LESSON 5. VITAL MANIFESTATIONS OF THE CELL (PART 1)_477b669b43f01bee354e64cdd6553820.pdf

Cytology and Histology (Academic course 2023/24) Verónica Mª Molina Hernández LESSON 5: VITAL MANIFESTATIONS OF THE CELL A. LYSOSOMES I. DEFINITION Lysosomes are membrane-surrounded organoids 0.25 to 0.50 µm in diameter containing enzymes. Their function is responsible for intracellular digestion...

Cytology and Histology (Academic course 2023/24) Verónica Mª Molina Hernández LESSON 5: VITAL MANIFESTATIONS OF THE CELL A. LYSOSOMES I. DEFINITION Lysosomes are membrane-surrounded organoids 0.25 to 0.50 µm in diameter containing enzymes. Their function is responsible for intracellular digestion and enzymatic degradation of intra- or extracellular soluble and solid substances. They may have ovoid or irregular morphology and their content may be homogeneous electrodense (primary lysosomes) or consist of accumulations of electrodense granules in a moderately electrodense matrix (secondary lysosomes). They were discovered and named by Christian de Duve, who eventually received the Nobel Prize in Physiology or Medicine in 1974. He succeeded in detecting the enzyme activity from the microsomal fraction. This was the crucial step in the serendipitous discovery of lysosomes. It became clear that this enzyme from the cell fraction came from membranous fractions, which were definitely cell organelles, and in 1955 De Duve named them "lysosomes" to reflect their digestive properties. Originally, De Duve had termed the organelles the "suicide bags" or "suicide sacs" of the cells, for their hypothesized role in apoptosis. However, it has since been concluded that they only play a minor role in cell death. Alex Novikoff successfully obtained the first electron micrographs of the new organelle. Using a staining method for acid phosphatase, de Duve and Novikoff confirmed the location of the hydrolytic enzymes of lysosomes using light and electron microscopic studies. It is not possible to identify (primary) lysosomes with certainty on morphological criteria alone. Histochemical demonstration of acid hydrolases or some other hydrolases is needed. So far, about 50 enzymes have been identified in lysosomes, such as proteases, glucosidases, lipases, phosphatases, phospholipases, nucleases and sulphatases. Because all these enzymes require an acidic environment for optimal functioning, lysosomal membranes possess proton pumps that actively transport H+ ions into the lysosome, maintaining a pH of 5.0. In addition to this characteristic, the lysosomal membrane has qualities that differentiate it from other cell membranes: the inner unit membrane has numerous glycoproteins (i.e. Lysosome-associated membrane glycoprotein LAMP-1 and LAMP-2) that protect it from acidity and enzyme activity. This membrane also contains transport proteins that facilitate the passage of the end products of digestion into the hyaloplasm. 1 Cytology and Histology (Academic course 2023/24) Verónica Mª Molina Hernández II. FUNCTIONS OF LYSOSOMES Lysosomes are organoids that are involved in two fundamental aspects of cell biology: nutrition and defence. In addition, they sometimes perform their function outside the cell. The functions of lysosomes can be summarised as follows: A) CELL NUTRITION. Lysosomes act by degrading complex molecules (carbohydrates, lipids, proteins and nucleic acids) into simpler ones that can be used by the cell by means of enzymes (lipases, nucleases, glycosidases, proteases). B) CELLULAR DEFENCE. Lysosomes destroy biological and non-biological structures that are harmful to the cell. They are therefore abundant in cells specialised in the defence of the organism, such as macrophages or polymorphonuclear neutrophils. We can distinguish between heterophagy and autophagy. -Heterophagy or phagocytosis: is the process by which a cell captures a particle or set of macromolecules (larger than 150 nm) and carries them from the outside to the inside of the cell. When a particle comes into contact with the cell membrane, either by binding to specific membrane receptors (for Fc regions of antibodies, complement or opsonins) or by physical properties, the cell emits cytoplasmic processes called pseudopodia through the mobilisation of microtubules, actin and myosin from the cytoplasm. These pseudopods engulf and surround the particle on all sides, so that it is contained in a membranous pocket called a phagocytic vesicle. This phagocytic vesicle detaches from the cell membrane, sinks into the cytoplasmic matrix and forms an endocytosis vacuole called phagosome. When a phagosome comes into contact with a primary lysosome, the membranes fuse, so that the