoli is fixed. After a cell divides, a nucleolus is formed when chromosomes are brought together into nucleolar organizing regions. During cell division, the nucleolus disappears. Some studies suggest that the nucleolus may be involved with cellular aging and, therefore, may affect the senescence of an organism. The Nuclear Envelope - The nuclear envelope is a double-layered membrane that encloses the contents of the nucleus during most of the cell's lifecycle. The space between the layers is called the perinuclear space and appears to connect with the rough endoplasmic reticulum. The envelope is perforated with tiny holes called nuclear pores. These pores regulate the passage of molecules between the nucleus and cytoplasm, permitting some to pass through the membrane, but not others. The inner surface has a protein lining called the nuclear lamina, which binds to chromatin and other nuclear components. During mitosis, or cell division, the nuclear envelope disintegrates, but reforms as the two cells complete their formation and the chromatin begins to unravel and disperse. Nuclear Pores - The nuclear envelope is perforated with holes called nuclear pores. These pores regulate the passage of molecules between the nucleus and cytoplasm, permitting some to pass through the membrane, but not others. Building blocks for building DNA and RNA are allowed into the nucleus as well as molecules that provide the energy for constructing genetic material. Chromatin and Chromosomes Packed inside the nucleus of every human cell is nearly 6 feet of DNA, which is subdivided into 46 individual molecules, one for each chromosome and each about 1.5 inches long. Collecting all this material into a microscopic cell nucleus is an extraordinary feat of packaging. For DNA to function when necessary, it can't be haphazardly crammed into the nucleus or simply wound up like a ball of string. Consequently, during interphase, DNA is combined with proteins and organized into a precise, compact structure, a dense string-like fiber called chromatin, which condenses even further into chromosomes during cell division. Each DNA strand wraps around groups of small protein molecules called histones, forming a series of bead-like structures, called nucleosomes, connected by the DNA strand (as illustrated in Figure 1). Under the microscope, uncondensed chromatin has a "beads on a string" appearance. The string of nucleosomes, already compacted by a factor of six, is then coiled into an even denser structure known as a solenoid that compacts the DNA by a factor of 40. The solenoid structure then coils to form a hollow tube. This complex compression and structuring of DNA serves several functions. The overall negative charge of the DNA is neutralized by the positive charge of the histone molecules, the DNA takes up much less space, and inactive DNA can be folded into inaccessible locations until it is needed. There are two basic types of chromatin. Euchromatin is the genetically active type of chromatin involved in transcribing RNA to produce proteins used in cell function and growth. The predominant type of chromatin found in cells during interphase, euchromatin is more diffuse than the other kind of chromatin, which is termed heterochromatin. The additional compression of heterochromatin is thought to involve various proteins in addition to the histones, and the DNA it contains is thought to be genetically inactive. Heterochromatin tends to be most concentrated along chromosomes at certain regions of the structures, such as the centromeres and telomeres. Genes typically located in euchromatin can be experimentally silenced (not expressed) by relocating them to a heterochromatin position. Throughout the life of a cell, chromatin fibers take on different forms inside the nucleus. During interphase, when the cell is carrying out its normal functions, the chromatin is dispersed throughout the nucleus in what appears to be a tangle of fibers. This exposes the euchromatin and makes it available for the transcription process. When the cell enters metaphase and prepares to divide, the chromatin changes dramatically. First, all the chromatin strands make copies of themselves through the process of DNA replication. Then they are compressed to an even greater degree as they undergo a 10,000-fold compaction into specialized structures for reproduction, the chromosomes (see Figure 2). As the cell divides to become two cells, the chromosomes separate, giving each cell a complete copy of the genetic information contained in the chromatin. The number of chromosomes within the nuclei of an organism's cells is a species-specific trait. Human diploid cells (those that are not gametes) characteristically exhibit 46 chromosomes, but this number can be as low as 2, as is the case for some ants and roundworms, or more than a thousand, as exemplified by the Indian fern (Ophioglossum reticulatum), which has 1,260 chromosomes. Accordingly, the number of chromosomes a species has does not correlate to the complexity of the organism. The Nucleolus The nucleolus is a prominent sub-nuclear structure that is not bound by a membrane and resides within the nuclear matrix. Though known to exist since the eighteenth century, the