Cytology - Cells and Cell Structure PDF

 Cytology PLASMA MEMBRANE The plasma membrane is a lipid-bilayered structure visible with transmission electron microscopy. It (cell membrane, plasma- lem...

 Cytology PLASMA MEMBRANE The plasma membrane is a lipid-bilayered structure visible with transmission electron microscopy. It (cell membrane, plasma- lemma) is a dynamic structure that actively participates in many physiologic and biochemical activities essential to cell function and survival. The total thickness of the plasma membrane is about 8 to 10 nm. Molecules are arranged asymmetrically: Complex sugars - On the outer portion 40/50% proteins Cholesterol 30/40% phospholipids with different residues depending on the tissue - Phosphoglycerides (based on glycerol) are most abundant in all cell membranes; Sphingolipids (based on the aminoalchool sphingosine) instead are contained in some membranes such as the neural tissues’ ones The plasma membrane is a selective barrier, susceptible to chemical and physical variation in the external environment, that must receive signals from the outside and send signal transduction inside. Also, it regulates cell-to-cell adhesion. Fluid mosaic model The plasma membrane is composed of an amphipathic lipid layer containing embedded integral membrane proteins with peripheral membrane proteins attached to its surfaces. The current interpretation of the molecular organisation of the plasma membrane is referred to as the modified fluid–mosaic model being a mosaic because the membrane consists primarily of phospholipid, cholesterol, and protein molecules. The lipid molecules form a lipid bilayer with an amphipathic character (it is both hydrophobic and hydrophilic). It is a fluid because individual phospholipids and proteins can move side-to-side within the layer. Proof is given by using a virus to infect both mouse and human cells and an antibody staining: scientists saw that, after some time after the cells’ fusion, the protein diffused. Temperature is directly proportional to fluidity Length of fatty acids chains, number of proteins and unsaturated fatty acids are inversely proportional to fluidity Cholesterol is inversely proportional to fluidity - It renders the lipid belayer less deformable decreasing its permeability and preventing the crystallisation of fatty acids Most lipids and some proteins can drift laterally in the plane of the membrane and rarely they can flip-flop from one layer to another: the distribution and movement of proteins within the lipid bilayer is not as random as once thought. Plasma membrane appears to be patchy with localised regions that are distinct in structure and function and vary in thickness and composition. These localised regions contain high concentrations of cholesterol and glycosphingolipids and are called lipid rafts. Owing to the high concentration of cholesterol and the presence of longer, highly saturated fatty-acid chains, the lipid raft area is thicker and exhibits less fluidity than the surrounding plasma membrane. Cholesterol is the dynamic “glue” that holds the raft together; its removal from the raft results in dispersion of raft-associated lipids and proteins. Lipid rafts contain a variety of integral and peripheral membrane proteins involved in cell signalling. They can be viewed as “signalling platforms” floating in the ocean of lipids. Each individual raft is equipped with all of the necessary elements (receptors, coupling factors, effector enzymes, and substrates) to receive and convey specific signals. Signal transduction in lipid rafts occurs more rapidly and efficiently because of the close proximity of interacting proteins. In addition, different signalling rafts allow for the separation of specific signalling molecules from each other. 1  Cytology Glycocalix Carbohydrates may be attached to proteins, thereby forming glycoproteins; or to lipids of the bilayer, thereby forming glycolipids. These surface molecules constitute a layer at the surface of the cell, referred to as the cell coat or glycocalyx. They help establish extracellular microenvironments at the membrane surface that have specific functions in metabolism, cell recognition, and cell association and serve as receptor sites for hormones. Molecular filter - In the intestine breaches in the pericellular matrix allows the passage of microbiota thus inflaming the intestine Cell-to-cell adhesion Site of enzymes Recognition of sugars - It is the first step of migration of leukocytes out of the bloodstream to reach the sites of infection Proteins In most plasma membranes, protein molecules constitute approximately half of the total membrane mass. Most of the proteins are embedded within the lipid bilayer or pass through the lipid bilayer completely: these proteins are called integral membrane proteins. The other types of protein is peripheral membrane proteins and they are not embedded within the lipid bilayer, but are associated with the plasma membrane by strong ionic interactions, mainly with integral proteins on both the extracellular and intracellular surfaces of the membrane. EGF-R in carcinomas: Epithelial cells express the receptor for the epidermal growth factor (EGF), a protein ligand of major importance in proliferation, differentiation and survival of the tissue. In carcinomas, EGF-R is constitutively active and stimulates proliferation without waiting for the ligand’s arrival, therefore oncologists use drugs targeting the aberrant EGF-R to reduce its activity. Integral membrane proteins have important functions in cell metabolism, regulation, integration, and cell signalling. Six broad categories of membrane proteins have been defined in terms of their function: pumps, channels, receptors, linkers, enzymes, and structural proteins. The categories are not mutually exclusive (a structural membrane protein may simultaneously serve as a receptor, an enzyme, a pump, or any combination of these functions). Transport within the plasma membrane Substances that enter or leave the cell must traverse the plasma membrane. Some substances (fat-soluble and small, uncharged molecules) cross the plasma membrane by simple diffusion down their concentration gradient. All other molecules require membrane transport proteins to provide them with individual passage across the plasma membrane: this mechanism is called facilitated diffusion. 