Cell Structure and Function Cytology PDF

LU | Faculty of Sciences Cell Structure and Function - Cell Biology - By Reem Dakhil M1 Biochemistry and founder of Unbeatable Chapter 1 Cell Biology Birth Definitions – Cell doctrine  Cell Biology vs Cytology Cell Biology...

LU | Faculty of Sciences Cell Structure and Function - Cell Biology - By Reem Dakhil M1 Biochemistry and founder of Unbeatable Chapter 1 Cell Biology Birth Definitions – Cell doctrine  Cell Biology vs Cytology Cell Biology Cytology The study of cells and their The study of cells and their components structurally and components structurally functionally  The Cell Doctrine Unit Division Organism Fundamental unit of Cells divide Cells + their products structure & function = organism Life Double-life Basis of life continuity One for its own, (mitosis – meiosis – one for the organism fertilization)  Chapter 2 Molecular Components of cells Inorganic and Organic Compounds What atoms and molecules are found in our cells?  Atomic level 96% 4% Very low C, H, N, O Na, K, Cl, S, P Trace elements Most abundant atoms B, F, Mn, Fe, Co, Cu, Zn, Se, I…  Molecular Level Organic molecules “Biomolecules” Inorganic molecules Proteins Water Carbohydrates Mineral salts Lipids Nucleic acids 1. Water  Basis of cell life  Most abundant  Electrically neutral  Extremely polar: partially (-) O & partially (+) H  Forms Hydrogen bonds (with H2O or with other molecules) & ionic bonds (solubilizes salts)  H2O is found free or interacting with molecules  2. Mineral Salts  Found dissolved & ionized by water (due to water polarity)  [ ] inside cell ≠ [ ] interstitial fluid  Balance in [ ] is essential for: membrane permeability, nerve impulse, muscle contraction, cell division …  Metal ions are essential for the activity of certain proteins (muscle contraction, O2 transfer, intercellular signaling…)  3. Organic Compounds Molecules Macromolecules +Vitamins amino acids proteins needed at low [ ] monosaccharaides polysaccharides can be carbs (vit. C) fatty acids lipids or lipids (vit. A, E, D) nucleotides DNA, RNA  I. Proteins 1. Introduction  Most abundant organic molecules  Most diverse (encoded by genes)  diversity in functions  Unbranched polymers of aminoacids  I. Proteins 2. Function ~ All functions for cell & organism life Morphology – cell’s molecular identity (HLA, blood groups) – gene expression & DNA replication – storage & transportation – intercellular & organ communication – immunity – senses – cell cycle – muscle contraction * but they are not energetic molecules  I. Proteins 3. Diverse Chemical Composition Although diverse, but chemically: proteins are a homogeneous class (1 unbranched chain of aa) They differ by number & sequence of aa, which are identical for a protein type Diverse sequences  diverse 3D structures  diverse functions  I. Proteins 4. The Amino Acids  Made of: C, H, N, O, S  Cα linked to: NH3, COOH, H, and side chain R  They differ by R only  4 groups: - Non-polar (Alkyl, benzyl) - Polar uncharged (NH2, CO, OH) - Polar positive / basic (NH3+) - Polar negative / acidic (COO-)  20 types, 9 of them are essential aa (can’t be synthesized by cells, must be supplied by diet)  Only L-stereoisomers form polypeptides  I. Proteins 4. The Amino Acids  All aa are soluble in water (due to amino & carboxyl groups) BUT not all proteins/peptides are soluble in water  aa ionization depends on: pH & R  Zwitterions: aa with uncharged R and ionized groups (NH2 & COOH) at pH=7  I. Proteins 5. Polypeptide  I. Proteins 5. Polypeptide  Linear unbranched chain of aa joined by covalent bonds “peptide bonds”  Formed by ribosome; digested by proteases  Reaction: nucleophile attack between NH2 of aa (n) & COOH of aa (n-1)  “amide bond” CO – NH  H2O is eliminated  Backbone: repeating N – Cα – C  polarized: N-terminus (Amino terminus NH2) & C-terminus (Carboxy terminus COOH)  I. Proteins 6. Polypeptide Flexibility Extended groups (R, H, =O) are not in the same plane, they rotate in different directions  confers flexibility  helps folding to adopt 3D conformations  I. Proteins 7. Structures of Proteins a. Primary Structure b. Secondary Structure c. Tertiary Structure d. Quaternary Structure  I. Proteins a. Primary Structure  Linear aa sequence  Determined by: nucleotide sequence in DNA  Stabilized by: covalent bonds between aa  Determines all of the other structures  its alteration impairs protein functions  All copies of the same protein have identical primary structures  I. Proteins b. Secondary Structure  Folding of aa portions into: helices & β-pleated sheets (+ turns & irregular random coiling)  Stabilized by hydrogen bonds denaturation by heating (except for supercoiling: covalent denaturation by chemical reaction bonds)  A specific aa sequence will acquire the same folding wherever it is found  I. Proteins c. Tertiary Structure  Helical & non-helical regions are folded back in precise positions  Determined by secondary structures  Hides hydrophobic aa in core & exposes charged aa  Brings distant segments together  active domains  Stabilized by: - hydrogen bonds - ionic bonds - hydrophobic interactions - salt bridges - disulfide bridges  I. Proteins c. Tertiary Structure  Protein Denaturation  Unfolding + segment separation of proteins  By: physical agents (low pH, high temp) & chemical agents (detergents, urea)  Protein loses its activity  Irreversible unless mild  I. Proteins d. Quaternary Structure  Some proteins need it  Assembly of ≥2 subunits (identical or different)  forms active domains  Stabilized by: weak bonds (+ sometimes covalent)  Protein activation / deactivation: by changing its 3D conformation: by: - Phosphorylation / dephosphorylating - Allosteric transition (ligand binding / dissociation)  I. Proteins 8. Classification of Proteins  By composition - Holoproteins / apoproteins: only aa - Heteroproteins: aa + other molecules ex: lipoproteins, glycoproteins, nucleoproteins, hemoproteins (ion)  By structure - Fibrous: insoluble in water (keratin, collagen) - Globular: soluble in water (globulin, albumin, histones)  By function Structural – defense – regulatory – transporter – catalytic – contraction…  II. Carbohydrates Functions  Energetic  Structural  Cell identity glycogen in animals cellulose in plant cell wall blood groups  II. Carbohydrates 1. Monosaccharides Definition  aka “simple oses”  Most are D-stereoisomers  CnH2nOn (3≤n≤6)  Less diverse than proteins since: - Only a few monosaccharaides can form polymers - Different types do not combine much (max 2) D-glucose C6H12O6  II. Carbohydrates 1. Classification of Monosaccharides  By functional group - Aldoses (aldehyde group) - Ketoses (ketone group)  By n 3: trioses – 4: tetroses – 5: pentoses – 6: hexoses Aldose pentose: ribose Aldose hexoses: glucose, galactose, mannose Ketose hexose: fructose  II. Carbohydrates 2. Cyclic Oses  n>4  straight chain or cycle  cycle is more stable  or  Pyranoses (6C) & furanoses (5C) α β  From intramolecular reaction between OH & aldehyde/ketone group  α & β isomers: differ by orientation of OH that replaced the aldehyde/ketone - α: downward - β: upward  Usually cyclic oses are switchable between α & β until polymerized.  II. Carbohydrates 4. Modification  Osamines addition of amine group (---NH2) eg: glucoseglucosamine. galactosegalactosamine  N-acetyl osamines addition of acetyl group (---COCH3) to the amine group eg: NAG: N-acetyl galactosamine  Uronic acids acidification of CH2OH into COOH eg: NANA: N-Acetyl Neuraminic Acid (from mannose)  II. Carbohydrates 5. Dimers and Polymers  Disaccharide: 2 oses joined by covalent bond “glycosidic bond” Its formation releases H2O - Saccharose = glucose + fructose - Lactose = glucose + galactose - Maltose = 2 glucose  Oligosaccharide: short polymer of monosaccharaides can be branched / unbranched can be linked to proteins / lipids for maturation  II. Carbohydrates 5. Dimers and Polymers  Polysaccharide (Glycan): long chain of monosaccharides branched / unbranched simple or modified oses Functions: Structural - cellulose in plant cell wall – peptidoglycans in bacterial cell wall Energetic - glycogen in muscles – starch in plants  II. Carbohydrates 5. Dimers and Polymers  Homopolysaccharide: polymer of the same molecule - Starch & glycogen = branched polymers of α-D-glucose - Cellulose = unbranched polymer of β-D-glucose - Chitin = polymer of N-acetyl glucosamine, in insects shell  Heteropolysaccharide: polymer of 2 diferent simple/modified oses - GAG = glucosamine + galactosamine - Hyaluronic acid = glucoronic acid + N-acetyl glucosamine - Chondroitin Sulfate = glucuronic acid + N-acetyl galactosamine - Keratan Sulfate = N-acetyl galactosamine + D-galactose  III. Lipids All are non-polar  insoluble in water, soluble in non-polar solvents Functions Structural: phospholipids: component of cell membrane Energetic: triglycerides: stored in plants seeds & animal adipose tissue Communication: steroid hormones, eicosanoids, phosphatidylinositol 2 Types Saponifiable: have f.a, can undergo saponification reaction Non-saponifiable: no f.a, can not undergo saponification  III. Lipids 1. Saponifiable Lipids a. Fatty Acids  Amphipathic: 1 polar hydrophilic head (COOH) & nonpolar hydrophobic tail (hydrocarbon chain)  Formula: CH3-(CH2)n-COOH (2≤n≤20) n is even  Rarely occur free  More energetic than carbs  Differ by: length of chain & unsaturation degree (nb of C=C)  both affect membrane fluidity  Saturated f.a.: no double bond, solid at room t◦ (Butyric acid, Myristic acid, Palmitic acid)  Unsaturated f.a.: ≥1 double bond, fluid at room t◦ (Arachidonic acid, Linoleic acid, Oleic acid)  III. Lipids 1. Saponifiable Lipids b. Triglycerides / Triacylglycerols / neutral fats  Neutral fats  no polar groups  Glycerol + 3 f.a joined by ester bonds  Diverse since many f.a types can form it  Function: stored energy reserve  Hydrolysis: by lipases or: by alkaline medium + heating  III. Lipids 1. Saponifiable Lipids c. Phospholipids  Function: Structural (found in cell membranes)  Amphipathic: 1 large polar head (phosphate) + 2 hydrophobic tails  Glycerol-derived: Glycerophosphatides: Phosphate + glycerol + 2 f.a  Sphingosine-derived: Sphingophospholipids: Phosphate + sphingosine + 1 f.a Found in myelin sheath (sphingomyelin) * Ceramide = sphingosine + f.a  Choline, Serine, Ethanolamine, or Inositol