primary function of the nucleolus was not discovered until the 1960s. It is now been determined that nucleoli manufacture the subunits that combine to form ribosomes, the cell's protein-producing factories. Accordingly, the size of nucleoli depends upon the ribosomal requirements of the type of cell in which they are found. In cells that produce large amounts of protein, and thus call for significant numbers of ribosomes, the size of the nucleolus is considerable, sometimes occupying as much as 25 percent of the total volume of the nucleus. Through the microscope, the nucleolus appears like a large dark spot within the nucleus (see Figure 2). Eukaryotic cells often contain a single nucleolus, but several are also possible. The exact number of nucleoli is fixed among members of the same species. Each diploid cell in the human body features only one nucleolus, though immediately after cell division ten tiny nucleoli appear before they coalesce into a single, large nucleolus. This is because nucleoli are formed at certain chromosome sites usually referred to as nucleolus organizer regions (NORs), and two copies of five different human chromosomes contain NORs. The DNA found at chromosomal NORs encodes the genes for ribosomal RNA (rRNA). At the onset of mitosis, the single nucleolus present in a human cell disappears, and subsequent to the process, the formation of the new nucleolus, which is created from the ten smaller nucleolus-like structures that develop from the NORs, can be observed. The nucleolus is comprised of granular and fibrillar components, as well as an ill-defined matrix, in addition to DNA. The granular material consists of ribosomal subunits that have already been formed but have not yet matured and are waiting to be exported to the cytoplasm. The threadlike fibrillar part of a nucleolus is predominantly composed of rRNA molecules and associated proteins that have joined together to form fibrils. It has not yet been determined exactly how the various components of the nucleolus are secured together and organized. Illustrated in Figure 2 is a fluorescence digital image of a Swiss mouse embryo fibroblast cell stained with fluorescent probes targeting the nucleus (blue), mitochondria network (red), Golgi complex (green) and nucleoli (magenta) to demonstrate the proximity of these structures. The nucleoli of this cell were targeted with an antibody to fibrillarin, a small nuclear ribonucleoprotein (SnRNP) involved in ribosomal RNA processing and centralized within the sub-organelle. Fibrillarin has a well conserved amino acid sequence that enables the protein to serve as an excellent marker for nucleoli in a wide variety of species. In recent years, scientists have been industriously investigating a possible connection between the nucleolus and cell senescence (aging). Most evidence for such a link thus far has come from studies with yeast cells, some of which suggest that cumulative damage to ribosomal RNA genes and fragmentation of the nucleolus may be central to the aging process. Support for the theory has also come from genetic research focused upon Werner syndrome, an inherited disease characterized by premature aging, but additional studies are needed to more precisely understand any possible role the nucleolus plays in senescence. The Nuclear Envelope The nuclear envelope is a double-layered membrane that encloses the contents of the nucleus during most of the cell's lifecycle. The outer nuclear membrane is continuous with the membrane of the rough endoplasmic reticulum (ER), and like that structure, features numerous ribosomes attached to the surface. The outer membrane is also continuous with the inner nuclear membrane since the two layers are fused together at numerous tiny holes called nuclear pores that perforate the nuclear envelope. These pores regulate the passage of molecules between the nucleus and cytoplasm, permitting some to pass through the membrane, but not others. The space between the outer and inner membranes is termed the perinuclear space and is connected with the lumen of the rough ER. Structural support is provided to the nuclear envelope by two different networks of intermediate filaments. Along the inner surface of the nucleus, one of these networks is organized into a special mesh-like lining called the nuclear lamina, which binds to chromatin, integral membrane proteins, and other nuclear components. The nuclear lamina is also thought play a role in directing materials inside the nucleus toward the nuclear pores for export and in the disintegration of the nuclear envelope during cell division and its subsequent reformation at the end of the process. The other intermediate filament network is located on the outside of the outer nuclear membrane and is not organized in such a systemic way as the nuclear lamina. The amount of traffic that must pass through the nuclear envelope on a continuous basis in order for the eukaryotic cell to function properly is considerable. RNA and ribosomal subunits must be constantly transferred from the nucleus where they are made to the cytoplasm, and histones, gene regulatory proteins, DNA and RNA polymerases, and other substances required for nuclear activities must be imported