2A Cell Biology PDF

02/08/2019 Cell Biology Cell is the smallest enclosed chemical system of thermodynamic nature, which forms the structural, functional and reproductive unit of life. Schleiden and Schwann Cell is the unit of structure, ph...

02/08/2019 Cell Biology Cell is the smallest enclosed chemical system of thermodynamic nature, which forms the structural, functional and reproductive unit of life. Schleiden and Schwann Cell is the unit of structure, physiology and organisation. Cell retains a dual existence as a distinct entity and building block in the construction of organisms. Cells form by spontaneous generation. (Later modified by Rudolf Virchow: Cells arise from pre- existing cells) Modern Cell Theory All living things are made up of cells Cell is the fundamental structural and functional unit Some are unicellular, some are multicellular All cells arise by division of pre-existing cells Cells contain hereditary information Cells are similar in chemical composition Energy flow occurs within cells Activity of organism depends on combined activity of independent cells Exception Viruses are considered living entities because they show biological activity, but they are not composed of cells The first cell did not originate from a pre-existing cell. Extra Cellular Matrix Jelly of Proteins and Polysaccharides Cells themselves produce and secrete the ECM Carbohydrate rich transmembrane molecules straddle the outer PM and enable adjacent cells to stick to one another ECM cushions and lubricates cells Chemicals circulate in ECM in the space between the trans membrane proteins — recognised by membrane associated receptors Collagen is a major component CAMs bind cell to cell & cell to ECM Gap junction: Structurally different but functionally similar, Connect cytosol of adjacent cells, Exchange of nutrients and signals PLASMA MEMBRANE 10 nm thick, lipid bilayer Elastic, Living, Dynamic Contains Amphipathic Lipids, Proteins, trace amounts of conjugated carbohydrates Physically separates cytoplasm from extra cellular environment file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 1/53 02/08/2019 Cell Biology Structure Lipids and Proteins in almost equal ratio. Carbohydrates are also found linked to proteins and lipids Assemblies are mostly non-covalent, but certain proteins are covalently attached to membrane lipids (e.g. Prenyl anchored protein) Not homogeneous, composition is dynamic: depends on functional characteristics on the cell Lipids are amphipathic (both hydrophilic and hydrophobic parts) — due to this they spontaneously form bimolecular sheets Specific proteins mediate distinctive functions of PM: pumps, channels, receptors, enzymes, signal transducers Lipid and protein molecules can diffuse from one location to another. Electrically polarised — Inside is negative -60 mV — Membrane Potential plays a key role in transport, energy conversion and excitability Relative proportion of proteins and lipids in any membrane depends on the physiological role played by the membrane. Proteins predominate in membranes that have an active metabolic role. Lipids predominate in membranes that have a primarily enveloping role Chemical composition Amphipathic Lipids — both polar and non-polar parts Proteins — Almost always globular Carbohydrates — Covalently attached to membrane lipid or protein Lipids Glycerophospholipids Phosphatidyl choline, Phosphatidyl inositol, Phosphatidyl glycerol 3 carbon glycerol backbone, 2 hydrocarbon fatty acid chains Due to presence of strongly polar phosphate group, and strongly non-polar fatty acid group, glycerophospholipids spontaneously form a bilayer in aqueous medium Fatty acid chains are mostly unbranched, and contain an even number of carbon atoms (C16 Palmitate, C18 Stearate) Presence of double bonds interferes with compact packing, as every double bond introduces a 45° bend, which enhances fluidity Sphingolipids Sphingomyelin is most common. Mainly found in the Myelin Sheath Phosphorylated head group, Sphingosine backbone Glycophingolipid: Sugar residue instead of the phosphorylated head group — Cerebrosides, Galactosides Sterols Absent in prokaryotes Fused ring structure — more rigid than other membrane lipids. Play an important role in stabilising membrane fluidity Membrane Raft: Sphingolipid associated with sterols in the PM that make a dense and floating lipid aggregate. file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 2/53 02/08/2019 Cell Biology Almost all proteins in the PM are globular Classification is on the basis of relative position and association with membrane lipids Integral Proteins: Intrinsic association, Associate with inner membrane, Mostly glycosylated, can only be separated by harsh enzymatic and detergent treatment External Proteins: Extrinsic association, Mostly bound to outer membrane, Weak association mostly by electrostatic forces Functional Basis Structural Proteins: Cytoskeleton anchoring proteins Enzymes like Adenylyl Cyclase Transport Proteins: Na/K pump Proteins Signal Proteins: Notch Signal Receptors: GPCR Coat Proteins: Clathrin/Caveolin Embedded antibodies Cell to Cell attachment proteins Present only in PM and ER membrane Short sugar chains (branched/unbranched) Carbohydrates Attached to exterior ectoproteins (glycoprotein) or to polar ends of phospholipids (glycolipids) Fluid Mosaic Model — Singer and Nicholson (1972) — For all bio-membranes Membrane as a structure in which a mosaic of proteins is discontinuously embedded in or attached to a fluid lipid bilayer Membrane proteins were considered discrete globular entities that associate with the membrane on the basis of their affinity for the hydrophobic interior file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 3/53 02/08/2019 Cell Biology Lipid bilayer is thermodynamically most stable when amphipathic lipids are subjected to an aqueous medium. A bilayer exposes polar hydrophilic parts to the aqueous exterior while protecting non-polar tail from water. Glycerophospholipids, being cylindrical, form bilayers most spontaneously Most of the lipid components of a membrane are in constant motion. Lipid Molecules readily exchange places with neighbour molecules within a monolayer Identifies two kinds of proteins based on nature of protein linkage to the lipid bilayer Integral Membrane Proteins: Cytosolic and exoplasmic domains have hydrophilic exterior surfaces. Membrane spanning domain contains many hydrophobic AA. Most transmembrane proteins are glycosylated. Peripheral Proteins: Almost entirely constituted by hydrophilic domains. Localised to cytosolic or ectoplasmic face. Bound to membrane indirectly by interactions with integral membrane proteins Lipid Anchored membrane protein: Bound covalently to one or more lipid molecules. Hydrophobic carbon chain of the lipid anchors the protein. Polypeptide chain itself does not enter the membrane. Regulation of membrane fluidity Unsaturated fats Double bonds increase fluidity of a lipid bilayer. Each double bond introduces a 45° bend, which interferes with compact packing. Cells of organisms living at low temperatures, have high proportions of unsaturated fatty acids. Certain membrane transport processes and enzyme activities cease when the viscosity of the lipid layer is too high If the lipid bilayer becomes too fluid, receptors for certain hormones are withdrawn, which hampers hormone action Cold acclimation in plants involves increase in ratio of phospholipids to sterols (mostly unsaturated fatty acids like Linoleic acid). Higher fluidity of lipid bilayer lowers the freezing point. PM can function at a lower temperature through this adaptation Cholestrol Cholestrol molecules orient themselves in the lipid bilayer in such a way that their hydroxyl groups remain close to the polar head groups of the phospholipids. Inhibits phase transition by preventing hydrocarbon chains from coming together and crystallising. Decreases permeability of lipid bilayers to small water soluble molecules. Enhance flexibility and mechanical stability of bilayer. Mobility of membrane molecules Lipids bound to integral membrane proteins have reduced mobility. (e.g. Lipids surrounding Cytochrome Oxidase) Some proteins are constrained by protein-protein interactions to form specialised structures Attachment to ectoplasmic skeleton Peripheral proteins may form a bridge like lattice between integral proteins and restrict their lateral mobility Functions Permeability Barrier: Regulates