Cell Biology Lecture Notes & Practical Slides PDF

Cell Biology Lecture Notes & Practical Slides For First Year Agriculture students Dietetic Program By Prof. Dr. Amal Mohamed Moustafa Former Head of Medical Histology & Cell Biology Department Faculty of Me...

Cell Biology Lecture Notes & Practical Slides For First Year Agriculture students Dietetic Program By Prof. Dr. Amal Mohamed Moustafa Former Head of Medical Histology & Cell Biology Department Faculty of Medicine- Mansoura University  Cell Biology Prof. Dr. Amal M. Moustafa Contents of Cell Biology Chapters Pages 1- Introduction, Types of Microscopes & Microtechniques. 2 2- Cell Structure 11 a) Cell Membrane 12 b) Membranous cell organelles 14 c) Non membranous cell organelles 19 d) Cell inclusion 24 e) Matrix 25 f) Nucleus 26 3- Cell cycle, Cell division & Karyotyping 32 4- Human chromosomes, Rules of inheritance & Chromosomal 38 anomalies 5- Intercellular junctions 45 6- Stem Cells 48 50 II- Practical Slides https://www.youtube.com/playlist?list=PLlaD_a0yIzNe6104fI_qVtU_Sl_2lKaIb 1  Cell Biology Prof. Dr. Amal M. Moustafa Cell Biology Histology is the science of tissues (GK. Histos: tissue, logy: study). It is concerned with the microscopic structure of cells (cell biology), tissues, organs, and systems in relation to their functions by using different micro-techniques. Cells: It is the smallest microscopic structural & functional unit in the body. Tissues: group of similar cells bounded together with extracellular matrix to form different tissues with specific function e.g., epithelium, connective tissue, muscular & nervous tissue. Four basic types of tissues: 1. ▪ Epithelial tissues – surface coverage 2. ▪ Connective tissues – to link or support other specialized tissues 3. ▪ Muscular tissues – contractile property 4. ▪ Nervous tissues – cells forming brain, spinal cord, and nerves Organs: two or more tissues are combined in varying proportions from the 4 basic tissues to give large organs e.g., stomach (is made of epithelium, smooth muscle, CT, & Nerves). Systems: several organs with related functions collect to form systems e.g., respiratory system& digestive system. 2  Cell Biology Prof. Dr. Amal M. Moustafa Types of Microscopes Microscope: is an instrument which magnifies the image & reveals fine details of the object. Types of Microscopes. A. Light Microscope (LM) B. Electron Microscope (EM) A. Light Microscope (LM) Definition: A light microscope is a laboratory tool that uses light to enhance the visualization and magnification of very small objects (cells & microorganisms). Parts of light microscope: 1. The stage - where the specimen is placed, allowing movement of the specimen around for better viewing with flexible knobs. 2. Microscopic lens - objective lens (magnification x4, x 20, x 40, x60, x 100) - eyepiece (ocular lens) (magnification x 5, x 10) 3  Cell Biology Prof. Dr. Amal M. Moustafa - They use lenses to focus light on the specimen, magnifying it thus producing Calculation of magnification of Light Microscope Final magnification = magnification of objective lens x magnification of the eyepiece lens Meter(m) = 100 centimeter (cm) Centimeter (cm) = 10 millimeter (mm) Millimeter (mm) = 1000 micrometers (µm) Micrometer (µm) = 1000 nanometers (nm) a magnifying image named virtual image. 3. The nosepiece has about three to five objective lenses with different magnifying power. It can move round to any position depending on the magnification power of the objective lens to focus on the image. 4. Two focusing knobs: - Fine adjustment knob - Coarse adjustment knob, - found on the microscopes’ arm, which can move the nosepiece to focus on the image & sharpen the image clarity. 5. A light illumination source: a mirror or electric lamp found at the base. 6. The condenser - The condenser is mounted below the stage which focuses a beam of light into the specimen. 7. A diaphragm with an aperture which controls the diameter of the beam of light that passes through the condenser. 4  Cell Biology Prof. Dr. Amal M. Moustafa II- Electron Microscope (EM) - There are two types: a- Transmission electron microscope (TEM) It is characterized by: Higher magnification : 1000 : 50 000. Needs extremely thin sections 0.08um. Stains: heavy metals (lead citrate & uranyl acetate). The image appears on the screen as white, black colors & shades of grey. Dark components in the electron micrograph are called electron dense. White components are called electron lucent. B. Scanning electron microscope (SEM) - It allows a 3-dimensional image which reveals the surface features of the tissue on the screen. - The outer surface of the tissues is covered with heavy metals (gold). 5  Cell Biology Prof. Dr. Amal M. Moustafa Micro-techniques Def: Techniques for preparing thin transparent sections and Staining of this tissue. Types: 1. Paraffin technique (Most common technique). 2. Frozen technique (Most rapid technique). 1. Paraffin technique Advantages of Paraffin Technique: 1- Gives thin sections (4- 6 μm). 2- Sections are stained easily by different colored stains. 3- Gives serial sections. Disadvantages of Paraffin Technique: 1- Take a few days to prepare. 2- Not suitable for demonstration of lipid, glycogen & enzymes. 6  Cell Biology Prof. Dr. Amal M. Moustafa 2. The Freezing technique The frozen section is the rapid tissue section by cooling the tissue with the help of cryostat to provide immediate report of the tissue sample. The cryostat is the instrument to freeze the tissue and to cut the frozen tissue for microscopic section. Advantages of Freezing Technique: 1-Very rapid method for rapid diagnosis of the lesion for intraoperative management, to know the extent of the lesion. 2- Suitable method to stain enzymes, lipid & carbohydrate in the tissue. Disadvantages of Freezing Technique: 1- Gives thick sections (10- 20 μm). 2- No serial sections. 