BIOL1025 I

• Format and content of the lectures on Cell Structure over the next 3 weeks


  • Cell membranes
  • Endomembrane system – Endoplasmic reticulum and Golgi
  • Cell components – mitochondria, nucleus, lysosomes, peroxisomes, ribosomes
  • Cytoskeleton
• Cell Membranes


  • Membranes are essential for living cells
  • Define the boundaries of the cell
  • Maintain differences between the cytosol and extracellular environment
  • Eukaryotic cells have membrane enclosed organelles such as the Golgi apparatus, endoplasmic reticulum and mitochondria
  • Allow for ionic gradients and selected movement of molecules and solutes
  • Generation of electrical signals
  • Proteins embedded in the membrane sense changes in the external environment generating signals affecting cellular behaviour
• Types of Membrane


  • Cell membrane
  • Endomembrane system
• Cell membrane


  • Delimits a semi-autonomous functional unit – the cell
  • Controls movement of ions and molecules into and out of the cell
  • Protection from external environment
  • Provides attachment sites
  • Functions in cell signalling
• Endomembrane system


  Internal membranes delimiting organelles
• The Cell Membrane


  Lipid bilayer with embedded or attached proteins held together via non-covalent interactions
• Phospholipids


  • Amphiphilic molecules
  • Hydrophilic (water loving) polar head group
  • Hydrophobic (water fearing) non-polar tail
  • Fatty acid tails usually 14-24 Carbons in length
  • One tail usually has one or more cis-double bonds (unsaturated)
  • Double bonds create a kink in the tail
  • Tail length and degree of saturation affects lipid packing
  • Lipid packing influences fluidity of the membrane
• Lipids


  • Constitute around 50% of the membrane mass
  • A small animal cell has around 10^9 (1 billion) lipid molecules organised in a bilayer
  • Lipids are self-organising
• Major phospholipids in mammalian plasma membranes

  • Phosphatidylethanolamine
  • Phosphatidylserine
  • Phosphatidylcholine
  • Sphingomyelin
  • Sphingosine
• Phosphatidylserine


  The only one to carry a negative charge
• Phospholipid bilayer


  • The polar nature means it energetically favours forming a sealed compartment
  • This means the phospholipid bilayer forms an enclosed cell
  • Artificial liposomes can be created with phospholipids in an aqueous solution
• Cholesterol


  • The polar hydroxyl head group inserts close to the polar group of the membrane phospholipids
  • Plant cells do not have cholesterol but instead related sterol compounds
  • Bacterial cells do not have cholesterol
• Lipid composition


  • Varies between different membranes
  • Not all membranes are the same
• Membrane Fluidity


  • At low temperature there is reduced energy and so the phospholipids move less and pack together tighter
  • Saturated hydrocarbons will allow for closer packing
  • At very low temperature a crystalline state is formed
  • At higher temperatures the phospholipids have increased kinetic energy and so move more and pack less tightly
  • Unsaturated hydrocarbons will create greater spacing thereby increasing fluidity
• Influence of Cholesterol on membrane fluidity

  • At low temperatures cholesterol increases the spacing between the hydrocarbons and increases fluidity
  • At high temperatures cholesterol pulls the hydrocarbon tails together and deceases fluidity (stabilises)
  • Cholesterol can place itself in the lipid bilayer
• Lateral Movement and Flip-Flopping

  • Phospholipids move rapidly laterally within the plane
  • Flip-flopping – moving between planes is a rare occurrence
  • Flexion – movement of the hydrocarbon tails
  • Rotation of the phospholipid
• cis-Double Bonds Affect Membrane Fluidity and Width

  • Cis-double bonds make chains more difficult to pack
  • Hydrocarbon chains are more spread out
  • Lipid bilayers are thinner
• Mobility of Proteins in the Plasma Membrane

  • Human and mouse cells were fused to form a hybrid cell
  • Distribution of cell surface proteins monitored using anti-mouse and anti-human antibodies labelled with red or green fluorescent dyes
  • At timepoint zero the dyes are in either half of the hybrid
  • After 40 minutes the dyes were distributed over the entire cell surface
  • The proteins must be mobile within the plasma membrane
• Lipid Rafts


  • Discrete membrane domains enriched in cholesterol and sphingolipids form rafts that move laterally
  • Lipid rafts may associate the specific membrane proteins
  • Roles may include cell signalling and uptake of extracellular molecules via endocytosis
• The Endomembrane System