passage of material between cell and surrounding In nerve cells, PM is involved in intercellular communication Microvilli: Portions of PM are modified to form finger like projections called Villi. Greatly increase the surface area of the cell and provide for increased passage of materials across the PM Junctions: Tight Junctions, Gap Junctions, Desmosomes In plants, it forms a scaffold for assembly of cell wall. Functions of PM proteins Transport proteins help to maintain ionic concentration and pH Junction between cells for cell-cell communication in a tissue file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 4/53 02/08/2019 Cell Biology Anchor points for cytoskeleton and for cell to attach to ECM MEMBRANE TRANSPORT Ions, Glucose, AA and Lipids enter the cell Metabolic Intermediates remain in the cell Waste compounds leave the cell Significance Maintain solute concentrations in the cytosol and membrane enclosed compartments in the cell Ingest essential nutrients Excrete metabolic wastes Regulate intracellular ionic concentrations Entry/Exit of signalling molecules Passive Transport Active Transport Consumes biological free energy No consumption of biological free energy Powered by ATP Hydrolysis or Proton Powered by electrochemical gradient Motive Force With or without transport proteins Almost always with aid of transport proteins Integral Proteins Pumps: Enzymes that utilise energy from ATP/light to move ions/solutes across membranes at relatively modest rates. Transporters: Provide pathways for solutes to move across membranes down their concentration gradients. Conformational change upon binding of solutes on one side of membrane transports the solutes to the other side. Ion gradients can be used as a source of energy for functions such as ATP synthesis. Translocation of an ion down its concentration gradient can drive another ion or solute up the gradient. Channels: Open and close transiently in a regulated manner. When a channel is open, flood of ions passes through the channel driven by electrical and concentration gradients. e.g. Nerves, Muscles PASSIVE TRANSPORT Uses KE of solutes in motion to drive the transport Affected by Permeability of cell membrane Size of substrate (smaller size, faster transport) Concentration/Electrochemical gradient (higher the gradient, faster the transport) Can be simple or facilitated Simple Diffusion Facilitated Diffusion file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 5/53 02/08/2019 Cell Biology Diffusion without the aid of any transport Molecules diffuse across membranes with protein the assistance of transport proteins. Diffusion occurs till the gradient is eliminated Mostly for hydrophobic nature of lipids (higher to lower free energy, till equilibrium is attained) Water flux in cell mostly by simple diffusion Carriers/Transporters Exchange of O2 and CO2 during respiration Bind a specific solute — Conformational Conditions Change — Solute passes to the other side. GLUT: Glucose transporter Substrate must be small — has to pass Band 3: Transport of bicarbonate and through lipid bilayer chloride ions Non Polar, Uncharged Transporters are solute specific and only Lipid Soluble transfer certain solutes Large general diffusion area Limits to number of solute molecules they Diffusion distance should not be too long can carry. Binding of certain molecules may inhibit their functioning. Advantages No saturation effect or toxin sensitivity as it does not depend on any protein Uniport: Only one substrate at a time Energetically favourable Co-transport: Two solutes are transported Entire cell surface acts as a diffusion surface simultaneously. Transport is coupled so that transport of one depends on the availability of the other. Disadvantages Symport: in the same direction Antiport: in opposite directions Slow and non-specific Cannot transport substances that are polar, charged or large Concentration gradient may at times be harmful (desiccation in hypertonic solutions) Facilitated Diffusion Ion Channels Hydrophilic pores through which certain types of solutes can pass through. Channels are specific for the type of solute they will transfer. Most allow passage of only one kind of ion. (Na+, K+, Cl-) Specificity is ensured through Ion Specific binding sites and Size Filter. Opening and closed by conformational changes in the protein. Voltage Gated Channels: Open and close in response to change in membrane potential. file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 6/53 02/08/2019 Cell Biology Ligand Gated Channels: Triggered by binding of specific substances to the channel protein. e.g. Acetyl Choline, GABA, cAMP, cGMP Mechanosensitive Channels: Respond to mechanical forces that act on membrane. e.g. sound waves bending cilia-like projections on the hair cells of the inner ear. Pores Larger than ion channels and less specific Transmembrane segments are closed cylindrical ß sheet called ß barrel. e.g. ß barrel has a water filled pore at its centre. Aquaporins Water specific channels Transfer of water and sometimes small solutes such as glycerol Completely impermeable to charged species Play a key role in water excretion by kidneys Vasopressin regulates aquaporin When vasopressin is expressed, duct cells bind more Vasopressin Expression of aquaporins on PM, increases water permeability of cells Allow increased return of water from nascent urine to blood. ACTIVE TRANSPORT Uses specific transport proteins called pumps Energy mostly comes from ATP hydrolysis but may also be derived from light or redox reactions Sodium Potassium Pump Cation Exchange pump driven by energy of one ATP molecule Exchange of 3 Na+ ions outside the cell, in exchange for import of 2 K+ into the cell. Dimer with two subunits — Concentration of Na+ is higher in blood, Concentration of K+ is higher inside the cell. file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 7/53 02/08/2019 Cell Biology ABC Transporters (ATP Binding Cassette) Transmembrane proteins that expose a ligand binding domain at one surface and a ATP binding domain on the other Ligand binding is restricted to a specific type of molecule ATP provides the energy to pump the ligand across the membrane ATP binding domains of ABC transporters are remarkably conserved across Archaea, Eubacteria, Plants, Animals CFTR: Cystic Fibrosis Trans Membrane Conductance Regulator TAP: Transporter associated with antigen processing. Secondary Active Transport ATP Hydrolysis is used to create a gradient The gradient then drives the passive flux of the substrate to be transported. e.g. S-GLUT Initially pump opens to the outside Na+ binds. This stimulates Glucose binding Conformational change — drives Na+ and Glucose inside Na+ dissociation causes Glucose to dissociate Conformation returns to original state Na+ is later pumped out in Na/K pump CYTOSKELETON Cellular Scaffolding within the Cytoplasm (found in prokaryotes as well) Maintains cell shape Imparts mechanical properties to cell Cellular Motion — Cilia, Flagella, Lamellopodia Intra Cellular transport Cell Division Microfilaments Intermediate Filaments Microtubules 7 nm, two inter twined 8-12 mm 25 nm actin chains More stable than actin 13 protofilaments which Resist tension and filaments are polymers of α and ß maintain cellular shape Maintain cell shape by tubulin Concentrated just beneath bearing tension Centrosome is formed the cellular surface from microtubules — file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 8/53 02/08/2019 Cell Biology Cytoplasmic Organise the internal 3D Centrioles are 9 triplet protuberances like structure sets Pseudopodia and Anchor organelles Cilia and Flagella: 9 Microvilli Cell-Cell and Cell-Matrix doublets oriented around Cell-Cell/Cell-Matrix junction 2 MTs. Each doublet is junctions: Role in signal Vimetins: Common structural connected to another by transduction support of many cells dyenein Important for cytokinesis Keratin: Skin cells, hair, Movement of organelles Actin + Myosin: nails Axoneme of Cilia and Muscular contraction, Neurofilaments: Nerve Cells Flagella Cytoplasmic streaming Lamin: Structural support to Synthesis of Cell Wall in nuclear envelope plants Mitotic Spindle ENDOPLASMIC RETICULUM Endomembrane system: ER + Golgi + Nuclear Envelope Secretory Pathway: ER — Transition Vesicles — GA — Discharge Vesicles — Exterior Physically continuous with Nuclear Envelope Cells which have a predominant transport function, like RBCs, do not have ER Cells active