7  Cell Biology Prof. Dr. Amal M. Moustafa Histology Stains Stain: a dye or substance used for staining of sections to differentiate different structures by different colours. Types of stains: 1. Acidic stain as Eosin (E): Has negative charge (anionic). Tends to stain the basic structures of the cell e.g. the cytoplasm. The substance stained with Eosin appears pink in colour and is called acidophilic 2. Basic stain as Haematoxylin (H): Has positive charge (cationic) Tends to stain the acidic structures of the cell e.g. the nucleus. The substance stained with Haematoxylin appears blue in colour and is called basophilic. 3. Neutral stain as Leishman stain (Methylene blue + Eosin) Gives two different colour. The acidic radical imparts its colour to the basic structures. The basic radical imparts its colour to the acidic structures. 8 Cell Biology Prof. Dr. Amal M. Moustafa *Steps for staining with Haematoxylin and Eosin (H &E) Stains* 9  Cell Biology Prof. Dr. Amal M. Moustafa *Special stains for specific components in the cell* 1. Lipids: by using frozen sections. Osmic acid: black colour. Oil Red O: Red colour. Sudan III: orange colour. 2. Glycogen: Best's carmine stain: gives red colour. 3. Carbohydrates: Periodic acid Schiff's reaction (PAS): gives magenta colour. 4. collagen fibers: Mallory stain: gives blue color. 5. Elastic fibers Van Gieson stain: gives yellow color. Orcin stain: gives brown color. Verhoeff’s stain: gives black color. 6. Nucleic acid can be demonstrated by Pyronin methyl green stain. RNA stained red by pyronin. DNA stained green by methyl green. 7. Enzymes: Succinic dehydrogenase enzyme, it is present in mitochondria. Acid phosphatase enzyme, it is present in the lysosomes. Other types of stain: 1. Vital stain: Staining of living cells in vivo e.g., staining of phagocytic cells in the body by injecting Trypan blue. 2. Supravital stain: Staining of living cells in vitro i.e., outside the body e.g., staining of mitochondria by Janus green B 3. Metachromatic stain: Staining of a tissue by a colour different from that of original stain e.g., granules of mast cells are colored reddish purple when stained with Toluidine blue. 10  Cell Biology Prof. Dr. Amal M. Moustafa The Cell Structure The animal cell is the structural and functional unit in the body. Interphase cell: this term is used to denote any cell that is not dividing, whether it is preparing for the next division or has lost its ability to divide. The interphase cell consists of: (1) Cytoplasm. (2) Nucleus. The human cells are variable in Size: - for example, the human ovum is the largest cell (150 µm) and the granule cell in the cerebellum is the smallest cell (4 µm). The human cells are variable in Shape: - for example, it could be squamous, cubical, columnar, rounded, oval, polyhedral, pyriform, pyramidal, or stellate. 11  Cell Biology Prof. Dr. Amal M. Moustafa Cell membrane Def: The plasma membrane envelops the cell and maintains its structural and functional integrity. LM: It is very thin to be resolved by H&E stain. Special stain: Silver or PAS stain can be seen. EM: Thickness: 7.5- 10 nm. It appears as 3 layers; outer and inner electron dense layers and a middle light electron lucent thus called tri-lamellar membrane or unit membrane. Chemical structure of the cell membrane: It is composed of: 1. Lipids (phospholipids, Cholesterol). 2. Proteins. 3. Carbohydrates. 12  Cell Biology Prof. Dr. Amal M. Moustafa lipids Proteins Carbohydrates A. Phospholipid molecules: 1- Intrinsic (integral) 1- Glycolipids protein: -They form the main part of the 2-Glycoproteins cell membrane & arranged in a- small proteins are -Located on the bilayer. inserted in each lipid external surface of the -Each phospholipid molecule bilayer. cell membrane only consists of: b- They are large proteins forming the cell coat 1- A rounded charged extend across the two-lipid or glycocalyx. hydrophilic head directed bilayer of the cell - it includes cell outward. membrane called receptors. 2- Two long non-charged tails (transmembrane protein) & directed inward forming a act as hydrophilic Function: hydrophobic layer in the channels. a- cell adhesion. middle. 2- Extrinsic (peripheral) b- cell immunity B. Cholesterol molecules: located protein: small protein between the tails of phospholipid form non continuous layer c- cell protection molecules in both layers of the outside the lipid bilayer & d- cell recognition membrane. loosely attached to both outer surface of the cell membrane Functions of cell membrane: 1. Isolate the cytoplasm from the external environment. 2. Regulate the exchange of substances (e.g., Sodium potassium pump). 3. Endocytosis: Phagocytosis (cell eating): Internalization of solid materials. Pinocytosis (cell drinking): Internalization of liquid materials. Receptor Mediated Endocytosis 4. Exocytosis: A process by which the cell expels out any waste product. 5. Modification of the cell membrane e.g., Microvilli, cilia, flagella, and formation of cell junctions & myelin sheath. 13  Cell Biology Prof. Dr. Amal M. Moustafa I- Membranous cell organelles 1- Mitochondria Definition Membranous cell organelles containing oxidation -reduction enzymes for aerobic respiration & energy production (powerhouse of the cells). LM, -Succinic dehydrogenase stain ► It reveals Special activity of succinic dehydrogenase enzyme stains in the mitochondria in the form of dark blue stained mitochondria -Janus green B (supravital stain) staining the mitochondria green color in mitochondrial culture Shape Rod-shaped, granules or filament. EM 1-The mitochondrion is surrounded Double membranes separated by intermembranous space: a- The outer membrane is smooth. b- The inner layer is folding forming shelf -like structures called cristae project into the matrix. 2 -Mitochondrial matrix within the inner membranes that contains: a) Oxidative enzymes of Kreb’s cycle. b) DNA & RNA (mRNA, tRNA, rRNA) c)Electron dense matrix granules rich in Ca2+ Functions 1- Generate energy needed to power the cell's biochemical reactions. The chemical energy produced by the mitochondria is stored in a small molecule called adenosine triphosphate (ATP). 2- -oxidation of short chain fatty acids. 