endocytosis 1

types of endocytosis

• phagocytosis

• pinocytosis

  • dynamin-independent

    • macropinocytosis

    • lipid-raft mediated

    • non-clathrin/non-caveolar

  • dynamin-dependent

    • caveolar

    • clathrin-mediated

major endocytic compartments

• endocytic vesicles

• early endosomes

• recycling endosomes

• late endosomes / multivesicular bodies

• lysosomes

endocytic vesicles

• clathrin

  • fuse with early endosomes

• non-clathrin

  • mostly deliver their contents to early endosomes

• phagosomes

• caveolae-mediated endocytosis

clathrin structure

triskelion: 3 heavy chains, 3 light chains

clathrin-mediated endocytosis

• clathrins form lattice coat, induce curvature to membrane for pit formation

• legs of clathrin are intertwined with neighbours

• clathrin is “outer coat”

• clathrin binds to adaptors (mostly AP-2 on cell surface) which recruit cargo receptors

• dynamin performs vesicle fission

• receptor-mediated, receptors can concentrate cargo

stages of vesicle formation in clathrin-mediated endocytosis

1. initiation

   • formation of clathrin-coated pit, clathrin coat assembly, cargo receptor recruitment

2. propagation

   • invagination of clathrin-coated pit

3. budding

   • continual invagination of clathrin-coated pit, scission of its neck

4. uncoating

   • disassembly of clathrin coat

clathrin adaptors

• AP (adaptor protein) complexes vs monomeric GGA (Golgi-localizing, Gamma-adaptin ear domain homology, ARF-binding proteins)

• AP complexes and GGA interact with N-terminal region of clathrin heavy chain

• at cell surface, AP-2 is important (no Arf interaction)

• on Golgi, other APs and GGA are important, as well as Arf1

• monomeric ARH (autosomal recessive hypercholesterolemia) for LDL receptor internalization, binds to N-terminal region of clathrin heavy chain

AP-2 complex

functions at the plasma membrane with clathrin to recognize cargo receptors

dynamin

• assemble to form helix at stalk of budding vesicle

• GTP hydrolysis causes conformation change, constricting neck

dynamin and synaptic vesicle recycling

• fruit fly dynamin called shibire has temperature sensitive allele

• at restrictive temperature (30°C), flies are paralyzed

• at permissive temperature (~20°C), paralysis is reversed, flies move

• at neuromuscular junction, synaptic vesicles of neurotransmitters are not being recycled

  • long necks, but no pinching off

early endosome / sorting endosome

• mildly acidic

• tubular and vacuolar domains

• sorting function

  • some ligands detach from their receptors due to low pH

  • ligands remain in the lumen and will eventually reach the lysosome

  • receptors and much of the internalized membrane are sorted into tubular extensions that detach and become recycling enzymes

• membrane contains small GTPase Rab5 and tether protein EEA1 as well as recycling and non-recycling receptors

• lumen contains cargo destined for lysosomal degradation

recycling endosome

• recycling branch of endocytic pathway

• less acidic than early endosome

• form from tubular extensions of early endosome

• return membrane and most receptors to cell surface

• in some cells, don’t immediately return to cell surface but form a tubular “recycling compartment”, sometimes concentrated near the centrosome

  • fast versus slow recycling

• contains small GTPase Rab11, recycling receptors

late endosome / multivesicular bodies

• degradative branch of endocytic pathway

• more acidic

• inward budding by ESCRT-0, -I, -II, and -III complexes → intraluminal vesicles

• membrane proteins targeted for degradation are budded into internal vesicles

• contains some degradative enzymes

• some proteins can cycle between MVBs and Golgi

  • includes mannose-6-phosphate receptor and sortilin

• small GTPase Rab7 in membrane and proteins recruited by Rab7 including HOPS tethering complex, dyneins, and kinesins

lysosome

• terminal component for degradation

• acidic (pH 4.5-5)

• heterologous population of electron dense organelles reflecting various stages of cargo breakdown

• low pH for degradative enzyme function

  • if it were to break, the enzymes would be harmless in cytoplasm

• digested components shuttled out by specialized channels and transporters

• degradative enzymes are capable of destroying lysosomal membrane

• membrane contains highly glycosylated proteins called LAMPs and LIMPs which provide protection

• exact relationship with late endosomes unclear

• small GTPase Rab7 present