in secretory functions, such as activated B lymphocytes, have very well developed and highly branched ER system. Structure Cistern: Flattened vesicles having a large surface area, Branched and inter connected, Enclose a lumen which is in continuity with lumen of nuclear envelope. Tubules: Elongated extensions from margins of cistern, Mostly smooth, branched and interconnected Vesicles: Surround ER system near margins, Mostly smooth Biochemical Properties If a tissue or cell is disrupted by homogenisation, ER breaks into fragments and reseals into small closed vesicles called microsomes. Lumen of ER is an oxidising environment Contains enzymes for protein glycosylation, lipid modification, cholesterol biosynthesis file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 9/53 02/08/2019 Cell Biology Membrane of ER is a lipid bilayer with integral proteins such as Ribophorins, Signal Peptidase, SNARE Post Translational Modifications in ER Importance: Correct 3D folding, enhances stability, cell-cell recognition, aids in targeting Glycosylation, subunit assembly, protein folding, disulphide formation In cytosol, cysteine residues are in reduced state. Oxidising conditions in the ER lumen provide for S-S bond formation (aided by Protein Disulphide Polymerase) Alpha helices are formed in RER Rough ER Smooth ER Synthesis of Lipids. Lipids are hydrophobic and are synthesised in association with membranes, rather than in the aqueous environment of the cytosol. Contains enzymes to make cholesterol and modify it into steroid hormones. Production of enzymes to detoxify lipid soluble drugs. The enzymes catalyse breakdown into water Studded with sites for protein soluble drugs, which then leave the cell and can be manufacturing ribosomes excreted in urine. Ribosomes bind to the ER once it Regulation of Calcium concentration — store calcium begins to synthesise a protein ions in muscle cells. SER has a high concentration of Produces lysosomal enzymes with a Calcium binding proteins. Mannose 6 Phosphate marker added Converts Glucose 6 Phosphate to Glucose (Step in in cis-Golgi network. Gluconeogenesis) Secretion Proteins Form the exit sites from where TV carrying nascent Constitutively with no tag polypeptides bud off for transport to GA Regulated secretion involving Allows increased surface area for action or storage of Cathrin and paired basic amino key enzymes acids Production site for Lipoproteins in Hepatocytes. Initial modification of proteins in the Enzymes that make the lipid component of Lumen. e.g. Glycosylation. lipoprotein are located in membrane of SER T Tubules: In striated muscle, ER is specially adapted to surround the myofibrils forming triads with invaginations of the plasma membrane called T Tubules. In conjunction with Calcium ion concentration modulation, it triggers contraction and relaxation. Continuous with the NE, followed by the GA Dispersed throughout the Cytosol More in number in cells actively More in number in cells involved in lipid synthesis involved in protein synthesis No ribosomes Ribosomes are present on the surface Tubule like appearance Stack like appearance GOLGI APPARATUS file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 10/53 02/08/2019 Cell Biology Most prominent in cells with a significant secretory function. e.g. Plasma B cells that secrete antibodies Flat sacs and Cisterns stacked parallel in a protein matrix (5-8 sacs) Cis Golgi Network, Cis Golgi, Medial Golgi, Trans Golgi, Trans Golgi Network — Located between ER and PM Vesicles from ER fuse with cis-Golgi network and progress through the stacks to trans Golgi network At trans Golgi network, they are packaged and sent to the required destination Each region contains different enzymes, which selectively modify the contents. Primary function — Process proteins targeted to the ectoplasm, PM, lysosomes or endosomes Protein delivery system: Processes proteins and sorts them within coated discharge vesicles Most transport vesicles that leave the rough ER, are transported to the Golgi apparatus Final processing and sorting centre for all those cellular proteins that are being targeted by the secretory pathways. GA processes them and sorts them within coated discharge vesicles Localisation of Golgi depends on microtubules. If microtubules are experimentally depolymerised, GA reorganises into individuals stacks, that are found throughout the cytoplasm. Disassembly Architecture of Golgi apparatus depends on microtubule cytoskeleton and cytoplasmic Golgi matrix proteins, which form a scaffold between the cisterns that gives GA its integrity Matrix proteins form long filaments that tether transport vesicles close to GA During cell division When cell starts to divide, protein kinases phosphorylate Golgi matrix proteins — causes Golgi apparatus to fragment and disperse in the Cytosol. Golgi enzymes are returned to ER in vesicles Golgi fragments are distributed between the two daughter cells where they are dephosphorylated, leading to reassembly of Golgi apparatus Organisation of Golgi Vesicle Transport Model Golgi cistern are static organelles, which contain an array of enzymes. Passing of molecules through Golgi is achieved by forward moving transport vesicles — bud from one cistern and fuse with the next in a cis to trans manner. Cisternal Maturation Model Each Golgi cistern matures as it moves outwards through a stack. At each stage, Golgi proteins that are carried forward in a cistern, are moved back to an earlier (younger, to the cis) compartment in COPI coated vesicles. Each cistern receives enzymes from the one ahead of it. file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 11/53 02/08/2019 Cell Biology Functions Targeting of proteins through the secretory pathway N linked carbohydrates are cited from semi processed carbohydrates. Glycosylation at O linked Ser/Thr Adds Mannose 6 Phosphate label to proteins of lysosomes Sulfonation, Adenylation, Proteolytic cleavage of zymogens in trans GA Sorts proteins into appropriate discharge vesicles Many Polysaccharides are made in Golgi. LYSOSOME Rene De Duve described both Lysosomes and Peroxisomes as 'micro bodies' due to small size. Single Membrane Filled with acid dependent hydrolytic enzymes used for intracellular digestion of macromolecules — Protease, Nuclease, Glycosidase, Lipase, Phospholipase Lipase — Digests lipids Carbohydrates — Digests carbohydrates Protease — Digests proteins Nuclease — Digests nucleic acids Phosphoric Acid mono-esters Can break down a variety of biomolecules — Proteins, Nucleic Acids, Lipids, Carbohydrates Lysosomes do not play a significant role in cell death. Accidental release of Lysosomal enzyme does not cause significant harm. Because cytosolic pH is moderately basic and lysosomal enzymes are active only at pH < 5. Hence, Lysosomes being characterised as suicide bags of the cell is unfounded. Lysosome refers to any Eukaryotic organelle if, It contains an acidic pH It contains acid hydrolase enzymes Enzymes show latency By this definition, Vacuoles (excluding storage vacuoles) are also lysosomes pH inside Lysosome H+ pump in Lysosomal membrane uses energy of ATP Hydrolysis to pump H+ into the Lysosome, maintaining an acidic pH in the lumen. Concentration of H+ inside the Lysosome = 100 x concentration outside. Cl- channel protein allows Chlorine ions to enter. This leads to formation of Hydrochloric Acid inside the Lysosome. Acidic pH helps to denature proteins, making them accessible to action of Lysosomal hydrolases. Lysosomal membrane proteins are highly glycosylated, which helps to protect them from lysosomal proteases in the Lumen. Lysosome Morphology Lysosome Morphology depends on Source cell or organism from where Lysosome has been isolated file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 12/53 02/08/2019 Cell Biology Functional status of lysosomes Primary Lysosomes: Newly formed, not yet active Secondary Lysosomes: Functional Lysosome acting on its substrate Residual Body: Lysosome which contains undigested parts of its substrate Lysosome Formation Primary Lysosome Pinches off from the trans-Golgi network. Contains Lysosomal enzymes and lysosome specific class H+ ATPase for proton pumping Primary Lysosome is not active — does not contain an active substrate to be acted upon Acid hydrolase are targeted to lysosomes by mannose-6-phosphate residues which are recognised by mannose-6-phosphate receptors in the trans Golgi network and packaged into Cathrin