can be added to increase head polarity  III. Lipids 1. Saponifiable Lipids d. Glycolipids  Function: cell identity (blood group – immunity – cell-cell recognition)  found on outer cell membrane  - Glycerol-derived: in plants & bacteria (Sugar + glycerol + 2 f.a ) - Sphingosine-derived: in animals (Sugar + sphingosine + 1 f.a)  - Cerebrosides: Sugar is a simple ose monosaccharide (eg: galactocerebroside in brain’s myelin sheath) - Gangliosides: Sugar is an oligosaccharide  III. Lipids 1. Saponifiable Lipids e. Cerides  Esters of f.a + “fatty alcohol” (long hydrocarbon chain ending with OH)  In bee wax, leaf cuticle, cork  III. Lipids 2. Non-saponifiable Lipids a. Terpenes  Polymers of propene with cyclization at one end & (maybe) polar OH at the other  moderately amphipathic  Vit. A, E, K & carotenoids (lycopene, carotene)  III. Lipids 2. Non-saponifiable Lipids b. Steroids cholesterol  Cyclic molecules carrying different functional groups  Functions: Communication: Steroid hormones (estrogen, progesterone, testosterone, corticosterone, adrenal hormones…) Vitamins: Vit. D for growth & bone development (synth in skin) Structural: Cholesterol in plasma membrane controls its fluidity (bidirectionally) *It’ a precursor for steroid hormones, vit. D, bile salts… (Found in animal products)  III. Lipids 3. Hydrophobic Interactions Lipids are amphipathic water insoluble mixing forms heterogeneous mixtures  Monolayers  Bilayers: polar hydrophilic surfaces, hydrophobic center that prevents diffusion of large hydrophilic molecules  Micelles: sphere of hydrophilic surface & hydrophobic core  Liposomes: long bilayer folded back  hydrophilic surfaces & lumen  IV. Nucleic Acids 1. Composition  Nucleic Acid = Unbranched chain of nucleotides (DNA/RNA)  Nucleotide = Nucleoside + phosphate (nucleoside monophosphate)  Nucleoside = Nitrogenous base + Pentose Nitrogenous bases: Purines (A, G) – Pyrimidines (C, U, T) Nucleosides: Adenosine, Guanosine, Thymidine, Uridine, Cytidine Nucleotides: AMP, ADP, ATP, GMP, GDP, GTP, CMP, CDP, CTP, TMP, TDP, TTP, UMP, UDP, UTP * Precede by d (deoxy) for DNA * NTP is used when building nucleotides  2 P used for energy  IV. Nucleic Acids 1. Composition Bonds: Nitrogenous Base & pentose: N-glycosidic bond (1’) Pentose & phosphate: phosphoester bond (5’) Adjacent nucleotides: phosphodiester bond (5’ & 3’) Facing nitrogenous bases: hydrogen bond  IV. Nucleic Acids 1. Composition DNA RNA Pentose Deoxyribose Ribose Nitrogenous Thymine Uracil base 2◦ structure Double-stranded Single-stranded Nucleus, mitochondria, Nucleus, mitochondria, Location chloroplast chroloplast + cytoplasm Stability More stable Less stable  IV. Nucleic Acids 2. DNA Structure  2 polynucleotide chains (strands) forming a double helix  - Backbone: phosphate & sugar - Nitrogenous bases: in the middle of the double helix  Nitrogenous bases pairing: - hydrogen bonds - complementary purine-pyrimidine (A&T, C&G) - antiparallel (one strand rotated 180◦ wrt the other)  Grooves (major & minor)  their functional groups bind to proteins for condensation, replication, & transcription  Dimensions: diameter=20 A, distance 2 bp=3.4 A, turn=34 A or 10 bp  Helix forms: A-helix (in a water-poor medium) ; B-helix (usual form) ; Z-helix (in vitro)  DNA denaturation: heating (100◦C) or alkaline medium  double strand unwinds & hydrogen bond is broken BUT it’s reversible  IV. Nucleic Acids 3. DNA replication  During S phase of interphase  Aim: transmission & preservation of genetic information  Replication complex = set of enzymes: - helicase - primase - topoisomerase (gyrase) - DNA polymerase - ligase  Requires a lot of energy (from precursors: dNTPs)  Semi-conservative: each new DNA molecule contains 1 old template strand and 1 newly synthesized strand  IV. Nucleic Acids 3. DNA replication Nuclease activity Some polymerases have the ability to degrade DNA Exonuclease activity: ability to degrade DNA from extremities. Some polymerases act 3’-5’ direction, others 5’-3’, others both  IV. Nucleic Acids 3. DNA replication 1. Helix unwinding: helicase separates the 2 strands at specific points (origin) * eukaryotes have many replication origins per chromosome * replicon: long segment of replicated DNA starting from origin 2. Primase (RNA polymerase) adds a primer (RNA sequence) to provide 3’OH end for DNA polymerase to start 3. DNA polymerase binds and starts synthesizing new strand using dNTPs and adding them in a complementary manner (A T, C G) template is read 3’-5’, new strand is synthesized 5’-3’ 4. Topoisomerase (gyrase): releases the tension  relaxes supercoiled DNA 5. DNA polymerase I removes primers (5’-3’ exonuclease) 6. Ligases join Okazaki fragments  IV. Nucleic Acids 4. RNA Structure  Single polynucleotide chain  Some intramolecular pairing snRNA mRNA tRNA rRNA Abundance - Least abundant intermediate Most abundant Diversity - Most diverse (encoded intermediate Least diverse by many genes) (2 genes) Structure Bound to proteins  Single-stranded with 2◦: trefoil (3 loops) Highly structured form snRNP some pairing * middle loop has (many pairs & folds) anticodon Binds to proteins  * ends in 5’CCA3’ forms ribosome which carries aa 3◦: L-shaped Function mRNA maturation by Translated into Translation of mRNA Translation of mRNA splicing proteins  IV. Nucleic Acids Replication vs. Transcription Replication Transcription Both DNA segments are templates Only 1 strand is a template The whole strand is replicated Only genes are transcribed Uses helicase, ligase.. + DNA Uses helicase, ligase.. + RNA Polymerase polymerase Chapter 3 Eukaryotes, Prokaryotes, and Viruses I. Definition of Eukaryotes & Prokaryotes Eukaryotes Prokaryotes Nuclear membrane No nuclear membrane (DNA & ribosomes separated) (DNA & ribosomes not separated) Compartmentalized (organelles) Non-compartmentalized (no organelles) Animals, plants, fungi, protists, protozoa Eubacteria, archaebacteria, mycoplasma (PPLO) Unicellular or multicellular unicellular Size: tens to hundreds of μm Size: 1-2 μm Viruses are not living things (can’t reproduce autonomously)  not cells Cells Viruses Size in μm Size in nm Visible by light microscopy Visible by electron microscopy  II. Eukaryotic Cells Organelles Only in animals Common Only in plants centrosomes nucleus, cytosol plastids lysosomes (hyaloplasm), ribosomes, vacuoles cilia cytoskeleton, ER, Golgi, cell wall flagella mitochondria, plasma plasmodesmata microvilli membrane, peroxisome II. Eukaryotic Cells 1. Animal Cells  Heterotrophs: feed on organic matter  Smaller than plant cells II. Eukaryotic Cells 2. Plant Cells  Photoautotrophs: produce their own organic matter by photosynthesis  Bigger than animal cells  II. Eukaryotic Cells 2. Plant Cells Cell Wall Thick, surrounds the plasma membrane Made of:  cellulose (polymer of β-D-glucose) + Matrix:  hemicellulose (gucose + pentoses)  pectin (rich in uronic acids  glycoproteins (small amounts)  II. Eukaryotic Cells 2. Plant Cells Cell Wall Functions:  Supportive: surrounds plasma membrane  Protective: - against fluctuations in osmotic pressure - against intruders (viruses, bacteria, fungi)  Structural: determines cell shape & growth direction  Adhesion between cells: through middle lamella (layer rich in pectin where neighboring cell walls fuse)  II. Eukaryotic Cells 2. Plant Cells Plasmodesmata Cytoplasmic bridges between neighboring cells that interrupt the cell wall  exchange molecules between cells  II. Eukaryotic Cells 2. Plant Cells Plastids Family of organelles Functions:  Photosynthesis  storage of organic compounds Example: chloroplast: photosynthesis in green pants: 𝑙𝑖𝑔ℎ𝑡 CO2 + H2O Glucose + O2  II. Eukaryotic Cells 2. Plant Cells Vacuoles  In mature plant cells: large: occupy major part of the cell  In young dividing cells: small vacuoles that will fuse together in mature cell  Single membrane: tonoplast  Role: - expand cell volume without diluting the cytoplasm - storage of water, cell products, metabolic intermediates  II. Prokaryotes Structurally: appear simple Biochemically: diverse and complex biochemical reactions Not all bacteria are harmful! Some are beneficial (lactic bacteria: ferments milk into yougurt) Some are harmful (tetanus toxin) Oxygen requirement - Aerobic: require oxygen to survive - Strict anaerobic: cannot survive in presence of oxygen - Facultative anaerobic: can survive in both presence or absence of oxygen  II. Prokaryotes Trophic mode - Heterotrophs: feed on organic nutrients - Photoautotrophs: make photosynthesis - Chemoautotrophs: make chemosynthesis Colonies Prokaryotes are unicellular. They can live in clusters or “colonies” BUT not multicellular, since no differentiation (all cells are identical) Growth  Asexual  By “fissiparity” or “binary fission”, not mitosis  Rapid (every 20 min)  II. Prokaryotes Do they need other cells? They grow freely if nutrients are available Some are intracellular parasites  infect eukaryotes Size & shape diversity  Genus names are according to shape.  