1. Passive - No energy is required: simple and facilitated diffusion 2. Active - Energy or ATP is needed: it happens against concentration gradient and can be driven by Na+- K+ pumps for depolarisation of the cell 3. Vacuoles or vesicles - For large molecules Resisting-therapy tumours: ATP-binding cassette (ABC) receptors are responsible for resistance in tumour cells to chemotherapy increasing the efflux at the expense of ATP hydrolysis: a consequence is the failure of DNA repair and cellular death. P-glycoprotein-1 and MOR1 use inhibitors to the pump to reinstate drug sensibility. 2  Cytology ROUGH ENDOPLASMIC RETICULUM The rough endosplasmic reticulum is made of tubules delimited by a membrane and intercommunicating. This membrane continues in the nuclear envelope since it is necessary when mRNA needs to exit the nucleus and arrive to the ribosomes on the rER. Its extension depends on the metabolic needs of the cell, therefore rER is abundant in cells undergoing high protein synthesis. Several ribosomes are bound on the membrane and they are involved in the synthesis of proteins placed in the lumen of rER: here is where mRNA arrives and translation can begin. Ribososomes With the TEM, the rER appears as a series of interconnected, membrane-limited, flattened sacs called cisternae, with particles studding the exterior surface of the membrane. These particles, called ribosomes, are attached to the membrane of the rER by ribosomal docking proteins. Ribosomes measure 15 to 20 nm in diameter and consist of a small and large subunit. Each subunit contains ribosomal RNA (rRNA) of different length as well as numerous different proteins. Groups of ribosomes form short spiral arrays called polyribosomes or polysomes in which many ribosomes are attached to a thread of messenger RNA (mRNA). In contrast, free ribosomes reside within the cytoplasm. They are not associated with any intracellular membranes and are structurally and functionally identical to polysomes of the rER. After the synthesised protein enters the rER’s lumen, the first 5-30 amino acids encode a signal peptide that is recognised and bound by a signal recognition peptide (SRP), that binds to a receptor of the ER. Then, it can enter through a channel. RER is also in command of post-translational modifications of protein such as: Proteolysis Glycosilation Phosphorylation Functions 1. Synthesis of proteins 2. Acquisition of protein steric conformations - Secondary and tertiary structures 3. Assemblage of polypeptides with quaternary structures 4. Beginning of protein glycosilation (continued in the Golgi apparatus) 5. Hydroxylation of lysine and proline residues 3  Cytology SMOOTH ENDOPLASMIC RETICULUM The sER consists of short anastomosing tubules that are not associated with ribosomes. Cells with large amounts of smooth-surfaced endoplasmic reticulum may exhibit distinct cytoplasmic eosinophilia (acidophilia) when viewed in the light microscope. The sER is structurally similar to the rER but lacks the ribosome-docking proteins. It tends to be tubular rather than sheet-like, and it may be separate from the rER or an extension of it. The sER is abundant in cells that function in lipid metabolism (cells that synthesize fatty acids and phospholipids), and it proliferates in hepatocytes when animals are challenged with lipophilic drugs. The sER is well developed in cells that synthesise and secrete steroids such as adrenocortical cells and testicular Leydig (interstitial) cells. Lipids are synthesised by enzymes on the sER and newly-synthesised molecules are inserted in the lipid bilayer on the cytoplasmic side. Then, the enzyme flippase catalyses the flip-flop movement to bring these molecules on the opposite side of the bilayer. Storage of ions In skeletal and cardiac muscle, the sER is also called the sarcoplasmic reticulum: it runs longitudinally and surrounds each myofibril. It sequesters Ca2, which is essential for the contractile process and is closely apposed to the plasma-membrane invaginations that conduct the contractile impulses to the interior of the cell. Cell detoxifying processes The sER is the principal organelle involved in detoxification and conjugation of noxious substances. It is particularly well developed in the liver and contains a variety of detoxifying enzymes related to cytochrome P450 that are anchored directly into sER plasma membranes. They modify and detoxify hydrophobic compounds such as pesticides and carcinogens by chemically converting them into water-soluble conjugated products that can be eliminated from the body. The degree to which the liver is involved in detoxification at any given time may be estimated by the amount of sER present in liver cells. The sER is also involved in: Lipid and steroid metabolism Glycogen metabolism Membrane formation and recycling Because of these widely disparate functions, numerous other enzymes, including hydrolases, methylases, glucose- 6-phosphatase, ATPases, and lipid oxidases, are associated with the sER, depending on its functional role. 4  Cytology GOLGI APPARATUS The Golgi apparatus was described more than 100 years ago by histologist Camillo Golgi. In studies of osmium-impregnated nerve cells in Pavia in 1898, he discovered an organelle that formed networks around the nucleus. This made him win the Nobel prize in 1906. It was also described as well-developed in secretory cells. Changes in the shape and location of the Golgi apparatus relative to its secretory state were described even before it was viewed with the electron microscope and before its functional relationship to the rER was established. It is active both in cells that secrete protein by exocytosis and in cells that synthesise large amounts of membrane and membrane-associated proteins such as nerve cells. Structure and function In EM, the Golgi apparatus appears as a series of stacked, flattened, membrane-limited sacs or cisternae and tubular extensions embedded in a network of microtubules near the microtubule-organising centre. Small vesicles involved in vesicular transport are seen in association with the cisternae. The Golgi apparatus is polarised both morphologically and functionally. The flattened cisternae located closest to the rER represent the forming face, or the cis-Golgi network (CGN). The cisternae located away from the rER represent the maturing face, or the trans-Golgi network (TGN). The cisternae located between the TGN and CGN are commonly referred as the medial-Golgi