from the cytoplasm. An active mammalian cell can synthesize about 20,000 ribosome subunits per minute, and at certain points in the cell cycle, as many as 30,000 histones per minute are required by the nucleus. In order for such a tremendous number of molecules to pass through the nuclear envelope in a timely manner, the nuclear pores must be highly efficient at selectively allowing the passage of materials to and from the nucleus. Nuclear Pores The nuclear envelope is perforated with tiny holes known as nuclear pores, which were first discovered in the mid-twentieth century. These pores regulate the passage of molecules between the nucleus and cytoplasm, permitting some to pass through the membrane, but not others. Building blocks for manufacturing DNA and RNA are some of the materials that are allowed into the nucleus, as are molecules that provide the energy for constructing genetic material. Ribosomal subunits, which are built in nucleosomes, are a prime example of materials that must be allowed to leave the nucleus and enter the cytoplasm. Nuclear pores are fully permeable to small molecules up to the size of the smallest proteins, but form a barrier keeping most large molecules out of the nucleus. Yet, some larger proteins, such as histones, are granted admission into the nucleus despite the fact that that the pores should be too small to let them through. It is generally thought that the elaborate protein structure called the nuclear pore complex (see Figure 1) that surrounds each pore plays a key role in allowing the active transport of a select set of large molecules into and out of the nucleus. The nuclear pore complex is comprised of several subunits. Surrounding the inside of the pore is a nonmembranous material organized into an annulus that extends spoke-like structures toward the center of the small channel. The actual pore wall is predominantly comprised of columnar subunits, and lumenal subunits, with the help of transmembrane proteins, anchor the entire pore complex into the nuclear envelope. Also, tiny fibrils usually extend form both sides of the complex and congregate into basket-like configurations on the nuclear side of the complex. The proteinaceous composition of these fibrils is different on opposing sides of the structure. In addition to their role in nuclear transport, nuclear pores are important as sites where the outer membrane and inner membrane of the nuclear envelope are fused together. Due to this fusion, the membranes can be considered continuous with one another although they have different biochemical characteristics and can function in distinctive ways. Since the outer nuclear membrane is also continuous with the membrane of the endoplasmic reticulum (ER), both it and the inner nuclear membrane can exchange membranous materials with the ER. This capability enables the nuclear envelope to grow bigger or smaller when necessary to accommodate the dynamic contents of the nucleus. Illustrated in Figure 2 is a fluorescence digital image of an adherent culture of Madin-Darby canine kidney cells (MDCK line) stained with fluorescent probes targeting the nucleus (blue), nuclear pore complex proteins (red), and the tight junctions formed between epithelial cells (green) to demonstrate the proximity of these structures. The nuclear pores of these cells were targeted with a wide-spectrum polyclonal antibody to a large family of nuclear pore complex proteins, which serves as a useful tool for studying the morphology and composition of the nucleus and nuclear envelope. The antibody mixture is also useful in studying changes in the nuclear structure during mitosis and meiosis (note the mitotic cell in the lower central portion of Figure 2). Pore density varies greatly and tends to be greatest among cells that are highly activate and differentiated, such as liver cells. A typical mammalian cell features approximately 3,000 to 4,000 pores along its nuclear envelope. The oocytes of certain amphibians, however, have such large nuclei and such a density of pores that the nuclear envelope of one of the cells may contain more than ten million pores. Consequently, these oocytes have been heavily utilized in studies of nuclear pore complexes and nuclear transport. Centrioles Found only in animal cells, these paired organelles are typically located together near the nucleus in the centrosome, a granular mass that serves as an organizing center for microtubules. Within the centrosome, the centrioles are positioned so that they are at right angles to each other, as illustrated in Figure 1. Each centriole is made of nine bundles of microtubules (three per bundle) arranged in a ring. Centrioles play a notable role in cell division. During interphase of an animal cell, the centrioles and other components of the centrosome are duplicated, though scientists are not yet sure how this duplication takes place. At first the two pairs of centrioles remain in close proximity to each other, but as mitosis initiates, the original centrosome divides and the pairs are split up so that one set of centrioles is located in each of the new microtubule-organizing centers. These new centers radiate microtubules in star-shaped clusters known as asters. As the asters