Clathrin-mediated Endocytosis

Define endocytosis

de novo production of internal membranes from the plasma membrane to internalise proteins, lipids and extracellular fluid

pinocytosis

non-specific process where the cell takes up ECF

purpose of endocytosis

• control membrane composition

• regulate cell interactions with environment

• nutrient uptake

• pathogen/debris destruction

• retrieval of vesicle proteins

• receptor endocytosis to regulate sensitivity to ligands

PIP2

nucleation point for clathrin-dependent process - many components of machinery bind it

epsin

binds PIP2 and inserts amphipathic helix into membrane, promoting membrane curvature

How does helix insertion induce membrane curvature?

Inserted into one leaflet → phospholipid splaying and increased curvature as one surface area greater than other

proteins containing BAR domains

dimerise → crescent-shaped homodimers. Positively charged residues in dimer’s membrane binding face bind the curved membrane’s negatively charged phospholipids, stabilising it

FCHo2

binds membranes with shallow curvature - required at early endocytosis stage

amphiphysin

binds membranes of high positive curvature, required at later endocytosis stage

adaptor protein complex II (AP2)

binds PIP2, recruits cargo proteins and clathrin. Cargo contains dileucine and tyrosine sorting signals

dileucine signal

[D/E]xxxLL

Tyrosine signal

Txx-hydrophobic AA

clathrin structure

• 3 legged (triskelion)

• 3 heavy chains (192 kDa) each bound to light chain (33 to 36 kDa)

• spontaneously assembles into clathrin cages which form vesicle

dynamin

GTPase which self-assembles into helical collar around neck of budding vesicles

What is dynamin GTP hydrolysis theorised to create?

lengthwise helical collar extension → vesicle being pinched/popped off membrane

Shibire

• temp-sensitive Drosophila phenotype

• mutated dynamin blocking GTPase activity

• no Vesicle scission → vesicles with collar still attached to membrane

What proteins mediate the uncoating of clathrin-coated vesicles?

Auxilin (tyrosine protein phosphatase) and HSC70 (heat shock protein)

auxilin-KO mice

increased ratio of clathrin-coated structures to synaptic vesicles

Why can transferrin receptor assays be used to diagnose anaemia?

Transferrin receptor level increases on cells when iron low as typically the receptor would be endocytosed and recycled

Why aren’t ferritin assays as effective as transferrin assays at diagnosing anaemia?

[ferritin] elevated in blood due to inflammation, autoimmune disease and some cancer

Clathrin light chain isoform (CLCb) in non-small-cell lung cancer (NSCLC)

upregulated. Increases endocytosis rates via dynamin 1→ activation of positive feedback of EGFR dependent PKB/GSK-3beta phosphorylation → increased metastasis (Chen et al, 2017)