network. Its functions are (different functions correspond to different enzymes): 1. Modification of proteins and lipids by glycosilation 2. Activation of peptides by preoteolysis or phosphorylation 3. Synthesis of complex carbohydrates: glycosaminoglycans and mucin 4. Selection of enzymes to be delivered to lysosomes 5. Production of micro-vesicles into which proteins or lipids are stored Destination of proteins rER - These proteins have a sequence of AA (Lys-Asp-Glu- Leu) called KDEL in order to be recognised by receptors on the ER membrane Golgi apparatus Lysosomes - They have a specific marker, a phosphorylated sugar called mannose-6-phosphate Exocitosis - The content of the vesicles leaving the Golgi apparatus follows two secretion ways: 1. Constitutive secretion: vesicles are coated by COPs continuously and immediately secrete proteins without any stimulation 2. Regulated secretion: vesicles coated by stronger clathrin-like proteins produce proteins under stimulation and they store those until maturation of fusion with other vesicles or the plasma membrane. 5  Cytology VESICULAR TRANSPORT Vesicular transport maintains the integrity of the plasma membrane and also provides for the transfer of molecules between different cellular compartments. It involves configurational changes in the plasma membrane at localised sites and subsequent formation of vesicles from the membrane or fusion of vesicles with the membrane. The major mechanism by which large molecules enter, leave, and move within the cell is called vesicle budding or gemmation. Vesicles formed by budding from the plasma membrane of one compartment fuse with the plasma membrane of another compartment. Within the cell, this process ensures intercompartmental transfer of the vesicle contents. Endocytosis is the general term for processes of vesicular transport in which substances enter the cell. In general, endocytosis controls the composition of the plasma membrane and the cellular response to changes in the external environment. It also plays key roles in nutrient uptake, cell signalling, and cell shape changes. Exocytosis is the general term for processes of vesicular transport in which substances leave the cell. COPs coating Coating of proteins favour the bending of the membrane during the formation of the vesicle and allows the selection of the components that have to be inserted and transported into the vesicle: these proteins are called coating proteins (COPs). Vesicles coated by COPs-II move from the rER to the Golgi (anterograde transport) Vesicles coated by COPs-I move from the Golgi to the rER (retrograde transport): KDEL Vesicles coated by clathrin move from the Golgi to the plasma membrane or endosomes Transported vesicles must specifically recognise the destination compartment in order to deliver their content into the correct place. In the cell, the corrrect address is recognised by Rab-GTPase bound to the membrane of the traveling vesicle. Rab-GTPase interacts with tethering proteins located on the target membrane. This initial interaction provides recognition of the vesicle and recruits the necessary number of tethering proteins to dock the incoming vesicle. The docking complex between Rab-GTPase and its receptor immobilises the vesicle near the target membrane. To ensure accurate targeting, each vesicle contains a vesicle-specific membrane protein called a v-SNARE. The target membrane also contains a specific membrane protein, t-SNARE, that interacts with v-SNARE to form the cis-SNARE complex. SNAREs are a family of transmembrane proteins that were originally grouped according to their location within the vesicle (v-SNARE) or target membrane (t-SNARE). 6  Cytology Receptor-mediated endocytosis Receptor-mediated endocytosis allows entry of specific molecules into the cell. In this mechanism, receptors for specific molecules, called cargo receptors, accumulate in well-defined regions of the cell membrane. These regions, which are represented by the lipid rafts in the plasma membrane, eventually become coated pits. The name coated pit is derived from these regions’ appearance in the electron microscope (EM) as an accumulation of electron-dense material that represents aggregation of clathrin molecules on the cytoplasmic surface of the plasma membrane. Cargo receptors recognise and bind to specific molecules that come in contact with the plasma membrane. Clathrin molecules then assemble into a basket-like cage that helps change the shape of the plasma membrane into a vesicle-like invagination. Clathrin interacts with the cargo receptor via another coating-protein complex, adaptin, which is instrumental in selecting appropriate cargo molecules for transport into the cells. Thus, selected cargo proteins and their receptors are pulled from the extracellular space into the lumen of a forming vesicle. The large (100 kDa) mechanoenzyme GTPase called dynamin mediates the liberation of forming clathrin-coated vesicles from the plasma membrane during receptor-mediated endocytosis. The type of vesicle formed as a result of receptor-mediated endocytosis is referred to as a coated vesicle, and the process itself is known as clathrin-dependent endocytosis. Clathrin-coated vesicles are also involved in the movement of the cargo material from the plasma membrane to early endosomes and from the Golgi apparatus to the early and late endosomes. Pinocytosis Pinocytosis is the nonspecific ingestion of fluid and small protein molecules via small vesicles, usually smaller than 150 nm in diameter. Pinocytosis is performed by virtually every cell in the organism, and it is constitutive (it involves a continuous dynamic formation of small vesicles at the cell surface). The mechanism proposed for vesicle formation in pinocytosis is associated with the presence of caveolin and flotillin proteins that are found in lipid rafts. Also, mechanoenzymes such as GTPase (dynamin) are involved in pinocytotic vesicle scission (the process of pinching off from the plasma membrane). Pinocytotic vesicles are visible with the TEM, and they have a smooth surface. These smooth pinocytotic vesicles are especially numerous in the endothelium of blood vessels and in smooth muscle cells. Because caveolin-1 forms complexes (of 14 to 16 monomers) that effect changes in membrane curvature leading to vesicle formation, pinocytosis does not require clathrin and therefore may be referred to as clathrin-independent endocytosis. Phagocytosis Phagocytosis is the ingestion of large particles such as cell debris, bacteria, and other foreign materials. In this nonselective process, plasma membrane sends out pseudopodia to engulf phagocytosed particles into large vesicles (larger than 250 nm in diameter) called phagosomes. Phagocytosis is performed mainly by a specialised group of cells belonging to the mononuclear phagocytotic system (MPS). Phagocytosis is generally a receptor-mediated process and is also triggered by recognition of pathogen- associated molecular patterns (PAMPs) that are commonly expressed on pathogen surfaces by Toll-like receptors. 