primary lysosome expels its contents (hydrolytic enzymes). The two vesicles become one sole digestive vacuole called heterophagolysosome or secondary lysosome. The digested content passes into the cytoplasm, although the non-soluble components remain after digestion inside the vacuoles constituting a residual body and is removed by exocytosis. Sometimes, if phagocytosis is very intense, the osmotic pressure is so great that the membranes can rupture and the enzymes are released into the cytoplasm, causing the autolysis of these cells, the remains of which form pus. -Autophagy: the process by which the digestive system of the lysosomes is used in the removal of organoids and inclusions in the normal turnover of cell components. Thus, certain mitochondria, excess of endoplasmic reticulum cisternae or unusable secretory granules are surrounded by a membrane to form an autophagic vacuole. The primary lysosome then fuses with this vacuole, releases enzymes into it that digest its contents, and an autophagolysosome or secondary lysosome is formed. If the enzymatic equipment of the lysosomes is used to destroy excess secretory granules, the process is called crinophagy. E.g. Disposal of excess secretory granules containing insulin by fusion of these granules with lysosomes in the beta cells in the pancreatic islets. 2 Cytology and Histology (Academic course 2023/24) Verónica Mª Molina Hernández If the residual bodies from autophagic activity are not removed by exocytosis but are stored in the cytoplasm of the cell, they give rise to the so-called lipofuscin or "wearand-tear pigment". Lipofuscin is a brownish, PAS+ pigment (structure with its own colour, it does not need to be stained). Ultrastructurally, it consists of protein accumulations together with lipid structures or droplets surrounded by a membrane unit. It is usually observed in cardiac muscle fibers and neurons of old animals. Cardiac muscle fibers and neurons are long-lived cells that must renew their organoids quite frequently. Figure 1. Lipofuscin. Detail of protein structures and lipid droplets. Transmission electron microscopy (TEM). C) EXTRACELLULAR FUNCTION. Although lysosomes almost always perform their functions inside the cells, there are situations in which enzymes are released outside the cells, for example, the acrosome of the spermatozoon by breaking the zona pellucida and membrane of the oocyte and the remodelling of bone by osteoclasts. III. FORMATION OF LYSOSOMES Enzymes and lysosomal membranes are synthesised in the rough endoplasmic reticulum and from there pass through different transfer vesicles to the forming or CIS face of the Golgi complex where the mannose residues are phophoriled. The enzymes and membranes subsequently bud off from the TRANS side of the Golgi complex to form coated vesicles (clathrin). Clathrin is a filamentous protein that promotes vesicle formation and detachment from the TRANS face of the Golgi complex. Clathrin is rapidly lost and both types of vesicles fuse with a pre-existing vesicle, called a late endosome, which maintains an acidic pH. This late endosome, which already has enzymes and a special membrane, matures into a primary lysosome. IV. CLASSIFICATION OF LYSOSOMES • Primary lysosomes are those that have not yet become involved in digestive activity and include lysosomes and multivesicular bodies. Multivesicular bodies can appear in some cell types, such as endothelial cells, membrane-surrounded vacuoles of 0.5 to 2 m containing many small vesicles are observed. These vacuoles are acid phosphatase positive and are considered a special form of primary lysosome. 3 Cytology and Histology (Academic course 2023/24) Verónica Mª Molina Hernández • Secondary lysosomes are the vacuolar structures in which there is or has been digestive activity and include heterophagolysosomes, autophagolysosomes and residual bodies. B. PEROXISOMES Peroxisomes (microbodies) are cell organelles that appear similar to lysosomes but are smaller (0.2 to 0.4 µm in diameter) spherical to ovoid, membrane-surrounded organoids that contain more than 40 oxidative enzymes such as urate oxidase (only in some species), amino oxidase, D-amino acid oxidase and catalase. These enzymes are oxidoreductases. Peroxisomes are stained by the peroxidase reaction, which serves to distinguish them from lysosomes. That is, they are acid phosphatase- and peroxidase+. The functions of peroxisomes include their role in the catabolism of fatty acids (beta oxidation), puric bases and glycolic acid and forming acetyl coenzyme A (CoA) and hydrogen peroxide (H2O2). Peroxisomes are abundant in the cells of the liver and kidney because they are organs