Cell Biology - Endosomes-Lysosomes Endocytosis PDF

Endosomes & Lysosomes [1] Paul J. McDermott, Ph.D. Office: (843) 792-3462 Email: [email protected] ENDOSOMES, LYSOSOMES & ENDOCYTOSIS A. MECHANISMS OF ENDOCYTOSIS 1. Phagocytosis 2. Pinocytosis 3. Receptor-mediated Endocytosis B. PHAGOCYTOSIS: 4 MAIN STEPS 1. Binding at Extracellular Surface 2. Devel...

Endosomes & Lysosomes [1] Paul J. McDermott, Ph.D. Office: (843) 792-3462 Email: [email protected] ENDOSOMES, LYSOSOMES & ENDOCYTOSIS A. MECHANISMS OF ENDOCYTOSIS 1. Phagocytosis 2. Pinocytosis 3. Receptor-mediated Endocytosis B. PHAGOCYTOSIS: 4 MAIN STEPS 1. Binding at Extracellular Surface 2. Development of Pseudopodia 3. Formation of Phagosomes 4. Formation of Phagolysosomes C. RECEPTOR-MEDIATED ENDOCYTOSIS 1. Overview 2. Formation of Clathrin-Coated Vesicles 3. Vesicular Trafficking 4. Types of Ligands 5. Multivesicular Bodies D. CAVEOLAE 1. Structure 2. Molecular Components 3. Functions E. STRUCTURE AND FUNCTION OF LYSOSOMES 1. Structure 2. Formation 3. Delivery of Digestible Materials to Lysosomes: 3 Main Routes 4. Trafficking Summary 5. Lysosomal Storage Diseases Suggested Reading: • Molecular Biology of the Cell: http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=mboc4 • Junqueira’s Basic Histology Text and Atlas, 16e, © 2021, McGraw-Hill, Chapter 2 “Eat not to dullness; drink not to elevation” Benjamin Franklin Endosomes & Lysosomes [2] OBJECTIVES 1. Describe the differences between micropinocytosis and macropinocytosis. 2. Describe the main steps involved in phagocytosis. 3. Specify the stages of receptor-mediated endocytosis and describe the roles of clathrin, AP2 and dynamin. 4. Specify the trafficking steps involved in receptor-mediated endocytosis of a ligand from the extracellular space to the lumen of a lysosome. 5. Explain the functional significance of membrane recycling. 6. Describe the structure and function of multivesicular bodies (MVB). 7. Describe the structure and possible functions of caveolae. 8. Specify the main types of lysosomal hydrolases. 9. Describe 3 specializations in lysosomal membranes and their functional significance. 10. Describe the details of lysosome formation including sorting of enzymes in the Golgi complex, trafficking to late endosomes and maturation into lysosomes. 11. Describe the 3 main routes for delivery of digestible materials to lysosomes, specifically, receptor-mediated endocytosis, phagocytosis and autophagy. 12. Explain why lysosomal storage diseases occur and know the defective enzymes associated with each of the following diseases: I-Cell, Gaucher, Tay-Sachs. Illustrations adapted from: • The Cell: A Molecular Approach © 2000 ASM Press and Sinauer Associates, Inc. • Medical Cell Biology, 3rd Ed. © 2008, Elsevier Inc. • Molecular Biology of the Cell © 2002, Garland Science • Junqueira’s Basic Histology, 12th Edition: Text and Atlas © 2010, McGraw-Hill Endosomes & Lysosomes [3] A. MECHANISMS OF ENDOCYTOSIS Endocytosis is a general term for internalization of material into the cell by vesicles derived from the plasma membrane. 1. Phagocytosis (cell eating): Ingestion of bacteria, large particles or other cells. It occurs mainly Org(2) in specialized cell types such as neutrophils and macrophages. 2. Pinocytosis (cell drinking): Non-specific ingestion of liquid and solutes such as proteins. Org(1) It occurs in all cell types by clathrin-independent mechanisms. a) Micropinocytosis: Uptake via small endocytic vesicles formed by aggregation of proteins such as flotillins and possibly caveolins in lipid rafts. b) Macropinocytosis: Uptake via large endocytic vesicles formed by extensions or “ruffles” of the plasma membrane. Formation of ruffles is dependent on cytoskeletal actin filaments. 