7  Cytology This process does not require clathrin for phagosome formation. However, because of initial pseudopodial extensions of plasma membrane that contribute to the formation of phagosome, the actin cytoskeleton must be rearranged in a process that requires depolymerisation and repolymerisation of the actin filaments. Thus, phagocytosis is referred to as clathrin-independent but actin-dependent endocytosis. Autophagy Autophagy represents the major cellular pathway in which a number of cytoplasmic proteins, organelles, and other cellular structures are degraded in the lysosomal compartment. This important process maintains a well-controlled balance between anabolic and catabolic cell functions and permits the cell to eliminate unwanted or unnecessary organelles. Digested components of organelles are recycled and reused for normal cell growth and development. Cytoplasmic proteins and organelles are substrates for lysosomal degradation in the process of autophagy. Autophagy plays an essential role during starvation, cellular differentiation, cell death, and cell aging. In the last few years, applying genetic screening tests originally developed for yeasts, researchers uncovered several autophagy-related genes (Atg genes) in mammalian cell genome. The presence of adequate nutrients and growth factors stimulates enzymatic activity of a serine/threonine kinase known as mammalian target of rapamycin (mTOR). High mTOR activity exerts an inhibitory effect on autophagy. The opposite is found in nutrient starvation, hypoxia, and high temperature, where lack of mTOR activity causes activation of Atg genes. This results in formation of an Atg1 protein-kinase autophagy–regulatory complex that initiates the process of autophagy. 1. Macroautophagy, or simply autophagy, is a nonspecific process in which a portion of the cytoplasm or an entire organelle is first surrounded by a double or multilamellar intracelular membrane of endoplasmic reticulum, called isolation membrane, to form a vacuole called an autophagosome. This process is aided by proteins encoded by several Atg genes. After targeted delivery of lysosomal enzymes, the autophagosome matures into a lysosome. The isolation membrane disintegrates within the hydrolytic compartment of a lysosome. Macroautophagy occurs in the liver during the first stages of starvation. 2. Microautophagy is also a nonspecific process in which cytoplasmic proteins are degraded in a slow, continuous process under normal physiologic conditions. In microautophagy, small cytoplasmic soluble proteins are internalised into the lysosomes by invagination of the lysosomal membrane. 3. Chaperone-mediated autophagy is the only selective process of protein degradation and requires assistance from specific cytosolic chaperones such as heat-shock chaperone protein called hsc73. This process is activated during nutrient deprivation and requires the presence of targeting signals on the degraded proteins and a specific receptor on the lysosomal membrane. Chaperone-mediated direct transport resembles the process of protein import to various other cellular organelles: hsc73 binds to the protein and assists in its transport through the lysosomal membrane into the lumen, where it is finally degraded. Chaperone-mediated autophagy is responsible for the degradation of approximately 30% of cytoplasmic proteins in organs such as the liver and kidney. 8  Cytology LYSOSOMES Lysosomes are digestive organelles that were recognised only after histochemical procedures were used to demonstrate lysosomal enzymes. They were discovered in 1955 by the Belgian scientist Christian de Duve, who observed that when cells were repeatedly frozen and thawed before centrifugation, they release an enzyme in larger amount: this enzyme was acid phosphatase. To explain this phenomenon, he suggested that this enzyme must have been encased in some sort of membrane-bound organelle within the cell. Structure Lysosomes are organelles about 0,2-0,5 μm in diameter, rich in hydrolytic enzymes (50 kinds) such as proteases, nucleases, glycosidases, lipases, and phospholipases. A lysosome represents a major digestive compartment in the cell that degrades macromolecules derived from endocytotic pathways as well as from the cell itself in a process known as autophagy (removal of cytoplasmic components, particularly membrane-bounded organelles, by digesting them within lysosomes). Proteins of the lysosomes are synthesised in the rER and tagged with mannose-6-phosphate to differentiate them from other enzymes. Sugar molecules cover almost the entire luminal surface of these proteins, thus protecting them from digestion by hydrolytic enzymes. Lysobisphosphatidic acids within the lysosomal membrane may play an important role in restricting the activity of hydrolytic enzymes directed against the membrane. In addition, lysosomes and late endosomes contain proton (H) pumps that transport H ions into the lysosomal lumen, maintaining a low pH (4.7). The lysosomal membrane also contains transport proteins that transport the final products of digestion (amino acids, sugars, nucleotides) to the cytoplasm, where they are used in the synthetic processes of the cell or are exocytosed. Functions They are the main site of cellular digestion of materials introduced in the cell to obtain useful compounds for the cell (present in all cells) and they destroy micro-organisms or damaged cells that have been phagocytosed: they are abundant in cells with intense phagocytes. Also, they act as a storage source: in the deep layers of the epidermis, for example, are evident some cells characterised by dark inclusions, they are the melanocytes. They produce a dark pigment (melanin) from tyrosine amino acid. Then, melanin is stored in the lysosomes that are named melanosomes. Lysosomal disorder: Lysosomal disorders are a group of genetic diseases that originates from an abnormal accumulation of substances in lysosomes due to defects in lysosomal enzymes that cannot correctly degrade molecules. Children are affected and they are incurable, thus fatal. Some of them are known as Tay-Sachs disease, Gauchet disease, Neumann-Pick disease and Hunter syndrome. 