Cell Membrane & Vesicular Transport PDF

1 Cell membrane& vesicular Transport ILOs By the end of this lecture, students will be able to 1. Relate the molecular structure of membrane lipids to its different membrane functions. 2. Correlate the variable peripheral & integral membrane molecules to membrane function. 3. Int...

1 Cell membrane& vesicular Transport ILOs By the end of this lecture, students will be able to 1. Relate the molecular structure of membrane lipids to its different membrane functions. 2. Correlate the variable peripheral & integral membrane molecules to membrane function. 3. Interpret the diagrammatic presentation of the fluid mosaic model 4. Interpret endocytosis & exocytosis as vital processes for transport across the cell membrane. 5. Differentiate between types of endocytosis in normal and pathological conditions. Cell Components The cell is composed of two basic parts: cytoplasm (Gr. kytos, cell, + plasma, thing formed) and nucleus. Individual cytoplasmic components are usually not clearly distinguishable in common hematoxylin and eosin-stained preparations; the nucleus, however, appears intensely stained dark blue or black. (Why?) Cytoplasm The outermost component of the cell, separating the cytoplasm from its extracellular environment, is the plasma membrane (plasmalemma). However, even if the plasma membrane is the external limit of the cell, there is a continuum between the interior of the cell and extracellular macromolecules. The cytoplasm is composed of a matrix, or cytosol, in which are embedded the organelles, the cytoskeleton, and deposits of carbohydrates, lipids, and pigments. Higher levels of organization Plasma Membrane All eukaryotic cells are enveloped by a limiting membrane composed of phospholipids, cholesterol, proteins, and chains of oligosaccharides covalently linked to phospholipids and protein molecules. The cell, or plasma, membrane functions as a selective barrier that Page 1 of 5 regulates the passage of certain materials into and out of the cell and facilitates the transport of specific molecules. One important role of the cell membrane is to keep constant the intracellular milieu, which is different from the extracellular fluid. Membranes also carry out a number of specific recognition and regulatory functions (to be discussed later), playing an important role in the interactions of the cell with its environment. Membranes range from 7.5 to 10 nm in thickness and consequently are visible only in the electron microscope (so, what is visible on the light microscope?) Molecular structure of the cell membrane (Figure 2) 1- Membrane phospholipids. Structure: two long, nonpolar (hydrophobic) hydrocarbon chains linked to a charged (hydrophilic) head group. (Why it appears black in electron micrograph? Is it a single line?) o Organization: the hydrophobic (nonpolar) chains directed toward the centre of the membrane and the hydrophilic (charged) heads directed outward. o Cholesterol is also a constituent of cell membranes. Role of cholesterol; (It will be discussed later with fluidity of the membrane). Note: ✔ This represents the fluid mosaic model of the cell membrane. ✔ The lipid composition of each half of the bilayer could be different according to its functional role. ✔ The trilaminar appearance are characteristic of all the internal cellular membranes (eg. Nuclear, mitochondrial, and endoplasmic reticulum) as well as the plasma membrane. ✔ Some of the lipids, known as glycolipids, possess oligosaccharide chains that extend outward from the surface of the cell membrane and thus contribute to lipid asymmetry. (Figure 2) 2- Proteins, About 50% of the plasma membrane components Types: Page 2 of 5  o Integral proteins are directly incorporated within the lipid bilayer. Some integral proteins span the membrane one or more times, from one side to the other. Other proteins are large enough to extend across the two lipid layers and protrude from both membrane surfaces (transmembrane proteins). Integrins are transmembrane proteins that are linked to cytoplasmic cytoskeletal filaments and to extracellular molecules. Through these linkages there is a constant exchange of influence, in both ways, between the extracellular matrix and the cytoplasm. o Peripheral proteins exhibit a looser association with membrane surfaces. They may protrude from either the outer or inner surface. o Organelle-specific membranes proteins confer unique functions on certain organelles. Correlate the functional forms of integral membrane protein in Fig. 2 with its type. 3- The carbohydrate moieties of glycoproteins and glycolipids exoplasmic domain; they are important components of specific molecules called receptors that participate in important interactions such as cell adhesion, recognition, and response to protein hormones. As with lipids, the distribution of membrane proteins