Cytology and Histology (2023/24) Past Paper PDF

Cytology and Histology (Academic course 2023/24) Verónica Mª Molina Hernández LESSON 3: CYTOPLASMATIC ORGANOIDS I. RIBOSOMES Ribosomes were first described by George Palade in 1953 and so they were called particle of Palade. These organoids are present in both animal and plant cells. They are mad...

Cytology and Histology (Academic course 2023/24) Verónica Mª Molina Hernández LESSON 3: CYTOPLASMATIC ORGANOIDS I. RIBOSOMES Ribosomes were first described by George Palade in 1953 and so they were called particle of Palade. These organoids are present in both animal and plant cells. They are made up of ribonucleic acids (RNA) and proteins and their function is to contribute to the synthesis of cellular proteins by translating information from nuclear DNA. Ribosomes have different dimensions and locations depending on whether they are in prokaryotic or eukaryotic cells. In the prokaryotic cells, they are located freely in the cytoplasm and have a sedimentation rate of 70S (Svedberg units), and in the eukaryotic cells, they can be found free or associated with each other or with cell membranes, with a sedimentation rate of 80S. The ribosomes of eukaryotic cells associate with the membranous structures of the endoplasmic reticulum. In some cases, these associations are visible under the light microscope as areas of strong basophilic: examples are the Nissl bodies of neurons and the basal area of acinar cells in the pancreas. The strong basophilic of ribosomes under the light microscope is due to the fact that these organoids have phosphate radicals and ribosomal RNA in their structure that have affinity for cationic or basic dyes. 1A 1B Figure 1. Diagram of ribosomes (A). Ribosomes and rough endoplasmic reticulum under the electron microscope (B). Ribosomes are 250 by 150Å in size and can be seen by negative staining to consist of two pieces or subunits of different sizes separated by a cleft (Figure 1). In the case of eukaryotic cells, the two ribosomal subunits have sedimentation rates of 60S and 40S, respectively. The ribosome is a piece consisting of an inverted dome-like base, which corresponds to the large subunit, perforated by a canaliculus through which the formed proteins are evacuated, and a second piece, the small subunit, which corresponds to the cap. Both subunits consist of 28S RNA, the large subunit, and 18S RNA, the small subunit, associated with proteins. This RNA is known as ribosomal RNA (rRNA), but there 1 Cytology and Histology (Academic course 2023/24) Verónica Mª Molina Hernández is another ribosome-associated RNA involved in protein synthesis, called transfer RNA (tRNA). In addition, a third filamentous RNA, messenger RNA (mRNA), is located in the cytoplasm and is associated with ribosome-free particles. During protein synthesis, numerous ribosomes associate with each other via a strand of mRNA, which is located between the two subunits, constituting polyribosomes or polysomes. These associations can be attached via the large subunit to membranes of endoplasmic reticulum cisternae to form the rough endoplasmic reticulum. Ribosomes can be considered as complex biological machines involved in protein synthesis by linking the three ribonucleic acids to specific enzymes and their structural proteins. Proteins are synthesised by the intervention of mRNA, which originates from the nucleus, and are associated with the large ribonucleoprotein particles that form the ribosomes, in particular tRNA. In the ribosome, the mRNA sequence is translated into the corresponding amino acid sequence of each protein. A significant proportion of cellular proteins are synthesised in the cytoplasm by isolated ribosomes, whereas most proteins, both structural and functional, are formed in polysomes, either in the cytoplasm or attached to membranes. II. ENDOPLASMIC RETICULUM Endoplasmic reticulum was first observed by Porter, Claude and Fulham in 1945 in cultured rat fibroblasts. Its name is due to its reticular constitution and location in the endoplasm, hence the name endoplasmic reticulum (ER). Subsequently, studies on different cell types showed that the endoplasmic reticulum may or may not be associated with ribosomes, so it was deduced that there are two classes: granular or rough endoplasmic reticulum, if it is associated with ribosomes, and agranular or smooth endoplasmic reticulum, if its wall is free of ribosomes. Both are made up of canaliculi, vesicles and cisternae of different arrangement and dimensions, which increase or decrease in proportion depending on their functional state and the degree of differentiation of the cell. 1. Rough (or granular) endoplasmic reticulum (RER) Studies of the acinar cells of the pancreas showed that the basal pole had an intense basophilia, and this was related to the protein synthesis activity of these