move to opposing poles of the cells, the microtubules, with the help of the centrioles, become organized into a spindle-shaped formation that spans the cell (see Figure 2). These spindle fibers act as guides for the alignment of the chromosomes as they separate later during the process of cell division. Though centrioles play a role in the mitosis of animal cells, plant cells are able to reproduce without them. Researchers have, therefore, been very interested in determining exactly how important the organelles really are. Studies have shown that certain animal cells, particularly female gametes (oocytes), can successfully divide even when their centrioles are destroyed. Some investigators have also found, however, that the absence of centrioles in animal cells is associated with an increased number of divisional errors and substantial delays in the mitotic process, especially before chromosome segregation. Consequently, it has been suggested that centrioles evolved as a refinement of the cell, making mitosis a much more efficient and less error-prone process. In cells that feature cilia or flagella, basal bodies, which exhibit the same structural form as centrioles, are present. These assemblies are located, however, near the cell surface at the base of each cilium or flagellum, rather than in the centrosome near the nucleus. Basal bodies are anchored in their cytoplasmic locations by what is called a rootlet system in the cell. In some organisms, such as the unicellular Chlamydomonas, basal bodies change their location and are functionally converted to centrioles before the mitotic process. Cilia and Flagella Cilia and flagella are motile cellular appendages found in most microorganisms and animals, but not in higher plants. In multicellular organisms, cilia function to move a cell or group of cells or to help transport fluid or materials past them. The respiratory tract in humans is lined with cilia that keep inhaled dust, smog, and potentially harmful microorganisms from entering the lungs. Among other tasks, cilia also generate water currents to carry food and oxygen past the gills of clams and transport food through the digestive systems of snails. Flagella are found primarily on gametes, but create the water currents necessary for respiration and circulation in sponges and coelenterates as well. For single-celled eukaryotes, cilia and flagella are essential for the locomotion of individual organisms. Protozoans belonging to the phylum Ciliophora are covered with cilia, while flagella are a characteristic of the protozoan group Mastigophora. In eukaryotic cells, cilia and flagella contain the motor protein dynein and microtubules, which are composed of linear polymers of globular proteins called tubulin. The core of each of the structures is termed the axoneme and contains two central microtubules that are surrounded by an outer ring of nine doublet microtubules. One full microtubule and one partial microtubule, the latter of which shares a tubule wall with the other microtubule, comprise each doublet microtubule (see Figure 1). Dynein molecules are located around the circumference of the axoneme at regular intervals along its length where they bridge the gaps between adjacent microtubule doublets. A plasma membrane surrounds the entire axoneme complex, which is attached to the cell at a structure termed the basal body (also known as a kinetosome). Basal bodies maintain the basic outer ring structure of the axoneme, but each of the nine sets of circumferential filaments is composed of three microtubules, rather than a doublet of microtubules. Thus, the basal body is structurally identical to the centrioles that are found in the centrosome located near the nucleus of the cell. In some organisms, such as the unicellular Chlamydomonas, basal bodies are locationally and functionally altered into centrioles and their flagella resorbed before cell division. Eukaryotic cilia and flagella are generally differentiated based on size and number: cilia are usually shorter and occur together in much greater numbers than flagella, which are often solitary. The structures also exhibit somewhat different types of motion, though in both cases movement is generated by the activation of dynein and the resultant bending of the axoneme. The movement of cilia is often described as whip-like, or compared to the breast stroke in swimming. Adjacent cilia move almost simultaneously (but not quite), so that in groups of cilia, wave-like patterns of motion occur. Flagella, however, exhibit a smooth, independent undulatory type of movement in eukaryotes. Prokaryotic flagella, which have a completely different structure built from the protein flagellin, move in a rotating fashion powered by the basal motor. Defects in the cilia and flagella of human cells are associated with some notable medical problems. For example, a hereditary condition known as Kartagener's syndrome is caused by problems with the dynein arms that extend between the microtubules present in the axoneme, and is characterized by recurrent respiratory infections related to the inability of cilia in the respiratory tract to clear away bacteria or other materials. The disease also results in male sterility due