9  Cytology PEROXISOMES Peroxisomes are single membrane–bounded organelles containing oxidative enzymes. These microbodies are small (0.5 μm in diameter), membrane-limited spherical organelles that contain oxidative enzymes, particularly catalase and other peroxidases. Detoxifying organelles Virtually all oxidative enzymes produce hydrogen peroxide (H2O2) as a product of the oxidation reaction. Hydrogen peroxide is a toxic substance. The catalase universally present in peroxisomes carefully regulates the cellular hydrogen peroxide content by breaking down hydrogen peroxide, thus protecting the cell. In addition, peroxisomes contain D-amino acid oxidases, β- oxidation enzymes, and numerous other enzymes. Oxidative enzymes are particularly important in liver cells (hepatocytes), where they perform a variety of detoxification processes. Peroxisomes in hepatocytes are responsible for detoxification of ingested alcohol by converting it to acetaldehyde. The β-oxidation of fatty acids is also a major function of peroxisomes. In some cells, peroxisomal fatty-acid oxidation may equal that of mitochondria. The proteins contained in the peroxisome lumen and membrane are synthesised on cytoplasmic ribosomes and imported into the peroxisome. A protein destined for peroxisomes must have a peroxisomal targeting signal attached to its carboxy-terminus. Although abundant in liver and kidney cells, peroxisomes are also found in most other cells. The number of peroxisomes present in a cell increases in response to diet, drugs, and hormonal stimulation. Central nervous system Oligodendrocytes have a fundamental role in the support of atonal integrity and their peroxisomes are important guardians of atonal vitality. If they are damaged there could be atonal loss and demyelination (tumour). Zellweger disease: Various human metabolic disorders are caused by the inability to import peroxisomal proteins into the organelle because of a faulty peroxisomal targeting signal or its receptor. Several severe disorders are associated with nonfunctional peroxisomes. In the most common inherited disease related to nonfunctional peroxisomes, Zellweger syndrome, which leads to early death, peroxisomes lose their ability to function because of a lack of necessary enzymes. The disorder is caused by a mutation in the gene encoding the receptor for the peroxisome targeting signal that does not recogniSe the signal Ser-Lys-Leu at the carboxy-terminus of enzymes directed to peroxisomes. Therapies for peroxisomal disorders have been unsatisfactory to date. 10  Cytology MITOCHONDRIA Mitochondria are organelles with a double- membrane system: The outer membrane is smooth and rich in lipids (50%) and enzymes for the lipid synthesis and fatty acids metabolism. The inner membrane is similar to bacteria membranes, containing cardiolipin and enzymes for the respiratory chain and ATP synthase for its production (energy source). The inner membrane is arranged in folds, named cristae, that can be lamellar (protein synthesis) or tubular (steroid- secreting cells). When the mitochondria are active they are tightly packed across the organelles. Between the two membranes there is the intermediate space containing cytochrome c, a component of the electron transport chain and really important for cell apoptosis. Inside, instead, there is the matrix which contains dense matrix granules that store Ca2 and other divalent and trivalent cations. These granules increase in number and size when the concentration of divalent (and trivalent) cations increases in the cytoplasm. Mitochondria can accumulate cations against a concentration gradient. Thus, in addition to ATP production, mitochondria also regulate the concentration of certain ions of the cytoplasmic matrix, a role they share with the sER. The matrix also contains mitochondrial DNA, ribosomes, and tRNAs. Enzymes for DNA synthesis and transcription, mRNA, rRNA, tRNA and ribosomes Owns set of nucleotides Ca2+ and Mg2+ ions Enzymes for fatty acids oxidation and Krebs cycle Specificities The number of mitochondria is heterogeneous and depends on the cell’s metabolism. However, usually they are 1,000-2,000 (in the oocytes they reach 30,000 per cell). Moreover, there is no fixed site, but mitochondria are localised in the cytoplasm where a high energy support is needed. Mitochondria are present in all cells except red blood cells and terminal keratinocytes. Striated skeletal muscle Striated cardiac muscle - Arranged in rows parallel to myofibrils Kidney tubular cells - Between invaginations Spermatozoa - They form a collar to grand mobility Ciliated cells - Needed for the microtubules of the cilia Presynaptic terminal of neurones Their functions are: 1. Energy production 2. Lipid and phospholipid metabolism - Krebs cycle 3. Synthesis of steroid hormones 4. Accumulation of cations in the matrix granules 5. Heat production during respiratory chain 6. Apoptosis - They decide to release cytochrome c from the mitochondrial intermembraneous space into the cell cytoplasm and this is regulated by apoptosis Bcl-2 protein family that regulates membrane permeability. 