is different in the two surfaces of the cell membranes. Therefore, all membranes in the cell are asymmetric. Glycocalyx (cell coat) In the electron microscope the external surface of the cell shows a fuzzy carbohydrate-rich region called the glycocalyx. This layer is composed of carbohydrate chains linked to membrane proteins and lipids and of cell-secreted glycoproteins and proteoglycans. Functional significance of glycocalyx The glycocalyx has a role in cell recognition and attachment to other cells and to extracellular molecules. Transport across the cell membrane Movement of molecules and ions across membranes occurs by different mechanisms including: a. Passive transport (simple and facilitated diffusion). b. Active transport (pump and cotransport carrier). (Fig 3) c. Bulk movement of materials into and out of the cells (endocytosis & exocytosis) which will be discussed in this section. Mass transfer of material also occurs through the plasma membrane. This bulk uptake of material is known as endocytosis. The corresponding name for release of material in bulk is exocytosis. However, at the molecular level, exocytosis and endocytosis are different processes that utilize different protein molecules. Page 3 of 5 Endocytosis: (Gr. endon, within, + kytos, cell) The process whereby a cell ingests macromolecules, particulate matter, and other substances from the extracellular space. Mechanisms of endocytosis: (Figure 3) 1. Phagocytosis (cell eating) It is a nonselective process of engulfing larger particulate matter, such as microorganisms, cell fragments, and degenerated cells (e.g., defunct red blood cells), by specialized cells known as phagocytes. The engulfed material is enclosed in a vesicle called the phagosome. 2. Pinocytosis Nonselective process where small invaginations of the cell membrane form and entrap extracellular fluid forming a pinocytotic vesicle that pinch off from the cell surface and may fuse with lysosomes (see the section on Lysosomes later in this chapter). Receptor-Mediated Endocytosis It is a selective process that involves engulfment of macromolecules. This process depends on interaction between two structures: ⮚ Receptor proteins (cargo receptors) in the cell membrane. Cargo receptors are transmembrane proteins. ⮚ Macromolecules (ligands), (What is ligand?) such as low-density lipoproteins and protein hormones that bind with its receptor on the cell membrane. The receptors are either originally widely dispersed over the surface or aggregated in special regions called coated pits. Binding of the ligand to its receptor causes widely dispersed receptors to accumulate in coated pits. The coated pit invaginates and pinches off from the cell membrane, forming a coated vesicle that carries both the ligand and its receptor into the cell. Page 4 of 5 Note: Caveolae is a special form of endocytosis where the coating protein is caveolin (will be discussed later) Fate of the endocytotic vesicle (Figure 4) ⮚ The coated vesicles soon lose their clathrin coat and fuse with early endosomes near the periphery of the cell, a system of vesicles and tubules located in the cytosol near the cell surface. The clathrin molecules separated from the coated vesicles are moved back to the cell membrane to participate in the formation of new coated pits. ⮚ If some contents of the early endosome require degradation, they are transferred deeper in the cytoplasm into the late endosome. This similar set of tubules and vesicles, located deeper in the cytoplasm near the Golgi apparatus, helps to prepare its contents for eventual destruction by lysosomes. ⮚ The membranes of all endosomes contain ATP-linked H+ pumps that acidify the interior of the endosomes by actively pumping H+ ions into the interior of the endosome. Fate of the Endosome contents ⮚ Receptors that are separated from their ligand by the acidic pH of the early endosomes may return to the cell membrane to be reused. For example, low-density lipoprotein receptors are recycled several times. ⮚ The ligands typically are transferred to late endosomes. However, some ligands are returned to the extracellular milieu to be used again. An example of this activity is the iron-transporting protein transferrin. Occasionally, both the receptor and the ligand (e.g., epidermal growth factor and its receptor) are transferred to the late endosome, and then to a lysosome, for eventual degradation. (Fig 4) Fig. 4 Formation of early & late endosomes Page 5 of 5 2 Lipid Structure & functions in bio-membranes: ILOs By the end of this lecture, students will be able to 1. Describe structure-function relationship of phospholipids 2. Describe the structure-function relationship of cholesterol 3. Interpret their importance in controlling cell membrane function Membranes can be regarded as a “fluid mosaic model” the fluid nature of the membranes allows greater