to the inability of sperm cells to propel themselves via flagella. Damage to respiratory cilia may also be acquired rather than inherited and is most commonly linked to smoking cigarettes. Bronchitis, for instance, is often triggered by a build-up of mucus and tar in the lungs that cannot be properly removed due to smoking-related impairment of cilia. The Endoplasmic Reticulum The endoplasmic reticulum (ER) is a network of flattened sacs and branching tubules that extends throughout the cytoplasm in plant and animal cells. These sacs and tubules are all interconnected by a single continuous membrane so that the organelle has only one large, highly convoluted and complexly arranged lumen (internal space). Usually referred to as the endoplasmic reticulum cisternal space, the lumen of the organelle often takes up more than 10 percent of the total volume of a cell. The endoplasmic reticulum membrane allows molecules to be selectively transferred between the lumen and the cytoplasm, and since it is connected to the double-layered nuclear envelope, it further provides a pipeline between the nucleus and the cytoplasm. The endoplasmic reticulum manufactures, processes, and transports a wide variety of biochemical compounds for use inside and outside of the cell. Consequently, many of the proteins found in the cisternal space of the endoplasmic reticulum lumen are there only transiently as they pass on their way to other locations. Other proteins, however, are targeted to constantly remain in the lumen and are known as endoplasmic reticulum resident proteins. These special proteins, which are necessary for the endoplasmic reticulum to carry out its normal functions, contain a specialized retention signal consisting of a specific sequence of amino acids that enables them to be retained by the organelle. An example of an important endoplasmic reticulum resident protein is the chaperone protein known as BiP (formally: the chaperone immunoglobulin-binding protein), which identifies other proteins that have been improperly built or processed and keeps them from being sent to their final destinations. There are two basic kinds of endoplasmic reticulum morphologies: rough and smooth. The surface of rough endoplasmic reticulum is covered with ribosomes, giving it a bumpy appearance when viewed through the microscope. This type of endoplasmic reticulum is involved mainly with the production and processing of proteins that will be exported, or secreted, from the cell. The ribosomes assemble amino acids into protein units, which are transported into the rough endoplasmic reticulum for further processing. These proteins may be either transmembrane proteins, which become embedded in the membrane of the endoplasmic reticulum, or watersoluble proteins, which are able to pass completely through the membrane into the lumen. Those that reach the inside of the endoplasmic reticulum are folded into the correct three-dimensional conformation, as a flattened cardboard box might be opened up and folded into its proper shape in order to become a useful container. Chemicals, such as carbohydrates or sugars, are added, then the endoplasmic reticulum either transports the completed proteins to areas of the cell where they are needed, or they are sent to the Golgi apparatus for further processing and modification. Most proteins exported from the endoplasmic reticulum exit the organelle in vesicles budded from the smooth portion, which has a more even appearance than rough endoplasmic reticulum when viewed through the electron microscope because of the lack of ribosomes. The smooth endoplasmic reticulum in most cells is much less extensive than the rough endoplasmic reticulum and is sometimes alternatively termed transitional. Smooth endoplasmic reticulum is chiefly involved, however, with the production of lipids (fats), building blocks for carbohydrate metabolism, and the detoxification of drugs and poisons. Therefore, in some specialized cells, such as those that are occupied chiefly in lipid and carbohydrate metabolism (brain and muscle) or detoxification (liver), the smooth endoplasmic reticulum is much more extensive and is crucial to cellular function. Smooth endoplasmic reticulum also plays a role in various cellular activities through its storage of calcium and involvement in calcium metabolism. In muscle cells, smooth endoplasmic reticulum releases calcium to trigger muscle contractions. Presented in Figure 2 is a fluorescence digital image taken through the microscope of the endoplasmic reticulum network in a bovine (cow) pulmonary artery endothelial cell grown in culture. It is the rough endoplasmic reticulum that is directly continuous with the nuclear envelope (as illustrated in Figure 1), which is also studded with ribosomes, and the two organelles are thought to have evolved simultaneously in ancient cells. Due to their physical membranous connection, the lumen of the endoplasmic reticulum and the space between the layers of the nuclear envelope comprise a single compartment. Accordingly, the nucleus has direct access to proteins (many of which are produced by the ribosomes upon its surface) and other materials present in the endoplasmic reticulum lumen, so that transport