Clathrin-mediated Endocytosis
3: Endocytosis is the morphological opposite of exocytosis and involves the de novo production of internal membranes from the plasma membrane. Internalises plasma membrane proteins, lipids and extracellular fluid.
4: Many endocytosis pathways occur in cells eg phagocytosis, pinocytosis and receptor-mediated endocytosis.
Phagocytosis is the removal of pathogens and cell debris via a phagosome which fuses with a lysosome in which pathogen digestion occurs.
Pinocytosis is a non-specific process where the cell takes up ECF.
Receptor mediated endocytosis is a form of clathrin mediated endocytosis where a receptor and its ligand are taken into the cell - regulated.
5: Endocytosis allows membrane composition to be controlled, regulates cell interactions with external environment, nutrient uptake, pathogen and debris destruction and allows retrieval of vesicle proteins following exocytosis. Endocytosis of receptors at membrane regulates cell sensitivity to ligands eg insulin binding its receptor triggers receptor endocytosis, reducing subsequent sensitivity and likelihood of over-response.
6: Clathrin-mediated endocytosis is a major endocytic pathway requiring a large number of proteins. Involves formation of clathrin-coated vesicles at membrane
7: Clathrin mediated endocytosis overview:
curvature of membrane to form membrane-attached vesicle
clathrin, cargo proteins and accessory proteins recruited
scission of clathrin-coated vesicle from membrane
removal of clathrin coat
8: PIP2 acts as nucleation point for clathrin-dependent endocytosis as many components of the machinery bind to it. Cells with reduced PIP2 take up less transferrin which enters cells via clathrin-mediated endocytosis.
9: Epsin binds PIP2 and inserts an amphipathic helix into membrane to promote membrane curvature (invagination). Membrane curvature recruits further proteins to stabilise it. It also recruits clathrin to the membrane.
10: Upon binding PIPs, epsin undergoes structural changes
11: Insertion of the amphipathic helix occurs into one leaflet of the bilayer, causing the phospholipids to splay, increasing curvature as surface area of 1 leaflet greater than the other.
12: Proteins containing BAR domains dimerise to form crescent-shaped homodimers which bind membranes via the concave face and help stabilise the curved membrane. The positively charged residues within the membrane binding face bind the negatively charged phospholipids.
13: BAR domain-containing proteins eg FCHo2, amphiphysin and endophilin are involved in membrane sculpting events in different stages of endocytosis.
14: FCHo2 binds membranes with shallow curvature and is required at an early stage of endocytosis.
Amphiphysin binds to membranes of high positive curvature and are required at a later step.
15: Adaptor protein complexes (AP) are hetero-tetramers with 4 main types: 1 to 4. AP2 is involve in endocytosis whereas 1, 3 and 4 are generally involved in budding events from intracellular membranes.
16: AP2 binds membrane PI 4,5 bisphosphate (PIP2) and recruits cargo proteins and clathrin. Cargo typically contains dileucine and tyrosine based sorting signals recognised by AP2. Dileucine signal: [D/E]xxxLL. Tyrosine = TxxF where F is hydrophobic
17: Clathrin is a 3 legged structure - triskelion - consisting of 3 heavy chains (192 kDa) each bound to a light chain (33 to 36 kDa). Able to spontaneously assemble into geodesic clathrin cages which can form a vesicle from the membrane. One of the principle roles of clathrin is to determine the overall size of the vesicle that’s going to be internalised.
18: GTPase dynamin self-assembles into a helical collar around the neck of a budding vesicle. GTP hydrolysis is theorised to create a lengthwise extension of the helical collar which results in vesicle being pinched/popped off the membrane
19: Shibire (Japanese for paralysed) is a temp-sensitive phenotype described in a mutant Drosophila line. Phenotype due to point dynamin mutations which block GTPase activity. Vesicle scission disturbed - vesicles with dynamin collar still attached to membrane
20: Uncoating clathrin-coated vesicles mediated by auxilin and HSC70. Auxilin is a tyrosine protein phosphatase and HSC70, or heat shock cognate, is a chaperone protein.
21: Auxilin-KO mice have increased ratio of clathrin-coated structures to synaptic vesicles, consistent with defect in vesicle uncoating.
22: Clathrin-mediated endocytosis allows cellular uptake of Fe^(3+). The transferrin receptor is endocytosed and constitutively recycled with or without bound transferrin.
Transferrin receptor assays can be used to diagnose anaemia as receptor level increased on cells when iron low. Ferritin assay usually used but [ferritin] can be elevated in blood due to inflammation, autoimmune diseases and some cancers.
23: Viruses and some other pathogens exploit endocytic pathways for cellular uptake eg influenza A, HSV01 and dengue virus.