Spherical: coccus, elongate: bacillus, spiral: spirochetes  Some bacteria change shapes depending on the medium  Average size: 1-2 μm  II. Prokaryotes Classifications Eubacteria (true bacteria):  live in mild conditions similar to eukaryotes Archaebacteria (ancient bacteria) extremophile:  live in extreme conditions (pH, temperature, salinity)  Chemoautotrophs Thermophile: live at high T◦ Halophile: live at high salt [ ] Methanogens: anaerobic – produce methane  II. Prokaryotes Mycoplasma “PleuroPneumonia-Like Organismsms” (PPLO)  Very small bacteria (0.2 μm)  Spherical or filamentous  Class: mollicutes  Can grow free + can be intracellular parasites  Cause diseases in humans, animals (respiratory & urogenital), and plants  No cell wall & no capsule  Lack a rigid envelope  highly polymorphic  Special plasma membrane: has lipids, cholesterol, & proteins  Some are eubacteria, others are archaebacteria (thermoplasma) II. Prokaryotes Cellular Structures Simpler than eukaryotes since no compartments BUT: cell wall is more complex  Nucleoid  Plasmid  Cytoplasmic structures  Plasma membrane  Cell wall  Capsule  Flagella  Pilli & fimbriae  Endospore  II. Prokaryotes a. Nucleoid  Dense region in cytoplasm  NOT separated by a membrane  Contains DNA (single circular chromosome) condensed by proteins  It separates the 2 DNA copies during cell division  No nucleolus nucleoid  II. Prokaryotes * Plasmid  Extrachromosomal genetic material  Found in some bacteria (and some eukaryotes)  Several small circular DNA molecules  Carry a few genes  Give advantages to bacteria (eg: resistance to antibiotics)  Can be transferred between bacteria by conjugation  Experimentally: used as vectors in molecular biology and genetic engineering  II. Prokaryotes b. Cytoplasmic Structures Bacterial cytoplasm = single compartment BUT: can perform many processes without organelles Example 1: mitochondria in eukaryotes: has enzymes for cell respiration and production of ATP. Bacteria have such enzymes for cell respiration in cell membrane and cytoplasm Example 2: chloroplast in eukaryotes: perform photosynthesis. Photosynthetic bacteria have photosynthetic lamellae: infoldings of plasma membrane. Can become independent structures (chromatophores). But not comparable to eukaryotic ER. Example 3: cyanobacteria: have gas vacuoles that control buoyancy & carboxysomes: enzymes that fix CO2 for photosynthesis Example 4: chlorobium: have chlorophyll-containing vesicles  II. Prokaryotes b. Cytoplasmic Structures  Ribosomes - translation - different from eukaryotic ribosomes - free in cytoplasm or attached to plasma membrane - none attached to lamellae  Granules / inclusions storage of organic & inorganic compounds (sulfur, phosphates, carbohydrates…)  II. Prokaryotes c. Bacterial Envelope Bacterial envelope = plasma membrane + cell wall (except mycoplasma) + capsule (if present)  II. Prokaryotes c. Bacterial Envelope 1* Plasma Membrane  Lipid bilayer, same as eukaryotes, but without steroids (except mycoplasma)  Selective and controlled permeability  essential for cell life  Contains proteins performing many metabolic reactions (transporters, ATP synthase, respiratory chain…)  Gives infolding: cytoplasmic lamella  Mesosome: not a real infolding, just an artifact of sample preparation  II. Prokaryotes c. Bacterial Envelope 2* Cell Wall  - Determines cell shape - Protects against changes in osmotic pressure  Bacterial cell wall = peptidoglycans = 3D network of heteropolysaccharides (glucose + N-acetylglucosamine) joined by peptide bridges  II. Prokaryotes c. Bacterial Envelope 2* Cell Wall Gram (+) Gram (-) Cyanobacteria Cell wall thickness thick thin thin peptidoglycans Cell wall components peptidoglycans peptidoglycans + teichoic acids 2nd lipid bilayer Enveloped by capsule (outer membrane) gelatinous layer + maybe capsule Outer membrane -  present -  II. Prokaryotes c. Bacterial Envelope 2* Cell Wall Gram (-) outer membrane Outer leaflet:  has LPS (lipopolysaccharide) endotoxin instead of phospholipids  has porins for transport Inner leaflet:  has special lipoproteins that anchor the outer membrane to peptidoglycan cell wall  II. Prokaryotes c. Bacterial Envelope 3* Capsule (glycocalyx)  The outer structure of cell envelope  In many bacteria (not all)  Made of polysaccharides  Functions: - adhesion (cell-cell & cell-support) - protects bacteria against phagocytosis  capsule increases the pathogenicity of bacteria  II. Prokaryotes d. Flagella  Thread-like appendages  1 or more per cell  At 1 pole, or distributed over the entire cell  Function: cell locomotion  3 parts: - basal body: anchored to in the envelope – generates rotation - hook: connects the filament to the basal body - filament: hollow cylinder – polymer of 1 protein: flagellin  Location: arises from plasma membrane, cell wall, and capsule (if present)  Eukaryotic flagellum: enveloped by an extension of plasma membrane. Bacterial flagellum: naked (not enveloped)  Cyanobacteria: lack flagella, move by gliding using their gelantinous layers. Occur as: free cells or small clusters/colonies or long filamentous chains  II. Prokaryotes e. Pili & Fimbriae  Pili = hair Fimbriae = fringes  Rigid appendages on cell surface  Shorter & finer than flagella  Functions: - bacteria-bacterial adhesion & conjugation (exchange of DNA between 2 bacteria through a channel: sexual pilus) - bacterial-host adhesion - receptors for viruses!  II. Prokaryotes f. Endospore  Formed by several bacteria (not all)  Formed in conditions of stress & nutritional depletion  A resting cell highly resistant to dessication (drought) – heat – chemical stress  Contains: DNA + some cell structures + membrane + special cell wall + coat  Resists harsh conditions for decades  When medium becomes favorable again, germinates the original bacterium  III. Viruses  Multimolecular complexes  Not cells = not living things  cannot divide by their own(even with nutrients)  Lack metabolic enzymes & pathways  Need host cells to multiply  Infect animals, plants, & bacteria Cause many diseases (AIDS – smallpox – chickenpox – rabies – poliomyelitis – mumps – measles hepatitis – mononucleosis – influenza – common cold) Some can cause cancers  III. Viruses Composition Virus = Nucleic acids (DNA/RNA, linear/circular, ss/ds) + Capsid (made of protein subunits: capsomeres) + Few proteins (polymerases, integrase) + Some: envelope (lipid bilayer) eg: HIV, influenza, smallpox + Some: tail linked to head  Viroid = only RNA molecule = very simple virus (no proteins)  III. Viruses Virus-Host Relationship  Viruses deviate / alter the cell metabolism (transcription, translation, replication) for their own benefit  Virus is specific: to a certain species, organ, tissue, cell, & even sub-cell type since: requires specific receptors on the cell BUT: Specificity is not absolute. Influenza (humans) may infect other species if the cell has the same receptors  Viruses change after leaving the host. Eg: influenza changes every year: H1N1: infects humans & pigs H5N1: infects humans & birds H: hemagglutinin, N: neuraminidase (antigens on envelope)  III. Viruses 1. Structure & Classification of Viruses Classifications according to: - Size: 20  200 nm - Nucleic acids: DNA (adenoviruses) / RNA (HIV, influenza) - Envelope: present or absent - Capsid: helical (TMV), polyhedral (adenovirus), complex (bacteriophage), or no symmetry  determines viral shape: rod (TMV), globular (HIV), polyhedral (adenovirus), helical, filamentous, or complex Bacteriophage: complex: = polyhedral head + tail (proteins arranged helical) + tail fibers (attachment to cell receptor) - Host cell: infecting prokaryotes (bacteriophages/phages) / eukaryotes  III. Viruses 2. Proliferation of Viruses 1. Viral capsid / membrane proteins recognize host cell receptor 2. Virus adheres to host surface 3. Virus injects its nucleic acid + proteins (polymerases…) into the host cytoplasm - enveloped virus: fuses its lipid bilayer with cell membrane  nucleocapsid enters the cytoplasm  capsid dissociates  nucleic acid released - non-enveloped virus: only nucleic acid is injected through a channel (capsid doesn’t enter) 4. Nucleic acid in cytoplasm performs lytic or lysogenic cycle 5. Thousands of new viruses are produced 6. Host cell dies  III. Viruses RNA Viruses Retroviruses: reverse transcriptase RNA DNA (provirus) No reverse transcriptase: RNA replication by “RNA-dependent RNA Polymerase” / “replicase” Lytic or Lysogenic cycle? Choice determined by: specific viral proteins – host cell conditions  III. Viruses Lysogenic Cycle Viral DNA is inserted into cell chromosome by “integrase”  DNA integrated is silent not active  cell seems normal As cell divides, cellular DNA & viral DNA is replicated  transmitted to daughter cells  silent reproduction of virus ! Lysogenic cycle can transform cells & cause cancer ! Lysogenic can transform to lytic cycle due to signals (eg: UV)  III. Viruses Lytic Cycle Cell metabolism is deviated  viral genes are preferably transcribed over cell genes 1. RNA is produced 2. translated to proteins by host cell equipment 3. viral nucleic acids produced by replication / transcription 4. assembly of viral particles: cage of capsid around nucleic acids 5. release of produced viruses - no lipid envelope: released by causing cell lysis using their enzymes - enveloped: released by budding: formed nucleocapsid interacts with host cell membrane (now has viral proteins)  takes a fragment of cell membrane as an envelope  detaches from cell ! Integration might occur  IV. Prions Unusual pathogens = only proteins (no nucleic acids)  Protein  Abnormal conformation  Can transform its abnormal conformation to other normal proteins  Protease resistant  accumulate in cell  cause toxicity  IV. Prions TSE (Transmissable Spongiform Encephalopathies)  Disease in animals & humans due to prions  Death of nerve cells (neurodegeneration) Disease examples: - Old: Scarpie (in sheep) - new: BSE (Bovine Spongiform Encephalopathy) Creutzfeldt-Jacob disease: from eating BSE-infected food  contains PrPres (resistant prion) PrPc (Prion-related Protein Cellular): normal protein – sensitive to proteolysis – found in nerve & blood cells PrPres (resistant): abnormal protein – resistant to proteases  forms insoluble fibrils o When normal PrPc interacts with abnormal PrPres  normal PrPc becomes also abnormal & resistant to proteases Chapter 4 Plasma Membrane and Other Cell Membranes  Cell Membranes “Biomembranes”  plasma membrane / cell membrane / cytoplasmic membrane.  