flexibility to the cell than it would if the membranes were rigid. It also allows the motion of membrane components, required for some types of membrane transport as membranes are dynamic structures. The cell membrane is an asymmetric structure with the two sides of membrane being structurally and functionally different. This difference is due to the difference in composition and orientation of lipids, proteins and carbohydrates. ❖ How can this membranes’ structure serve its function? o Phospholipids: Phospholipids are ionic compounds. Like fatty acids (FA), phospholipids are amphipathic in nature. That is, each has a hydrophilic head, which is the phosphate group plus whatever alcohol is attached to it and a long, hydrophobic tail containing FA. There are two classes of phospholipids, those that have glycerol as a backbone (GLYCEROPHOSPHLIPIDS or PHOSPHOGLYCERIDES ) and those that have sphingosine as a backbone (SPHINGOPHOSPHOLIPIDS). ✓ Glycerophosphlipids (Phosphoglycerides): Of the two major phospholipid classes present in membranes, phosphoglycerides are more common and consist of a glycerol backbone to which are attached two fatty acids in ester linkage and a phosphorylated alcohol. Phosphoglycerides structurally similar to Figure (10-1): Triglycerides Vs Phospholipids triglycerides except that on the third carbon of glycerol instead of a fatty acid there is a phosphate attached to a polar head group. The fatty acid constituents are usually even-numbered carbon molecules, most commonly containing 16 or 18 carbons. They can be saturated or unsaturated with one or more cis double bonds. Page 1 of 5  The simplest phosphoglyceride is Phosphatidic acid, which is 1,2- diacylglycerol 3-phosphate, a key intermediate in the formation of other phosphoglycerides In most phosphoglycerides present in membranes, the phosphate is esterified to an alcohol such as ethanolamine and choline or glycerol. Lecithin (phosphatidyl choline): - It is the most abundant phosphoglyceride. The phosphorylated alcohol here is choline. - It represents a large proportion of the body’s store of choline. - Choline is important in nervous transmission. - Lecithin is also a major constituent of the surfactant preventing adherence, due to surface tension of the inner surfaces of the lungs. - Its absence from the lungs of premature infants causes respiratory distress syndrome. - There is a certain enzyme called lecithinase enzyme present in the venum of cobra. It splits the unsaturated FA from lecithin in cell membrane giving rise to lysolecithin. This substance can produce lysis of red cell membrane and haemolysis. Cephalin (phosphatidyl ethanolamine): It is another abundant phosphoglycerols (phosphoglycerides) which is also found in cell membranes. The base is ethanolamine. Cephalin is one of the important blood clotting factors. ✓ Sphingophospholipids: The second major class of phospholipids is composed of Sphingomyelin, which contains a sphingosine backbone rather than glycerol. A fatty acid (usually longer and less saturated than that of phophoglycerides) is attached by an amide linkage to the amino group of sphingosine, forming ceramide. The primary hydroxyl group of sphingosine is esterified to phosphorylcholine, forming sphingomyelin. As the name implies, sphingomyelin is prominent in myelin sheaths. They play a major role in signal transmission and cell recognition. Figure(10-2): Structure of Sphingomyein Page 2 of 5  ❖ What is the Structure-Function Relationship of phospholipids? Phospholipids are the predominant lipids of cell membranes. In membranes, the hydrophilic (polar) head of the phospholipid extends outward, interacting with the intracellular or extracellular aqueous environment. While, the hydrophobic portion of a phospholipid molecule is associated with the nonpolar portions of other membrane constituents, such as glycolipids, proteins, and cholesterol. The hydrophobic tail containing the fatty acids plays a major role in maintaining the fluidity of the Figure (10-3): Structure of membrane membrane, unsaturated fatty acids add to membrane fluidity. These unsaturated fatty acids form kinks that increase the space between the phospholipids increasing the membrane’s fluidity. In addition, the increased space allows certain small molecules to cross the membrane quickly and easily. In the same time, they form weak non-covalent bonds with each other, holding the bilayer together. Therefore, phospholipid molecules are responsible for the semi-permeability of the cell membrane. They prevent passage of water soluble substances and ions; however they allow passage of small non-polar and fat soluble substances. The amounts and fatty acid compositions of the various phospholipids vary among the different cellular membranes. Clinical Implications: There are many diseases associated with problems in the ability of the phospholipid