coated vesicles. Following removal of Cathrin, these transport vesicles act as primary lysosome. Active Lysosome: Primary lysosome fuses with a vesicle containing the substrate. Primary Lysosome + Endosome: Fuses with endosome which contains substances taken up by endocytosis, in Cathrin coated endocytotic vesicles. Membrane components are recycled to PM and as the endoscope matures, internal pH lowers to 5.5 which plays a role in activity of lysosomal acid hydrolases. Primary Lysosome + Phagosome: Specialised cells such as macrophages take up and degrade large particles, including bacteria, cell debris and aged cells. Lysosome of this type are large and heterogeneous, depending on the content of material engulfed. Primary Lysosome + Autophagosome: Turnover of cell’s own components. Organelle is enclosed in a membrane derived from ER, which fuses with the primary lysosome. Melanosome: Lysosome which contain melanin pigment produced by melanocytes. By exocytosis, they release the pigment into extra cellular space. This is taken up by keratinocytes leading to colour of the skin. When release from melanosomes is blocked, it leads to albinism. Functions Endocytosis — digested by lysosome and useful components are released into the cytosol Phagocytosis — Foreign material (potentially harmful) is digested by Lysosomes and the debris is excreted. file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 13/53 02/08/2019 Cell Biology Autophagocytosis — Damaged organelles, proteins, excessive enzymes are degraded and usable substances such as sugars, AA and nucleotides are released back into the Cytosol. Apoptotic Bodies — In late stages of Apoptosis, Lysosomes clear cellular debris. After apoptosis, degraded components are packed into apoptotic bodies which are engulfed by neighbouring cells. Lysosomes of these cells bring about the final digestion. Sperm — Lysosomal content of sperm helps in fertilising the egg by degrading the outer layer of egg. Once the sperm and egg fuse, paternal mitochondria are degraded by lysosomes of the egg cell. Sperm derived mitochondria tend to accumulate genetic mutations due to high metabolic activity of sperms. Cell membrane repair — Lysosome fuses with cell membrane at a location close to the damaged patch. Sphingomyelinase degrades the damaged membrane lipids. Fusion of Lysosomes with the membrane provides extra lipids which prevents constriction of the membrane. Digest food particles (Endocytosis), Engulf virus/bacteria (Phagocytosis), consume damaged organelles (Autophagy) Helps to kill cells that are no longer wanted. e.g. tail of tadpole. Defects Tay Sachs Disease: Gangliosides cannot be broken down. Nerve cells are greatly enlarged with swollen lipid filled Lysosomes. Hunter’s Disease: Defect in enzyme for breakdown of Glycosaminoglycans Gaucher's Disease: Glycolipids cannot be broken down due to mutation in a gene that encodes a lysosomal enzyme Inclusion Cell Disease: lack of mannose-6-phosphate leads to failure of lysosomal enzyme targetting PEROXISOMES Discovered by Christian De Duve as micro bodies. Microbodies that participate in metabolism of fatty acids and other metabolites Contain enzymes that rid the cell of peroxides Single lipid bilayer membrane Catalase and Oxidase enzymes at very high concentrations form a crystalline core. This crystalline core can be detected using electron microscopy. Digestion by peroxisomes produces peroxides which is corrosive. Catalase degrades peroxides into oxygen and water Contain membrane proteins critical for importing proteins into the organelles and aiding in proliferation Generally self-replicate by enlarging and then dividing. Transport: For maintenance , Peroxisomes import all their proteins from the Cytosol, because they do not have a genome. Short signal sequence of 3 AA directs the import for proteins into Peroxisomes. It involves soluble receptor proteins in Cytosol and Peroxisomal membrane proteins called Peroxins Zellweger syndrome: Mutation in gene encoding for Peroxin (integral peroxisomal membrane protein) — leads to defects in importing proteins. Cells end up with empty Peroxisomes leading to defects in brain, liver and kidney. Functions Oxidation of Fatty Acids file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 14/53 02/08/2019 Cell Biology Peroxisomal oxidation of Fatty Acids yields Acetyl groups, which can act as a precursor in biosynthetic and metabolic pathways (ß oxidation) Acetyl groups are transported into Cytosol, where they are used in synthesis of Cholesterol and other metabolites. Adrenoleukodystrophy: ADL gene codes for a receptor that transports into the peroxisomes, the enzymes needed for oxidation of very long chain fatty acids. Due to this fatty acids cannot be converted to Acetyl CoA In liver and kidney cells, toxic molecules enter peroxisomes and are degraded producing harmless products. Formaldehyde, Alcohol etc. are reduced to form non toxic products using peroxides. Glyoxisomes: Organelles in plant seeds that oxidise stored lipids as a source of carbon and energy for growth. Contain enzymes that convert fatty acids to glucose. Some theories suggest that Peroxisomes were probably the first organelles involved in Oxygen utilisation. Catalyse the first reactions in formation of Plasmalogens (most abundant phospholipids in myelin). Peroxisomal disorders thus lead to neurological disorders. MITOCHONDRIA Double membrane enclosed thread or rod shaped cytosolic organelle Supply cellular energy as ATP through oxidative breakdown of organic substrates. Role in: Signalling, cellular differentiation, cell death and control of cell cycle Mitochondria are variable in their shape and appearance Distribution and number of mitochondria depend on kinds of functions a cell performs. For cells that need more energy, large number of mitochondria with many Cristae are found (Pseudopodia of Amoeba, between muscle fibres, inner segment of rod and cone cells of retina) Structure of Mitochondria Two membranes, each composed of a lipid bilayer. They have distinct physicochemical properties, which decides the biochemical function. The membranes are fluid mosaic in ultrastructure. Outer membrane Freely permeable to small molecules file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 15/53 02/08/2019 Cell Biology Protein to Phospholipid ratio similar to eukaryotic Plasma Membrane. Proteins like Translocons and Porins Porins — allow molecules 5000 Daltons or less in molecular weight to freely diffuse from one side of the membrane to the other. Porins form aqueous channels through the lipid bilayer Inter membrane space Space between outer membrane and inner membrane. Freely permeable outer membrane means the concentration of small molecules in cytosol and inter membrane space is in equilibrium Inner membrane Very high protein to phospholipid ratio. Contain 1/5 of total protein in the mitochondria Has many folds called Cristae — hence, has a much larger surface area which is ideal for anchoring proteins and as a site for reactions Much less permeable — electrical insulator and chemical barrier Proteins perform redox reactions of oxidative phosphorylation ATP synthase generates ATP in the matrix Facilitates generation of a proton gradient, being impermeable to H+ ions 4 Electron Transport Chain Complexes, used by ATP synthase to form ATP from ADP and Pi NADH Q Oxido Reductase: NADH to Quinone Succinate Q Reductase: FADH2 to Quinone Q Cytochrome Oxido Reductase: Quinone to Cyt C Cytochrome Oxidase: Cyt C to O2 Specific transport proteins regulate metabolite passage into and out of the matrix Proteins import machinery Transporters in the inner mitochondrial membrane help to integrate mitochondrial and cytosolic metabolic pathways Matrix Space enclosed by the PM 2/3 of total protein in a mitochondrion Important in production of ATP using ATP synthase of the inner membrane Oxidation of pyruvate, Citric Acid Cycle Enzymes, Ribosome, tRNA, mt genome NUCLEUS Visible only during interphase of cell cycle Contains genomic material and serves as site of initial stages of gene expression Shape is mostly spherical or oblong which minimises the surface area required to enclose a specific volume Nucleus is larger in cells that have less need for cytoplasmic functions. Cells specialised for transport (RBCs) have no nucleus. But such cells cannot divide Presence of Nucleus differentiates Eukaryotic cell from Prokaryotic cell Nuclear Envelope = Membranes + Lamina + NPC