Cytomembranes: membranes of: nucleus – ER – Golgi – mitochondria – vacuole – plastids… Visible by electron (not light) microscopy Less developed in prokaryotes than eukaryotes  Functions  Physical: - Cell membrane: separates the cytoplasm from the surrounding medium  cell integrity - Organelle membranes: separate organelles from the cytosol  Biochemical: Signal transduction – phosphorylation & cell respiration (prokaryotes)  Communication with neighboring cells  Selective & controlled permeability: regulates the passage of solutes in & out Permeability Hydrophobic Hydrophilic Large Molecules Molecules Molecules most can easily pass require channels / by vesicles transporters (exocytosis, (membrane proteins) endocytosis, phagocytosis)  Composition  Phospholipids (glycerol/sphingosine-derived)  Glycolipids (eg: gangliosides) on outer leaflet  antigens  Steroids (eg: cholesterol)  Proteins (most membrane functions)  Carbphydrates (linked to proteins / lipids) * glycocalyx: outer layer abundant in carbohydrates – gives a (-) charge  functions: protection against proteolytic enzymes – endocytosis – adhesion – recognition ** Quantities differ by cell types  I. The Lipid Bilayer A study on hydrophobic interactions: - Polar heads directed outward & inward - Non-polar tails directed to one another Bilayer  stabilized by hydrophobic interactions Staining by osmium tetroxide (polar): Trilaminar appearance 2 outer dark lamina  osmophilic  polar 1 central clear lamina  osmophobic  nonpolar Thickness: Variable according to f.a & proteins Osmophilic lamina = 20->25 A Osmophobic lamina = 25->35 A Total = 65->85 A  II. Fluid Mosaic Model 1. Definition & protein types  Bimolecular lipid layer E: exoplasmic: outer layer P: protoplasmic: inner layer  Composed of phospholipids + little neutral fats & steroids + proteins interrupting the surface in a mosaic manner Mosaic: irregular distribution of proteins in the bilayer  II. Fluid Mosaic Model Membrane Proteins Types  Peripheral/extrinsic proteins: at inner & outer surfaces attached by weak bonds (not covalent) to lipids or other proteins  Peripheral/extrinsic proteins: at inner & outer surfaces attached by covalent bonds (tight) via a lipid  Integral / intrinsic proteins: penetrate the membrane - partially (1 layer): “monotopic” - entirely (both layers): “polytopic” -- single-pass polytopic (bitopic): cross the membrane once -- multipass: cross it many times * all have: segment of 20 hydrophobic a.a (α helix) to interact with the hydrophobic tails. Many spans  many hydrophobic segments  II. Fluid Mosaic Model Membrane Proteins Connections Interactions with: - cytoskeleton components - cytosolic proteins - extracellular matrix proteins Constitutive (permanent) or induced (ligand-receptor eg. Hormone)  II. Fluid Mosaic Model Membrane Proteins Families 1. Immunoglobulins superfamily (IgSF) Functions: immunity – calium-independent cell-cell adhesion (eg. between Lymphocytes & macrophages) Examples: anitbodies – T-cell receptors 2. Cadherins: glycoproteins Functions: - calcium-dependent cell adhesion – communication with the medium (cell/matrix) On many animal cells  II. Fluid Mosaic Model Membrane Proteins Families 3. Selectins: glycoproteins Calcium-dependent acitivity Function: recognize & bind specific sugar motifs (eg: interaction between WBC & endothelial cells of blood vessels during inflammation) 4. Integrins: attach to basement membrane components CAM (Cell Adhesion Molecules) = immunoglobulins + cadherins + selectin SAM (Substrate Adhesion Molecules) = integrins  II. Fluid Mosaic Model Carbohydrates  Most bound to lipids (glycolipids) or proteins (glycoproteins)  Most on the outer surface  In organelle membranes; carbohydrates are exposed away from the cytosol (inner layer)  Function: cell identity (eg: blood group) catalytic & structural functions (transporters – channels – linked to cytoskeleton – receptors)  II. Fluid Mosaic Model 2. Membrane asymmetry of proteins & lipids Asymmetry = not uniformly distributed Protein asymmetry - Some proteins present in only 1 layer (outer or inner) - Some present in both but different quantities Location according to function: - Outer: attachment to ECM – disulfide bonds - Inner: attachment to cytoskeleton  II. Fluid Mosaic Model 2. Membrane asymmetry of proteins & lipids Carbohydrate asymmetry Always outwardly (away from cytosol) Lipid asymmetry Different polar heads between outer & inner layer Different f.a. (quantity & quality) Flip-flop movement Phospholipids and glycolipids cannot flip from one layer to another  they are flipped by enzymes: flipases  II. Fluid Mosaic Model 3. Fluidity of Membranes – Mobility of Lipids & Proteins a. Factors affecting membrane fluidity Membrane appears static. BUT: it’s highly dynamic  fluid - Addition & removal of components - Continuous movement of components (no covalent bonds)  II. Fluid Mosaic Model 3. Fluidity of Membranes – Mobility of Lipids & Proteins a. Factors affecting membrane fluidity 1) Unsaturation of f.a Double bonds (C=C) ↗ fluidity. Saturated f.a: rigid crystalline structure 2) Length of f.a As length of f.a ↗, fluidity ↘ 3) Temperature As T◦ ↗, fluidity ↗ (fats & waxes melt by heating) 4) Cholesterol Bidirectional regulator Low T◦: cholesterol ↗ fluidity High T◦: cholesterol ↘ fluidity by intercalating between phospholipids  prevents their clustering & hardening  II. Fluid Mosaic Model 3. Fluidity of Membranes – Mobility of Lipids & Proteins a. Factors affecting membrane fluidity Importance of fluidity Membrane too fluid  components dissociate  structure lost Membrane too viscous  prevents interaction & movement  function blocked Cells adjust fluidity by adjusting: f.a unsaturation & cholesterol amount Ex: cells below 37◦ promote unsaturating enzymes  compensate loss of fluidity Plants: rich in unsaturated f.a to maintain fluidity at night & in winter (T◦ ↘) Plants that thrive in winter  high % of unsaturated f.a  II. Fluid Mosaic Model 3. Fluidity of Membranes – Mobility of Lipids & Proteins b. Mobility of Membrane Components Protein mobility experiment Heterokaryon = large hybrid cell from fusion of 2 diploid cells from different species (eg: human & mouse) 1. Antigens of mouse & human cell were labelled by different colors 2. Fusion was induced human + mouse  heterokaryon 3. After fusion: antigens were at opposite poles 4. 1h later: both antigens uniformly distributed on the entire heterokaryon membrane  Proteins move laterally in the lipid layer (not static)  II. Fluid Mosaic Model 3. Fluidity of Membranes – Mobility of Lipids & Proteins b. Mobility of Membrane Components  Lipid mobility > protein mobility  Exclusively lateral  within the same layer: no flip-flop without lipases  May be controlled by cytoskeleton components – ECM – basal lamina  Limited to 1 pole of membrane in a polarized cell (due to tight junctions)  II. Fluid Mosaic Model 4. Polarization & Functional Domains Polarization = Membrane composition & function is not uniform through the entire cell surface  Distinct poles  Distinct proteins & lipids  Distinct properties & functions  II. Fluid Mosaic Model 4. Polarization & Functional Domains Epithelial Cell - apical: transporters & channels for nutrient absorption from lumen - basal: adhesion to basement membrane – transport of nutrient to blood vessels - lateral: adhesion to neighboring cell Hepatocyte: Parenchymal liver cell - pole facing endothelial cell: exchanges substances (sugars, aa, insulin, gases) with blood - pole facing neighboring hepatocyte: exchange substances – make junctions (desmosomes) - pole in contact with bile canaliculi: discharge of bile pigments & salts into gallbladder  II. Fluid Mosaic Model 5. Microdomains: “Lipid Rafts” Subdomains (local differentiation) in plasma membrane  Role: signal transduction  Contain: specific phospholipids (sphingomyelin – glycosphingolipids) & proteins (kinase-like signaling proteins)  Caveoli: lipid rafts at an invagination of plasma membrane  Contain: caveolin (protein)  Role: endocytosis  III. Specialization of Plasma Membrane  Specialized junctional regions  Function: - Adhesion (cell-cell & cell-matrix) - Intercellular transport & communication - Wound healing  Formed: in embryonic life & remain stable  Involve: SAM & CAM membrane proteins – cytoskeleton components  Proteins that function in adhesion  function in signaling III. Specialization of Plasma Membrane 1. Tight Junctions Narrowed / plugged intercellular space  Function: Cell-cell adhesion + plugs intercellular space  barrier to flow of materials between cells  selective absorption by intestine (selective permeability at apical pole)  Location: belt between the apical & lateral pole  Cell membranes of neighboring cells fuse  like 2 halves of a zipper  belt surrounds the cell  Protein family involved: occludins  Cytoskeleton component it’s associated with: actin filaments  determines cell shape  Tissues: epithelia (eg: intestine enterocyte) – seminiferous tubules (sertoli cells) – endothelial cells (blood-brain)  Control: dissociated by immune cells during an immune response to pass through epithelia III. Specialization of Plasma Membrane 2. Intermediate Junctions / Belt Desmosomes / “Zonula Adherens”  Function: adhesion of cells BUT: leaves intercellular space (doesn’t plug it)  Location: just below tight junction belt – encircles the cell  Protein: cadherins  Cytoskeleton component: actin filaments – linked to cadherin by catenin  dense thick zone (network of protein filaments)  Tissues: many, especially epithelia III. Specialization of Plasma Membrane 3. Spot Desmosomes “Macula Adherens” Punctual junctions  button-like attachment points  strongest junction  Location: everywhere between 2 neighboring cells  Function: connect intermediate filaments (keratin) by a 3D network  internal support  strength & resistance to pressure  Proteins: cadherins  Cytoskeleton component: intermediate filaments  interact with cadherins at a discoid dense region “cytoplasmic plaque” (anchorage proteins)  Tissues: all, abundant in those subjected to mechanical pressure: skin epithelium – uterus – cardiac muscle III. Specialization of Plasma Membrane 4. Hemidesmosomes Half-desmosomes  Location: basal pole of epithelia  Function: attachment of epithelia to basement membrane (laminins)  adhesion between epithelia & connective tissue  Proteins: integrins  Cytoskeleton component: intermediate filaments attach to plectin plaques III. Specialization of Plasma Membrane 5. Synaptic Junctions  Function: signal transduction  Location: contact point between axon & cell  Protein involved: cadherin  Function: transmission of action potential from presynaptic to postsynaptic 1. Action potential in presynaptic membrane 2. Change in presynaptic membrane permeability  release of neurotransmitters into synaptic cleft 3. Neurotransmitters bind to receptors in postsynaptic membrane 4. Change in postsynaptic membrane permeability 5. Postsynaptic message (action potential) III. Specialization of Plasma Membrane 6. Gap Junctions Pipline-like structures  Location: between neighboring cells  extend from the cytosol of 1 cell to that of the other  narrow intercellular space (tightened)  Proteins: connexins  Function: transportation: exchange of molecules between neighboring cells  Tissues: all except skeletal muscle & nerve cells III. Specialization of Plasma Membrane 6. Gap Junctions Structure:  Aqueous channels in clusters  1 channel = 1 connexon = 6 connexin