involved in remove toxic substances (i.e. alcohol) from the body. In some species, peroxisomes contain an electrodense inclusion, called a nucleoid, suspended in a homogeneous matrix of lower density. Nucleoids contain urate oxidase. Peroxisomes of primates, birds, reptiles and some breeds of dogs (Dalmatian, English bulldog) lack nucleoid and thus urate oxidase. The biological importance of nucleoid is due to its urate oxidase activity, an enzyme involved in metabolising uric acid to allantoin; therefore, animals lacking nucleoid in their peroxisomes only metabolise puric bases to uric acid and are called uricotelic animals. The inability to metabolise or eliminate all uric acid ingested from food or produced in the body is the cause of the metabolic disease called "gout". In contrast, non-uricotelic animals, those with nucleoid in their peroxisomes, degrade the puric bases to allantoin and therefore cannot suffer from this disease. C. MECHANISMS OF CELLULAR SUBSTANCE UPTAKE AND DIGESTION In this section, we will study the three types of mechanisms of substance uptake by the cell with modification of the cellular plasma membrane, thus ignoring passive (diffusion) or active (ATP-consuming) ion transport mechanisms. The process by which a cell ingests large molecules or macromolecules (enzymes, nucleic acids, histones, etc.), particulate material and other substances from the extracellular space by the formation of vesicles (phagosomes and endosomes) is known as endocytosis. Moreover, sometimes exist the degradation of intracellular substances (autosomes or cytosomes) by the enzymes of lysosomes. In both cases, the material to be internalized is surrounded by an area of cell membrane, which then buds off inside the cell to form a vesicle containing the ingested material. We speak of phagocytosis when the incorporated particles are visible with the optical microscope and pinocytosis when they are liquid particles or dissolved substances only visible with the electron microscope. Both processes require energy expenditure and are preceded by the attachment of the 4 Cytology and Histology (Academic course 2023/24) Verónica Mª Molina Hernández particle to the glycocalyx (sometimes located in a specific area of the membrane, where there are receptors). The digestion of extra- or intracellular material always occurs within a vacuole surrounded by a membrane to prevent the enzymes from passing into the cytoplasm and digesting the cell, so the union of the vacuoles that contain the substances to be digested are called secondary lysosomes, which depending on their origin can be: phagolysosome, endolysosome and autolysosome or cytolysosome. After degradation, if the resulting products are water-soluble, they dissolve in the cytoplasm. If they are not, they can be eliminated from the cell by exocytosis or remain inside the vacuole as a permanent residual body, giving rise to telolysosomes or myelin figures. There are three types of endocytosis: 1) Phagocytosis: previously discussed and in which phagocytic vesicles larger than 250 nm are formed. 2) Pinocytosis: which is considered the ingestion of liquids by the cell with modification of the plasma membrane, forming vesicles smaller than 250 nm. The substances that are incorporated by pinocytosis are protein molecules and other substances, including viruses, with a size no greater than 150 nm. Pinocytosis vacuoles form at specific membrane sites lined by a filamentous material called clathrin (coated vesicles). Once in the cytoplasm, clathrin disassembles from the vacuole and heads toward the membrane, the vacuoles being called endosomes. The endosomes fuse with the lysosomes forming endolysosomes that degrade the substances that finally pass into the hyaloplasm. The presence of specific receptors in the membrane allows the incorporation of specific substances. The transport of fluids through the cell is known as cytopempsis or transcytosis and serves to nourish the underlying cells. In this case, macromolecules (IgA, transferrin or insulin) are captured in vesicles on one side of the cell, drawn across the cell, and ejected on the other side. It occurs frequently in blood capillaries (endothelial cells) and muscle cells, but also in epithelial cells, neurons, osteoclasts and M cells of the intestine. 3) Micropinocytosis: the process is similar to the previous one, but the vesicles formed are between 75 to 90 nm. In addition, clathrin-coated vesicles, called "coated vesicles", are often formed. The formation of coated vesicles appears to be related to the receptor-mediated introduction of particles and is therefore also referred to as "receptor-mediated endocytosis". 5