3. Receptor-Mediated Endocytosis (clathrin-dependent): Ingestion of receptor-bound ligands into clathrin-coated vesicles for transport to the early endosome compartment. B. PHAGOCYTOSIS: 4 MAIN STEPS 1. Binding at Extracellular Surface: interaction of material with the plasma membrane 2. Development of Pseudopodia: extensions of plasma membrane by a microfilament-dependent mechanism 3. Formation of Phagosomes: large intracellular vesicles formed by fusion of pseudopodia 4. Formation of Phagolysosomes: fusion of phagosomes with lysosomes for enzymatic digestion 1 2 Bacterium Fig 3 Pseudopodium 4 Plasma Membrane Lysosome Neutrophil 1 µM Macrophage injecting red blood cells C. RECEPTOR-MEDIATED ENDOCYTOSIS 1. Overview • Ligands are internalized selectively at specified sites on the plasma membrane. • Ligands bind to cell surface receptors concentrated in clathrin-coated pits. • Approximately 1-2% of the cell surface is occupied by clathrin-coated pits. • Budding of the pits from the plasma membrane to form clathrin-coated vesicles requires the membrane protein dynamin, which is a GTPase required to “pinch off” vesicles. • Clathrin-coat dissociates and vesicle fuses with early endosomes. • Sorting in early endosomes: receptors are recycled back to the plasma membrane while the ligands remain in vesicles that are transported to late endosomes. • Late endosomes mature into lysosomes that degrade the ingested material. C. RECEPTOR-MEDIATED ENDOCYTOSIS 2. Formation of Clathrin-Coated Vesicles a) Stages Endosomes & Lysosomes [4] Schematic diag(4) b) Structure of Clathrin Coats: Clathrin molecules assemble into basket-like structure or cage. Clathrin has two main functions: 1) distorts the plasma membrane by budding to produce a vesicle; 2) capture membrane receptors and bound molecules. Fxn(2) Molecular structure of Clathrin (triskelion) Scanning EM of Clathrin-Coated Pits Fig c) Adaptor Protein (AP): Consists of adaptin subunits located on inner layer of the clathrin-coat. It provides specificity by coupling of the receptor to the clathrin-coated pit. Dynamin Fxn Ligand Fig Clathrin Steps(3) Receptor Adaptor Protein 2 (AP2) Clathrin-Coated Vesicle • Ligand binds to a receptor on the extracellular surface of the plasma membrane. • Adaptor Protein 2 (AP2) binds to a specific AA sequence in the cytoplasmic tail domain of the receptor and to the clathrin coat. • The clathrin-coated vesicle buds off with assistance of the GTPase dynamin. C. RECEPTOR-MEDIATED ENDOCYTOSIS Endosomes & Lysosomes [5] 3. Vesicular Trafficking Dynamin Plasma Membrane Ligand Receptor AP2 Clathrin Recycling to Plasma Membrane Internalization Clathrin-Coated Vesicle Transport Vesicle Lysosome Dissociation of Coat e a Fusion Early Endosome b c d Late Endosome From trans Golgi Network trans Golgi Network Recycling of Mannose-6-P receptors Mannose-6-P tagged lysosomal enzymes a) Clathrin coat dissociates after internalization and vesicles fuse with early endosomes. b) Early endosome membrane contains H+ pump that lowers pH of the lumen to approximately 6.0, which causes dissociation of ligand from the receptor. c) Sorting occurs to produce 2 types of vesicles that bud from the early endosome: • Transport (recycling) vesicles that carry receptors & membrane back to the plasma membrane • Endocytic vesicles that carry ligand to the late endosome mannose-6-p tagged lys enzymes d) Late endosomes have pH of approximately 5.5 and fuse with transport vesicles carrying lysosomal hydrolases from the trans Golgi network. e) Late endosomes mature into lysosomes by a process that involves: • Sorting of hydrolytic enzymes • Recycling of endosomal membrane with integral proteins back to trans Golgi network • Lowering pH to approximately 5.0 for enzyme activation Nota Bene: Vesicular trafficking is dependent on microtubules for intracellular transport using either dynein motor proteins (towards minus end of MTs) or kinesins (towards plus end of MTs). C. RECEPTOR-MEDIATED ENDOCYTOSIS 4. Types of Ligands: Examples Endosomes & Lysosomes [6] a) Hormones: Insulin, Glucagon, Luteinizing Hormone (LH), Growth Hormone, Prolactin, Thyroid Stimulating Hormone (TSH), Thyroid Hormone b) Growth factors: Epidermal Growth Factor (EGF) and Nerve Growth Factor (NGF) c) Lipoproteins: LDL, oxidized LDL, VLDL and HDL d) Antibodies: IgE, polymeric IgA, IgG via Fc receptors e) Vitamins: A, D, B12 f) Iron Binding Proteins: Transferrin•Fe2+ g) Toxins: Diptheria toxin, Cholera toxin h) Viruses: Rous Sarcoma Virus, Adenovirus 5. Multivesicular Bodies Multivesicular bodies are a special type of late endosome in which proteins such as receptors are Def pinched off as part of smaller vesicles into the lumen. For example, during receptor-mediated endocytosis of EGF, the EGF receptor is not recycled because it is tagged for degradation by covalent linkage of a small protein called ubiquitin to its cytoplasmic tail domain. Ubiquitination Ubq def enables sorting of integral membrane proteins such as the EGF receptor into vesicles that form by invagination of the membrane and bud off into the lumen. The accumulation of these small vesicles gives rise to a Multivesicular Body (MVB). MVBs eventually fuse with lysosomes, which results in degradation of the vesicles. MVBs EGF Receptor with Ubiquitin tag Vesicles Fig Multivesicular Body Functions of MVBs: • Sorting and degradation of integral membrane proteins • Hormone desensitization by down-regulation of the receptor D. CAVEOLAE 1. Structure: Caveolae are invaginations of the plasma membrane approximately 50-80 nm in diameter. They are abundant in vascular endothelial cells, smooth muscle cells, fibroblasts and adipocytes. Found in cells(4) Plasma Membrane Caveolae Lysosome RER Endosomes & Lysosomes [7] D. CAVEOLAE 2. Molecular Components Plasma membrane a) Caveolins: Family of integral membrane proteins b) Lipid rafts: Enriched in cholesterol and sphingolipids Caveola Def Caveolae are specialized, morphologically distinct sphingolipidcholesterol microdomains, which are stabilized by caveolins. 3. Functions The functions of caveolae have not been determined on the basis of definitive experimental proof. Several functions have been proposed Fxn(2) including micropinocytosis, transcytosis or formation of membrane scaffolding for cell signaling molecules. Recent evidence indicates that the primary function of caveolae could be to prevent mechanical Primary damage to the plasma membrane, particularly in endothelial and fxn muscle cells. Caveolae flatten in cells exposed to extreme mechanical stress, which is hypothesized to prevent cell rupture by increasing surface area of the plasma membrane. Nat Rev Mol Cell Biol 8:185, 2007 E. STRUCTURE AND FUNCTION OF LYSOSOMES 1. Structure a) Size: Variable, 0.2 - 0.6 microns in diameter b) Lumen: Contains approx. 40 types of Acid Hydrolases • Nucleases • Phosphatases • Proteases • Phospholipases • Lipases • Sulfatases • Glycosidases c) Membrane: Specializations required for proper function • Proton pump lowers pH of lumen to 5.0 for enzyme activation. • Glycosylation of membrane proteins on luminal surface protects lysosome from self-digestion. • Membrane transporters release products of enzymatic digestion into cytosol, e.g. AAs, sugars and lipids. d) Appearance in EM: Lysosomes are electron dense organelles that are heterogeneous in size and shape. The electron density varies in intensity and uniformity, depending on composition of the digested material in the lysosome. sugars lipids AA Acid Hydrolases pH ~ 5.0 H+ ATP H+ ADP•Pi Endosomes & Lysosomes [8] E. STRUCTURE AND FUNCTION OF LYSOSOMES Lysosomal enzyme 2. Formation Lysosomal enzymes contain N-linked oligosaccharides that are phosphorylated in the cis Golgi network to generate a mannose-6-P tag. These proteins are sorted as they move through the Golgi complex. Mannose-6-P a) Mannose-6-P tagged proteins are “trapped” and bud from the trans Golgi