vesicles are not needed to obtain them. The close association between the endoplasmic reticulum and the nucleus also enables the organelles to share information in a very efficient manner. For instance, if the endoplasmic reticulum begins to undergo functional problems and unfolded proteins accumulate within the organelle, which can be extremely hazardous to the cell, the organelle quickly sends a signal to the nucleus (as well as to the cytoplasm). The nucleus responds by slowing ribosomal translation through a several-step process, thereby giving the endoplasmic reticulum extra time to catch up on its protein folding, thus maintaining cellular health. Endosomes and Endocytosis Endosomes are membrane-bound vesicles, formed via a complex family of processes collectively known as endocytosis, and found in the cytoplasm of virtually every animal cell. The basic mechanism of endocytosis is the reverse of what occurs during exocytosis or cellular secretion. It involves the invagination (folding inward) of a cell’s plasma membrane to surround macromolecules or other matter diffusing through the extracellular fluid. The encircled foreign materials are then brought into the cell, and following a pinching-off of the membrane (termed budding), are released to the cytoplasm in a sac-like vesicle. The size of vesicles varies, and those larger than 100 nanometers in diameter are typically referred to as vacuoles. Three primary mechanisms of endocytosis that are exhibited by a typical cell are illustrated in Figure 1. On the far left of the figure, receptor mediated endocytosis, which is the most specifically-targeted form of the endocytic process, is presented. Through receptor mediated endocytosis, active cells are able to take in significant amounts of particular molecules (ligands) that bind to receptor sites extending from the cytoplasmic membrane into the extracellular fluid surrounding the cell. These receptor sites are commonly grouped together along coated pits in the membrane, which are lined on their cytoplasmic surface with a bristle-like collection of coat proteins. The coat proteins are thought to play a role in enlarging the pit and forming a vesicle. Note, as shown in Figure 1, vesicles produced via receptor mediated endocytosis may internalize other molecules in addition to ligands, though the ligands are usually brought into the cell in higher concentration. A less specific mechanism of endocytosis is pinocytosis, which is illustrated in the central section of Figure 1. By means of pinocytosis, a cell is able to ingest droplets of liquid from the extracellular fluid. All solutes found in the droplets outside of the cell may become encased in the vesicles formed via this process, with those present in the greatest concentration in the extracellular fluid also becoming the most concentrated in the membranous sacs. Pinocytic vesicles tend to be smaller than vesicles produced by other endocytic processes. The final type of endocytosis, termed phagocytosis (see Figure 1), is probably the most wellknown manner in which a cell may import outside materials. In many school science labs, children observe amoebas under the microscope and watch the single-celled organisms eat by stretching out pseudopodia and encircling any food particles they find in their paths. This engulfment and subsequent packaging of the particles into vesicles, which are usually large enough to be correctly referred to as vacuoles, is phagocytosis. Though commonly associated with amoebas, phagocytosis is practiced by many organisms. In most multicellular animals, phagocytic cells chiefly function in bodily defense rather than as a means to gain nourishment. For example, leukocytes in the human body often phagocytose protozoa, bacteria, dead cells, and similar materials in order to help stave off infections or other problems. Once freed into the cytoplasm, several small vesicles produced via endocytosis may come together to form a single entity. This endosome generally functions in one of two ways. Most commonly, endosomes transport their contents in a series of steps to a lysosome, which subsequently digests the materials. In other instances, however, endosomes are used by the cell to transport various substances between different portions of the external cell membrane. This latter function is particularly important among epithelial cells, such as those that compose the outer layer of the skin, because they exhibit polarity (one side of the cell is different from the other side). Illustrated in Figure 2 is a fluorescence digital image of a single African green monkey kidney fibroblast cell (CV-1 line) transfected with a fluorescent protein fused to a targeting amino acid sequence for endosomes (green). The nucleus, plasma membrane, and endosome components are marked in the figure. An endosome that is destined to transfer its contents to a lysosome generally goes through several changes along its way. In its initial form, when the structure is often referred to as an early endosome, the specialized vesicle contains a single compartment. Over time, however, chemical changes in the vesicle take place and the membrane surrounding the endosome folds in upon itself in a way that is similar to