11  Cytology Autonomy and biogenesis Mitochondria are believed to have evolved from an aerobic prokaryote (Eubacterium) that lived symbiotically within primitive eukaryotic cells. This hypothesis received support with the demonstration that mitochondria possess their own genome, increase their numbers by segmentation, and synthesise some of their structural (constituent) proteins. Mitochondrial DNA is a closed circular molecule that encodes 13 enzymes involved in the oxidative phosphorylation pathway, two rRNAs, and 22 transfer RNAs (tRNAs) used in the translation of the mitochondrial mRNA. Moreover, they are maternally inherited and this leads to little opportunity for genetic recombination between lineages: mtDNA is a useful source of information for scientists involved in population genetics and evolutionary biology. After a brief growing period, they divide into smaller mitochondria with a mean life 9/10 days (5/6 in more active cells). Aged mitochondria are eliminated through autophagy by lysosomes that degrade them with their lyric enzymes. Myoclonus epilepsy with ragged red fibres (MERRF): Several mitochondrial defects are related to defects in enzymes that produce ATP. Metabolically active tissues that use large amounts of ATP such as muscle cells and neurones are most affected. For example, myoclonic epilepsy with ragged red fibres (MERRF) is characterised by muscle weakness, ataxia, seizures, and cardiac and respiratory failure. Microscopic examination of muscle tissue from affected patients shows aggregates of abnormal mitochondria, providing a ragged appearance of red muscle fibres. MERRF is caused by mutation of the mitochondrial DNA gene encoding tRNA for lysine. This defect produces two abnormal complexes in the electron-transport chain of respiratory enzymes affecting ATP production. 12  Cytology CYTOSKELETON The cytoskeleton is an intricate network of protein filaments throughout the cytoplasm and supporting the structure of the cell and its components giving the cytoplasm resistance and flexibility. Its functions are: 1. Providing and maintaining cell shape 2. Driving vesicle trafficking and determining organelle position 3. Supporting the plasma membrane and providing mechanical links allowing the cell to bear stress or tension 4. Allowing chromosome separation during mitosis and cell division 5. Allowing neurones to maintain shape and spatial conformation 6. Allowing cell motility Microfilaments Microfilaments are actin filaments and they have a key role in proving the cell scaffold and allowing cell movement. They are ubiquitous (2-15% of cell proteins) and occur in five isoforms: they are formed by polymerisation of G-actin (globular actin) arranged in a double helix structure of a 7 nm diameter. Functions of these membrane-associated actin filaments include the following. 1. Anchorage and movement of membrane protein - Actin filaments are distributed in three-dimensional networks throughout the cell and are used as anchors within specialised cell junctions such as focal adhesions. 2. Formation of the structural core of microvilli on absorptive epithelial cells - Actin filaments may also help maintain the shape of the apical cell surface (the apical terminal web of actin filaments serves as a set of tension cables under the cell surface). 3. Locomotion of cells - Locomotion is achieved by the force exerted by actin filaments by polymerisation at their growing ends. This mechanism is used in many migrating cells, in particular, on transformed cells of invasive tumours. As a result of actin polymerisation at their leading edge, cells extend processes from their surface by pushing the plasma membrane ahead of the growing actin filaments. The leading-edge extensions of a crawling cell are called lamellipodia; they contain elongating organised bundles of actin filaments with their plus ends directed toward the plasma membrane. 4. Extension of cell processes - These processes can be observed in many other cells that exhibit small protrusions called filopodia, located around their surface. As in lamellipodia, these protrusions contain loose aggregations of 10 to 20 actin filaments organised in the same direction, again with their plus ends directed toward the plasma membrane. 13  Cytology Invadopodia: Actin is involved in the invasion by carcinoma cells of the surroundings for metastasis. Small cytoplasmic projections containing F-actin extend beyond the leading-edge of lamellipodia in migrating cells. Invadopodia are lamellipodia on the ventral surface of invading cells and they can be equipped with proteases to degrade the ECM. Intermediate filaments Intermediate filaments are rope-shaped proteins that provide mechanical strength to cells. Single protein molecules (monomers) assemble in dimeric coiled structures: dimers are head-to-tail arranged, forming tetramers, then many tetramers associate to form filaments with a diameter of 10 nm. They are tissue-specific: Keratin: epithelial tissue Vimentin: connective tissue Desmin: muscle tissue Glial fibrillation protein: neural tissue Lamina: nucleus Tissue-specificity is used in tumour diagnosis to determine the tissue origin and the appropriate choice of therapy. Intermediate filaments are involved in supporting specific cell structures because their assembly and arrangement provide mechanical strength and resistance to extra-cellular forces: cell-cell junctions and cell-extracellular matrix junctions use them. Also, they are localised beneath the nuclear envelope as to form the nuclear lamina, a structure providing a link for chromatin (different from the dynamic role of actin), and crucial in mitosis during cell division. Microtubules Microtubules are elongated polymeric structures composed of equal parts of α-tubulin and β-tubulin. Microtubules measure 20 to 25 nm in diameter. The wall is approximately 5 nm thick and consists of 13 circularly arrayed globular dimeric tubulin molecules. The dimers polymerise in an end-to-end fashion, with molecule of one dimer bound to the molecule of the next dimer in a repeating pattern. Longitudinal contacts between dimers link them into a linear structure called a protofilament. They can rapidly dissemble and assemble to create a system of connection within the cell. They grow from an area called microtubule-organising centre (MTOC) located near the nucleus and extend toward the cell periphery. It controls the number, the position and the orientation of microtubules. Microtubules are polar structures because all of the dimers in each protofilament have the same orientation. Each microtubule possesses a nongrowing (-) end that corresponds to α-tubulin; in the cell, it is usually embedded in the MTOC and often stabilised by actin-capping proteins. The growing (+) end of microtubules corresponds to β-tubulin and extends the cell periphery. Microtubules mediate motion of organelles and vesicles in the cell: dynein towards centre, kinesin towards periphery. Also, they are important for axonic transport in neurones. 