bilayer to perform these functions. One of these is Alzheimer’s disease, characterized by brain shrinkage and memory loss. One idea explaining why Alzheimer’s disease occurs is the formation of plaque sticking to the phospholipid bilayer of the brain neurons. Interference with the membrane’s phospholipids block communication between the brain neurons, eventually leading to neuron death and in turn causing the symptoms of Alzheimer’s, such as poor short-term memory. One of the facts about the Fluid-Mosaic membrane model is that the components of the bilayers are free to move. Phospholipids have several types of movements, rotational where phospholipid rotates on its axis to interact with its immediate neighbors. lateral, where the phospholipid moves around in one leaflet. Finally, it is possible for phospholipids to Page 3 of 5  move between both leaflets of the bilayer in transverse movement, in a “flip-flop” manner, these movements are important for cell signalling. o Cholesterol: Compounds containing 27 carbon structures with four rings. A Cholesterol is a very important steroid to the body. When cholesterol binds to a fatty acid it forms a Cholesteryl ester. Cholesteryl esters have a lower solubility in water due to their increased hydrophobicity. In the memebrane cholesterol can be present on it’s own or combined B with proteins Figure (10-4): A: Cholesterol Cholesterol Helps control the Fluidity of Cell Membranes: B:Cholesteryl - Cholesterol is an amphipathic molecule, meaning, like Ester phospholipids, it contains a hydrophilic and a hydrophobic portion. - The cholesterol molecules are randomly distributed across the phospholipid bilayer. - Cholesterol's hydroxyl (OH) group aligns with the phosphate heads of the phospholipids. The remaining portion of it tucks into the fatty acid portion of the membrane. - Because of the way cholesterol is shaped, part of the steroid ring is closely attracted to part of the fatty acid chain on the nearest phospholipid. This helps slightly immobilize the membrane (decrease the fluidity) and make it less soluble to very small water- soluble molecules that could otherwise pass through more easily. Figure (10-5): Cholesterol in - Without cholesterol, cell membranes would membrane be too fluid, not firm enough, and too permeable to some molecules. - However, in cold temperature Cholesterol increases the fluidity as it helps separate the hydrophobic tails of phospholipids so that the fatty acid chains can't come together and cyrstallize. Cholesterol Helps Secure Important Proteins in the Membrane: - The plasma membrane contains many proteins that perform important functions, because certain proteins' size or shape requires a thicker phospholipid bed to sit in, and because certain proteins need to stick together to function properly, the fluidity of the cell membrane where the molecules are constantly moving randomly, could pose a problem. Page 4 of 5 - In these areas the plasma membrane contains high concentrations of cholesterol and glycosphingolipids which aggregate more making these parts of the membrane thicker, and making it ideal for accommodating certain proteins. - The previous association of cholesterol and glycosphingolipids together with proteins form “lipid rafts” which are microdomains of the plasma membrane that function to organize and regulate membrane signalling. - A special type of lipid rafts is the “Caveolae” which are flask like invaginations in the plasma membrane (while lipid rafts are generally flat regions) they are mainly present in fat cells and muscle cells. - Caveolae plays an important role in cell signalling, they can bud from the plasma membrane to form a vesicle for endocytosis (cellular process in which substances are brought into the cell) or can flatten into the membrane to help cells with stand mechanical stress. Page 5 of 5  7 Dynamics of Drug Actions ILOs By the end of this lecture, students will be able to: 1. Appraise the importance of efficacy versus potency in therapeutic selection. 2. Compare the quantitative distinction in response of different drugs when either acting on the same receptors or on different ones. 3. Explain the importance of potentiation & antagonism in fields of therapy. 4. Predict relative drug safety and drugs to be monitored upon analysing the quantal dose-frequency curves considering its effective and toxic responses. 5. Appraise implications of variation of drug response, in fields of therapy. GRADED DOSE-RESPONSE CURVE IS USED FOR:  Quantitative Comparison of Effect of Different Drugs Acting on SAME RECEPTOR: E A B  Comparing agonistic action of B, C, D, E, F to the full agonist “A” C as shown in Figure 1 which reveals: a. Drugs B, C, E are of same efficacy as “A” i.e., Full Agonists. D F b. It also reveals that potency of E> “A” while “A”>B>C in potency. c. Drugs D & F have less efficacy than “A” i.e., Partial Agonists. D>F in efficacy, while F> D in potency.  