cells. Garnier, in 1897, found that this area consisted of associations of tubules with ribosomes, i.e. cisternae of rough endoplasmic reticulum. These are structures similar to the Nissl bodies of neurons, which are considered to be focal areas of rough endoplasmic reticulum. This reticulum is made up of tubules that can take various forms: parallel tubules, concentric stratifications, anastomosing canaliculi and more or less extensive cisternae. The tubules or cisternae are bounded by a 50Å to 70Å thick membrane to which ribosomes are attached by the large subunit. The arrangement is variable and may depend not only on the cell type but also on the protein synthesis activity of the cell at any given time; thus, the presence of parallel tubules and concentric stratifications is 2 Cytology and Histology (Academic course 2023/24) Verónica Mª Molina Hernández usually a consequence of high protein synthesis activity. Furthermore, in these cells, the lumen of the reticulum is dilated, indicating a high degree of activity (Figure 2). The essential components of the reticulum are its membrane unit, which is similar to the cytoplasmic membrane, although thinner, and the ribosomes. The essential difference between the membrane of the rough endoplasmic reticulum and that of the smooth endoplasmic reticulum and other membranous organoids is the presence of functional proteins that form a complex consisting of two glycoproteins called ribophorins. This complex is the active point where ribosomes are coupled by their large subunit and are arranged like a small canaliculus through which the proteins formed by the polysomes are evacuated towards the lumen of their cisternae. Ribophorins are located in the thickness of the membranes, passing completely through them, and are attached to the structural proteins by mean of fibrous proteins. As an integral part of the cytoplasmic vacuolar system, the reticulum is related in particular to the structures that make up this system, and in general to all the membranous elements of the cell, even if only at specific times, as is the case with the cytoplasmic membrane. Its relationship with the different components of the cytoplasmic vacuolar system can be direct, as in the case of the reticulum with the nuclear envelope, and indirect, with the formation of transfer vesicles between the reticulum and the Golgi complex. The rough or granular endoplasmic reticulum is involved in cellular activity either with general functions such as mechanical, participating in the formation of the cytoskeleton, osmotic between the reticulum compartment and the cytoplasm, and those of substance transport through its lumens, or specific ones as a fundamental part of protein synthesis. A B C Figure 2. RER of normal (A) and dilated (B) cisterns. Images of RER at transmission EM (C). 3 Cytology and Histology (Academic course 2023/24) Verónica Mª Molina Hernández 2. Smooth endoplasmic reticulum (or agranular) endoplasmic reticulum (SER) Although many of the generalities described for the granular or rough endoplasmic reticulum are applicable to the agranular or smooth endoplasmic reticulum, this modality differs from the former in that it does not present associations with ribosomes and in its arrangement in the cell, since it forms vesicles and short anastomosing sinuous canaliculi that occupy a large part of the cytoplasmic volume (Figure 3). As a rule, however, much of this reticulum corresponds to terminal areas of the rough reticulum. The smooth endoplasmic reticulum is sometimes arranged in concentric circles around liposomes and mitochondria, as is the case in steroidproducing cells (adrenocortical cells, Leydig cells and corpus luteum of the ovary). However, it may adopt a peculiar arrangement in the form of anastomosed canaliculi that envelop the sarcomeres of striated muscle cells where it is known as sarcoplasmic reticulum, and in the case of sensory cells of the organ of Corti it is arranged as cisternae that relate to the cytoplasmic membrane. They are also present in adipocytes of brown adipose tissue. One of the fundamental functions of the smooth endoplasmic reticulum is the degradation of toxic substances (e.g., alcohol and barbiturates). On the other hand, it can also store protein substances from the rough endoplasmic reticulum or synthesised by polyribosomes. Proteins formed in the rough endoplasmic reticulum are introduced into the cisternae of the smooth endoplasmic reticulum by internal transport between the two reticula, while for the rest of the proteins, as these membranes lack ribophorins, large amphipathic proteins are activated by a mechanism known as post-translational import or protein formation. The smooth endoplasmic reticulum has general functions in common with those of the RER, such as mechanical support, osmosis, and transport of substances. Specific functions include conduction and storage of Ca2+ ions involved in muscle contraction, synthesis of steroids, triglycerides and phospholipids, degradation of toxic substances and glycogen (glycogenolysis), and demarcation and detachment of platelets in the megakaryocyte. A B Figure 3. REL in sacs (A). REL or sarcoplasmic reticulum in skeletal muscle (B). 