14  Cell Biology Prof. Dr. Amal M. Moustafa 2- Endoplasmic reticulum (ER) Def: ❖ It is a membranous organelle formed of a communicating tubular channel forming a membranous reticulum in the cytoplasm. ❖ It extends from the cell membrane to the nuclear membrane, so it is responsible for transport and exchange of material through various regions of the cytoplasm “intracellular highway “ 15  Cell Biology Prof. Dr. Amal M. Moustafa Rough endoplasmic reticulum Smooth endoplasmic reticulum (rER) (sER) Definition -Membranous cell organelles are Membranous cell organelles are well developed in protein well developed in steroid hormones synthesizing cells e.g. plasma cells. forming cells e.g., liver & adrenal -It extends from the nuclear cells. membrane to the cell membrane. -It is mostly located nearby the cell “Intracellular highway “ membrane. LM, H&E stain: rER gives the H&E stain: sER when abundant, cytoplasm basophilic color (due to it gives the cytoplasm acidophilic Special the presence of polyribosomes on color. stains its cytosolic surface). EM -It appears as regular parallel - It appears as irregular flattened membranous tubules anastomosing tubules & sacs enclose a space i.e. lumen called (vesicles) with smooth outer surface cisternae. that lack attached polyribosomes. -It shows attached electron dense - They are continuous with rER i.e. polyribosomes on its cytosolic “Intracellular highway “ surface. Functions 1- Proteins synthesis by attached 1. Glycogen breakdown to glucose. polyribosomes. 2. Lipid synthesis, e.g., cholesterol 2- segregation of the formed & phospholipid for cell proteins inside the cisternae. membrane. 3-Initial glycosylation of proteins. 3. Formation of oil droplets. 4-Transport of proteins: by pacing 4. Steroid hormones synthesis e.g., estrogen, testosterone & of the formed proteins in a cortisone. membranous vesicle that bud off 5. Storage of Ca2+ & pump it from the cisternae to be delivered during muscle contraction. to the Golgi apparatus (Transfer Vesicles) 6. Detoxification of hormones, drugs & alcohol in the liver. 16  Cell Biology Prof. Dr. Amal M. Moustafa 3- Golgi Apparatus Definition It is a membranous cell organelle. Proteins received from the rER are further modifying, packaging and sorts of proteins in vesicles in secretory cells. LM, By H&E stain: It appears as a pale unstained area in the deeply stained Special basophilic cytoplasm of protein synthesizing cells. This area is called stains negative Golgi image as it lacks ribosomes. By Silver stain: It appears as network of brown granules & fibrils. EM 1-Golgi saccules: 4-10 parallel flat curved membranous saccules arranged one above the other forming stacks. Each saccule has a narrow central part and expanded periphery. Saccules are interconnected with each other. Each stack has two faces: Convex surface called immature entry face, cis face. It receives transfer vescicles from rER. Concave surface called mature exit face or trans face. 2-Micro-vesicles (transfer vesicles): Tiny vesicles present at the immature face (cis face) originate by budding from the rER. 3-Macro-vesicles (secretory vesicles): Large vesicles present at the mature face (trans face). They are bud off from the mature face of Golgi stack then: Migrate to the cell membrane, fuse with it, and discharge their content outside the cell (exocytosis). Remain in the cell cytoplasm as lysosomes. Functions 1. Concentration & packaging of the secretory products received from rER. 2. Modification of proteins by adding carbohydrate (glycosylation of proteins). or sulfate 3. Formation of lysosomes. 4. Renewal and maintain the cell membrane & cell coat. 17  Cell Biology Prof. Dr. Amal M. Moustafa 4- Lysosomes Definition membranous cell organelles which contain hydrolytic enzymes for intra- cytoplasmic digestion (suicide bags). LM, Special Histochemical stain: Acid phosphatase activity stain. stains EM Two types of lysosomes: 1. Primary lysosomes: They are not involved in intra-cellular digestion. Small spherical membranous vesicles with a homogenous electron dense appearance. Contain many acid hydrolases enzymes. 2. Secondary lysosomes: They are involved in intra-cellular digestion. They are large spherical membranous vesicles with heterogenous electron dense core. Types of 2nd lysosomes: 1-Heterolysosomes: Primary lysosomes + phagosome (containing solid phagocytosed particles) 2-Multi-vesicular body: Primary lysosomes + pinocytotic vesicles (containing fluid) 3-Autolysosomes: Primary lysosomes + worn out organelles (mitochondria, ER) which maintain the cell healthy Functions 1-intra-cytoplasmic digestion of food, waste products, damaged organelles & damaged cells, so they are present in all cells specially in macrophages 2- Activation of thyroxin hormone 3- Help sperm to penetrate the ovum (Fertilization) 18  Cell Biology Prof. Dr. Amal M. Moustafa 5.Peroxisomes Definition Membranous cell organelles that contain oxidation enzymes (like catalase in human & uricase in rodents) which converts hydrogen peroxide into water H2O2 -----→ H2O+O2 Origen - It arises as a bud (membranous vesicles) from rER. Special Histochemical stain: catalase enzyme activity. stains EM a- They are small (0.5um) finely granular single membrane bounded vesicles in human. b- They show an electron dense semi-crystalline core contains uricase enzyme in rodents. Functions 1. B-oxidation of long chain fatty acids. 2. Detoxification of hydrogen peroxide (H2O2) by catalase enzyme. 3. Formation of bile acids & detoxification of alcohol in the liver. II-Non-membranous cell organelles 1. Cytoplasmic filaments Def: - non-membranous cell organelles. - They are thread-like structures that act as part of the cytoskeleton. Thin filaments Thick filaments Intermediate filaments (Actin) (Myosin) (Many types) Diameter 5 nm 15 nm 10 nm. Contractility Contractile Contractile Non-contractile Distribution & 1.Actin & myosin located in muscle for contraction. 1-Supporting and function 2- in cell division, actin & myosin filaments encircle the maintaining the shape of cell equator of the cells form a contractile ring; its the cell. contraction leads to separation of the two daughter cells. 