Comparing the effect of addition of another drug to “A” a. If this drug causes a slope shift to the left “like the effect of E”: it is called “ POTENTIATION “. b. If this drug causes a slope shift to the right“like the effect of B” it is called “ANTAGONISM”.  Comparing the effect of addition of an antagonist to “A” as shown in Figure 2 which reveals: a. If it causes a right parallel shift and appears to decrease potency of an agonist as in “B” and can be overcome by increasing concentration of the agonist, it is a Competitive Reversible Antagonist. b. If it causes a nonparallel shift to the right and appears to decrease efficacy of an agonist as in “C” and cannot be overcome by increasing concentration of the agonist, it is either a Competitive Irreversible Antagonist or a Non- Competitive Antagonist. A B Competitive – Potency Antagonism Efficacy  Quantitative Comparison of Effect of Different Drugs C Irreversible - Acting on DIFFERENT RECEPTORS: Competitive Antagonism Non--Competitive Antagonism Fig 1: Comparing effects of Different Agonists. Fig 2: Dose-Response-Curve of Different Antagonists  Comparing the action of drugs, A, B, C, D, on different receptors, shown in Figure 3 reveals: They can vary in efficacy; Drug B >A >D >C in efficacy. They could not be compared in potency as they do not act on same receptor. N.B. If one drug acting on a receptor increases the action of another drug acting on a different receptor; this is termed “SYNERGISM” or “ SUMMATION” , the new curve induced by both drugs will be more efficacious than that of the first drug alone. This is to differentiate from the forestated “POTENTIATION”, where the new curve induced by both drugs will be of more potency than that of the first drug alone. N.B. The Graded-Dose-Response-Curve gives information about the relation of drug concentration/dose in a particular tissue or whole body, but it does not reflect the relation between the drug dose and the proportion of population that therapeutically responded or that developed side effects. Alternatively, a QUANTAL DOSE-RESPONSE-CURVE (figure 4) has become of major clinical importance in justifying that. It is quantal because for any individual in the population the response is always all or none, i.e., - Therapeutically [a drug for sleep; induce sleep or not / a drug lowering cholesterol; dropped it to target level or not] - Adversely, e.g., hypoglycaemia, hepatic injury, hypertension, etc. or not]. QUANTAL DOSE-RESPONSE CURVE IS USED FOR:  Predicting the relative DRUG SAFETY by: 1. Determining from this dose-response-frequency curve: Median-Effective-Dose, ED50: the drug dose that induces a specific therapeutic response in half the population. Median-Toxic -Dose, TD50: the drug dose that induces a special (adverse) toxic response in half the population. 2. Calculating the relative measure of drug safety, termed “THERAPUTIC INDEX” [TI] = TD50 / ED50 whereby if: TI is low  drug is = not safe, as Digoxin. TI is high  drug is = safe, as Penicillin (regarding the high doses).  Determining Drugs that need THERAPEUTIC MONITORING: In clinical practice, determination of blood drug concentration is recommended for certain therapeutics. This is termed Therapeutic Drug Monitoring and is indicated when a drug has narrow therapeutic window, i.e., when the difference between Fig 3: Comparable Dose-Response of Different Fig. 4: Quantal Dose-Response-Curve the dose causing Drugs acting on different receptors.  toxicity and therapeutic effect is very small, i.e., unsafe drugs as Warfarin. Drugs with wide therapeutic window, are safe and do not need monitoring as Ampicillin as shown in Figure 5. Fig. 5: Narrow versus Wide Therapeutic Window of drugs. VARIATION IN DRUG RESPONSE In certain instances, the response of drugs may become reduced, increased, or altered.  If responsiveness to a drug becomes REDUCED gradually, in consequence to repeated administration, this is “TOLERANCE”. It indicates a need to increase the dose of a drug, to maintain the attained response. It could be caused by down regulation of receptors, or decrease in response effectiveness. “TACHYPHYLAXIS” is an acute rapidly developed tolerance, when doses of a drug are repeated in quick succession. N.B. “REFRACTORINESS” signifies the loss of therapeutic efficacy of a drug. “RESISTANCE” signifies the complete loss of effectiveness to antibiotics or anticancer…etc.  If responsiveness to a drug becomes INCREASED: as the exaggeration in vasodilatation produced by Nitrates when it induces syncope; this is “HYPER-SUSCEPTIBILITY” (DRUG INTOLERANCE).  If responsiveness to a drug becomes ALTERED:  When an abnormal response to a therapeutic dose of a drug develops due to a genetic defect, this is “IDIOSYNCRASY” as with Sulphonamide developing haemolytic anaemia in patients with glucose-6- phosphate deficiency.  