4 Cytology and Histology (Academic course 2023/24) Verónica Mª Molina Hernández III. GOLGI COMPLEX Golgi complex was first observed by Camillo Golgi when studying cerebellum nerve cells from an owl (Bubo bubo) by means of the Golgi silver impregnation technique. He noticed, next to the nucleus, a network or reticulum blackened by the reduced silver, which he called the internal reticular apparatus because of its location in the endoplasm. It was later observed that this organoid was involved in multiple functions of the cell, so it is more correctly called a complex rather than an apparatus. Golgi complex, together with the endoplasmic reticulum and the nuclear envelope, constitute a membranous system whose structures are directly or indirectly related, forming the cytoplasmic vacuolar system. The location of the Golgi complex is not specific for each cell type; however, we can generalise that in cells of ectodermal origin it is usually located between the apical pole and the nucleus, in a supranuclear situation; in exocrine cells it is located between the excretory pole and the nucleus, and in endocrine cells its location, on the other hand, is not well defined. There are other cell types, such as neurons, where they are distributed fractionally around the nucleus. In this case, the Golgi complex is formed by a set of sacs and vesicles integrated as a single system, but there are exceptions to this rule, since several fractions can be dispersed throughout the cytoplasm, each fraction being called a dictyosomes. Golgi complex consists of a set of stacked flattened sacs and cisternae surrounded by vesicles that can be either transfer vesicles containing synthesised material or secretory vesicles, so that these vesicles are the expression of the manifest polarity of this complex: a forming or cis-face and a maturating or trans-face. The flattened sacs are stacked with a curvature that determines a convex and a concave face. The membrane of the sacs is generally thicker than that forming the endoplasmic reticulum, between 60 and 70 Å thick, and delimits an inner lumen of about 200 to 300 Å. The number of sacs varies between 4 and 8. Golgi sacs are fenestrated discs whose convex face is the forming or cis-face, while the concave face is the maturating or transface. As a general rule, the sacs that are concentrated on the concave side tend to have a more dilated lumen. Towards the ends, the saccules tend to dilate like cisterns and correspond to areas that have partially lost their functionality and eventually detach and form vacuoles, which are usually waste vesicles (Figure 4). Transfer vesicles originate from the endoplasmic reticulum, mainly from the rough, but also from the smooth endoplasmic reticulum. They are very uniform and contain the synthesised substances to be further processed and transformed in the Golgi complex. These vesicles are called coated vesicles, due to the presence of the protein clathrin. Clathrin is a low molecular weight polypeptide that promotes the separation of vesicles from the Golgi complex. The clathrin-coated vesicles are protein-like, so the membrane is clearly visible and filaments are associated with it. The coated vesicles attach to the convex fenestrated or forming cis-face and are then called transfer vesicles. As a rule, these transfer vesicles are about 200Å in diameter and have a moderately electrodense content. 5 Cytology and Histology (Academic course 2023/24) Verónica Mª Molina Hernández The sacs of the concave face give rise to vesicles of about 100-150Å and 200300Å in diameter that are located towards the ends of the sacs or over the entire surface of the maturating or trans-face. In glandular cells, these condensation vesicles form the so-called secretory granules, which have very different size and a highly variable content. In some cell populations, such as plasma cells, the region of the Golgi complex is sharply defined as an area adjacent to the nucleus and colourless after routine light microscopy staining techniques, so this phenomenon is referred to as Golgi negative staining. However, by using supravital dyes, such as methylene blue and Nile blue sulphate, the Golgi complex can be directly observed under the light microscope. In addition, the Golgi complex can accumulate certain dyes such as trypan blue and ferric and cupric compounds. The vacuoles and vesicles are the most easily visible by histochemical and histoenzymological techniques, with PAS and nucleoside phosphatase positive reactions. They also