  Includes - Golgi apparatus, Endoplasmic reticulum, Vesicles, Nuclear envelope, Cell membrane, Vacuoles, Lysosomes

  
• Nuclear Envelope


  • Lipid bilayer (inner and outer layers)
  • The nuclear membrane is contiguous with the endoplasmic reticulum
• Nuclear Pore


  • Not simply a hole in the membrane
  • Complex of proteins regulating movement in and out of the nucleus
• Nuclear Pore Complex (NPC)


  • Comprises ~ 30 NPC proteins (nucleoporins)
  • Present in multiple copies
  • Octagonal symmetry
  • Around 3000-4000 NPCs per cell
  • Transport ~500 macromolecules per second
  • Bidirectional movement
• What Can Move In , What Can Move Out?

  • IN - Building blocks for DNA & RNA synthesis, Molecules used to provide energy, Ribosomal proteins
  • OUT - Ribosomal subunits synthesised by the nucleolus
• Nuclear Localisation Signal (NLS)


  Amino acid sequence that tags a protein for entry into the nucleus
• Nuclear Export Signal (NES)


  Amino acid sequence that tags a protein for exit from the nucleus
• Why is the Function of the NPC Important?

  • Small molecules (5 Kda) rapidly move freely in and out
  • Larger proteins move more slowly
  • Proteins >60 Kda barely able to enter by passive diffusion
  • Allows nucleus and cytoplasm to maintain distinct populations of proteins
• Mitosis and the Nuclear Envelope

  The nuclear envelope breaks down during prophase
• The Nuclear Membrane is Contiguous with the Endoplasmic Reticulum (ER)


  • ER constitutes more than 50% of total cellular membrane
  • Key role in protein and lipid synthesis
  • Ca++ store in the cell – important in signalling processes
  • Synthesis of transmembrane proteins and lipids for cell organelles
  • Most proteins destined for secretion or the ER lumen, Golgi or lysosomes are initially delivered to the ER
• Rough Endoplasmic Reticulum (RER) and Smooth Endoplasmic Reticulum (SER)

  • RER - Synthesis of proteins
  • SER - Synthesis of hormones, lipids, detoxification, conversion of glycogen to glucose
• Co-translational Protein Import Into The ER


  • Ribosome binds to the ER membrane
  • Protein imported into ER lumen as it is translated
• Protein Import Into the ER


  • Transmembrane proteins
  • Water soluble proteins
  • Only partially translocated across the membrane
  • Fully translocate across the membrane
  • Some will function in the ER – others transported to other locations
• Cotranslational Translocation Into ER Lumen

  • Translocator is closed until ribosome binds
  • N-terminal signal peptide initiates passage of protein through the translocator
  • Signal peptide cleaved by signal peptidase
  • Mature protein located in the ER lumen
  • Ribosome released and translocator closes
  • Requires an ER signal sequence
• Polysomes


  • A single mRNA can be simultaneously bound by several ribosomes
  • Sucrose gradient to separate mRNA with different numbers of attached ribosomes
• Protein Glycosylation in the ER

  • N-Linked glycosylation to asparagine residues (Asn-X-Ser and Asn-X-Thr)
  • Precursor oligosaccharide transferred from a dolichol lipid anchor catalysed by a transmembrane oligosaccharyl transferase enzyme
  • Trimming of the oligosaccharides occurs in the Golgi
• Golgi Apparatus


  • Major site of carbohydrate synthesis - Pectin and hemicellulose in plants, Glycosaminoglycans in animals
  • Glycosylation of proteins
  • Part of the secretory pathway – sorts and dispatches proteins made in the ER
  • Interconnected flattened cisternae
  • cis face and trans face either side of the medial cisternae
• Movement of Vesicles from the ER to the Golgi


  • Vesicles bud off the ER at specialised exit sites with a COPII coat
  • COPII plays a role in recruiting proteins with 'exit or transport' signals
  • Incorrectly folded proteins retained in the ER
  • Cystic fibrosis – slightly incorrectly folded plasma membrane protein produced, this would function but it is retained in the ER
• Getting from the ER to the Cis Golgi


  • COPII vesicles shed the COPII coat and fuse to form the vesicular tubular cluster
  • This fuses with the cis-Golgi
  • KDEL receptor retrieves proteins back to the ER
• Movement of Proteins Through the Golgi

  • 2 proposed routes – cisternal maturation and vesicle transport
  • May be a combination of both mechanisms
• Complex Glycosylation of Proteins in the Golgi


  The addition of complex sugar side chains is important for the function of many proteins