2-Tumour identification 3- Actin is located in blood platelets for clot retraction. 4. Microvilli formed of Actin. 5. Amoeboid movement of certain cells 19  Cell Biology Prof. Dr. Amal M. Moustafa 2. Microtubules Def: thin tubular-like structure of unfixed length but are of uniform diameter (25um). Distribution: All over the cytoplasm. EM: Microtubules appear as hollow tubules. In cross section, appear as small circles. The wall consists of 13 protofilaments that are composed of tubulin dimers. Functions: 1. Determination shape of the cell (cytoskeleton). 2. Formation of mitotic spindle during cell division. 3. Formation of centrioles, cilia & flagella. 4. Intracellular cellular transport of macromolecules. NB: Cytoplasmic Cytoskeleton Structure: It is formed of a complex network formed of: Microfilaments, intermediate filaments & Microtubules bind together with some proteins to link them to the cell membrane forming framework called micro-trabecular lattice. Function of cytoskeleton: a- Maintain shape of the cell, bear stress & keep the cell from collapsing. b- Hold the cell organelles in their right position. c. Dynamic intercellular movements 20  Cell Biology Prof. Dr. Amal M. Moustafa 3. Centrioles Definition centrioles are non-membranous organelles important for cell reproduction. LM, Iron Hematoxylin stain: can demonstrate the centrioles as two dark bodies near the nucleus. Special stains EM Each cell has two short cylindrical centrioles lying at right angle with each other and surrounded by a matrix of tubulin protein (centrosome). The wall of each cylinder is composed of 27 microtubules longitudinally arranged in 9 bundles; each bundle consists of 3 microtubules (triplets). Functions 1. Play an important role in cell division. 2. Form the basal bodies of cilia & flagella. 21  Cell Biology Prof. Dr. Amal M. Moustafa 4. Cilia Definition Cilia are hair-like processes that arise from the external surface of some cells. Origen Centrioles duplicate several times in ciliated cells & migrate to the free surface of the cell form the basal bodies of the cilia. Then, the shaft of the cilia grows up from each centriole (basal bodies). LM H. & E.: They appear as hair like striation on the free border of the cells. EM The cilia composed of shaft, basal body & rootlets. 1. The shaft (axoneme): -It is a finger-like projection surrounded by cell membrane. -It is composed of 18 microtubules longitudinally arranged in 9 bundles; each bundle consists of 2 microtubules (doublets) + 2 singlets microtubules surrounded by a central sheath in the center. 2. The basal body i.e. centrioles. 3. The rootlets: - They are growth fibers from the 3rd microtubules of the basal bodies which fix the basal bodies & shaft to the cytoplasm. Functions 1. Motile cilia beat in one direction to move a thin film of fluid or mucous which is present in the respiratory & female genital systems. 2. Non motile cilia (Stereocilia) become: a. Modified receptors to receive light stimuli, e.g., rods and cones in the retina. b. Non modified stereocilia e.g., epididymis. NB. Flagellum: it is the motile tail of the sperm used for forward movement of the sperm toward the ova. - It is the same structure as the axoneme of the cilia, but extremely long. 22  Cell Biology Prof. Dr. Amal M. Moustafa 5) Ribosomes Definition non-membranous organelles responsible for protein synthesis in the cell. LM, They are stained blue with Hematoxylin or methylene blue (basic Special stain) stains when present in large number in the cytoplasm, their site exhibits basophilia. Shape They are very small spherical bodies, 20-30 nm in diameter structure They are formed of ribosomal rRNA & proteins EM -They appear as electron dense granules. - Each ribosome is formed of two subunits; the large subunit is twice the size of the small. - They held together by messenger RNA forming polyribosomes. Types 1- Free ribosomes: scattered individually or attached to mRNA forming polyribosomes 2- Attached ribosomes: attached to the outer surface of rER. Functions 1-Free ribosomes: responsible for translation of the message carried by mRNA in the cytoplasm & link amino acids together to make proteins for the use of the cell. 2-Attached ribosomes: responsible for synthesis of secretory proteins & lysosomal proteins. 23  Cell Biology Prof. Dr. Amal M. Moustafa II- Cytoplasmic Inclusions Def: non-living, temporary product in the cytoplasm of the cells result from cell metabolism as carbohydrates, lipids, pigments & crystals. 1- Carbohydrates: 2- Lipids LM: Stored food in the cell e.g., glycogen Stored food in the form of granules in the liver & muscle. variable sized lipid globules in different cells e.g., adrenal, liver & fat cells. H&E the cytoplasm appears vacuolated. the cytoplasm shows empty stain spaces. Special PAS: → magenta granules. Oile red O: red globules. stains Best’s Carmine: → red granules. Sudan III: orange globules. Osmic acid: black globules. EM: 1- Alpha glycogen granules: rosettes - Appear as variable sized shape electron dense granules e.g. liver electron lucent or electron cells. dense lipid globules. 2- Beta glycogen granules: single electron dense granules e.g. muscle. 24  Cell Biology Prof. Dr. Amal M. Moustafa 3-Pigment Def: They are colored elements in the cytoplasm of specific cells. Types: Endogenous pigments Exogenous pigments 1.Myoglobin --red color (muscle fiber 1. Tattoo marks: certain dyes are 2.Hemoglobin-- red color (red blood injected in the skin by needle & taken corpuscles). by macrophages in the dermal tissue. 2. Lead mineral intoxication gives grey 3.Bilirubin is yellow brown. line in gingiva 4.Melanin is brown or black (skin, hair & eyes). 3. Silver mineral intoxication gives black color in the skin 5.Lipofuscin pigment is golden brown (↑↑↑ by age in the cardiac, 4. Carbon & dust particles are black muscle & nerve cells). color. 