When an immune response develops due to formation of antigen-antibody reaction, this is “HYPERSENSITIVITY REACTION” as with Penicillin developing skin reaction, bronchial asthma, or even anaphylaxis.  When an adaptive state develops to repeated drug administration and upon its cessation, withdrawal manifestations appear, this is “DEPENDENCE” as with Habituation; developing to Nicotine in Cigarettes or Cannabis or as Physical Dependence “Addiction”; developing to Diazepam or Morphine.  8 Nucleus and phases of cell cycle ILOs By the end of this lecture, students will be able to 1. Correlate structure of different components to the nucleus to its function. 2. Differentiate between functional forms of the chromatin. 3. Interpret structural organization of the chromosome. 4. Discuss the nuclear and cellular changes during the phases of the cell cycle The Cell Nucleus: Introduction The nucleus contains a blueprint for all cell structures and activities, encoded in the DNA of the chromosomes. It also contains the molecular machinery to replicate its DNA and to synthesize and process the three types of RNA; ribosomal (rRNA), messenger (mRNA), and transfer (tRNA). (Are there DNA in the cell outside the nucleus?) The nucleus does not produce proteins; the numerous protein molecules needed for the activities of the nucleus are imported from the cytoplasm. Structure of the nucleus as seen by LM The nucleus frequently appears as a rounded or elongated structure, usually in the center of the cell (Figure.1A). Its main components are the nuclear envelope, chromatin, nucleolus, and nuclear matrix (Figure.1B). The size and morphological features of nuclei in a specific normal tissue tend to be uniform. In common hematoxylin and eosin-stained preparations; the nucleus, however, appears intensely stained dark blue or black. (Why?) Ultrastructure of the nucleus Three components are recognized: 1. Nuclear Envelope Electron microscopy shows that the nucleus is surrounded by two parallel membranes separated by a narrow space called the perinuclear cisterna. Together, the paired membranes and the intervening space make up the nuclear envelope. (Fig 1B) Polyribosomes are attached to the outer membrane, showing that the nuclear envelope is a in continuity with the endoplasmic reticulum. (Why?) At sites at which the inner and outer membranes of the nuclear envelope fuse, there are gaps, the nuclear pores (Figure 1B), that provide controlled pathways between the nucleus and the cytoplasm. Because the nuclear envelope is impermeable to ions and molecules of all sizes, the exchange of substances between the nucleus and the cytoplasm is made only through the nuclear pores. Ions and Page 1 of 5 molecules with a diameter up to 9 nm pass freely through the nuclear pore without consuming energy. But molecules and molecular complexes larger than 9 nm are transported by an active process, mediated by receptors, which uses energy from adenosine triphosphate (ATP). 2. Chromatin o Chromatin is composed mainly of coiled strands DNA bound to basic proteins (histones). o The basic structural unit of chromatin is the nucleosome (Figure 2), which consists of a core of four types of histones, wrapped around DNA base pairs. (What is the role of histones?) o Linker DNA; An additional DNA segment forms a link between adjacent nucleosomes, and another type of histone is bound to this DNA. This organization of chromatin has been referred to as "beads-on-a-string." Nonhistone proteins are also associated with chromatin, but their arrangement is less well understood. o Functional forms of Chromatin; in nondividing nuclei, is in fact the chromosomes in a different degree of uncoiling. According to the degree of chromosome condensation, two types of chromatin can be distinguished with both the light and electron microscopes (Figure 3). Heterochromatin (Gr. heteros, other, + chroma, color), which is electron dense, appears as coarse granules in the electron microscope and as basophilic clumps in the light microscope. It represents the inactive form of chromatin and acts as a reserve in less active cells. Euchromatin is the less coiled portion of the chromosomes, visible as a finely dispersed granular material in the electron microscope and as lightly stained basophilic areas in the light microscope. It represents the active form of chromatin and more abundant in active cells. The proportion of heterochromatin to euchromatin accounts for the light-to-dark appearance of nuclei 0in tissue sections as seen in light and electron microscopes. The intensity of nuclear staining of the chromatin is frequently used interpret the functional state of the nucleus. (How?) Figure 2 Nucleosome structure Page 2 of 5  Figure 3 Electron micrograph of a nucleus showing the heterochromatin (HC) and euchromatin (EC). Unlabeled arrows