Lysosomes

Lysosomes The main function of these microscopic organelles is to serve as digestion compartments for cellular materials that have exceeded their lifetime or are otherwise no longer useful. In this regard, the lysosomes recycle the cell's organic material in a process known as autophagy. Lysosomes break down cellular waste products, fats, carbohydrates, proteins, and other macromolecules into simple compounds, which are then transferred back into the cytoplasm as new cell-building materials. To accomplish the tasks associated with digestion, the lysosomes utilize about 40 different types of hydrolytic enzymes, all of which are manufactured in the endoplasmic reticulum and modified in the Golgi apparatus. Lysosomes are often budded from the membrane of the Golgi apparatus, but in some cases they develop gradually from late endosomes, which are vesicles that carry materials brought into the cell by a process known as endocytosis. Like other microbodies, lysosomes are spherical organelles contained by a single layer membrane, though their size and shape varies to some extent. This membrane protects the rest of the cell from the harsh digestive enzymes contained in the lysosomes, which would otherwise cause significant damage. The cell is further safeguarded from exposure to the biochemical catalysts present in lysosomes by their dependency on an acidic environment. With an average pH of about 4.8, the lysosomal matrix is favorable for enzymatic activity, but the neutral environment of the cytosol renders most of the digestive enzymes inoperative, so even if a lysosome is ruptured, the cell as a whole may remain uninjured. The acidity of the lysosome is maintained with the help of hydrogen ion pumps, and the organelle avoids self-digestion by glucosylation of inner membrane proteins to prevent their degradation. The discovery of lysosomes involved the use of a centrifuge to separate the various components of cells. In the mid-twentieth century, the Belgian scientist Christian René de Duve was investigating carbohydrate metabolism of liver cells and observed that that the cells released an enzyme called acid phosphatase in larger amounts when they received proportionally greater damage in the centrifuge. To explain this phenomenon, de Duve suggested that the digestive enzyme was encased in some sort of membrane-bound organelle within the cell, which he dubbed the lysosome. After estimating the probable size of the lysosome, he was able to identify the organelle in images produced with an electron microscope. Lysosomes are found in all animal cells, but are most numerous in disease-fighting cells, such as white blood cells. This is because white blood cells must digest more material than most other types of cells in their quest to battle bacteria, viruses, and other foreign intruders. Several human diseases are caused by lysosome enzyme disorders that interfere with cellular digestion. TaySachs disease, for example, is caused by a genetic defect that prevents the formation of an essential enzyme that breaks down complex lipids called gangliosides. An accumulation of these lipids damages the nervous system, causes mental retardation, and death in early childhood. Also, arthritis inflammation and pain are related to the escape of lysosome enzymes. Microfilaments Common to all eukaryotic cells, these filaments are primarily structural in function and are an important component of the cytoskeleton, along with microtubules and often the intermediate filaments. Microfilaments range from 5 to 9 nanometers in diameter and are designed to bear large amounts of tension. In association with myosin, microfilaments help to generate the forces used in cellular contraction and basic cell movements. The filaments also enable a dividing cell to pinch off into two cells and are involved in amoeboid movements of certain types of cells. Microfilaments are solid rods made of a protein known as actin. When it is first produced by the cell, actin appears in a globular form (G-actin; see Figure 1). In microfilaments, however, which are also often referred to as actin filaments, long polymerized chains of the molecules are intertwined in a helix, creating a filamentous form of the protein (F-actin). All of the subunits that compose a microfilament are connected in such a way that they have the same orientation. Due to this fact, each microfilament exhibits polarity, the two ends of the filament being distinctly different. This polarity affects the growth rate of microfilaments, one end (termed the plus end) typically assembling and disassembling faster than the other (the minus end). Unlike microtubules, which typically extend out from the centrosome of a cell, microfilaments are typically nucleated at the plasma membrane. Therefore, the periphery (edges) of a cell generally contains the highest concentration of microfilaments. A number of external factors and a group of special proteins influence microfilament characteristics, however, and enable them to make rapid changes if needed, even if the filaments must be completely disassembled in one region of the cell and reassembled somewhere else. When found directly beneath the plasma membrane, microfilaments are considered part of the cell cortex, which regulates the shape and movement of the cell's surface. Consequently, microfilaments play a key role in