4 -Crystals Reinke crystals: They are present in interstitial cells of Leydig in the testes. III- Cytoplasmic matrix (cytosol) a. It is the gel like structure contains: b. Lipids, proteins, carbohydrates, enzymes, minerals, ions, RNA, metabolites, O2 & CO2. c. Located in between Microfilaments, intermediate filaments & microtubular network filling the intra-cellular spaces between cytoplasmic organelles and inclusions. Function: 1. Media for transport of the intracytoplasmic substance. 2. Site of chemical reactions. 3. Microfilaments: maintain shape of the cell & form cellular cortex 4. intermediate filaments: Hold the cell organelles in their right position. 25  Cell Biology Prof. Dr. Amal M. Moustafa Nucleus Def: large rounded or elongated structure found in most of the cells and plays important role in all cellular metabolic activities, cell division and heredity. Sites: It is present in all cells except mature RBCs and blood platelets. Position: It may be central, eccentric, peripheral, or basal. Eccentric Basal Central Peripheral Shape: May be rounded, oval, flattened, kidney shaped, bilobed, segmented or lobulated. Number: Mononucleated: containing one nucleus e.g., most human cells. Binucleated: containing two nuclei, e.g., liver cells. Multinucleated: containing more than 2 nuclei, e.g., osteoclast & skeletal muscle. Size: Small, medium sized or large. LM: In H&E-stained sections the nucleus appears basophilic due to its content of nucleic acids. The nucleus may be: 26  Cell Biology Prof. Dr. Amal M. Moustafa 1-Vesicular (open-face) nucleus: Lightly stained. Looks like a vesicle, its details could be identified. In metabolically active cells e.g., nuclei of nerve cells and liver cells. 2-Condensed nucleus: Deeply stained nucleus. Its details could not be identified. In metabolically inactive cells e.g. small lymphocyte. EM: Consists of four components: 1. Nuclear membrane 2. Chromatin. 3. Nucleolus. 4. Nuclear sap. 27  Cell Biology Prof. Dr. Amal M. Moustafa 1- Nuclear Membrane (Nuclear Envelope) Def: the membrane which encloses the nucleus and separates it from the surrounding cytoplasm. LM: Appears as a single basophilic line surrounding the nucleus due to the presence of peripheral chromatin on its inner surface & ribosomes on its outer surface. EM: Formed of two membranes; inner and outer nuclear membranes separated by perinuclear space (30-50 nm wide). The outer membrane is rough due to attached ribosomes& continuous with rER cisternae. The inner membrane is fibrillar due to attached chromatin peripheral heterochromatin. The nuclear membrane is interrupted by many circular nuclear pores. Functions: 1. Separates the chromosomes in the nucleus from the surrounding cytoplasm. 2. Allows the exchange of materials between the nucleus & the surrounding cytoplasm through the nuclear pores. 28  Cell Biology Prof. Dr. Amal M. Moustafa 2- Chromatin Def: basophilic material from which chromosomes are formed. Functions: 1. Carry genetic information. 2. Directs & control protein synthesis in the cell. 3. Formation of RNA. Structure: - It is formed of DNA + Histone protein. Types: - Two types of chromatins 1- Euchromatin 2- Heterochromatin Structure Extended parts of chromosomes Coiled parts of chromosomes. LM Appears as clear fine granular Appear as basophilic coarse areas. chromatin granules. Site Occupies the more central 1- Nucleolus associated chromatin. regions of the nucleus. 2- Chromatin islands. 3- Peripheral chromatin. Function Directs protein synthesis -------------------------- Content Active genes Inactive genes 29  Cell Biology Prof. Dr. Amal M. Moustafa 3-Nucleolus Def: one or more rounded basophilic bodies present in the interphase nuclei. LM: They are prominent in cells actively synthesizing protein. Site: central or eccentric in the nuclear sap. Staining: basophilic due to: The surrounding nucleolar associated chromatin. Its RNA content. EM: Spongy appearance, not limited by membrane & bounded by nucleolus associated chromatin. Functions: - Formation of ribosomal RNA & ribosomal subunit (rRNA + protein) which pass through the nuclear pores to the cytoplasm. 30  Cell Biology Prof. Dr. Amal M. Moustafa 4-Nuclear sap Def: A colloidal solution that fills the spaces between the chromatin & nucleolus. Content: nucleoproteins, sugar, enzymes, ca2+, potassium, phosphorus ions. EM: Appears as clear electron lucent areas. Vesicular nucleus: contains large amount of nuclear sap. Condensed nucleus: contains small amount of nuclear sap. Vesicular nucleus Condensed nucleus Function: Acts as a medium through which RNA moves towards the nuclear pores. ***Nuclear changes indicating cell death*** 1-Pyknosis: the nucleus becomes condensed, homogenous, smaller in size, darker in staining & eccentric in position. 2- Karyorrhexis: the nucleus is broken into small fragments. 3. Karyolysis: the nucleus dissolves & finally disappears. 31  Cell Biology Prof. Dr. Amal M. Moustafa Cell cycle Definition: The cell cycle is the series of events that take place in a cell leading to its division and duplication of its DNA (diploid cell; 2n) to produce two daughter cells. Stages of cell cycle includes: - 1. interphase & 2. Cell division. Interphase: -It refers to the rest of the cell (the period between two successive cell divisions). -It includes 3 stages: - 1. Cell grows (G1), 46-S chromosomes 2. Replication of DNA (S): 92 s-chromosomes (46-D chromosomes) 3. Preparation for mitosis (G2). Cell division Types: (1) Amitosis (direct division): It is a simple fission of nucleus & cytoplasm into two daughter cells. It occurs in amoeba & mitochondria. (2) Mitosis (indirect cell division): It results in two daughter cells; each one has the same 46-s chromosomes (diploid number). It occurs in somatic cells (which enable regeneration of body cells). (3) Meiosis (reduction cell division): It results in four cells; each one has 23-s chromosomes (haploid number). It occurs in germ cells (sperm & ova) for human reproduction. 32  Cell Biology Prof. Dr. Amal M. Moustafa Mitosis Definition: It occurs in somatic cells. It is the division of the nucleus of the cell (46 d- chromosomes) to produce of two daughter cells, has the same number of chromosomes as the parent cell (46 s- chromosomes) Stages: 4 stages 1- Prophase 1. The nuclear membrane, cell organelles break down to form a large number of small vesicles. 