subunits  Conexin: integral polytopic protein (4 transmembrane α-helices)  Hydrophilic sides at channel center (2nm diameter)  passage of any hydrophilic moledule [Ca2+] cytosol due to calcium-binding-proteins in SER Functions: - Fusion of secretory vesicles with cell membrane - Muscle contraction III. Endoplasmic Reticulum Roles 1. SER Functions d. Sequestering calcium ions Muscle contraction: 1. Action potential reaches muscle cell 2. Transverse tubules transport this potential to the SER 3. Calcium channels in SER open 4. Calcium released  binds troponin 5. Triggers interaction: troponin/tropomyosin/actin/myosin  contraction 6. Nerve impulse ceases (stops) 7. Channels close 8. Pumps transport calcium actively back into SER 9. Contraction stops III. Endoplasmic Reticulum Roles 1. SER Functions e. Other SER functions  Production & proliferation of peroxisome membrane  Production of HCl in stomach (for digestion)  III. Endoplasmic Reticulum Roles 2. RER Functions a. Synthesis of proteins by membrane-bound ribosomes Free & attached ribosomes: same structure, but give proteins of different destinations Free ribosomes Attached ribosomes cytosolic – peroxisomal – most integral proteins of plasma membrane & mitochondrial – most chloroplast – all the endomembrane system – Produce nucleoplasm - & extrinsic plasma proteins inside the endomembrane membrane proteins (cytosolic side) system – proteins secreted outside the cell After translation during translation Delivery (post-translational delivery) (co-translational delivery) Signal Address code Address code  III. Endoplasmic Reticulum Roles 2. RER Functions a. Synthesis of proteins by membrane-bound ribosomes Mechanism of attachment & insertion 1. Ribosome starts translation (initiation & early elongation) in cytosol 2. After 20-30 are polymerized: N-terminus from ribosome 3. N-terminus includes “signal peptide” (6-15 nonpolar aa)  attracts ribosome to RER membrane (or bacterial cell membrane) 4. Signal peptide is recognized by SRP (signal recognition particle) SRP = 6 protein subunits + 7S rRNA 5. SRP binds ribosome + signal peptide 6. Elongation stops temporarily  RER membrane (or bacterial cell membrane) 4. Signal peptide is recognized by SRP (signal recognition particle) = 6 protein III. Endoplasmic Reticulum Roles subunits + 7S rRNA 2. SRP Functions 5. RER binds ribosome + signal peptide a. Elongation of 6. Synthesis stops temporarily proteins by membrane-bound ribosomes 7. SRP binds to its receptor on RER membrane 8. SRP receptor is coupled to the translocon (protein aqueous channel)  ribosome binds it 9. SRP leaves the complex 10. Signal peptide binds to the inside of the translocon 11. Elongation continues 12. Growing peptide is gradually inserted into the lumen via translocon Actors are recycled for another use  III. Endoplasmic Reticulum Roles 2. RER Functions a. Synthesis of proteins by membrane-bound ribosomes Integral membrane proteins During insertion: a “stop-transfer sequence” (~15 hydrophobic aa) is recognized by a site in translocon  forms the helix that spans the lipid bilayer  insertion stops  sequence leaves the translocon by lateral opening  protein is in 3 compartments: RER lumen (N-terminus) – lipid bilayer – cytosol (C-terminus) ! inverted orientation (N in cytosol) is also possible  III. Endoplasmic Reticulum Roles 2. RER Functions a. Synthesis of proteins by membrane-bound ribosomes Maturation: Immediately after insertion, even before translation ends - Signal peptide removed by “signal peptidase” (enzyme attached to translocon on lumen side) - Carbs are added at different aa positions by “oligosaccharyl transferase” (integral protein in RER membrane) - Formation of disulfide bridges between certain cysteine residues by “protein disulfide isomerase” (PDI) - Protein folding into correct 2◦ & 3◦ structures Processing starts in RER & is completed in Golgi  III. Endoplasmic Reticulum Roles 2. RER Functions b. Glycosylation of proteins & maturation  Glycosylation = branching sugar motifs on proteins/lipids  For ~ all proteins by attached ribosomes  Necessary for protein maturation & activity  An oligosaccharide is added to specific aa at specific positions RER Golgi Added to … group Amino (N) OH Amino acid Asparagine Serine / threonine Name N-glycosylation O-glycosylation  III. Endoplasmic Reticulum Roles 2. RER Functions b. Glycosylation of proteins & maturation 1. Monosaccharides are supplied by donors (nucleotides) to acceptor (dolichol phosphate) 2. “Glycosyl-transferase” (RER integral membrane protein) builds the oligosaccharide on dolichol 3. “Oligosaccharyl-transferase” (RER integral membrane protein) transfers the oligosaccharide to the growing peptide  III. Endoplasmic Reticulum Roles 2. RER Functions c. Protein folding  Parallel to glycosylation  To adopt 2◦ & 3◦ structures  Monitored by: chaperones (calnexin & calreticulin)  If misfolded: other proteins bind it, unfold it, and give it a 2nd chance for correct folding  Repeated many times  If failed  rejected through translocon into cytosol  degraded by proteasome  III. Endoplasmic Reticulum Roles 2. RER Functions d. Biosynthesis of lipids and proliferation of membranes Membrane synthesis: not de novo , but addition of components to preexisting membranes  increase in area - Most membrane lipids: made fully in RER & SER - Sphingomyelin & glycolipids: synthesis starts in RER, is completed in Golgi - Some mitochondrial & chloroplast membrane lipids: synthesis in situ (by organelle enzymes) Lipid-synthesizing enzymes: integral proteins of RER - 1st: lipid inserted in cytosolic layer - 2nd: moved to luminal layer by flipase - Exported: by transport proteins to other cell membranes (some as walls of vesicle  III. Endoplasmic Reticulum Roles 2. RER Functions e. Addition of lipid molecules to proteins Glypation: covalent bonding of GPI (glycosyl phosphatidyl insositol) to peripheral protein that’s covalently linked to a membrane Myristoylation: … of myristic acid (f.a) Palmitoylation: … of palmitic acid (f.a) Isoprenylation: … of isoprene (non-f.a lipid)  III. Endoplasmic Reticulum Roles 2. RER Functions f. Export of vesicles to Golgi apparatus Components in RER pass between cisternae (interconnected)  small vesicles  fuse to larger vesicles & sacs  cis Golgi network  matured and sorted  leave to destinations ! No ribosomes in this region – transitional elements ! RER receives vesicles from Golgi (retrograde movement) to compensate the lost membrane fragments  Chapter 7 Golgi Complex  I. General Structure Golgi complex = Golgi apparatus = Golgi body  Named after its discoverer (Camillo Golgi)  Made of: many cisternae (flat, disk-like, dilated rims) + vesicles + tubules  Location: toward cell center (near centrioles), between RER & cell membrane  Structure: curved – resemble shallow bowl – arranged by microtubules  Dictyosome = stack of cisternae - Nb of cisternae per dictyosome in plants > animals - Nb of dictyosomes varies by cell type & physiology - Golgi is extensive in cells actively secreting proteins & carbs  I. General Structure Dictyosome is polarized: (diff functions, composition, & membrane thickness) - Cis-forming face (concave): oriented to ER - Middle - Trans-maturing face (convex): oriented to cell membrane Tubular networks: - CGN (cis-golgi network): sorts molecules to be sent back to RER from those to continue to cis - TGN (trans-golgi network): sorts proteins into different vesicles to be sent to different cell compartments ! Golgi is not static  continuous state of flux  vesicles are added to cis & detached from trans & edges  II. Functions  Maturation & sorting of proteins, glycoproteins, & carbs  Maturation of glycolipids Maturation examples:  Trimming by proteolytic enzymes (inactive  active polypeptide)  Modification of N-glycosylation: adding/removing monosaccharides – phosphorylating mannose  Hydroxylation at lysine by hydroxylases  Sulfation by sulfotransferases  O-glycosylation  II. Functions 1. Golgi complex roles in glycosylation  Protein glycosylation: Glycosylation starts in RER & continues in Golgi, by specific enzymes Removes & replaces some N-linked monosaccharides O-glycosylation only in Golgi  essential for protein maturation / folding / sorting / targeting  Lipid glycosylation: Ex: glycolipids for blood group: Produced in Golgi  transported as parts of secretory vesicles  integrated in cell membrane  II. Functions 2. Polysaccharides synthesis Most carbs are made in Golgi. Examples: GAG of ECM – hemicellulose & pectin of cell wall During plant cell division: Golgi is extensive: 1. Synthesizes cell wall components (hemicellulose & pectin) 2. Sorts them in vesicles 3. Vesicles migrate via microtubules of the phragmoplast to the equatorial plate 4. Vesicles fuse to form a plate 5. Plate grows toward old cell wall 6. Edges fuse with old cell all  daughter cells separated Cellulose is synthesized in situ by “cellulose synthase” (enzyme of cell membrane)  II. Functions 3. Sorting Process  Golgi is extensive in cells performing active secretion  It matures, sorts, and packs secretory products + makes lysosomes  Different vesicle types  different protein coats (coats interact with target membrane components) COP-II coated: vesicles from RER to Golgi COP-I coated: vesicles sent back from Golgi to RER (retrograde movement) + vesicles within Golgi Clathrin-coated: target lysosome + plant vacuole enzymes  ! Retrograde movement: Golgi to RER / cell membrane to Golgi  II. Functions 4. Proliferation of cell membranes By releasing vesicles from trans face & edges of middle cisternae  Vesicles fuse with plasma membrane  Increase its area  Proliferation Chapter 8 Lysosomes  I. General Description  Size: small  Shape: spherical/oval – dense in EM  Membrane: 1 lipid bilayer + contains acid phosphatase (marker)  Contains: hydrolytic enzymes  Functions: intracellular digestion – sorting  defense, nutrition, organelles renewal - Animal cells: have lysosomes - Plant cells: no lysosomes, but: plant vacuoles (regulate osmotic pressure – some intracellular digestion – storage) - Bacteria: no lysosomes/vacuoles, except cyanobacteria (gas vacuole) But: they have hydrolases in periplasmic space (between cell membrane & cell wall)  I. General Description Lysosomal enzymes  Location: lumen + membrane  Types: 50 different types  