14  Cytology The MTOC corresponds to the centrosome, composed of two orthogonally arranged centrioles, each made up of nine sets of triplets microtubules. The minus end of the microtubule remains attached to the MTOC, and the plus end represents the growing end directed toward the plasma membrane. Centrioles provide basal bodies for cilia and flagella and align the mitotic spindle during cell division. The known functions of centrioles can be organised into two categories: 1. Basal body formation - One of the important functions of the centriole is to provide basal bodies, which are necessary for the assembly of cilia and flagella. 2. Mitotic spindle formation - During mitosis, the position of centrioles determines the location of mitotic spindle poles. Centrioles are also necessary for the formation of a fully functional MTOC, which nucleates mitotic spindle–associated microtubules, crucial in establishing the axis of the developing mitotic spindle. Thus, the primary role of centrioles in mitosis is to position the mitotic spindle properly by recruiting the MTOC from which astral microtubules can grow and establish the axis for the developing spindle. Anti-cancer drugs: Some anti-cancer drugs are potent inhibitors of the polymerisation or depolymerisation of the mitotic spindle, and therefore inhibit cell division disrupting the assembly of microtubules. 15  Cytology NUCLEUS The nucleus is a membrane-limited compartment that contains the genome (genetic information) in eukaryotic cells, together with the machinery for DNA replication and RNA transcription and processing. It presents different shapes, related to the shape and content of the cell, and sometimes is irregular. Also, it is not present during the whole cell cycle and in all cells (red blood cells). Nonetheless, some cells have more nuclei, as osteoclasts (macrophages in bone). Structure It is enveloped by a double-bilayer membrane delimiting the perinuclear space called nuclear envelope. It consists of an inner and an outer membrane separated by a perinuclear cisternal space and perforated by nuclear pores. The outer membrane of the nuclear envelope is continuous with that of the rough-surfaced endoplasmic reticulum (rER) and is often studded with ribosomes. Inside the nucleus, there is chromatin. It is nuclear material organised as euchromatin or heterochromatin and contains DNA associated with roughly an equal mass of various nuclear proteins (histones) that are necessary for DNA to function. The nuclear lamina is formed by intermediate filaments and lies adjacent to the inner nuclear membrane. In addition to its supporting or “nucleoskeletal” function, nuclear lamina is essential in many nuclear activities such as DNA replication, transcription, and gene regulation. The major components of the lamina, as determined by biochemical isolation, are nuclear lamins, a specialised type of nuclear intermediate filament, and lamin-associated proteins. At numerous sites, the paired membranes of the nuclear envelope are punctuated by 70- to 80- nm “openings” through the envelope. These nuclear pores are formed from the merging of the inner and outer membranes of the nuclear envelope. The nucleoplasmic ring complex anchors a nuclear basket. The cylinder-shaped central framework encircles the central pore of the NPC, which acts as a close-fitting diaphragm or gated channel. In addition, each NPC contains one or more water-filled channels for transport of small molecules. Proteins directed to the nucleus present specific signal sequences for receptors for nuclear import: NLS (nuclear localisation signal). The nucleolus is a nonmembranous region of the nucleus with a granular appearance that surrounds transcriptionally active rRNA genes: it is free in the nucleoplasm. It is the primary site of ribosomal production and assembly. The nucleolus varies in size but is particularly well developed in cells active in protein synthesis. Some cells contain more than one nucleolus. 16  Cytology CELL CYCLE The cell cycle is the period of time between two cell divisions, that can last from hours to days: Yeast and bacteria: 2-4 hours Intestinal, epithelial cells, erythroblast: 12 hours Hepatocytes: 1 year Neurones and muscle tissue never divide during adult life, however they contain stem cells that in case of need can produce new cells Cycle phases 1. G0 - Quiescence, resting phase for undefined time. Cell division can be activated via internal stimuli or exogenous growth factors (damages for example). 2. G1 (8 hours) - Cellular growth, protein synthesis and duplication of organelles. The cell is preparing for division. 3. S (7-10 hours) - DNA duplication 4. G2 (2-5 hours) - Microtubules and mitotic spindle appearing, synthesis of cytoplasm components 5. M (1-2 hours) - Mitosis Mitosis Mitosis is a process of chromosome segregation and nuclear division followed by cell division that produces two daughter cells with the same chromosome number and DNA content as the parent cell. 1. Prophase begins as the replicated chromosomes condense and become visible. As the chromosomes continue to condense, each of the four chromosomes derived from each homologous pair can be seen to consist of two chromatids. In late prophase or prometaphase, the nuclear envelope begins to disintegrate into small transport vesicles and resembles the sER. The nucleolus, which may still be present in some cells, also completely disappears in prometaphase. Microtubules of the developing mitotic spindle attach to the chromosomes. 2. Metaphase begins as the mitotic spindle, consisting of three types of microtubules, becomes organised around the microtubule-organising centres (MTOCs) located at opposite poles of the cell. Microtubules are pulled toward the MTOC, where additional microtubules will attach. Microtubules and their associated motor proteins direct the movement of the chromosomes to a plane in the middle of the cell, the equatorial or metaphase plate. 3. Anaphase begins at the initial separation of sister chromatids. This separation occurs when the cohesins that have been holding the chromatids together break down. The chromatids then begin to separate and are pulled to opposite poles of the cell by the molecular motors (dyneins) sliding along the kinetochore microtubules toward the MTOC. 4. Telophase is marked by the reconstitution of a nuclear envelope around the chromosomes at each pole. The chromosomes uncoil and become indistinct except at regions that will remain condensed in the interphase nucleus. The nucleoli reappear, and the cytoplasm divides (cytokinesis) to form two daughter cells. The separation at the cleavage furrow is achieved by a contractile ring consisting of a very thin array of actin filaments positioned around the perimeter of the cell. Myosin II molecules are assembled into small filaments that interact with the actin filaments, causing the ring to contract. As the ring tightens, the cell is pinched into two daughter cells, genetically identical and containing the same kind and number of chromosomes. The daughter cells are (2d) in DNA content and (2n) in chromosome number. 