development of various cell surface projections (as illustrated in Figure 2), including filopodia, lamellipodia, and stereocilia. Illustrated in Figure 2 is a fluorescence digital image of an Indian Muntjac deer skin fibroblast cell stained with fluorescent probes targeting the nucleus (blue) and the actin cytoskeletal network (green). Individually, microfilaments are relatively flexible. In the cells of living organisms, however, the actin filaments are usually organized into larger, much stronger structures by various accessory proteins. The exact structural form that a group of microfilaments assumes depends on their primary function and the particular proteins that bind them together. For instance, in the core of surface protrusions called microspikes, microfilaments are organized into tight parallel bundles by the bundling protein fimbrin. Bundles of the filaments are less tightly packed together, however, when they are bound by alpha-actinin or are associated with fibroblast stress fibers (the parallel green fibers in Figure 2). Notably, the microfilament connections created by some cross-linking proteins result in a web-like network or gel form rather than filament bundles. Over the course of evolutionary history of the cell, actin has remained relatively unchanged. This, along with the fact that all eukaryotic cells heavily depend upon the integrity of their actin filaments in order to be able to survive the many stresses they are faced with in their environment, makes actin an excellent target for organisms seeking to injure cells. Accordingly, many plants, which are unable to physically avoid predators that might want to eat them or harm them in some other way, produce toxins that affect cellular actin and microfilaments as a defensive mechanism. The death cap mushroom, for example, produces a substance called phalloidin that binds to and stabilizes actin filaments, which can be fatal to cells. Microtubules These straight, hollow cylinders are found throughout the cytoplasm of all eukaryotic cells (prokaryotes don't have them) and carry out a variety of functions, ranging from transport to structural support. Microtubules, which are about 25 nanometers in diameter, form part of the cytoskeleton that gives structure and shape to a cell, and also serve as conveyor belts moving other organelles throughout the cytoplasm. In addition, microtubules are the major components of cilia and flagella, and participate in the formation of spindle fibers during cell division (mitosis). The length of microtubules in the cell varies between 200 nanometers and 25 micrometers, depending upon the task of a particular microtubule and the state of the cell's life cycle. Microtubules are biopolymers that are composed of subunits made from an abundant globular cytoplasmic protein known as tubulin, as illustrated in Figure 1. Each subunit of the microtubule is made of two slightly different but closely related simpler units called alpha-tubulin and betatubulin that are bound very tightly together to form heterodimers. In a microtubule, the subunits are organized in such a way that they all point the same direction to form 13 parallel protofilaments. This organization gives the structure polarity, with only the alpha-tubulin proteins exposed at one end and only beta-tubulin proteins at the other. By adding or removing globular tubulin proteins, the length of polymeric microtubules can be increased or decreased. Because the two ends of a microtubule are not the same, however, the rate at which growth or depolymerization occurs at each pole is different. The end of a polarized filament that grows and shrinks the fastest is known as the plus end and the opposing end is called the minus end. For all microtubules, the minus end is the one with exposed alpha-tubulins. In an animal cell, it is this end that is located at the centriole-containing centrosome found near the nucleus, while the plus end, comprised of exposed beta-units, is projected out toward the cell's surface. Microtubules are continuously being assembled and disassembled so that tubulin monomers can be transported elsewhere to build microtubules when needed. Presented in Figure 2 is a digital image of the microtubule network found in an embryonic mouse cell as seen through a fluorescence optical microscope. The extensive intertwined network is labeled with primary antibodies to alpha-tubulin, which are then stained with secondary antibodies containing a green fluorescent dye. The nucleus was counterstained with a red dye to note its location in relation to the microtubule network. Fluorescence microscopy is an important tool that scientists use to examine the structure and function of internal cellular organelles. In addition to their structural support role, microtubules also serve as a highway system along which organelles can be transported with the aid of motor proteins. For instance, the microtubule network interconnects the Golgi apparatus with the plasma membrane to guide secretory vesicles for export, and also transports mitochondria back and forth in the cytoplasm. Another example is the translocation of vesicles containing neurotransmitters by microtubules to the tips of nerve cell axons. The motor proteins involved in organelle transport operate by altering their threedimensional conformation using adenosine triphosphate (ATP) as fuel to