the invagination of the plasma membrane. In this case, however, the membrane is not pinched off. Consequently, a structure with multiple compartments, termed a multivesicular endosome, is formed. The multivesicular endosome is an intermediate structure in which further chemical changes, including a significant drop in pH level, take place as the vesicle develops into a late endosome. Though late endosomes are capable of breaking down many proteins and fats, a lysosome is needed to fully digest all of the materials they contain. Frequently the contents of late endosomes are conveyed to a lysosome through fusion (joining together) of their membranes. Under some circumstances, late endosomes are able to further mature into lysosomes through additional chemical and structural modifications, in which case fusion is not necessary for digestion to be completed. The Golgi Apparatus The Golgi apparatus (GA), also called Golgi body or Golgi complex and found universally in both plant and animal cells, is typically comprised of a series of five to eight cup-shaped, membrane-covered sacs called cisternae that look something like a stack of deflated balloons. In some unicellular flagellates, however, as many as 60 cisternae may combine to make up the Golgi apparatus. Similarly, the number of Golgi bodies in a cell varies according to its function. Animal cells generally contain between ten and twenty Golgi stacks per cell, which are linked into a single complex by tubular connections between cisternae. This complex is usually located close to the cell nucleus. Due to its relatively large size, the Golgi apparatus was one of the first organelles ever observed. In 1897, an Italian physician named Camillo Golgi, who was investigating the nervous system by using a new staining technique he developed (and which is still sometimes used today; known as Golgi staining or Golgi impregnation), observed in a sample under his light microscope a cellular structure that he termed the internal reticular apparatus. Soon after he publicly announced his discovery in 1898, the structure was named after him, becoming universally known as the Golgi apparatus. Yet, many scientists did not believe that what Golgi observed was a real organelle present in the cell and instead argued that the apparent body was a visual distortion caused by staining. The invention of the electron microscope in the twentieth century finally confirmed that the Golgi apparatus is a cellular organelle. The Golgi apparatus is often considered the distribution and shipping department for the cell's chemical products. It modifies proteins and lipids (fats) that have been built in the endoplasmic reticulum and prepares them for export outside of the cell or for transport to other locations in the cell. Proteins and lipids built in the smooth and rough endoplasmic reticulum bud off in tiny bubble-like vesicles that move through the cytoplasm until they reach the Golgi complex. The vesicles fuse with the Golgi membranes and release their internally stored molecules into the organelle. Once inside, the compounds are further processed by the Golgi apparatus, which adds molecules or chops tiny pieces off the ends. When completed, the product is extruded from the GA in a vesicle and directed to its final destination inside or outside the cell. The exported products are secretions of proteins or glycoproteins that are part of the cell's function in the organism. Other products are returned to the endoplasmic reticulum or may undergo maturation to become lysosomes. The modifications to molecules that take place in the Golgi apparatus occur in an orderly fashion. Each Golgi stack has two distinct ends, or faces. The cis face of a Golgi stack is the end of the organelle where substances enter from the endoplasmic reticulum for processing, while the trans face is where they exit in the form of smaller detached vesicles. Consequently, the cis face is found near the endoplasmic reticulum, from whence most of the material it receives comes, and the trans face is positioned near the plasma membrane of the cell, to where many of the substances it modifies are shipped. The chemical make-up of each face is different and the enzymes contained in the lumens (inner open spaces) of the cisternae between the faces are distinctive. Illustrated in Figure 2 is a fluorescence digital image taken through a microscope of the Golgi apparatus (pseudocolored red) in a typical animal cell. Note the close proximity of the Golgi membranes to the cell nucleus. Proteins, carbohydrates, phospholipids, and other molecules formed in the endoplasmic reticulum are transported to the Golgi apparatus to be biochemically modified during their transition from the cis to the trans poles of the complex. Enzymes present in the Golgi lumen modify the carbohydrate (or sugar) portion of glycoproteins by adding or subtracting individual sugar monomers. In addition, the Golgi apparatus manufactures a variety of macromolecules on its own, including a variety of polysaccharides. The Golgi complex in plant cells produces pectins and other polysaccharides specifically needed by for plant structure and metabolism. The products exported by the Golgi apparatus through the trans