2. The nucleolus disintegrates. 3. The chromosomes (46 d-chromosomes) become visible as thin threads. 4. The centrioles duplicate & each pair moves to each pole of the cell by growth of the cytoplasmic microtubules (mitotic spindles) from the microtubule organizing center (MTOC) around the centrioles. 2- Metaphase 1. Well-developed cytoplasmic Microtubules lead to elongating of the cell. 2. At the centromere of each chromosome a dense plaque named kinetochore appears & acts as chromosomal MTOC. The chromosomal microtubules start to grow toward each pole of the cell result in well arrangement of the chromosomes. 3. The 46 d–chromosomes become short, thick & align on the equatorial plane (Middle line) due to the activity of mitotic spindles. 3- Anaphase - Each d-Chromosome splits longitudinally at the centromere allowing its two sister chromatids to separate and migrate toward the opposite pole of the cell. - Separation & migration is carried out by the interaction of the mitotic spindle. 33  Cell Biology Prof. Dr. Amal M. Moustafa 4- Telophase - A constriction (cleavage furrow) develops at the equatorial plane of the parent cell. This constriction is produced by contraction of Actin & Myosin microfilaments. It progresses until the cytoplasm and its organelles are divided into two daughter cells (cytokinesis). - The new daughter cells contain 46s-chromosomes. - Nuclear envelope, nucleoli & cell organelles reappear. NB: - Mitotic figures: refer to the cell when detected at any phase of mitosis. It can be seen normally in growing tissue, or abnormally in malignant tumors. Meiosis - Definition: It is a special type of cell division in which the diploid mother cells in the testis & ovary undergo 2 successive divisions to produce the haploid in germ cells (sperm & ova). - Stages: 1. First Meiotic Division (Reduction Division) - Each daughter cell has 23 d-chromosomes. 2. Second Meiotic Division (Mitotic Division) - It is mitosis – like division. - It occurs rapidly after the first division. - Each daughter cell has 23 S-chromosomes. 34  Cell Biology Prof. Dr. Amal M. Moustafa Differences between mitosis & 1st meiosis Mitosis 1st Meiosis Site - Occurs in somatic Occurs in germ cells (Ova & sperm) only cells during growth during sexual reproduction. Prophase - or Norepair. Crossing over. Crossing over & exchange of genetic information take place ( ) homologous pairs of 23 d-chromosomes (bivalent). Metaphase - 46-d chromosomes align the homologous pairs of 23 d- on the equatorial plane. chromosomes (bivalent) align on either side of the equatorial plane (middle line). Anaphase - 46-d chromosomes split The spindle fibers contract & pull each longitudinally into 92 s- 23-d chromosomes, away from each other chromosomes, each 46 s- and toward each pole of the cell. chr. migrates to a pole. Telophase Each daughter cell is Each daughter cell is haploid and has only diploid number 23pairs of one half the total number of chromosomes s-chromosomes = (46 S- of the original cell (23 d-chromosomes). chromosomes). What is the result of 2nd meiosis (Mitotic division) in both males and females? -In males → Production of 4 viable spermatids of 23 s-chromosomes {Two (22+X) & Two (22+ Y)}. -In females → Production of 1 viable ovum of 23 s-chromosomes (22+X)/ month & 3- non-viable polar bodies. 35  Cell Biology Prof. Dr. Amal M. Moustafa Karyotyping - Def.: Karyotyping is the study of the number and morphology (shape) of chromosomes in individuals. - Def.: Karyotype is a map of paired identical chromosomes (homologues) from photomicrographs of metaphase chromosomes. Indication of Karyotyping 1. karyotyping is used to screen parents before marriage or before they conceive. 2. Infants or children with congenital anomalies which suggest to have chromosomal abnormality to be diagnosed. 3. Infertility: women with Turner syndrome or a man with one of the variants of Klinefelter’s are infertile. 4. Recurrent miscarriages: such as trisomy 16, are the cause of at least 50% of miscarriages. 5. Stillbirth: A karyotype is done as part of the testing following a stillbirth. 6. Leukaemia: Karyotype help diagnose of leukaemia e.g., the Philadelphia chromosome found in some people with chronic myeloid leukaemia or acute lymphocytic leukaemia. 36  Cell Biology Prof. Dr. Amal M. Moustafa Technique of Karyotyping: 1. A sample of venous blood is obtained from the examined person. Heparin is added to prevent blood clotting. 2. Blood is centrifuged to separate the white blood cells (Lymphocytes) taken to represent somatic cells. 3. The cells are cultured in a medium contain phytohaemagglutinin. It acts a mitogen stimulating lymphocytes to divide). 4. The cultured cells are incubated for about three days to obtain a suitable number of cells. 5. The process of mitosis (cell division) is arrested at metaphase by adding colchicine (inhibit mitotic spindle formation at metaphase). 6. The cultured cells are put in a hypotonic medium that makes the cell swell and separate the chromosomes from one another. 7. Samples from the cultured cells are spread on glass slides. 8. Cells are flattened and broken between the glass slide and a coverslip. 9. The chromosomes are fixed and stained by Giemsa stain. 10. The individual chromosomes on the Photograph are cut, matched into identical pairs, and mounted on a hard paper. The chromosomes are then counted and individually studied. This step is now easily done by the computer, using an image analyser system. 11. In the end, the final karyotype arranged according to banding pattern, number, length, shape, position of centromere of the chromosomes. -The karyotype shows the total number of chromosomes, the sex, and any structural abnormalities with individual chromosomes. 