move back and forth along a microtubule. With each step, the motor molecule releases one portion of the microtubule and grips a second site farther long the filament. Motor proteins, which are grouped into several distinct classes, attach to organelles through specialized receptors. Since eukaryotic cells greatly depend upon the integrity of microtubules and other cytoskeletal filaments to maintain their structure and essentially to survive, many plants produce natural toxins aimed at disrupting the microtubule network as a means of self-defense. Taxol, for example, is a toxic substance produced by a species of yew trees that increases microtubule polymerization (building a macromolecule) by binding to the filament and stabilizing it. Other natural toxins, such as the colchicine produced by the meadow saffron, destabilize microtubules and hinder their polymerization. Both kinds of events can be fatal to the affected cell, though in some circumstances, this can be beneficial to animals, as demonstrated by taxol, which is commonly used as a cancer medication Mitochondria Mitochondria are rod-shaped organelles that can be considered the power generators of the cell, converting oxygen and nutrients into adenosine triphosphate (ATP). ATP is the chemical energy "currency" of the cell that powers the cell's metabolic activities. This process is called aerobic respiration and is the reason animals breathe oxygen. Without mitochondria (singular, mitochondrion), higher animals would likely not exist because their cells would only be able to obtain energy from anaerobic respiration (in the absence of oxygen), a process much less efficient than aerobic respiration. In fact, mitochondria enable cells to produce 15 times more ATP than they could otherwise, and complex animals, like humans, need large amounts of energy in order to survive. The number of mitochondria present in a cell depends upon the metabolic requirements of that cell, and may range from a single large mitochondrion to thousands of the organelles. Mitochondria, which are found in nearly all eukaryotes, including plants, animals, fungi, and protists, are large enough to be observed with a light microscope and were first discovered in the 1800s. The name of the organelles was coined to reflect the way they looked to the first scientists to observe them, stemming from the Greek words for "thread" and "granule." For many years after their discovery, mitochondria were commonly believed to transmit hereditary information. It was not until the mid-1950s when a method for isolating the organelles intact was developed that the modern understanding of mitochondrial function was worked out. The elaborate structure of a mitochondrion is very important to the functioning of the organelle (see Figure 1). Two specialized membranes encircle each mitochondrion present in a cell, dividing the organelle into a narrow intermembrane space and a much larger internal matrix, each of which contains highly specialized proteins. The outer membrane of a mitochondrion contains many channels formed by the protein porin and acts like a sieve, filtering out molecules that are too big. Similarly, the inner membrane, which is highly convoluted so that a large number of infoldings called cristae are formed, also allows only certain molecules to pass through it and is much more selective than the outer membrane. To make certain that only those materials essential to the matrix are allowed into it, the inner membrane utilizes a group of transport proteins that will only transport the correct molecules. Together, the various compartments of a mitochondrion are able to work in harmony to generate ATP in a complex multi-step process. Mitochondria are generally oblong organelles, which range in size between 1 and 10 micrometers in length, and occur in numbers that directly correlate with the cell's level of metabolic activity. The organelles are quite flexible, however, and time-lapse studies of living cells have demonstrated that mitochondria change shape rapidly and move about in the cell almost constantly. Movements of the organelles appear to be linked in some way to the microtubules present in the cell, and are probably transported along the network with motor proteins. Consequently, mitochondria may be organized into lengthy traveling chains, packed tightly into relatively stable groups, or appear in many other formations based upon the particular needs of the cell and the characteristics of its microtubular network. Presented in Figure 2 is a digital image of the mitochondrial network found in the ovarian tissue from a mountain goat relative, known as the Himalayan Tahr, as seen through a fluorescence optical microscope. The extensive intertwined network is labeled with a synthetic dye named MitoTracker Red (red fluorescence) that localizes in the respiring mitochondria of living cells in culture. The rare twin nuclei in this cell were counterstained with a blue dye (cyan fluorescence) to denote their centralized location in relation to the mitochondrial network. Fluorescence microscopy is an important tool that scientists use to examine the structure and function of internal cellular organelles. The mitochondrion is different from most other organelles because it has its own circular DNA (similar to the DNA of prokaryotes) and reproduces independently of the cell in