show ADP-ase, ATP-ase-Mg-dependent, thiaminopyrophosphatase, glucosyl and galactosyltransferase positive reactions. Golgi complex is involved in numerous cellular functions: (i) concentration of different substances, e.g. polysaccharides (during spermatogenesis, subsequently constituting the acrosome), enzymes in the GERL region (Golgi, endoplasmic reticulum and lysosomes), which is located in a particular area where the Golgi sacs are related to the rough endoplasmic reticulum for the formation of lysosomes; (ii) polysaccharide synthesis, especially in mucous and cartilaginous cells; (iii) formation of the membranes of secretory granules; and, (iv) cell membrane turnover. A B Figure 4. Schematic of the Golgi complex (A). Electron microscopic images of Golgi complexes with different morphologies (B). IV. MITOCHONDRIA Mitochondria is considered a semi-autonomous membranous structure whose main function is energy production. In 1894, Altmann first described mitochondria as elementary forms of life and called them bioblasts. Subsequently, DNA was detected in their matrix, as well as the possibility of self-duplication. The current name 6 Cytology and Histology (Academic course 2023/24) Verónica Mª Molina Hernández "mitochondria" was given by Benda in 1897, due to their more or less elongated morphology: mito (filament) and chondria (particle). This organoid can be observed in both living and fixed cells, but special visualisation methods have to be used because of its low refractive index, which is similar to that of the cytoplasm. Mitochondria are not static elements within the cell, but have extrinsic movements, either produced by cytoplasmic currents or due to their internal biochemical reactions, which induce rotational and oscillatory movements. These movements facilitate the transport of mitochondria to places where energy is needed, although it is also true that this is not always the case, but they are sometimes located constantly in places where energy is highly needed, as in the case of muscle fibers, where they are arranged in a ring around the sarcomeres, or in the cones and rods of the retina, where they are located in the inner segment. Mitochondria average approximately 0.5-7 µm in length by 0.2-0.5 µm in width, although the size of mitochondria varies greatly from cell to cell type. In terms of shape, they can be broadly divided into two types: lamellar and tubular (Figure 5), although irregular shapes can also be seen. The membranous structure of mitochondria consists of the association of two membranes, a smooth outer membrane (40-70Å thick), and a folded inner membrane (50-60Å thick) forming numerous folds or cristae increasing greatly the surface area of the membrane. These cristae can have different shapes: villous, tubular, vesicular, lamellar, digitiform and some other irregular shapes that increase or decrease in number depending on cellular activity. Between the two membranes there is a space or intermembrane space, and the space enclosed by the inner membrane is termed the matrix space, which is more or less electrodense and constitutes the "mitochondrial matrix". Inside the matrix space, on the inner membrane, hypotonic treatments and negative staining show particles with a morphology similar to a drumstick in which the ATP synthase enzyme units are located. These structures are called oxysomes, elementary particles, F particles or Fernández-Morán particles. The mitochondrial matrix is one of the most important parts because it is where most of the mitochondrial activities take place. The matrix contains: (a) Mitochondrial DNA: identifiable as branched strands of varying thickness and located in the less dense regions of the matrix. It can also be observed as a ring. It differs from nuclear DNA in its composition of purine and pyrimidine bases. (b) Mitochondrial RNA: granules about 120Å diameter. Ribonucleic acid occurs as rRNA and tRNA and has a sedimentation rate of 55S. (c) Self ribosomes with a sedimentation rate of 55S. (d) Protein inclusions: has an important enzymatic equipment, which is located at specific sites in the mitochondria depending on the cell type. e) Dense granules (matrix granules) 300-500Å in diameter: are clusters of ions located close to the mitochondrial cristae. 7 Cytology and Histology (Academic course 2023/24) Verónica Mª Molina Hernández Mitochondria have numerous functions derived from their complex constitution: (1) they are involved in oxidative phosphorylation, beta-oxidation of fatty acids, glucose degradation and the Krebs cycle; and (2) they are involved in fatty acid and protein synthesis, ion secretion and concentration, and protein substance secretion and concentration. A B Figure 5. Schematic of mitochondria (A) with lamellar and tubular cristae. (B) Electron microscopic images of mitochondria with lamellar and tubular cristae. 8