Function: hydrolyze ~all biomolecules (proteins/lipids/nucleic acids/carbs)  Optimal pH: acidic “acid hydrolases”  Lysosomal lumen pH ~5  By: ATP-dependent proton pumps: pump H+ into lumen  Aim: protection of the cell: if hydrolases leak to cytoplasm  inactivated at cytosolic pH = 7  How is the luminal phase of lysosome membrane protected against hydrolases? By its high glycosylation state  I. General Description Substrates to be digested  In endosomes / phagosomes or: in cytosol entering by permeases  Digested into: small molecules  Pass into: the cytosol by: active/passive transport ! Digestion may be incomplete  produces wastes  expelled by exocytosis  I. General Description 1. Structure & Forms  Heterogeneous  different sizes & shapes  Number: varies by cell physiology  Membrane: 1 lipid bilayer – similar to cell membrane – less cholesterol  Integral membrane proteins: 1. ATPase proton pumps: transport H+ into lumen  acidic pH (5) 2. Permeases: - entry of substrates cytosollumen for digestion - export of digestion products lumencytosol 3. Enzymatic glycoproteins: e.g. acid phosphatase 4. Non-enzymatic glycoproteins: e.g. “lysosome-associated membrane proteins” lamp1 & lamp2. Function: lysosome genesis  I. General Description 1. Structure & Forms  Lysosome types a. Primary Lysosomes “protolysosomes” origin: TGN Small – 1 membrane – digestive enzymes not yet digesting b. Secondary Lysosomes “digestive vacuoles” = fusion of: ≥1 1◦ lysosome + vesicles containing substrates for digestion c. Residual bodies & lipofuscin pigment granules - residual bodies: Larger than 1◦ – irregular shape – contain wastes from incomplete digestion – expelled by exocytosis - Lifofuscin pigment granules: accumulated (kept) residual bodies in long-lived cells (eg: neurons) – brown-green pigment – their accumulation disturbs cell metabolism  I. General Description Secondary lysosomes origin Outside the cell Cell itself heterophagosome / Autophagosome / name of vesicle heterophagic vacuole autophagic vacuole name after Heterolysosome / Autophagosome / fusion heterophagolysosome autophagolysosome e.g. pathogens contain Organelle to be destroyed (virus/bacteria…) process phagocytosis macroautophagocytosis Organelle turnover function e.g. defense (destruction & replacement)  I. General Description Secondary lysosomes * Microautophagocytosis: for molecules (eg: peptides released by proteasomes)  enter lysosome via permeases * Organelle turnover is common during: cell growth – tissue repair – cell differentiation/dedifferentiation – metamorphosis – uterus size reduction after delivery – nutrient deficiency – endocrine glands controlling intracellular hormone levels  according to metabolic state & function: cell adjusts its organelle content * Substrates are totally or partially recycled  products are released to cytosol by active/passive transport **Some cells secrete lysosomal enzymes: macrophages – eosinophils …  I. General Description 2. Origin of lysosomes 1. In RER: Enzymes synthesized by attached ribosomes  maturation 2. In Golgi: TGN receptors recognize “address code”: mannose-6-phosphate  packed in clathrin-coated vesicles 3. In cytosol: clathrin coat is removed to become primary lysosome  mannose-6-phosphate receptors return to TGN II. Plant Vacuoles “Phytolysosomes” Plant cells: rigid cell wall  no endocytosis/phagocytosis  no lysosomes - Dividing plant cells (meristems): many small vacuoles - Mature cell (stops dividing): they fuse into 1 large vacuole  90% of cell volume Membrane: 1: tonoplast, containing pumps Transportation into vacuole: via pumps or intravacuolar pinocytosis [ions]vacuole > [ions]cytosol  Hypertonic  water enters vacuole (osmosis)  turgor pressure  functions: – mechanical support for soft tissues – stretches cell wall during cell growth II. Plant Vacuoles “Phytolysosomes” pH: acidic due to proton pumps other functions: - temporary storage of most molecules - storage of toxic compounds for defense against pests - storage of unwanted molecules (no exocytosis)  leaves fall to eliminated unwanted molecules - intracellular digestion by acid hydrolases synthesis: RER attached ribosomes  Golgi maturation  Chapter 9 Peroxisomes (Microbodies)  I. Introduction to Peroxisomes 1. Structure Properties Peroxisomes are like lysosomes in size & basic structure ONLY.  Functions: oxidation reactions – sorting – detoxification of toxic substances  Present in: animals & plants – NOT in bacteria  Abundant in: liver – kidney – many plant cell types *abundance depends on cell activity  Structure: - Membrane poor in cholesterol - Crystalline core of oxidative enzymes (depends on species) - Can be interconnected by thin canaliculi - Independent of endomembrane system  I. Introduction to Peroxisomes 1. Structure Properties  Proliferation: by fission (like mitochondria)  Origin:  Proteins not from TGN, not from attached ribosomes in RER  From free ribosomes  delivered to peroxisomes via “address code”  Most proteins are not glycosylated  I. Introduction to Peroxisomes 2. Peroxisome Function “Peroxisome” = synthesis & degradation of hydrogen peroxide (H2O2) = very toxic, highly reactive oxidizing agent > 50 enzyme types+ peroxins: for peroxisome biogenesis Enzymes:  catalase: most abundant: 𝒄𝒂𝒕𝒂𝒍𝒂𝒔𝒆 Converts excess H2O2 into water & oxygen: 2H2O2 2H2O + O2 Uses H2O2 produced by other enzymes to oxidize xenobiotics: 𝒄𝒂𝒕𝒂𝒍𝒂𝒔𝒆 “peroxidative reaction” H2O2 + XH2 X + 2 H 2O ! important in liver & kidneys  for toxins entering bloodstream  I. Introduction to Peroxisomes 2. Peroxisome Function Other enzymes:  aa-oxidase  urate oxidase (uricase)  peroxidase  f.a oxidation (β-oxidation) enzymes  glycolate oxidase: photorespiration in leaves  alanine/glyoxylate aminotransferase: photorespiration in leaves * enzymes vary by species & cell type. Eg: - hepatocyte: has enzymes that oxidize cholesterol  bile acids - primates: have no uricase  I. Introduction to Peroxisomes 2. Peroxisome Function Other functions: Synthesis of “plasmogens”: in brain special lipids, linking f.a + glycerol by ether bond (not ester) Enzyme “luciferase” in firefly: 𝑙𝑢𝑐𝑖𝑓𝑒𝑟𝑎𝑠𝑒 transforms luciferin luminescent product  insect emits light.  II. Glyoxysomes  Special form of peroxisomes  In plant seeds + seedlings  Enzymes: the above + glyoxylate cycle enzymes + Krebs cycle enzymes  Function: gluconeogenesis: glucose synthesis from lipids  Provide energy for germination, since photosynthesis has not yet started  Reactions: 𝑜𝑥𝑖𝑑𝑖𝑧𝑒𝑑 𝑜𝑥𝑎𝑙𝑜𝑎𝑐𝑒𝑡𝑎𝑡𝑒 𝑔𝑙𝑦𝑜𝑥𝑦𝑙𝑎𝑡𝑒 𝑐𝑦𝑐𝑙𝑒 f.a acetyl-coA citric acid glucose Chapter 10 Mitochondria  I. General Definition  Size: large  visible by LM. BUT ultrastructure: visible by EM  Present in eukaryotes – not in prokaryotes  In humans: mitochondria are maternal (mitochondria of sperm does not enter the oocyte) BUT: not 100% (contains proteins encoded by nucleus)  Variable: size – number – structure  Between: organisms – tissues – physiological state in same cell type  Low nb  large size, high nb  small size  Nb per cell: 1, 100s, or 1000s Nb in actively dividing cell > cell blocked at interphase Nb in moving cell > immobile cell Nb in cell cultured in O2-rich medium > in absence of O2  I. General Definition  Abundant in liver & muscle  Abundant in metabolically active regions In epithelia: near apical surface (active transport of nutrients) In sperm: base of flagellum (movement) In muscle: parallel to contractile fibrils (contraction)  Movement: by motor proteins on microtubules – slowed during mitosis  Lifespan: ½ life = several days – degraded by autophagy  Proliferation: by fission: doubles its content (import of proteins & lipids – DNA replication)  increases in size  divides into 2 smaller mitochondria  I. General Definition  Structure: 2 membranes (different areas – functions – properties) Matrix (organic & inorganic compounds)  Functions: Oxidation of organic compounds  CO2 + H2O Aerobic cell respiration “oxidative phosphorylation”  ATP (energy) ! Prokaryotes: no mitochondria, but have enzymes in cytoplasm & membrane e.g. ATP synthase in inner face of cell membrane  II. Structure  Most common shape: ovoid  Other shapes: filamentous (tubular)  network / spiral  4 sub-compartments: OMM – IMM – intermembrane space – matrix 1. Mitochondrial Membranes - Intermembrane space: narrow but expanded during respiration contains: enzymes – H+ – cytochrome C  II. Structure 1. Mitochondrial Membranes - Outer Mitochondrial Membrane (OMM): Thinner Less surface area (no cristae) High permeability (due to porins) 50% proteins in weight Proteins: aa-oxidation – f.a elongation – porins/channels – channels to import mitochondrial proteins made by free ribosomes – proteins to import cholesterol – cytochrome b5 – NADH-cytochrome-b-reductase – phosphatidase – phosphatase – phospholipase  II. Structure 1. Mitochondrial Membranes - Inner Mitochondrial Membrane (IMM): Thicker Bigger surface area  has cristae (inward folds)  their density varies by tissue & aerobic activity Restricted permeability >75% proteins in weight No cholesterol Has special phospholipid: cardiolipin = 2 P + 3 glycerol + 4 f.a(only in mitochondria) Low fluidity Proteins: ATP synthase + respiratory chain + prots controlling cytosolic Ca2+ +transporters (export ATP – import ADP&P) + transporters (pyruvate & H+) + Mitochondrial attached ribosomes  II. Structure 1. Mitochondrial Membranes * Respiratory chain:  4 enzyme complexes  Metabolic not physical chain (not covalently bound)  Act sequentially  Proteins: cytochromes (hemoproteins) & flavoprotein (FAD+proteins) for oxidation-reduction reactions  Function: oxidation of coenzymes reduced by glycolysis, Krebs cycle, β- oxidation + transfers e-s from coenzymes to O2 (final acceptor)  generates proton gradient  II. Structure 1. Mitochondrial Membranes ATP synthase = F0-F1 complex:  globular protein  several subunits  inner face of IMM  coupled to integral protein channel  II. Structure 1. Mitochondrial Membranes - Mitochondrial lipids: Synthesized by RER enzymes  transported to IMM & OMM ! No vesicles (Mitochondria & RER are in proximity) Some: produced/modified by OMM enzymes  II. Structure 2. Mitochondrial Matrix  Gel-like = rich in proteins, organic & inorganic compounds  Several circular DNA  ribosomes  Enzymes for DNA replication & gene expression  Enzymes for oxidation of organic compounds – Krebs cycle – β oxidation  Dense inclusions (Ca2+ - Mg2+ – ADP – phosphate)  Proteins: 13 are encoded by mitochondrial DNA All others: encoded by nuclear DNA  translation by cytosolic free ribosomes  delivered after translation  II. Structure 3. Import of Proteins 1. Peptides synthesized by free ribosomes in cytosol 2. Post-translational delivery to mitochondria Address code: “presequence” or “targeting sequence” near N-terminus ( (+) charged aa) 3. Translocation into mitochondria by presequence + chaperones HSP through: TOM in OMM and TIM in IMM (multisubunit channels) 4. Presequence is removed by mitochondrial protease  III. Mitochondrial Functions 1. Definition of oxidation & its general description Oxidation = loss of e-s & protons (H+) by organic compounds e-s & H+ pathway: organic compounds  coenzymes (NAD / FAD)  respiratory chain proteins  O2 (final acceptor)  becomes H2O During respiratory chain: H+ is pumped outside IMM  proton gradient (more H+ in intermembrane space)  used by ATP synthase to produce ATP “Oxidative Phosphorylation”: Oxidation supplies the energy to phosphorylate ADPATP Full oxidation: Organic compound  CO2 + H2O + energy Anaerobic conditions: no oxygen  no respiration  Fermentation  incomplete oxidation  produces organic compounds (lactate – ethanol)  less energy produced  III. Mitochondrial Functions 2. Carbohydrate oxidation 1. Digestion: Polysaccharides  monosaccarides in intestine.  