which it is found; an apparent case of endosymbiosis. Scientists hypothesize that millions of years ago small, free-living prokaryotes were engulfed, but not consumed, by larger prokaryotes, perhaps because they were able to resist the digestive enzymes of the host organism. The two organisms developed a symbiotic relationship over time, the larger organism providing the smaller with ample nutrients and the smaller organism providing ATP molecules to the larger one. Eventually, according to this view, the larger organism developed into the eukaryotic cell and the smaller organism into the mitochondrion. Mitochondrial DNA is localized to the matrix, which also contains a host of enzymes, as well as ribosomes for protein synthesis. Many of the critical metabolic steps of cellular respiration are catalyzed by enzymes that are able to diffuse through the mitochondrial matrix. The other proteins involved in respiration, including the enzyme that generates ATP, are embedded within the mitochondrial inner membrane. Infolding of the cristae dramatically increases the surface area available for hosting the enzymes responsible for cellular respiration. Mitochondria are similar to plant chloroplasts in that both organelles are able to produce energy and metabolites that are required by the host cell. As discussed above, mitochondria are the sites of respiration, and generate chemical energy in the form of ATP by metabolizing sugars, fats, and other chemical fuels with the assistance of molecular oxygen. Chloroplasts, in contrast, are found only in plants and algae, and are the primary sites of photosynthesis. These organelles work in a different manner to convert energy from the sun into the biosynthesis of required organic nutrients using carbon dioxide and water. Like mitochondria, chloroplasts also contain their own DNA and are able to grow and reproduce independently within the cell. In most animal species, mitochondria appear to be primarily inherited through the maternal lineage, though some recent evidence suggests that in rare instances mitochondria may also be inherited via a paternal route. Typically, a sperm carries mitochondria in its tail as an energy source for its long journey to the egg. When the sperm attaches to the egg during fertilization, the tail falls off. Consequently, the only mitochondria the new organism usually gets are from the egg its mother provided. Therefore, unlike nuclear DNA, mitochondrial DNA doesn't get shuffled every generation, so it is presumed to change at a slower rate, which is useful for the study of human evolution. Mitochondrial DNA is also used in forensic science as a tool for identifying corpses or body parts, and has been implicated in a number of genetic diseases, such as Alzheimer's disease and diabetes. Peroxisomes Microbodies are a diverse group of organelles that are found in the cytoplasm of almost all cells, roughly spherical, and bound by a single membrane. There are several types of microbodies, including lysosomes, but peroxisomes are the most common. All eukaryotes are comprised of one or more cells that contain peroxisomes. The organelles were first discovered by the Belgian scientist Christian de Duve, who also discovered lysosomes. Peroxisomes contain a variety of enzymes, which primarily function together to rid the cell of toxic substances, and in particular, hydrogen peroxide (a common byproduct of cellular metabolism). These organelles contain enzymes that convert the hydrogen peroxide to water, rendering the potentially toxic substance safe for release back into the cell. Some types of peroxisomes, such as those in liver cells, detoxify alcohol and other harmful compounds by transferring hydrogen from the poisons to molecules of oxygen (a process termed oxidation). Others are more important for their ability to initiate the production of phospholipids, which are typically used in the formation of membranes. In order to carry out their activities, peroxisomes use significant amounts of oxygen. This characteristic of the organelles would have been extremely important millions of years ago, before cells contained mitochondria, when the Earth's atmosphere first began to amass large amounts of oxygen due to the actions of photosynthetic bacteria. Peroxisomes would have been primarily responsible at that time for detoxifying cells by decreasing their levels of oxygen, which was then poisonous to most forms of life. The organelles would have provided the cellular benefit of carrying out a number of advantageous reactions as well. Later, when mitochondria eventually evolved, peroxisomes became less important (in some ways) to the cell since mitochondria also utilize oxygen to carry out many of the same reactions, but with the additional benefit of generating energy in the form of adenosine triphosphate (ATP) at the same time. Peroxisomes are similar in appearance to lysosomes, another type of microbody, but the two have very different origins. Lysosomes are generally formed in the Golgi complex, whereas peroxisomes self-replicate. Unlike self-replicating mitochondria, however, peroxisomes do not have their own internal DNA molecules. Consequently, the organelles must import the proteins they need to make copies of themselves from the surrounding cytosol. The importation process of peroxisomes is not yet well understood, but it appears to