17  Cytology Meiosis Meiosis involves two sequential nuclear divisions followed by cell divisions that produce gametes containing half the number of chromosomes and half the DNA found in somatic cells. It consists of two successive mitotic divisions without the additional S phase between the two divisions. During the S phase that precedes meiosis, DNA is replicated forming sister chromatids (two parallel strands of DNA) joined together by the centromere. The DNA content becomes (4d), but the chromosome number remains the same (2n). The cells then undergo a reductional division (meiosis I) and an equatorial division (meiosis II). It is carried out by specialised organs, gonads, that have the only cells that propagate genetic information to the next generation. Reduce chromosome number from diploid (2n) to haploid (n) Ensure that each daughter cell has one full set of chromosomes Promote genetic diversity Cell renewal Static cell populations consist of cells that no longer divide (postmitotic cells), such as cells of the central nervous system and skeletal or cardiac muscle cells. Under certain circumstances, some of these cells (cardiac myocytes) may enter mitotic division. Stable cell populations consist of cells that divide episodically and slowly to maintain normal tissue or organ structure. These cells may be stimulated by injury to become more mitotically active. Periosteal and perichondrial cells, smooth muscle cells, endothelial cells of blood vessels, and fibroblasts of the connective tissue may be included in this category. Renewing cell populations may be slowly or rapidly renewing but display regular mitotic activity. Division of such cells usually results in two daughter cells that differentiate both morphologically and functionally or two cells that remain as stem cells. Daughter cells may divide one or more times before their mature state is reached. The differentiated cell may ultimately be lost from the body. Slowly renewing populations include smooth muscle cells of most hollow organs, fibroblasts of the uterine wall, and epithelial cells of the lens of the eye. Slowly renewing populations may actually slowly increase in size during life, as do the smooth muscle cells of the gastrointestinal tract and the epithelial cells of the lens. Rapidly renewing populations include blood cells, epithelial cells and dermal fibroblasts of the skin, and the epithelial cells and subepithelial fibroblasts of the mucosal lining of the alimentary tract. These are differentiated cells, that differ from stem cells: after a stem cell becomes activated, it replicates into two daughter cells, one will go back to the rest (maintaining the pool stable) and the one will actually differentiate. Also, the maintenance of the number of cells in a tissue is based on a balance of different processes: proliferation, differentiation, cell death. 18  Cytology Apoptosis Apoptosis is programmed cell death and is a mode of cell death that occurs under normal physiologic conditions. The cell is an active participant in its own demise (“cellular suicide”). This process is activated by a variety of extrinsic and intrinsic signals. Cells undergoing apoptosis show the following characteristic morphologic and biochemical features: DNA fragmentation occurs in the nucleus and is an irreversible event that commits the cell to die. DNA fragmentation is a result of Ca2-dependent and Mg2- dependent activation of nuclear endonucleases. These enzymes selectively cleave DNA, generating small oligonucleosomal fragments. Nuclear chromatin then aggregates, and the nucleus may divide into several discrete fragments bounded by the nuclear envelope. Decrease in cell volume is achieved by shrinking of the cytoplasm. The cytoskeletal elements become reorganised in bundles parallel to the cell surface. Ribosomes become clumped within the cytoplasm, the rER forms a series of concentric whorls, and most of the endocytotic vesicles fuse with the plasma membrane. Loss of mitochondrial function is caused by changes in the permeability of the mitochondrial membrane channels. The integrity of the mitochondrion is breached, the mitochondrial transmembrane potential drops, and the electron-transport chain is disrupted. Proteins from the mitochondrial intermembrane space, such as cytochrome c are released into the cytoplasm to activate a cascade of proteolytic enzymes responsible for dismantling the cell. Membrane blebbing results from cell membrane alterations. One alteration is related to translocation of certain molecules from the cytoplasmic surface to the outer surface of the plasma membrane. Formation of apoptotic bodies, the final step of apoptosis, results in cell breakage. These membrane- bounded vesicles originate from the cytoplasmic bleb containing organelles and nuclear material. They are rapidly removed without a trace by phagocytotic cells. Necrosis Necrosis, or accidental cell death, is a pathologic process. It occurs when cells are exposed to an unfavourable physical or chemical environment (hypothermia, hypoxia, radiation, low pH, cell trauma) that causes acute cellular injury and damage to the plasma membrane. Under physiologic conditions, damage to the plasma membrane may also be initiated by viruses, or proteins called perforins. Rapid cell swelling and lysis are two characteristic features of this process. Necrosis begins with impairment of the cell's ability to maintain homeostasis. As a result of cell injury, damage to the cell membrane leads to an influx of water and extracellular ions. Intracellular organelles such as the mitochondria, rER, and nucleus undergo irreversible changes that are caused by cell swelling and cell membrane rupture (cell lysis). As a result of the ultimate breakdown of the plasma membrane, the cytoplasmic contents, including lysosomal enzymes, are released into the extracellular space. Therefore, necrotic cell death is often associated with extensive surrounding tissue damage and an intense inflammatory response. 19