Two concentric membranes that surround the nucleus and its underlying intermediate filament lattice, called Nuclear Lamina Nuclear Lamin Intermediate filament lattice around nucleus, under the inner NM, which provides structural support. NPCs are anchored to Nuclear Lamin. Association nuclear lain to inner NM is facilitated by Prenyl groups and binding to lamina associated proteins file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 16/53 02/08/2019 Cell Biology Lamina bind to chromatin through Nuclear Matrix proteins. This interaction may be important for DNA replication. Nuclear membranes are Glycophospholipid bilayers with integral membrane proteins Penetrated by Nuclear Pore Outer membrane is continuous with RER. Perinuclear space is continuous with lumen of RER Nucleolus: Distinct region of nucleus where ribosomes are produced Nuclear Matrix: Filamentous network to which chromatin attach (at Matrix Attachment regions). Enables localisation of nuclear processes Nucleoplasm Nucleus except nucleolus Contains replication and transcription machinery Slightly acidic, Contains nucleoprotamines and Histones, inorganic elements like P, K, Na, Ca Enzymes like polymerases for synthesis of DNA and RNA Nuclear Matrix: Short fibres of the size of intermediate filaments and proteins form a mesh. This enables localisation of nuclear processes. Matrix Attachment Regions (MARs): Regions where chromatin binds Constituents of Nucleoplasm Basic Proteins Nucleoprotamines: Simple proteins that remain bound to DNA by salt linkage. Common amino acid is Arginine Nucleohistones: high molecular weight proteins bound to DNA by ionic linkage. Common amino acids are Arginine, Lysine, Histidine Enzymes: DNA and RNA Polymerases, Co Factors and Co Enzymes Lipids: Very little lipid content Minerals: Inorganic elements like P, K, Na, Ca. Chromatin has more inorganic content than nucleoplasm Nuclear Pore Complex file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 17/53 02/08/2019 Cell Biology Cells which are more active transcriptionally and translationally have a greater density of NPCs NPC is a large structure about 15x the size of ribosomes. 8 fold symmetry around a large central channel. The pore is wider when the complex is active. Histones, Non Histone and Ribosomal proteins are imported. mRNA and Ribosomal subunits are exported. Structure Large Proteinaceous Structure that extends through the nuclear envelope Provides a channel for bidirectional transport between nucleus and cytosol Columnar Subunit: Bulk of the pore wall Annular Subunit: Extend towards centre of the pore Lumenal Subunit: Have transmembrane proteins that anchor the complex to the NM Cytoplasmic ring, Nuclear ring: Formed by 8 large proteins. Fibrils: On both cytoplasmic and nuclear side. On nuclear side they converge to form Nuclear fibrillar basket Advantages of having a nucleus Protects DNA from exposure to damaging agents Decoupling of transcription and translation — gene expression control both spatially and temporally Gene regulation as Heterochromatin/Euchromatin Protects DNA from damage by cytoskeleton — Dynamic cytoskeleton in stages of cell cycle create shear forces that can break the DNA Heterochromatin Euchromatin Regions of the genome that are highly condensed and are not transcribed Genome apart from heterochromatin Human Genome is 60% heterochromatin Less tightly coiled than heterochromatin Centromeric, Telometric, Clustered, Contains active genes Intercalary (between Euchromatin) e.g. Y chromosome in humans Dark Stain: +ve Heteropycnotic Light stain: -ve Heteropycnotic Compact DNA organisation DNA organisation not compact Concentrated in periphery of nucleus Concentrated in core of nucleus Proteins for gene expression cannot access Proteins for gene expression can access heterochromatin Euchromatin Gene Deserts: Low gene density High gene density file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 18/53 02/08/2019 Cell Biology Structural proteins and RNAs such as XIST Histones are the only structural proteins that represent more compact levels Many CpG islands as the gene density is No CpG islands, as it does not contain genes. higher. Rich in AT Condensation during cell division and Remains condensed throughout interphase extension during interphase Does not get acetylated Acetylated prior to gene expression May contain highly repetitive DNA Mostly single copy DNA Genetically stable, less mutations Genetically dynamic, many mutations Euchromatin and Heterochromatin differ in biophysical conformation and metabolic expression of genes, but not in their basic structure of DNA arrangement in chromosomes. Constitutive Heterochromatin Facultative Heterochromatin Permanent Heterochromatin Euchromatin that becomes compact during More common (5-10%) some stage No Euchromatinisation Less common (2.5%) Structural function — spacer between genes. Euchromatinisation occurs Generally, found as blocks in both Maybe active in one homolog and inactive in homologous chromosomes. another. e.g. X chromosome Replicate but don’t transcribe Contain actively transcribing genes Highly repetitive Mostly single copy Short term Heterochromatin Long term Heterochromatin DNA methylation after preliminary Histone De-acetylation inactivation has been carried out. Histone Methylation Molecular caging by structural RNAs such as Protein binding to Heterochromatin. e.g. XIST. XIST copies coat the second X Polycomb proteins in Drosophila chromosome, followed by Hypermethylation. Purpose: Gene silencing Histone Replacement 2A to 2A1 Significance of differential chromatinisation X chromosome silencing for dosage compensation Control temporal and spatial patterns of gene expression. Cellular differentiation in multi cellular organisms Protects parts of genome from DNA damage and mutagenesis Proper functioning of centromeres and telomeres Position Effect Variegation Variegation caused by silencing of a gene in some cells through its abnormal juxtaposition with heterochromatin via rearrangement/transposition Shows that Heterochromatin is dynamic and can spread into a region and retract from it later. State of chromatin — Euchromatin or Heterochromatin — is heritable. e.g. Mottled appearance of eye. Nucleolus Most striking and well studied component of the nucleus — can be visualised with a light microscope by applying acidophilic stains. file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 19/53 02/08/2019 Cell Biology Highly dynamic and undergo rounds of disassembly and reassembly Site of rRNA transcription and biogenesis of ribosomal subunits. Associated with Nucleolar Organiser Region Upon entry into Mitosis — Nucleolus disintegrates in Prophase and reappears in Telophase. M phase disassembly is triggered by CDK1 + Cyclin B complex Nucleolus is composed of RNA and Proteins Sub Nucleolar Domains Morphological Units Perinucleolar Compartment: RNA Fibrillar Centre: rRNA genes in tandem metabolism, rRNA processing arrays Cajal Bodies: snRNA biogenesis and Dense Fibrillar Component: Actively trafficking transcribing rRNA genes and rRNA Sam 68: RNA metabolism transcripts. SPECKLES: Centre for various RNA Granular Component: Site of late processing enzymes processing events in biogenesis of rRNAs. Paraspeckles: RNA processing + Splicing Gemini Bodies: mRNA modifications Functions of Nucleolar Proteins rRNA formation Ribosome biogenesis Genesis of translation factors Genesis of Chaperones Genesis of Cell Cycle regulators Function Storage site or reservoir for proteins that do not have roles in rRNA metabolism Nucleolar localisation of proteins is important in cell cycle regulation. Mutation of Nucleolar Protein Genes leads to disorders. CHROMOSOMES Chromosome is a discrete physical unit of genetic transmission. It is essentially a Nucleoprotein complex with particular structure and organisation Consists of Nucleic Acids + Histones Chromosomes are fundamental in hereditary transmission, mutation, selection and evolution. Chromosome number and Ploidy vary across species. Centromere — Region of chromosome to which spindle fibres attach during cell division. Appears to be constricted. Position of the constriction defines the ratio of the two chromosome arm lengths (Telocentric, Acrocentric, Metacentric) Karyogram Staining profile of a given dye for a set of chromosomes is called a Karyogram. Different dyes give distinct staining patterns Heterochromatin is generally densely stained while euchromatin is lightly