be heavily dependent upon peroxisomal targeting signals composed of specific amino acid sequences. These signals are thought to interact with receptor proteins present in the cytosol and docking proteins present in the peroxisomal membrane. As more and more proteins are imported into lumen of a peroxisome or are inserted into its membrane, the organelle gets larger and eventually reaches a point where fission takes place, resulting in two daughter peroxisomes. Illustrated in Figure 2 is a fluorescence digital image of an African water mongoose skin fibroblast cell stained with fluorescent probes targeting the nucleus (red), actin cytoskeletal network (blue), and peroxisomes (green). Since the early 1980s, a number of metabolic disorders have been discovered to be caused by molecular defects in peroxisomes. Two major categories have been described so far. The first category consists of disorders of peroxisome biogenesis in which the organelle fails to develop normally, causing defects in numerous peroxisomal proteins. The second category involves defects of single peroxisomal enzymes. Studies indicate that approximately one in every 20,000 people has some type of a peroxisomal disorder. The most serious of these disorders is Zellweger syndrome, which is characterized by an absence or reduced number of peroxisomes in the cells. Present in patients at birth (congenital), Zellweger syndrome has no cure or effective treatment and usually causes death within the first year of life. Plasma Membrane All living cells, prokaryotic and eukaryotic, have a plasma membrane that encloses their contents and serves as a semi-porous barrier to the outside environment. The membrane acts as a boundary, holding the cell constituents together and keeping other substances from entering. The plasma membrane is permeable to specific molecules, however, and allows nutrients and other essential elements to enter the cell and waste materials to leave the cell. Small molecules, such as oxygen, carbon dioxide, and water, are able to pass freely across the membrane, but the passage of larger molecules, such as amino acids and sugars, is carefully regulated. According to the accepted current theory, known as the fluid mosaic model, the plasma membrane is composed of a double layer (bilayer) of lipids, oily substances found in all cells (see Figure 1). Most of the lipids in the bilayer can be more precisely described as phospholipids, that is, lipids that feature a phosphate group at one end of each molecule. Phospholipids are characteristically hydrophilic ("water-loving") at their phosphate ends and hydrophobic ("water-fearing") along their lipid tail regions. In each layer of a plasma membrane, the hydrophobic lipid tails are oriented inwards and the hydrophilic phosphate groups are aligned so they face outwards, either toward the aqueous cytosol of the cell or the outside environment. Phospholipids tend to spontaneously aggregate by this mechanism whenever they are exposed to water. Within the phospholipid bilayer of the plasma membrane, many diverse proteins are embedded, while other proteins simply adhere to the surfaces of the bilayer. Some of these proteins, primarily those that are at least partially exposed on the external side of the membrane, have carbohydrates attached to their outer surfaces and are, therefore, referred to as glycoproteins. The positioning of proteins along the plasma membrane is related in part to the organization of the filaments that comprise the cytoskeleton, which help anchor them in place. The arrangement of proteins also involves the hydrophobic and hydrophilic regions found on the surfaces of the proteins: hydrophobic regions associate with the hydrophobic interior of the plasma membrane and hydrophilic regions extend past the surface of the membrane into either the inside of the cell or the outer environment. Plasma membrane proteins function in several different ways. Many of the proteins play a role in the selective transport of certain substances across the phospholipid bilayer, either acting as channels or active transport molecules. Others function as receptors, which bind informationproviding molecules, such as hormones, and transmit corresponding signals based on the obtained information to the interior of the cell. Membrane proteins may also exhibit enzymatic activity, catalyzing various reactions related to the plasma membrane. Since the 1970s, the plasma membrane has been frequently described as a fluid mosaic, which is reflective of the discovery that oftentimes the lipid molecules in the bilayer can move about in the plane of the membrane. However, depending upon a number of factors, including the exact composition of the bilayer and temperature, plasma membranes can undergo phase transitions which render their molecules less dynamic and produce a more gel-like or nearly solid state. Cells are able to regulate the fluidity of their plasma membranes to meet their particular needs by synthesizing more of certain types of molecules, such as those with specific kinds of bonds that keep them fluid at lower temperatures. The presence of cholesterol and glycolipids, which are found in most cell membranes, can also affect molecular dynamics and inhibit phase transitions. In prokaryotes and plants, the plasma membrane is an inner layer of protection since a rigid cell wall forms