37  Cell Biology Prof. Dr. Amal M. Moustafa Human Chromosomes & Cytogenetics - Genome: is the collection of all human genetic information located on 46 chromosomes in the nucleus of a human cell (Human genes ↑ 25000 genes). - Gene: is the basic unit of genetic information and formed of a specific sequence and length in DNA threads. It occupies a fixed position (locus) on a chromosome. Genes achieve their effects by directing the synthesis of proteins. - Locus: location of a gene on the chromosome. - Allele: one variant form of a gene at a particular locus (Aa, Bb,……….). - DNA: is a nucleic acid that contains the code of genetic information specifying the biological development and determine the inherited characters of all cellular forms of life. DNA forms the Chromosomes as double helix around a core of histone. - Chromosomes (DNA+Protein): storage units of genes. During the process of mitosis, each chromosome is formed of a pair of two exactly identical chromatids joined at the central point of each chromatid, called the centromere. Function of chromosomes: - a. Carries the genetic information. - b. Directs protein synthesis by formation of three types of RNA (ribosomal rRNA, Messenger mRNA, Transfer tRNA) 38  Cell Biology Prof. Dr. Amal M. Moustafa - Human somatic cells contains 46 chromosomes. Half of this chromosome number 23 comes from the mother ovum & the other half 23 comes from father sperm. - The nucleus of somatic cells contains - 44 chromosomes are called autosomes - 2 chromosomes are called sex chromosomes. - Human ovum usually contains one sex chromosome (X chromosome). - Human sperm may contains either X or Y chromosome. - → a Female with 44 autosomes and 2X chromosomes in each somatic cell (44, XX). - → a Male with 44 autosomes and XY sex chromosomes in each somatic cell (44, XY). - The normal human female karyotype →46, XX {Ovum 22X + sperm 22X} - The normal human male karyotype → 46, XY {Ovum 22X + Sperm 22Y}. 39  Cell Biology Prof. Dr. Amal M. Moustafa Rules of inheritance Genotypes: At each locus (except for sex chromosomes) there are 2 genes. These constitute the individual’s genotype at the locus (Aa, Bb,……….). Phenotypes: The expression of a genotype is termed a phenotype. For example, hair & skin color, weight, or the presence or absence of a disease. Dominant gene: A dominant allele is expressed even if it is paired with a recessive allele (AA, Aa). Recessive gene: A recessive allele is only visible when paired with another recessive allele (aa). X-linked Inheritance: The gene responsible for certain traits is located on X-chromosome. Females have 2 X-chromosomes, while males have 1 X-chromosome and 1 Y- chromosome. General patterns of inheritance 1. Autosomal recessive The disease appears in male and female children of unaffected parents, e.g. albinism, Sickle cell anemia & cystic fibrosis. 2. Autosomal dominant Affected males and females appear in each generation of the pedigree. Affected mothers and fathers transmit the phenotype to both sons and daughters, e.g., brown eye, Polydactyly & Huntington disease. 40  Cell Biology Prof. Dr. Amal M. Moustafa 3. X--linked recessive Many more males than females show the disorder. All the daughters of an affected male are “carriers”. None of the sons of an affected male show the disorder or are carriers, e.g. hemophilia. 4. X-linked dominant Affected males pass the disorder to all daughters but to none of their sons. Affected heterozygous females married to unaffected males pass the condition to half their sons and daughters, e.g. fragile X syndrome. 5. Codominant inheritance Two different versions (alleles) of a gene can be expressed, and each version makes a slightly different protein. Both alleles influence the genetic trait or determine the characteristics of the genetic condition, e.g. ABO system. 6. Mitochondrial inheritance This type of inheritance applies to genes in mitochondrial DNA. Mitochondrial disorders can appear in every generation of a family and can affect both males and females, but fathers do not pass mitochondrial traits to their children, e.g. Leber's hereditary optic neuropathy (LHON). 41  Cell Biology Prof. Dr. Amal M. Moustafa Chromosomal Anomalies Definition: - A chromosomal anomaly is a deviation from the normal number or from the normal morphology (shape) of chromosomes. - Chromosomal aberrations may be: 1- Numerical Aberration: Abnormality in the number of chromosomes. 2- Structural aberration: Abnormality in the shape of chromosomes. - Both abnormalities may affect autosomes or sex chromosomes. Causes of Chromosomal anomalies: 1- Genetic diseases that run in family. 2- late maternal age pregnancy. 3- Auto-immune disorders. 4- Exposure to radiation causes chromosomal mutations or damage. 5- Exposure to Infection by viruses during pregnancy e.g., German measles. Types of chromosomal Aberrations: A. Variations in the chromosome number 1. Aneuploidy ▪ -Addition or loss of one or more chromosomes ▪ -Trisomy (2n+1), monosomy (2n-1) The main cause of aneuploidy is nondisjunction due to failure of separation of homologous chromosomes during mitosis, and to lesser extent: simple loss and failure of duplication. Examples: A. Trisomy in Autosomes 1. Trisomy 21: Down Syndrome (47, 21+) 2. Trisomy 18: Edwards Syndrome (47, 18+) 3. Trisomy 13: Patau Syndrome (47,13+) 42  Cell Biology Prof. Dr. Amal M. Moustafa B. Aneuploidy of Sex Chromosomes 1. Turner Syndrome (45, XO) 2. Klinefelter Syndrome (47, XXY) 3. Jacobs Syndrome (47, XYY) 4. Metafemale (47, XXX) 2- Polyploidy ▪ Addition of more than 2 sets of chromosomes. ▪ Triploidy (3n), tetraploidy (4n) B. Alterations in the chromosome structure 1. Deletion – loss of part of a chromosome. -Wolf syndrome; due to deletion at short arm (p) of chromosome 4 (deletion at 4p) -Cri-du-chat Syndrome; due to deletion at short arm (p) of chromosome 5 (deletion at 5p) 2. Translocation – part of a chromosome breaks off and attaches to another, non- homologous chromosome. 