energy released 2. Delivery: monosaccharides (mainly glucose) to cells 3. In cytosol: glycolysis: 10 reactions: glucose (6C)  2 pyruvate (3C) + release of energy 4. Pyruvate enters mitochondrial matrix − 𝐶𝑂2 + 𝑐𝑜𝐴 5. Pyruvate acetyl acetyl coA 6. Krebs cycle (TCA cycle): 8 reactions: Acetyl coA  CO2 + reduction of coenzymes: FAD + NAD Protein catabolism: - 13 of 20 aa are oxidized into acetyl-coA - others degraded into metabolites of Krebs cycle  III. Mitochondrial Functions 3. Fatty acids oxidation  Acetyl-coA origins: glycolysis – f.a oxidation – aa oxidation – glycerol  f.a oxidation = β oxidation: in mitochondrial matrix & in peroxisome 1. f.a is degraded into many acetyl coA + e-s & H+ 2. e-s & H+ reduce coenzymes NAD 3. Acetyl-coA enters Krebs cycle  more energy 4. All reduced coenzymes are reoxidized by respiratory chain … O2… H+ gradient… ATP synthesis ! Acetyl-coA is an intermediate metabolite in most anabolic & catabolic pathways  III. Mitochondrial Functions 4. Other mitochondrial functions Cooperation with SER: - Synthesis of steroid hormones: + 𝑂𝐻 in IMM: cytochrome P450: cholesterol pregnenolone  used by SER/mitoch to make steroids - Synthesis of some phospholipids for membranes / lipoprotein particles Synthesis of non-essential aa: from Krebs cycle metabolites (precursors of aa) Storage of ions (Ca2+, Na+, K+…) / organic compounds (lipoproteins) Chapter 11 Plastids I. General Description of Plastids  Family of organelles (many types)  Cells: higher plants & algae  Structure: 2 membranes that enclose a stroma: several circular DNA – ribosomes – enzymes for DNA replication & gene expression (transcription – translation) – membrane-bound sacs (have photosynthetic chain & light- absorbing pigments e.g. chlorophyll)  Visible by: LM, but ultrastructure by EM  Proliferation: division after doubling their content  Proplastid Etioplast Leucoplast Chromoplast Small, 2 membranes: Enlarged Shape inner one origin of Ellipsoid Elongate irregular proplastid thylakoid membrane Meristems (young & Plant leaves that internal tissue Found in Roots – petals – fruits dividing plant cells) grow in the dark + epidermis Carotenoids (as lipid droplets in Protochlorophyll stroma) yellow / red / orange (yellow - Pigment Colorless - E.g. β-carotene in carrot roots chlorophyll (orange) – lycopene (a terpene) in precursor) tomato primitive thylakoids primary thylakoids (irregular (prolamellar body Thylakoids - - membranous + flattened structures) vesicles) into chloroplasts if into other plastid Differentiation suitable light - - types intensity I. General Description of Plastids Amyloplast: example of leucoplast:  In storing tissues (roots)  Function: storage of starch granules in stroma as concentric layers response to gravity  growth direction  Ultrastructure: destroyed by starch – only outer membrane left Outer membrane is also destroyed sometimes by bigger granules e.g. potato  Other less common plastids: elaioplast (stores oil) – proteoplast (stores proteins)  II. Chloroplast Structure  Found in: mosses (bryophytes) – algae – higher plants: green tissues (abundant in leaf mesophyll)  Pigments: rich in chlorophyll (green) + others  Variable: size – shape – number  By: species – cell type – physiological condition (e.g. light intensity)  Higher plants: lens-shaped, many chloroplasts – Algae: irregular shape, 1 or few  Vascular (flowering plants): more chloroplasts  Distribution in cytoplasm: uneven  Movement: due to flow of cytoplasm: Brownian movement (cyclose)  II. Chloroplast Structure 1. Chloroplast Membranes  2 membranes: OCM & ICM  Similar thickness, different functions & properties  Rich in galactolipids  Minor % of proteins in membranes  most in stroma & thylakoids OCM: has porins (permeable) + other proteins ICM: far less permeable  regulates transport between cytoplasm & stroma – has diverse transporters  II. Chloroplast Structure 2. Stroma = lamella suspended in granular fluid matrix Variation: in Structure – organization – density Depending on: species – cell type – physiological condition 6 sub-compartments: - OCM - ICM - intermembrane space - stroma - thylakoid membrane - thylakoid lumen  II. Chloroplast Structure 2. Stroma Contains: Starch granules: polymer of glucose produced by photosynthesis Lipid droplets + proteins  plastoglubules (functions since thylakoid biogenesis  senescence) Several circular DNA + enzymes for DNA replication & gene expression e.g. ribosomes & tRNA Rubisco: (ribulose biphosphate carboxylase) the most abundant enzyme on earth. Fixes CO2 for photosynthesis Proteins: linked by titin to Z line -> interact with actin in a higher order than other myosins  I. Cytoskeleton Components 1. Microtubule-Organizing Centers “MTOCs”  Centrosomes & basal bodies  Plant: no centrosome, but have MTOC proteins in nuclear envelope & cell cortex  Have γ-tubulin  Function: MT nucleation (initiate polymerization) Control MT nb, length, polarity, nb of protofilaments Control time & location of assembly  Same structure & components  regenerate each other E.g. in spermatid: centriole  basal body  MTs of cilia & flagella (internal support + movement)  I. Cytoskeleton Components 3. Microtubule-Organizing Centers “MTOCs” a. Centrosome  Centrosome = 2 centrioles at 90◦ wrt each other  Location: near cell center  Dimensions of centriole: diameter = 0.2μm, length = 0.4 μm  No membrane, but enveloped by pericentriolar material (dense amorphous matter of proteins)  Structure: γ-tubulin + 9 triplets of MTs + appendages (radially arranged)  I. Cytoskeleton Components 3. Microtubule-Organizing Centers “MTOCs” a. Centrosome  MT triplets: A: central MT – complete – connected to center by radial spokes B: incomplete – shares protofilaments with A C: incomplete – shares protofilaments with B  Tubulins: α, β, γ, ε  γ-tubulin: Location: in the core / in pericentriolar material Function: MT nucleation: provides an open-ring template to polymerize the 1st row of α-β dimers  I. Cytoskeleton Components 3. Microtubule-Organizing Centers “MTOCs” a. Centrosome  Function: o During G1S transition: Centrioles duplicate. Nucleation (2 new centrioles at 90◦ wrt old parent ones) in pericentriolar region, then elongation o During prophase: 1. nucleation of MTs  asters 2. elongation of MTs  from centriole to periphery  (-) at centriole, (+) at periphery 3. migration to cortex  generate spindle of division  I. Cytoskeleton Components 3. Microtubule-Organizing Centers “MTOCs” b. Basal Bodies  At basis of cilia & flagella  Same cross-section as centrioles (9 triplets of MTs, interconnected, connected to center)  Only some MTs from basal body extend to cilia & flagella  form axoneme  I. Cytoskeleton Components 4. Cilia & Flagella a. Structure  2 versions of the same organelle  Membrane-enclosed – on cell surface – motile  Cell: some eukaryotes. e.g. in most spermatozoa & protozoa – some algae Eukaryotic flagella: covered by plasma membrane Prokaryotic: not covered  naked spiral filament  same monomer: flagellin  I. Cytoskeleton Components 4. Cilia & Flagella a. Structure Cilia Flagella Number numerous Few, usually 1 Length Shorter 5-10 μm Longer 150 μm Diameter 0.2 μm Function Cell movement / particle Cell movement movement on cell surface location All around / apical surface  I. Cytoskeleton Components 4. Cilia & Flagella a. Structure Central core “axonemme” + covered by extension of plasma membrane  Axonemme MTs: 9 peripheral doublets + 2 central singlets  “9+2” arrangement doublets: A (complete) & B (incomplete) singlets: both complete – interconnected – enclosed in central sheath connections: - radial spokes: connect “A” MTs to central sheath - nexin: elastic protein – makes periodical bridges – connects MT “A” of a doublet to MT “B” of an adjacent doublet - dynein arms: motors for movement – periodical projections from “A” to “B” of an adjacent doublet  I. Cytoskeleton Components 4. Cilia & Flagella a. Structure  Polarity: same for all MTs: (-) at base (+) at periphery  Basal body: Structure same as centrioles  9 triplets MT A & B from the triplets prolonged to make doublets of axoneme ! Can regenerate cilia & flagella if they’re broken  I. Cytoskeleton Components 4. Cilia & Flagella b. Modes of Movement Cilia  Like oars: coordinated & synchronized  Push against the medium  cell moves in a direction perpendicular to cilia axis  Beating in 2 phases: 1) Rigid, pushing opposite to direction of motion 2) Flexible, recover to initial position Flagella  Diverse beating patterns (wave, spiral…) according to cell type  I. Cytoskeleton Components 4. Cilia & Flagella b. Modes of Movement Motion (in both) is generated by axoneme & requires ATP  even if isolated, cilia/flagella continue beating if supplied by ATP  locomotion resides within the axoneme Dynein hydrolyze ATP  mechanical energy Mechanism: Central MTs rotate  dyneins hit radial spokes  activated  move (limited) on doublet