face eventually fuse with the plasma membrane of the cell. Among the most important duties of the Golgi apparatus is to sort the wide variety of macromolecules produced by the cell and target them for distribution to their proper location. Specialized molecular identification labels or tags, such as phosphate groups, are added by the Golgi enzymes to aid in this sorting effort. Intermediate Filaments Intermediate filaments are a very broad class of fibrous proteins that play an important role as both structural and functional elements of the cytoskeleton. Ranging in size from 8 to 12 nanometers (in diameter; see Figure 1), intermediate filaments function as tension-bearing elements to help maintain cell shape and rigidity, and serve to anchor in place several organelles, including the nucleus and desmosomes. Intermediate filaments are also involved in formation of the nuclear lamina, a net-like meshwork array that lines the inner nuclear membrane and governs the shape of the nucleus. Although all eukaryotes contain the common cytoskeletal elements actin and tubulin (both free in the cytoplasm and polymerized in the form of microfilaments and microtubules), intermediate filaments are found only in some metazoan species, including vertebrates, nematodes, and molluscs. In vertebrates, intermediate filament presence and composition is not only speciesdependent, it also varies with the tissue type. For example, most animal epithelial cells contain keratins, a diverse family of intermediate filaments consisting of more than 50 members, while mesenchymal and muscle cells are rich in the fibrous proteins, vimentin and desmin, respectively. Intermediate filaments found in neurons and glial cells include peripherin, neurofilaments, and glial fibrillary acidic protein (GFAP). A variety of associated proteins bind to intermediate filaments, either to improve stability (through crosslinking) or to provide attachment sites for other protein assemblies, such as actin filaments and microtubules. Another highly specialized class of intermediate filaments are the nuclear lamins, which constitute the fibrous protein network that lines the inside of the nuclear membrane, as discussed above. Due to their close proximity to the membrane, nuclear lamins help attach the chromosomes to the nuclear membrane and provide anchorage points for the nuclear pores. Many scientists believe that nuclear lamins are the evolutionary ancestor of cytoplasmic intermediate filaments, which evolved through duplication and translocation of the gene product to the cytoplasm. The rigidity bestowed on cells by intermediate filament networks is especially useful to soft-bodied animals that do not possess an exoskeleton. Because intermediate filaments are very abundant in cells that are often subjected to high mechanical stress in vivo, it appears that their primary role is to provide physical strength to cells and tissues. Presented in Figure 2 is a digital image of the keratin intermediate network found in a rat kangaroo (PtK2 line) epithelial cell as seen through a fluorescence optical microscope. The extensive intertwined network was labeled with primary antibodies to several cytokeratin proteins, which were then stained with secondary antibodies containing a green fluorescent dye. The nucleus was counterstained with a blue dye to note its location in relation to the intermediate filament network. Fluorescence microscopy is an important tool that scientists use to examine the structure and function of internal cellular organelles and the cytoskeleton. As illustrated in Figure 1, intermediate filament monomer peptides are an elongated fibrous class of proteins with a central alpha-helical region capped with globular ends at both the amino and carboxylic acid termini. Two of the monomer units form a coiled-coil dimer that self-associates in an anti-parallel arrangement to form a staggered tetramer, which is the analogous soluble subunit for the globular actin monomer and the tubulin heterodimer (existing free in the cytoplasm). Tetramer units pack together laterally to form a sheet of eight parallel protofilaments that are supercoiled into a tight bundle. Each tightly coiled intermediate filament cross section reveals 32 individual alpha-helical peptides, which renders the filament easy to bend but quite difficult to break, thus accounting for the extreme structural rigidity. Although less is understood about the mechanism of intermediate filament assembly and disassembly, it is clear that some classes are highly dynamic structures with a significant rate of turnover in many cell types. Mutations in intermediate filament genes lead to a host of rather uncommon diseases. For example, defective keratins in skin tissue lead to a disorder known as epidermolysis bullosa simplex, manifested by skin blisters produced with even a slight mechanical stress. Similar blistering diseases due to keratin mutations in other tissues affect the esophagus, eyes, and mouth. Several neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS or Lou Gehrig's Disease), are associated with malfunctions in the intermediate filament (neurofilament) network, and defects in desmin intermediate filaments produce muscle disorders.