3. Reciprocal translocation – an exchange of fragments between two non-homologous chromosomes (reciprocal translocation). The Philadelphia Chromosome: - It means part of the long arm of chromosome 22 translocated to chromosome 9 which becomes long. - The Philadelphia chromosome (22) becomes short & causes a malignant disease in bone marrow cells called chronic myeloid leukemia. - Philadelphia Chromosome Karyotype: 46, XX, t (9; 22) 46, XY, t (9; 22) 43  Cell Biology Prof. Dr. Amal M. Moustafa 4. Duplication – segment of a chromosome is repeated due to attachment of a fragment that got cut off from one chromosome to its homologue, thus duplicating certain genes on it. 5. Inversion – part of a chromosome is oriented in the reverse of its usual direction where the fragment that got cut off from one chromosome is able to reattach to it, but in the reverse orientation. ***Important Karyotype: a. Normal female karyotype → 46, XX b. Normal male karyotype → 46, XY c. Klinefelter’s syndrome karyotype → 47, XXY d. Turner’s syndrome karyotype → 45, XO e. Down’s syndrome (Mongolism or Trisomy 21) karyotype → 47, XY+21(male Mongol) 47, XX+21(female Mongol) f. Philadelphia chromosome Karyotype: → 46, XX, t (9; 22) → 46, XY, t (9; 22) 44  Cell Biology Prof. Dr. Amal M. Moustafa Intercellular junctions “Cell- cell connections” Definition: Membrane associated structures that join adjacent cells and provide communication between them. Classification: B. Cell-Cell Junctions Types: 1- Tight (Occludens) junctions 2- Adherens junctions 3- Gap junctions 45  Cell Biology Prof. Dr. Amal M. Moustafa (1) Tight (Occludens) junctions Def. : The adjacent 2 cell membranes are tightly fused (zipper-like junctions) at certain sites. ◼ Types: 1- Zonula occludens: completely encircles the cells. ◼ e.g., GIT (intestinal cells) & Kidney cells. 2- Fascia occludens: - Patchy fusion between adjacent cell membranes e.g., blood capillary. Function: It plays a key role in tight sealing of the cells together that prevents intercellular transport of fluid e.g., blood capillary, intestinal & Kidney cells. (2) Adherent junctions Def. : They are intercellular junctions that provide strong intercellular binding material (cadherins adhesion) between two adjacent cells. ◼ Types: 1-Zonula adherens (Belt Desmosome): ◼ Structure: ❖ The intercellular space filled with binding substance (cadherin) ❖ Inside the cell, actin filaments join up the adhesion junctions. ◼ Site: In epithelial cells, especially in the skin & the GIT canal usually appear below tight junctions. 46  Cell Biology Prof. Dr. Amal M. Moustafa 2-Desmosomes (Macula adherens): ◼ Def: They are button-like spots adhering to neighboring cells with cadherin proteins & intermediate filaments. ◼ Structure: by EM it is formed of: - intercellular space of about 20 nm filled with an electron dense binding cadherin proteins. - Two cell membranes on either side. -Thickened electron dense plaques on the cytoplasmic sides of the cell membranes. -Intermediate filaments (tonofilaments) inserted in each plaque and loop back again into cytoplasm. ◼ Function & Sites: They hold & prevent separation of the epithelial cells e.g., between skin keratinocytes and between the cardiac muscle fibers. 3- Hemidesmosomes: - Def.: They are half desmosome. - Function: They join the base of epithelial cells to the basement membrane. Hemidesmosome Desmosome 3- Gap junction (Nexus) Def. : It is formed of cylindrical structures consisting of integral proteins of the cell membranes that are arranged side by side in the junction. It can open and close in response to changes in calcium levels, and PH. Function & Sites 1. Permits direct passage of ions & nutrition between the cells e.g., between osteocytes. 2. Permits the wave of depolarization to spread in the proper sequence e.g., between the cardiac muscle’s fibers & smooth muscles of intestine. 47  Cell Biology Prof. Dr. Amal M. Moustafa Stem cells Definition: - Stem cells (SC) are those cells which retain the ability to renew themselves through repeated mitotic cell division for an indefinite period of time and can differentiate into a wide variety of specialized cell types with different functions (developmental plasticity). Stem cell properties: a) Self-renewal: the ability to go through mitotic cell division while maintaining numerous cycles of the undifferentiated state. b) Potency: the capacity to differentiate into specialized cell types with different functions. Sources of stem cells 1- Embryonic stem cells. are found in blastocysts, in a developing embryo, stem cells can differentiate into all the specialized embryonic tissues e.g. Excess fertilized eggs from In vitro fertilization (IVF) clinics. 2- Adult stem cells are a few cells found in some adult tissues acting as a repair system for the body cells e.g. - Pericytes sc. -Bone marrow (Hematopoietic) sc. -Adipose tissue sc. - Epithelial sc. -Skin sc. -Intestinal sc. – skeletal muscle sc. - 48  Cell Biology Prof. Dr. Amal M. Moustafa Types of stem cells: 1. Totipotent stem cells are produced from the fusion of an egg & sperm cell & able to differentiate to all embryonic & extraembryonic cell types including placenta e.g., only the morula's cells are totipotent cells. 2. Pluripotent stem cells are the descendants of totipotent cells. - originate as inner mass cells within a blastocyst & able to become any tissue in the body except the placenta. 3. Multipotent stem cells can produce only cells of a closely related family of cells as haemopoietic undifferentiated mesenchymal stem cells (UMC). They differentiate into red blood cells, white blood cells and platelets. These are adult stem cells that persist in the corresponding organ for a varying period. As well as pericytes act as multipotent stem cells. Unipotent stem cells can produce only one cell type, e.g. Satellite cells (muscle stem cells). Function of stem cells: 1. Gives rise to all tissue cells. 2. Responsible for normal growth. 3. Responsible for normal regeneration & repair. 4. Stem cell therapy or regenerative medicine: - stem cells have a great role in the new medical therapies for replacing the damaged cells or tissues resulting from a variety of diseases or injuries. 49 Cell Biology Prof. Dr. Amal M. Moustafa 50 Cell Biology Prof. Dr. Amal M. Moustafa 51 Cell Biology Prof. Dr. Amal M. Moustafa 52 Cell Biology Prof. Dr. Amal M. Moustafa 53 Cell Biology Prof. Dr. Amal M. Moustafa 54 Cell Biology Prof. Dr. Amal M. Moustafa 55