network in clathrin-coated transport vesicles by binding to the mannose-6-P receptor which, in turn, binds to AP1 and clathrin on the cytosolic side of the Golgi membrane. Clathrin Adaptor Protein 1 (AP1) Fig Mannose-6-P Receptor Lysosomal Enzyme tagged with Mannose-6-P cytosol Golgi membranes lumen of trans Golgi Network b) Clathrin coat sheds and the transport vesicle fuses with the late endosome. c) Low pH (5.5) of late endosome dissociates mannose-6-P receptor from lysosomal enzyme. d) Mannose-6-P receptors are recycled back to the trans Golgi via transport vesicles. e) Late endosomes mature into lysosomes by: • Sorting of hydrolytic enzymes • Recycling of endosomal membrane with integral proteins back to trans Golgi network • Lowering pH to approximately 5.0 for enzyme activation Maturation e Lysosome d c Late Endosome Fusion b Shedding of Clathrin-Coat Transport Vesicle Membrane recycling of Mannose-6-P receptors Golgi Complex a Budding of clathrin-coated vesicle from the trans Golgi Networkcontains lysosomal enzymes Lysosomal enzyme bound to receptor Endosomes & Lysosomes [9] E. STRUCTURE AND FUNCTION OF LYSOSOMES 3. Delivery of Digestible Materials to Lysosomes: 3 Main Routes a) Receptor-Mediated Endocytosis and Pinocytosis: Endocytic vesicles are routed through early endosomes → late endosomes → lysosomes. b) Phagocytosis: Phagosomes fuse with lysosomes to form phagolysosomes. c) Autophagy: Self-digestion of organelles and proteins by the cell. • Cytosolic proteins and organelles are enclosed in an expanding isolation membrane (phagophore) that eventually fuses to form an autophagosome. • Autophagosomes fuse with lysosomes to form autolysosomes for digestion of materials. Cytosolic proteins and organelles Fig Autophagosome Hydrolases Isolation membrane (Phagophore) Lysosome Nature Cell Biology 9:1102-1109, 2007 fusion Autolysosome TEM of an autophagosome (arrow). Note the double membrane. 4. Trafficking Summary: Putting It All Together Dynamin Plasma Membrane Clathrin Ligand Receptor AP2 Internalization Phagocytosis Fusion Phagosome Recycling to Plasma Membrane Clathrin-Coated Vesicle Dissociation of Coat Early Endosome Lysosome Transport Vesicle Autophagosome Late Endosome Fusion Sorting Recycling COPI Clathrin-coated vesicles with AP1 Lysosomal enzymes tagged with Mannose-6-P TVN RER COPII Golgi Apparatus Endosomes & Lysosomes [10] E. STRUCTURE AND FUNCTION OF LYSOSOMES 5. Lysosomal Storage Diseases Autosomal recessive disorders caused by deficiency in a lysosomal enzyme. More than 40 lysosomal storage diseases have been identified. Defects in lysosome function result in accumulation of undegraded substrates. This causes adverse effects on cell function. Severity of each disease is characterized by age of onset, clinical course and CNS involvement. Disorder Defective Enzyme Cellular Defects N-acetylglucosamine-1phosphotransferase Defective in processing of Mannose-6-P tag- lysosomal enzymes are not sorted by Golgi into clathrin-coated vesicles bound for lysosomes- deficiency of multiple enzymes causes substrates to accumulate in cells and form Inclusion bodies Tay-Sachs Disease a-Hexosaminidase A Accumulation of GM2 Gangliosides in brain- causes increase in Glial cells and abnormal growth of dendritic processes of neurons Gaucher's Disease b-Glucocerebrosidase Accumulation of Glucocerebroside in macrophages- leads to enlarged spleen and liver Fabry Disease a-Galactosidase Accumulation of Glycosphingolipids in vascular endothelium of multiple organ systems- disrupts function of caveolae Pompe Disease a-D-Glucosidase I-Cell Disease Niemann-Pick Disease Sphingomyelinase Accumulation of Glycogen- causes enlargement of glycogen storage vesicles and appearance of autophagic vacuoles Accumulation of Sphingomyelin- impairs formation of lipid rafts in association with cholesterol- disrupts membrane functions of neurons in the brain