stained. Positions and sizes of bands are highly chromosome specific Karyograms can be used to characterise species file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 20/53 02/08/2019 Cell Biology Centromere Normal chromosomes have a single centromere that is seen under the microscope as the primary constriction. Centromere is essential for segregation during cell division. Chromosomes without a centromere do not attach to the spindle and fail to be segregated into either of the daughter nuclei Centromeric DNA is mostly repeat units. In humans, it is called alphoid DNA (171 bp long) Centromeric Protein CEN P-A is very similar to H3. It confers special properties to the centromeric nucleosome. In dividing cells, plate like regions called kinetochores are present on the surface of the chromosome. They are attachment points for microtubules that rise from the spindle pole bodies. Region of DNA where replication begins — can be identified by enzymes that Origin of form a part of the replication machinery Replication DNA sequences that control replication are cis acting elements Proteins that control replication are trans acting Structures with DNA and protein that cap the ends of eukaryotic chromosomes. Maintain structural integrity of chromosome. In absence of telomeres, the ends would recombine in a random manner and would also degrade faster. Ensure complete replication of extreme ends of chromosomes using telomerase because there is no template for the ends. Telomeres Ends are tethered to the nuclear membrane for positioning the chromosomes Eukaryotic telomeres consist of a long array of tandem repeats. Sequences of telomeres has remained highly conserved. Synthesis of leading strand is extended using the enzyme telomerase. Telomerase is a RNA-protein complex where the RNA component carries a short sequence that acts as a template for DNA polymerase to carry out complete replication of the lagging strand Chromosome Organisation DNA — Nucleosome — Chromatin — Loop — Mitotic Condensation file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 21/53 02/08/2019 Cell Biology DNA wound around a protein core to produce a bead like structure. DNA is wrapped around a core octamer of small basic proteins called Histones. Nucleosome arrangement was discovered by Roger Kornberg. When chromatin is isolated from nucleus of a cell, treated with a very low salt concentration solution and viewed with an electron microscope, it looks like beads on a string. Histone Octamer Core Octamer contains H2A, H2B, H3, H4 — (H2A-H2B)2/(H3-H4)2 H1 + Histone Core = Chromatosome Around 160 bp of DNA is wrapped around a histone Octamer Linker DNA between two octamers is about 15 to 105 bp long. Linker length varies across species and within species according to region of genome and stage of development. Histones are Lysine-Arginine rich positively charged proteins. They combine with negatively charged DNA molecule (phosphate backbone) through Nucleosome electrostatic interactions. Histones mostly bind to the DNA in the minor groove. Histones are the most abundant protein in chromatin. Major histones are H1, H2A, H2B, H3 and H4. Minor types are variants of the major types. Non Histone Proteins Almost 1/2 of chromosomes proteins — Lamina, Nuclear Matrix Proteins, Topo Isomerase, Condensin, Make up kinetochores, Cap the ends, Molecular Motors Structural Role Scaffold proteins to which DNA is attached. When chromatin is treated with concentrated salt solution, histones and most chromosomal proteins are removed, leaving only scaffold proteins Scaffold during interphase includes Lamin and Nuclear Matrix Proteins, and during metaphase includes Topoisomerase and Condensin Functional Role Components of replication machinery and transcription machinery RNA/DNA polymerases, TFs, Acetylases Nucleosomes fold upon themselves to form chromatin Adjacent nucleosomes are condensed along the length of the linker DNA Positively charged histone tails attach to the negatively charged surface of DNA 10 nm nucleosomal fibril is spontaneously supercoiled with 6 or 7 nucleosomes Chromatin per turn to form 30nm chromatin Nuclear Matrix: Intra nuclear framework observed in the nucleus analogous to the cytoskeleton, on which chromosomes and other components of the nucleus are organised. Loop 30 nm structure folds into loops. At MARs, the loops anchor to the scaffold comprising of Nuclear Lamina and matrix proteins file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 22/53 02/08/2019 Cell Biology When gene expression is needed, these loops can decondense and RNA polymerase can carry out transcription Base of the loops are rich in topoisomerase, which allow the DNA to swirl when anchored Loops are packed further to form the chromosome During cell division, chromosomes become highly condensed. DNA in a metaphase chromosome is compacted to about 1/10000 of its stretched out length. The two daughter DNA nuclei from sister chromosomes are held together by cohesins. For kinetochore assembly, certain histones such as H3 in the centromeric regions are modified. Chromatin is tightly bound as loops to a scaffold. Scaffold contains non histone proteins such as condensations and topoisomerase. Loop-Scaffold complex is further compacted by coiling Condensins Chromosome Large protein complexes that contain SMC proteins Use ATP to coil DNA — make large right handed loops in DNA SMC are large dimeric proteins hinge at the centre with globular domains Globular domains bind DNA and hydrolyse ATP Condensins Large protein complexes that contain SMC proteins — use ATP to coil DNA SMC are long dimeric protein molecules hinged at the centre with globular domains at the end that bind DNA and hydrolyse ATP Condensins make large right handed loops in the DNA Chromosomal abnormalities (Abnormal Karyotype) file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 23/53 02/08/2019 Cell Biology Occur when there is a defect in a chromosome, or in the arrangement of the genetic material on the chromosome. Chromosome abnormalities give rise to specific physical symptoms, however, the severity of these can vary from individual to individual Any increases or decreases in chromosomal material interfere with normal development and function. Chromosomal abnormalities can occur during meiosis and fertilization Normal Karyotype: Number of chromosomes is designated as 2n. 44 autosomal and 2 sex chromosomes. Autosomal chromosomes have two homologous sets (one from each parent) Numerical Aberrations: Caused by a failure of chromosome division, which results in cells with an extra chromosome or a deficiency in chromosomes. Down syndrome (47 chromosomes instead of 46) Turner syndrome (45 chromosomes instead of 46) Klinefelter’s Syndrome (extra Y chromosome) Triploidy Trisomy Monosomy Mosaicism Structural Aberrations: Occur due to a loss of genetic material, or a rearrangement in the location of the genetic material Deletions: Portion of the chromosome is missing or deleted. e.g. Wolf-Hirschhorn syndrome, Jacobsen syndrome Duplications: Portion of the chromosome is duplicated, resulting in extra genetic material Translocations: When a portion of one chromosome is transferred to another chromosome Inversions: A portion of the chromosome has broken off, turned upside down and reattached, therefore the genetic material is inverted Rings: A portion of a chromosome has broken off and formed a circle or ring. This can happen with or without loss of genetic material. Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere Chromosomal instability and breakage: Fragile X syndrome. Boys are worse affected by this because they only have one X-Chromosome GIANT CHROMOSOMES Chromosomes are decondensed during interphase. Exceptions being lamp brush chromosome of vertebrate and polytene chromosome of insect In giant chromosomes, regions that are actively synthesising RNA are the least condensed. Giant chromosomes are very long and thick during metaphase. Polytene Chromosome Polytene Chromosome is a giant chromosome found in interphase nuclei. It has multiple chromatids attached at a common centrosome Specialised cells undergo a unique way of gene amplification and carry out multiple rounds of DNA replication without chromatid separation (endoreplication) — become large banded chromosomes First discovered by E.G. Balbini Most regions of a chromosome participate in Polytenisation, but a few sequences such as those near the centromere are not amplified. file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 24/53 02/08/2019 Cell Biology Cells with polytene chromosome do not divide further, because the proliferation of such a cell would lead to formation of an aberrant cell line within the organism. e.g. Salivary glands in Drosophila. Polytene chromosome has about 1000 DNA molecules which are arranged side by side and arise from bout 10 rounds of DNA replication. Visible during Interphase and Prophase of Mitosis It is different from polyploidy, where the daughter DNA molecules separate but the cell does not divide. Features (After staining Drosophila salivary glands with Feulgen stain) Bands and interbands (85% bands) Chromosome Puffs: Localised swelling of the chromosome where the genetic material is in a more relaxed state Somatic Pairing: Chromosomes show homologous pairing, even in interphase. Chromocentre: Chromatids are joined at a common centre Genes are found in both bands and irterbands Appearance of puffs can be stimulated by addition of inducers such as Ecdysone — shows that chromatin structure undergoes dynamic change associated with gene activity Radioactively labelled Uridine (RNA precursor) — radioactivity accumulates in the puff, indicating they are sites of intense RNA transcription Some proteins bind to polytene chromosomes for puff formation. They participate in ATP dependent re-modelling of chromatin during puffing Significance Metabolic Advantage: High level of gene expression (Large amounts of glue before pupation in Drosophila) Chromosome mapping using banding pattens Suitable for in situ hybridisation Understand correlation between chromosome packaging and gene expression Relation between environmental stimulus and looping pattern (e.g. Ecdysone induction) Lamp brush Chromosome Giant chromosome found in mature oocytes of most of the vertebrates. Gall and Callan interpreted the structure in functional terms. It is a homologous pair of chromosomes that occurs during Diplotene of Prophase in Meiosis I 2 chromosomes, 4 chromatids, form brush like stiff loops along the main axis of the structure, all containing the same DNA. Maternal and Paternal chromosomes are held together by chiasmata at those sites where crossing over has previously occurred. file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 25/53 02/08/2019 Cell Biology Loop structure is made possible by histone modifying enzymes, chromatin remodelling complexes and other proteins needed for gene expression. RNA Polymerases and RNA transcripts are present near these loops. These are transitory structure that exist during the extended diplotene of first meiotic division in female gametocytes Leads to widespread RNA transcription from thousands of transcription units Transcription (Electron microscopy of the loops) RNA polymerase molecules are attached to the principal axis (DNA) of the loop from which RNA fibrils of increasing length extends Each loop of Lampbrush chromosome is found to perform intense transcription of hnRNA (precursors for of mRNA for various ribosomal proteins/cytoplasmic ribosomes, histone protein) Each lateral loop is covered by a matrix that consists of RNA transcripts with hnRNA (Heterogeneous RNA molecules) binding protein attached to them RNA synthesis starts at the thinner end and progresses towards the thicker end. The loops can be categorized by size thickness and other morphological characteristics. As transcription continues the DNA strands loop, the fibril of RNA that is hnRNA lengthens Actin filaments may be involved in extending the lampbrush chromosomal loop away from the chromomeric axis As meiosis proceeds further, the number of loops gradually decreases and the loops ultimately disappear due to reabsorption back into the chromosomes. Significance Metabolic advantage Chromosomal mapping using consistent pattern of chromatin loops Correlation between chromosome packaging and gene expression Relation between environmental stimulus and looping pattern Useful to study chromosome organisation and gene expression and also molecular and supramolecular morphology of RNA transcription file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 26/53 02/08/2019 Cell Biology RIBOSOMES Ribosome = 75% RNA, 25% proteins Ribosomes are organelles that are responsible for protein synthesis Hold the complex of mRNA and tRNA during synthesis Catalyse peptide bond formation Ensure accuracy of protein synthesis mRNA surveillance: Nuclear process for detection and destruction of mRNAs that contain premature termination codons by the process of nonsense mediated decay. Carried out by 80S type Ribosomes in the Eukaryotic nucleus. Ribosomes have a small and large subunit Prokaryotes: 70S (30S + 50S) — freely floating in the cytoplasm Eukaryotes: 80S (40S + 60S) — freely floating in the cytoplasm or attached to RER S: Size of ribosomes is measured in Svedberg units. It is the rate at which particles sediment in a gradient under centrifugal force and takes into account size, shape and density Applications Prokaryote and Eukaryote ribosomes look similar in structure. But there are differences in subunits and rRNA between them. Hence, protein synthesis is an ideal target for antibiotics as drugs that affect bacterial ribosomes would not hamper eukaryotic cells rRNAs are among the most conserved sequences, and hence a target in phylogenetic analyses. Structure of Ribosome rRNA are crucial for both structure and function of ribosomes. They are folded into well defined conserved structures with many short duplex regions. Proteins interact with the RNA mostly at the surface level. Precise 3D folding within the RNA molecules gives rise to functional domains and catalytic centres mRNA binding site: Located in smaller subunit’s solitary RNA. 16S in prokaryotes, 18S in eukaryotes A site (Amino Acyl site): New tRNA arrives and binds the codon. P site (Peptidyl site): Where charged tRNA is placed when the peptide bond has been formed between the newly arrived AA and the last AA of the nascent peptide chain. The tRNA holding the growing polypeptide chain of AA binds here. E site (Exit site): tRNA transiently binds the ribosome, before it leaves the complex after adding AA to the polypeptide bond Peptidyl Transferase Centre: Catalytic domain of the ribosome. Entirely located in the larger subunit. The 3D structure brings the AA together and catalyses the formation of a peptide bond between them. Peptide Tunnel: Tunnel composed of rRNA domains which cannot form any association with the nascent peptide chain. Newly growing PP chain grows through this tunnel. It is located near the P site. Factor Binding Centre: In the large subunit near the A site. Translation factors which bind to GTP interact with this domain. file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 27/53 02/08/2019 Cell Biology Ribosome Biogenesis Nucleolar Organising Region (NOR) has genes for different ribosome sub units. They are separated by multiple non transcribing spacer DNA 5S rRNA is synthesised outside the NOR 5.8S, 18S and 28S are transcribed together as a long RNA Long precursor RNA is spliced with the help of Nucleolin Proteins involved in Ribosome assembly are synthesised in the Cytosol, and then migrate to the Nucleolus Assembly of 40S and 60S subunits. 