indicate the nucleolus-associated chromatin around the nucleolus (NU). Arrowheads indicate the perinuclear cisterna. Underneath the cisterna is a layer of heterochromatin, the main component of the so-called nuclear membrane seen under the light microscope. X 26,000. Careful study of the chromatin of mammalian cell nuclei reveals a heterochromatin mass that is frequently observed in female cells but not in male cells. This chromatin clump is the sex chromatin and is one of the two X chromosomes present in female cells. The X chromosome that constitutes the sex chromatin remains tightly coiled and visible, whereas the other X chromosome is uncoiled and not visible. Evidence suggests that the sex chromatin is genetically inactive. The male has one X chromosome and one Y chromosome as sex determinants; the X chromosome is uncoiled, and therefore no sex chromatin is visible. In human epithelial cells, sex chromatin appears as a small granule attached to the nuclear envelope. The cells lining the internal surface of the cheek are frequently used to study sex chromatin. Blood smears are also often used, in which case the sex chromatin appears as a drumstick-like appendage to the nuclei of the neutrophilic leukocytes. 3. Nucleolus o The nucleolus is a spherical structure (Figure 17-5) that is rich in rRNA and protein. It is usually basophilic when stained with hematoxylin and eosin. o Significance of the nucleolus; it contains DNA that codes for rRNA (type of RNA present inside ribosomes). The nucleolus is the site of synthesis of ribosomal subunits (to be explained later). Ribosomal proteins, synthesized in the cytoplasm, become associated with rRNAs in the nucleolus; ribosome subunits then migrate into the cytoplasm. Heterochromatin is often attached to the nucleolus (nucleolus-associated chromatin), but the functional significance of the association is not known. The rRNAs are synthesized and modified inside the nucleus. In the nucleolus they receive proteins and are organized into small and large ribosomal subunits, which migrate to the cytoplasm through the nuclear pores. (Figure 17-4) Page 3 of 5  4. Nucleoplasm: The protoplasm within the nucleus, consisting of a fluid portion, a proteinaceous matrix, and various ribonucleoproteins particles. Nuclear & cellular changes during cell cycle: Cell cycle The cell cycle is a series of events within the cell that prepare the cell for dividing into two daughter cells. Phases of the cell cycle: I- Interphase; long period of time during which the cell increases its size and content and replicates its genetic material. It includes three stages: a) G1 (gap) phase, synthesis of macromolecules essential for DNA duplication begins & cell growth. b) S (synthetic) phase, DNA is duplicated. c) G2 phase, the cell undergoes preparations for mitosis. II- Mitosis, a shorter period of time during which the cell divides its nucleus and cytoplasm, giving rise to two daughter cells. The cell cycle may be thought of as beginning at the conclusion of the telophase stage in mitosis, after which the cell enters interphase. (Figure 6) The interphase Gap 1 Daughter cells formed during mitosis enter the G1 phase. During this phase, the cells synthesize RNA, regulatory proteins essential to DNA replication, and enzymes necessary to carry out these synthetic activities. Thus, cell growth occurs restoring cell size to normal. The centrioles (a cell organelle involved in cell division) begin to duplicate themselves, a process that is completed by the G2 phase. S Phase During the S phase, the synthetic phase of the cell cycle, the genome is duplicated. The cell now contains twice the normal complement of its DNA. Autosomal cells contain the diploid amount of DNA before the synthetic (S) phase of the cell cycle that becomes doubled after S phase in preparation for cell division. Page 4 of 5 G2 Phase The gap 2 phase (G2 phase) is the period between the end of DNA synthesis and the beginning of mitosis. During the G2 phase, the RNA and proteins essential to cell division are synthesized, the energy for mitosis is stored. Duplication of centrioles and formation of the needed microtubules are completed. DNA replication is analyzed for possible errors, and any of these errors is corrected. Cells that become highly differentiated (What is meant by differentiation?) after the last mitotic event may stop to undergo mitosis either permanently (e.g., neurons, muscle cells) or temporarily (e.g., peripheral lymphocytes) and return to the cell cycle at a later time. Cells that have left the cell cycle are said to be in a resting stage, the G0 (outside) phase, or the stable phase. Figure 6. The cell cycle in actively dividing cells. Nondividing cells, such as neurons, leave the cycle to enter the G0 phase (resting stage). Other cells, such as lymphocytes, may return to the cell cycle. Page 5 of 5