BIOL340 Cell & Molecular Biology Lecture Notes PDF

BIOL340: Cell & Molecular Biology Secretion & Endocytosis Week 2 Lecture 4 4th March, 2024 Anuk Indraratna [email protected] 1 Learning outcomes 1. Define the components of the endomembrane system in relation to one another. 2. Explain the mechanisms underlying vesicular transport between organelles....

BIOL340: Cell & Molecular Biology Secretion & Endocytosis Week 2 Lecture 4 4th March, 2024 Anuk Indraratna [email protected] 1 Learning outcomes 1. Define the components of the endomembrane system in relation to one another. 2. Explain the mechanisms underlying vesicular transport between organelles. 3. Describe the role of the Golgi apparatus in processing and dispatching biosynthetic products, including regulated secretion. 4. Describe the different modes of endocytosis and their purposes. 2 Lecture outline 1. Overview of endomembrane system and transport 2. Vesicular transport 3. The Golgi complex (aka Golgi apparatus) 4. Regulated secretion 5. Endocytosis 3 Lecture outline 1. Overview of endomembrane system and transport 2. Vesicular transport 3. The Golgi complex (aka Golgi apparatus) 4. Regulated secretion 5. Endocytosis 4 The endomembrane system ▪ Chemical and metabolic processes within a eukaryotic cell are compartmentalised — E.g. glucose formation will occur distinctly to glucose breakdown elsewhere — Destructive processes need to be contained (lysozymes, peroxisomes) ▪ Compartmentalisation occurs through organelles — Separates metabolic activity of one area to all others by at least one semi-permeable membrane ▪ Materials need to be transported between compartments — Endoplasmic reticulum, Golgi complex, endosomes, lysosomes, vacuoles — Individual components function as a co-ordinated network 5 Essential Cell Biology, 3rd edn, Alberts, Bray et al, Chapter 15 6 The endomembrane system 7 8 Transport to/from the plasma membrane ▪ Exocytosis (secretion) — Delivery of newly synthesised material — To the plasma membrane — Or the extracellular space ▪ Endocytosis — Capture extracellular molecules — Remove / recycle plasma membrane components — Deliver them to endosomes or lysosomes 9 ▪ PINK ARROW = SECRETORY — — ▪ Constitutive Regulated (stored; release is triggered) GREEN ARROW = ENDOCYTOSIS — — — Phagocytosis Pinocytosis Receptor-mediated endocytosis 10 Lecture outline 1. Overview of endomembrane system and transport 2. Vesicular transport 3. The Golgi complex (aka Golgi apparatus) 4. Regulated secretion 5. Endocytosis 11 Vesicular transport ▪ Proteins and lipids move through multiple compartments — — — — ▪ Protein synthesis at rough ER Modified at the Golgi Travel through cytosol Reach their fate (organelle, cytoplasm, membrane, secretion) This transport is handled primarily through vesicles — Budding off, movement, and fusion of small, mobile, membrane-enveloped packages of molecules — They travel non-randomly, directed by motor proteins along the cytoskeleton 12 Vesicular transport 13 Vesicular transport 14 Vesicular transport ▪ Vesicles are carried by motor proteins ▪ Move along ‘tracks’ of microtubules of the cytoskeleton (later lectures) 15 Protein coats 16 Protein coats 17 1. Cargo molecule binds to its receptor within the trans-Golgi membrane 2. Adaptor protein recruits clathrin, initiating a lattice structure (coated pit) with curvature of the membrane 3. Membrane-bending and fission proteins are recruited to the neck of the budding vesicle 4. The clathrin-coated vesicle pinches off, enclosing cargo 5. Clathrin disassembles Protein coats 18 RED ARROW = SECRETORY GREEN ARROW = ENDOCYTIC BLUE ARROW = RETRIEVAL 19 Lecture outline 1. Overview of endomembrane system and transport 2. Vesicular transport 3. The Golgi complex (aka Golgi apparatus) 4. Regulated secretion 5. Endocytosis 20 Golgi apparatus ▪ Primary site of glycans synthesis and attachment to proteins and lipids — Glycan labels direct specific proteins towards certain fates ▪ Highly ordered processing and modification of proteins and lipids ▪ Sorting & dispatch station for products of the ER — Receives material from ER — Also sends material back for recycling purposes (retrograde transport) 21 Glycosylation at the Golgi ▪ Glycosylation is an important post-translational modification of proteins ▪ N-linked glycosylation is common in eukaryotes — — — ▪ Important roles in protein maturation — — — ▪ Oligosaccharides are attached to asparagine Begins with high-mannose glycans in ER Complexity continues in Golgi Improves solubility Mediates binding to chaperone proteins Provides resistance against proteolytic enzymes Glycans can target proteins to their destination — — Mannose-6-phosphate-tagged proteins will be sequestered in the lysosome More on this later 22 23 Lecture outline 1. Overview of endomembrane system and transport 2. Vesicular transport 3. The Golgi complex (aka Golgi apparatus) 4. Regulated secretion 5. Endocytosis 24 Regulated secretion ▪ Secretion happens continually in all cells — Constitutive secretion ▪ Or, can be triggered — Regulated secretion — Only occurs in cells specialised for secretion — E.g. hormones, mucus, digestive enzymes ▪ In regulated secretion, secretory vesicles await extracelluar signals — Accumulate near plasma membrane — E.g. increase in blood glucose stimulates secretion of insulin from pancreas 25 26 Lecture outline 1. Overview of endomembrane system and transport 2. Vesicular transport 3. The Golgi complex (aka Golgi apparatus) 4. Regulated secretion 5. Endocytosis 27 Endocytosis ▪ Continuous, multi-modal process ▪ Internalise molecules from the environment — Phagocytosis: “cellular eating”; selective — Pinocytosis: “cellular drinking”; non-selective — Receptor-mediated endocytosis: highly selective ▪ Endocytosis occurs through vesicular transport and has many roles — — — — Uptake essential nutrients and molecules Regulate the abundance and activity of membrane proteins and receptors Remove pathogens and debris from environment Recycle membrane components ▪ Often ends with lysosome-mediated destruction 28 Lysosomes ▪ Small membrane-bound organelle where material is degraded ▪ Inhospitable space that must be compartmentalised from the cell — — — — Acidic pH (~4.5 – 5) is optimal pH for hydrolytic enzymes Enzymes: nucleases, lipidases, proteases, glycosidases Production can be upregulated as needed Targeted to lysosome via M6P tag by Golgi ▪ Employed in the digestion of intra- and extracellular material — — — — Pathogens and pathogenic debris Damaged or obsolete organelles (autophagy) Misfolded, surplus, aggregated proteins Metabolic elements to be recycled 29 30 Phagocytosis ▪ Performed by specialised cells, such as macrophages ▪ Targets pathogens, dead / dying cells, cell debris ▪ Triggered when a particle binds to a surface receptor — Antibodies – produced by adaptive immune system Pathogen-associated molecular patterns (PAMPs) — Damage-associated molecular patterns (DAMPs) — Phosphatidylserine exposed on apoptotic cells ▪ Material is enveloped, ingested, and then destroyed — Extensions of plasma membrane, called pseudopuds, engulf material and form phagosome — Phagosome fuses with lysosome — Acidification, enzyme digestion, ROS — Material stored or exocytosed 31 32 Pinocytosis ▪ Performed constantly and rapidly by all cells ▪ Indiscriminate ingestion of cell’s own plasma membrane along with extracellular fluid ▪ Enables turn-over of membrane and consistent sampling of environment ▪ Begins with clathrin-coated vesicles forming at the plasma membrane — — — — Pinches off, shedding of clathrin Delivered to endosome Here, material is sorted and recycled Material destined for degradation remains and eventually fuses with lysosome 33 Receptor-mediated endocytosis ▪ Performed by all cells ▪ Many essential metabolites such as B12, iron ▪ Mechanically similar to pinocytosis, but far more selective ▪ Cholesterol is an important example ▪ Allows for concentrated internalisation of target molecules, 1000-fold more efficiently — Cholesterol alone is extremely insoluble — Bound in blood by low-density lipoproteins (LDL) — LDL binds to receptors, which are then ingested by receptormediated endocytosis 34 Lecture outline 1. Overview of endomembrane system and transport 2. Vesicular transport 3. The Golgi complex (aka Golgi apparatus) 4. Regulated secretion 5. Endocytosis 35 Summary ▪ Chemical and metabolic processes are compartmentalised into membrane-bound organelles ▪ The endomembrane system consists of ER, Golgi, lysosomes, endosomes, which work in concert to receive, dispatch, and process cellular materials ▪ Transfer of material between these compartments relies on vesicular transport — — — Formed via the activity of coat proteins (and other associated proteins) Membrane-bound units which are moved along the ‘tracks’ of cytoskeleton by motor proteins Ends with fusion of vesicles with target compartment ▪ Golgi is responsible primarily for processing, modifying, and dispatching ER-synthesised proteins and lipids ▪ Secretion (export / exocytosis) of material can be constitutive or regulated ▪ Endocytosis = intake of materials in a targeted or untargeted manner — — — — Allows turnover of plasma membrane components, sampling of environment, clearance of debris Different modes: phagocytosis, pinocytosis, receptor-mediated endocytosis Endosome is involved in sorting and recycling materials Lysosome is involved with degradation of material 36 Learning outcomes 1. Define the components of the endomembrane system in relation to one another. 2. Explain the mechanisms underlying vesicular transport between organelles. 3. Describe the role of the Golgi apparatus in processing and dispatching biosynthetic products, including regulated secretion. 4. Describe the different modes of endocytosis and their purposes. 37 Example quiz questions 1. Explain, with the aid of a diagram, the steps involved in vesicular transport. [10 marks] 2. Explain, with examples, the importance of post-translational modifications that occur at the ER and/or Golgi apparatus. [5 marks] 3. Compare and contrast the three major modes of endocytosis. [10 marks] 4. Explain, with examples, the differences between constitutive and regulated secretion. [4 marks] 38