MTs by conformational change  doublets slide on each other in alternation  spiral beating  II. Cytoskeleton Functions 1. Structural support –> determines & maintains cell shape e.g. flat cell = radially arranged MTs 2. Resistance against mechanical pressure 3. Determine specific features of membrane e.g. microvilli (actin) 4. Movement of organelles, vesicles, molecules (on MTs as rails for motor proteins) 5. Positioning of organelles (by MTs) 6. Cell movement by crawling (pseudopods/lamellipods) & by propelling (cilia & flagella) only in certain cell types 7. Form axon of nerve cell (MTs, thin & intermediate filaments) 8. Muscle contraction  organism movement (thin & thick filaments) 9. Mitosis: anaphase: chromosome migration by spindle fibers (MTs) 10. Mitosis: cytokinesis: contractile ring by actin filaments & myosin  III. Plant Cell Cytoskeleton  Plant: no intermediate filaments – no centrosome But: has MTOC proteins in cytosolic face of nuclear envelope & cell cortex  MTs & actin filaments: Determine cell shape & cell wall growth Intercellular transport e.g. chromosome migration Pollen tube formation & movement of nuclei for fertilization (actin)  III. Plant Cell Cytoskeleton MTs distribution during different cell stages: 1. Interphase: - Location: in cortex under cell envelope - Orientation: horizontal transverse - Function: drive cellulose-synthesizing enzymes of plasma membrane during synthesis - Produced cellulose becomes parallel to MTs  rope-like transverse ties  resist turgor pressure laterally  plant cell grows only longitudinally, not laterally -  MTs determine cell shape indirectly  III. Plant Cell Cytoskeleton MTs distribution during different cell stages: 2. Early prophase: all MTs disappear except a bundle  encircles equatorial region  future plane of division  “preprophase band” 3. Metaphase: all MTs disappear except spindle of division  segregates chromosomes 4. Telophase: - mitotic spindle disassembles - MTs of “phragmoplast” drive vesicles (contain cell wall components) from TGN to equatorial plate  vesicles fuse  new cell wall between daughter cells  Chapter 13 Organization of the Nucleus  I. Role of the Nucleus - Stores & organizes DNA (genetic material) -  contains all program for cell life & organelle life - Controls all physiological processes in a cell Cloning Take nucleus from a cell + insert into oocyte whose nucleus is removed  evolves into a whole individual Conclusion: nucleus contains all genes necessary for development into adult organism  I. Role of the Nucleus Gene expression  Different tissues: have the same genes, but each cell type expresses only the genes necessary for its function  Housekeeping genes: expressed in al cell types Merotomi experiment Cut the cell  fragment that contains nucleus survives, the other fragment degrades Conclusion: nucleus is indispensable for cell metabolism & life ! Nucleus is not independent: e.g. transcription in nucleus, but translation in cytosol  II. Structural Overview Nuclear envelope  Belongs to endomembrane system  2 membranes - Lipid bilayer – fluid mosaic model - Different properties & lipid/protein ratios - Merge at specific points: nuclear pores  controlled transport  Intermembrane space: narrow – “perinuclear space” – stores Ca2+  Role: barrier for ions / solutes / macromolecules  Outer membrane: continuous with RER membrane – may have attached ribosomes (cytoplasmic)  II. Structural Overview Nuclear proteins  Free ribosomes (cytoplasm)  nucleus.  Signal sequence: NLS (nuclear localization signal): basic (+) aa Nuclear lamina / lamina densa  Location: inner face of inner membrane  Role: supports nuclear envelope – site of attachment of chromatin  Made of: intermediate filaments “lamins”  Disassembled: before cell division, phosphorylation Cage of intermediate filaments  Location: around nucleus  Role: external support & localization  II. Structural Overview Nucleoplasm Viscous amorphous mass. Contains: 1. Chromatin: uncondensed chromosomes. Seem tangled, but occupy specific positions 2. Nuclear matrix: fibrillar 3D network of proteins. Functions: Skeleton: determines nuclear shape (oval-spherical) Chromatin distribution & localization RNA movement & maturation Anchorage of transcription & replication machinery Non-histone proteins: form scaffold  determines chromosome size & shape ! disassembles when chromatin is condensed into chromosome 3. Nucleoli: ≥1 , oval dense region Role: rRNA production + assembly with ribosomal proteins  large & small subunits  III. DNA / Chromosomes / Chromatin 1. Chromosome = “colored body” appears during cell division  1 chromosome = 1 DNA molecule + many proteins (e.g. histones) + some RNA as RNP  Visible by LM during division  Differ in size & shape  Structure: Linear – 1 centromere (primary constriction) – 2 telomeres (ends) 𝑝 2 arms: p (short) & q (long)  centromeric index: 𝐼𝑐 = 𝑝+𝑞 shows centromere position Centromeres: specific repeated sequences  bind kinetochore  binds MTs of spindle of division Telomeres: specific sequences  recognized by telomerase  lengthens chromosome at each cell cycle  compensates shortening due to lagging strand (incomplete replication) III. DNA / Chromosomes / Chromatin Number of chromosomes:  Differs between organisms  Some organisms have the same nb of chromosomes but NOT the same genes (different primary structure)  Chromosome nb is NOT related to complexity of species  SINCE: majority of DNA is non-coding sequences  Eukaryotes: diploid = pairs = 2 homologous copies: paternal & maternal  Homologous = same size, shape, centromere position, banding pattern, same genes & same loci, but different alleles  Autosomes: n-1 pairs + Sex chromosomes: 1 pair  Humans: 22 autosomes & 1 XX/XY III. DNA / Chromosomes / Chromatin Metaphase chromosomes: Highest condensation state of DNA  Inactive (no transcription/replication) 2 chromatids, identical due to DNA replication, joined by cohesin (protein) End of Anaphase chromosomes: 1 chromatid only III. DNA / Chromosomes / Chromatin Karyotyping: 1. Block cells in metaphase using drugs that prevent MT polymerization (e.g. colchicine) 2. Extract chromosomes 3. Stain chromosomes with dyes 4. Observe & photograph 5. Classify them in pairs Aim: look for abnormalities (monosomy, trisomy, deletion, translocation) III. DNA / Chromosomes / Chromatin 2. Chromatin Chromatin = DNA during interphase = from partial uncondensation of chromatid in telophase Filaments – seem tangled – occupy specific locations in nucleus Euchromatin Heterochromatin stain light dark condensation mild high contains expressed inactive genes + noncoding DNA genes of centromeres & telomeres types - Constitutive: permanent Facultative: transient  IV. Levels of DNA Condensation Length of the 46 DNA molecules in 1 cell = 2 meters !  Cannot fit inside the cell  Needs folding & condensation Highest condensation: metaphase chromosomes Lowest condensation: euchromatin expressed genes (interphase) - Proteins guiding condensation: histones & non-histones  Histones: 5 types: H1, H2A, H2B, H3, H4 Similar in ~ all eukaryotes Encoded by repeated genes Expressed in S phase only Rich in (+) aa  binds the (-) DNA  IV. Levels of DNA Condensation 1. Beads on a string / chromatin fibers / nucleofilament: winding of DNA around nucleosome core (146 bp/turn) *Nucleosome = octamer (8) histones: 2 H2A, 2 H2B, 2 H3, 2 H4 *DNA linker = DNA separating 2 nucleosomes  2-3 fold reduction of DNA length 2. Solenoid shape: H1 binds DNA at nucleosome  H1 histones interact, & consecutive nucleosomes interact  stabilize 2nd condensation  6 nucleosomes / turn  40-50 fold length reduction 1. Beads 2. IV. Levels Solenoid of DNA Condensation 3. Non-histone (chromosomal) proteins: chromosomal proteins form a scaffold  chromatin are looped & attached (at regions unoccupied by nucleosomes)  scaffold determines chromosomes size & shape  chromatid. Prokaryotes & viruses: also condense DNA, by other proteins (NOT histones) Plasmid DNA: supercoiled around itself Mitochondrial & plastid DNA: coiled like plasmid  V. Nucleolus  Dense structures in nucleus  No membrane  Contains rNRA & proteins  Visible by: LM & EM  Disappear: during cell division. Reappear: late telophase  Arise from: NOR “Nucleolar Organizing Regions”: rDNA = rRNA-encoding genes *rDNA: repeated sequence encoding 28S, 18S, 5.8S rRNA *Human rDNA: on 5 pairs of chromosomes  10 loci  10 small nucleoli  merge into 1 after telophase  V. Nucleolus Function: Active transcription of rDNA Maturation of RNA  rRNA Assembly of rRNA + proteins  small & large subunits Zones: FC: fibrillar centers: the rDNA DFC: dense fibrillar components: surround FC, actively transcribes DNA  rRNA precursor G: granular region: rRNA mature & assemble with proteins ! Small & large subunits are exported independenty to cytosol.  VI. Nuclear Pores Nuclear envelope functions:  Delimit the nucleoplasm  Barrier to components between nucleus & cytosol  Allows exchange of specific molecules: - enter nucleus: ribosome subunits (after synthesis in cytosol) to assemble with rRNA in nucleolus - leave nucleus: mature tRNA & mRNA - both directions: snRNP (for RNA maturation)  VI. Nuclear Pores NPC “nuclear pore complex” Pores where the 2 nuclear membranes fuse together  Size: large enough to pass the large ribosomal subunit  Location: in clusters, especially facing euchromatin  Structure: basket-like apparatus  Number: variable according to cell type & physiology  Function: strict control of passing molecules – transport in both directions  Proteins: 50 types “nucleoporins”  VI. Nuclear Pores NPC “nuclear pore complex” Structure: Nucleoporins in symmetrical octamers  form 2 rings: cytosolic & nuclear sides Radial spokes from rings  central plug transporter in pore center Another smaller ring deep in nucleus  basket shape Cytoplasmic filaments project from cytosolic ring  cytoplasm Transport: Small molecules: diffuse through basket slots Large molecules: active transport by the central transporter (active  energy  GTP) / exportins Proteins destined to the nucleus: (+) charged aa at C  recognized by importins Chapter 14 The Cell Cycle  I. Overview Cell cycle = series of biological events that are repeated = interphase (G1-S-G2) + Mitosis (prophase-metaphase-anaphase-telophase) Duration:  varies by organism – tissue – age  Generally: mitosi