40S subunit exported out of nucleolus earlier than 60S subunit. This lag is important for correct protein synthesis Initiation: rRNA transcription in FC Production: rRNA + protein assembly in DFC Maturation: Processing + 3D alignment in Granulocyte component. CELL DIVISION Before division of a cell into daughter cells Daughter cell receives Enlarges 1 complete set of genetic material Genome Replication Sufficient amount of protoplasm Proliferates membrane component Genetically semi-autonomous organelles Proliferate autonomous organelles Membrane components CELL CYCLE Stages through which a cell passes from one cell division to the next (Replication of genetic material — separation of DNA molecules — Cytokinesis) file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 28/53 02/08/2019 Cell Biology Interphase (I Phase): 75-90% of the total generation time. Prepares the nucleus and cell to divide. Karyokinesis (M Phase): Division of nucleus Cytokinesis (D Phase) Mitogen: Stimulus which induces cell division. Exact stimulus for cell division varies from cell to cell. e.g. Nerve Growth Factor, Fibroblast Growth Factor, Interleukins Reversible Phosophorylation: Dephosphorylation of target proteins by specific kinases and cyclins exerts control on various stages of the cell cycle. Interphase: Changes that take place in a newly formed cell and its nucleus, before it develops the ability to divide. G1 phase Preconditions: Minimum cell size, Optimum supply of nutrients Cells grow in size, Nucleus enlarges slightly RNA and proteins are synthesised Large pool of nucleotides, AA and energy rich compounds are formed towards the end of G1 phase. Checkpoint - Go At the close of G1 phase, decision to divide further has to be taken — check for integrity of DNA and sufficient nutrients If the cell does not divide any further it is arrested in the Go phase — Quiescent Cell Represents absence of mitogens or active suppression of genes or both These cells are physiologically active and carry out their normal functions file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 29/53 02/08/2019 Cell Biology Sometimes, they are terminally differentiated and will never enter the cell cycle again. Lymphocytes become active and enter the cell cycle on encountering an appropriate mitogen (antigen in this case) Cancer Cells cannot enter this phase and repeat the cell cycle indefinitely. Chromosomes duplicate. DNA content doubles Each chromatid duplicates and the resultant chromosome has two chromatids attached at the centromere through proteins called cohesins. S phase Histone synthesis Euchromatin areas replicate earlier than heterochromatin areas Centrosome is replicated. Newly formed centriole is small in the beginning and is called pro centriole. It becomes mature around mid-prophase. Preparation for mitosis attains final stage. RNA and Protein synthesis G2 phase Synthesis of energy rich macro molecules Entry of G2 to M phase requires phosphorylation of H1 histones of chromatin fibres. MITOSIS Mitosis: Type of cell division in which chromosomes replicate and become equally distributed to daughter nuclei, so that the daughter nuclei have the same number and type of chromosomes Protoplasm shows increased viscosity Chromosomal condensation to form sister chromatids Cytoskeletal elements are reorganised through phosphorylation Nuclear envelope starts to disintegrate Prophase Centrosomes migrate to respective poles and begin to form spindle fibres. Calcium containing protein Calmodulin is involved in organisation of spindle poles. Spindle fibres are made up of tubulin. 90-95% tubulin, 3-5% actin + myosin + lipids NE completely disintegrates Microtubules span through the cell and connect to a few kinetochores. Prometaphase Gradually, chromosomes attach to opposite MTOCs on both kinetochores Tightening of MT brings chromosomes towards equator of the spindle Metaphase Chromosomes are shortest and thickest. Arranged on equatorial plane of spindle. Centromeres of all chromosomes form the metaphase plate. Chromosome in the Metaphase file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 30/53 02/08/2019 Cell Biology plate is connected to a MT from both the spindle poles. Tension in the MT maintains the position at the metaphase plate Assembly of metaphase plate activates the Anaphase Promoting Complex (APC) Cohesin hold the sister chromatids together form the time they are formed in DNA replication, until the onset of anaphase. APC (is a ubiquitin ligase complex) hydrolyses Securin, which is an inhibitory protein for Separin. Activated Separin hydrolyses Cohesin. Removal of Cohesin allows the division of each chromosome into into two sister chromatids. Splitting occurs even when Spindle assembly is disrupted using MT toxins. Daughter chromosomes move towards the poles Anaphase along the MT. At the end of Anaphase, there are two groups of chromosomes, one at each pole. Spindle begins to degenerate after Anaphase Mitotic Apparatus: Temporary structure in a dividing cell that allows chromosomes to move to opposite poles of the cell so that they can form the nuclear material in the resulting daughter nuclei. Components: Microtubules, Centrioles, Proteins involved in mitosis such as Cohesin, Separin, Securin etc. Chromosomal groups at the end of Anaphase reorganise themselves into nuclei. Chromosomes elongate and form chromatin fibres. Telophase Nucleoli are formed from pre nucleolar bodies by nucleologenesis Nucleoplasm collects in area of chromatin NE appears on the outside from pieces of older NE Cytokinesis M Phase = Mitosis + Cytokinesis Cytokinesis is division of protoplast into two daughter cells, after karyokinesis so that each daughter cell comes to have its own nucleus. Starts at the mid phase of anaphase and completes simultaneously with the telophase. Occurs along the metaphasic plate. (In animals by cleavage, in plants by formation of cell plate) Dense fibrous and vesicular mid develops in the central equatorial region from remains of spindle. Microfilaments collect below the cell surface in the equatorial plane which produces a contractile ring. The contraction produces a furrow which further develops and cleaves the cell into two daughters. file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 31/53 02/08/2019 Cell Biology Importance of Mitosis Growth and Development — somatic cell division Maintains cell surface area to volume ratio by inducing overgrown cells to divide. Maintains chromosome number Wound healing and regeneration Asexual reproduction — primary mode of proliferating in bacteria, yeast etc. Wound healing and replacing old cells CELL CYCLE REGULATION Why Cell Cycle Regulation? DNA replication must alternate with mitotic division Coordination with protoplasmic growth so that there is no progressive loss or gain of protoplasm Events of cell cycle in a linear manner Stages of the cell cycle occur in a linear manner Halts if there is any persistent damage to genetic material Checkpoints: Regulatory mechanisms sensitive to both intrinsic and extrinsic signalling systems which can sense progress of the cell cycle and inhibit it. Functions of checkpoints is to check Entire genome is replicated prior to mitosis Only one round of genomic replication per cell cycle Corresponding growth in protoplasm so that cytokinesis occurs Cell has achieved necessary maturity Environment is favourable for growth and reproduction There are enough nutrients for the process to go through At the end of G1 phase Commitment step — most important checkpoint G1/S Ensures that previous mitosis completed correctly, optimum environmental conditions are available for mitosis, protoplasm has attained enough mass and nutrients to support cell division S Ensure DNA integrity Ensure that DNA has replicated correctly DNA damages have been repaired End of G2 Protoplasm volume is sufficient Immediate environment is favourable Precise alignment of chromosomes on the spindle apparatus, so that genetic material is M divided equally between the daughter cells. Molecular Mechanisms of Cell Cycle Control file:///Users/sashibhusanmishra/Documents/CellBio/Cell Biology.html 32/53 02/08/2019 Cell Biology Experimental observations S x G1: Both nuclei replicate — S phase activator induces replication S x G2: S nucleus replicates, G2 nucleus waits — S phase activator cannot make DNA replicate twice M x Interphase: Both cells divide — M promoter can induce premature division G1 x G2: G1 stays in interphase, G2 waits — S phase and M phase promoters exist transiently, not active during G1 and G2 phases. Mechanism Cell cycle regulation takes place by synthesis of new proteins and degradation of earlier proteins Fundamental control is by Cyclin-CDK complexes, whose activity is modulated by Phosphorylation and De-phosophorylation G1 — S: Phosphorylation of DNA polymerase S — M: Phosphorylation of mitotic proteins Protein kinases are constitutively present. Cyclins are not constitutively present. They are synthesised when needed and get destroyed when their use is over. G1 cyclins: Mediate G1/S transition, Form G1/S cyclin-CDK complex S cyclins: Govern DNA replication through S phase cyclin-CDk complex M cyclins: Mediate G2/M transition, M phase cyclin-CDK complex Anaphase Promoting complex: Triggers events leading to destruction of cohesions. Degrades M phase cyclins Transition is mediated by a sudden increase in CDK activity that phosphorylates key proteins needed for the transition Specific cyclin synthesis for a particular cell cycle transition Degradation of other cyclins by Ubiquitin mediated proteolysis Selective phosphorylation of kinases Direct inhibition of Cyclin