the outside boundary for their cells. The cell wall has pores that allow materials to enter and leave the cell, but they are not very selective about what passes through. The plasma membrane, which lines the cell wall, provides the final filter between the cell interior and the environment. Eukaryotic animal cells are generally thought to have descended from prokaryotes that lost their cell walls. With only the flexible plasma membrane left to enclose them, these primordial creatures would have been able to expand in size and complexity. Eukaryotic cells are generally ten times larger than prokaryotic cells and have membranes enclosing interior components, the organelles. Like the exterior plasma membrane, these membranes also regulate the flow of materials, allowing the cell to segregate its chemical functions into discrete internal compartments. Ribosomes All living cells contain ribosomes, tiny organelles composed of approximately 60 percent ribosomal RNA (rRNA) and 40 percent protein. However, though they are generally described as organelles, it is important to note that ribosomes are not bound by a membrane and are much smaller than other organelles. Some cell types may hold a few million ribosomes, but several thousand is more typical. The organelles require the use of an electron microscope to be visually detected. Ribosomes are mainly found bound to the endoplasmic reticulum and the nuclear envelope, as well as freely scattered throughout the cytoplasm, depending upon whether the cell is plant, animal, or bacteria. The organelles serve as the protein production machinery for the cell and are consequently most abundant in cells that are active in protein synthesis, such as pancreas and brain cells. Some of the proteins synthesized by ribosomes are for the cell's own internal use, especially those that are produced by free ribosomes. Many of the proteins produced by bound ribosomes, however, are transported outside of the cell. In eukaryotes, the rRNA in ribosomes is organized into four strands, and in prokaryotes, three strands. Eukaryote ribosomes are produced and assembled in the nucleolus. Ribosomal proteins enter the nucleolus and combine with the four rRNA strands to create the two ribosomal subunits (one small and one large) that will make up the completed ribosome (see Figure 1). The ribosome units leave the nucleus through the nuclear pores and unite once in the cytoplasm for the purpose of protein synthesis. When protein production is not being carried out, the two subunits of a ribosome are separated. In 2000, the complete three-dimensional structure of the large and small subunits of a ribosome was established. Evidence based on this structure suggests, as had long been assumed, that it is the rRNA that provides the ribosome with its basic formation and functionality, not proteins. Apparently the proteins in a ribosome help fill in structural gaps and enhance protein synthesis, although the process can take place in their absence, albeit at a much slower rate. The units of a ribosome are often described by their Svedberg (s) values, which are based upon their rate of sedimentation in a centrifuge. The ribosomes in a eukaryotic cell generally have a Svedberg value of 80S and are comprised of 40s and 60s subunits. Prokaryotic cells, on the other hand, contain 70S ribosomes, each of which consists of a 30s and a 50s subunit. As demonstrated by these values, Svedberg units are not additive, so the values of the two subunits of a ribosome do not add up to the Svedberg value of the entire organelle. This is because the rate of sedimentation of a molecule depends upon its size and shape, rather than simply its molecular weight. Protein synthesis requires the assistance of two other kinds of RNA molecules in addition to rRNA. Messenger RNA (mRNA) provides the template of instructions from the cellular DNA for building a specific protein. Transfer RNA (tRNA) brings the protein building blocks, amino acids, to the ribosome. There are three adjacent tRNA binding sites on a ribosome: the aminoacyl binding site for a tRNA molecule attached to the next amino acid in the protein (as illustrated in Figure 1), the peptidyl binding site for the central tRNA molecule containing the growing peptide chain, and an exit binding site to discharge used tRNA molecules from the ribosome. Once the protein backbone amino acids are polymerized, the ribosome releases the protein and it is transported to the cytoplasm in prokaryotes or to the Golgi apparatus in eukaryotes. There, the proteins are completed and released inside or outside the cell. Ribosomes are very efficient organelles. A single ribosome in a eukaryotic cell can add 2 amino acids to a protein chain every second. In prokaryotes, ribosomes can work even faster, adding about 20 amino acids to a polypeptide every second. In addition to the most familiar cellular locations of ribosomes, the organelles can also be found inside mitochondria and the chloroplasts of plants. These ribosomes notably differ in size and makeup than other ribosomes found in eukaryotic cells, and are more akin to those present in bacteria and blue-green algae cells. The similarity of mitochondrial and chloroplast ribosomes to prokaryotic ribosomes is generally considered strong supportive evidence that mitochondria and chloroplasts evolved from ancestral prokaryotes.