BS2092 - Endocytosis

Endocytosis

the movement of vesicles into the cell with cargo

Endosome maturation

the process where early endosome convert into late endosome to transport cargo for lysosomal degradation

Key sorting compartment in the endocytic pathway

early endosome

Endocytic pathway

• endocytic vesicles (from the plasma membrane) fuse with early endosome

• in the early endosome, there are long tubular projections which form vesicles

• as the endosome matures, the projections are lost and the endosome acquires intralumenal vesicle bodies (vesicles within vesicles)

• the late endosome can fuse with the lysosome to form the endolysosome

• where the proteins and cargo are broken down

• as the contents are broken down it becomes the lysosome again

Types of endocytosis

• phagocytosis

• pinocytosis (clathrin mediated endocytosis, caveolin mediated endocytosis)

• macropinocytosis

Common features of phagocytosis and macropinocytosis

• triggered in response to stimuli

• engulfment of large cargo or vacuole

• involves localised actin polymerisation

• involves activation of Rho family GTPases

Features of clathrin-mediated endocytosis

• active in nearly all cells

• occurs through plasma membrane invaginations

• clathrin coated pits (CCPs) occupy about 2% of cell surface area

• short lifetime

Size of clathrin mediated plasma membrane invaginations

~100nm

Cells which do not undergo clathrin-mediated endocytosis

mature mammalian red blood cells

Number of CCPs per cell

~1000

Approximate CCP lifetime

1-2 mins

Time taken for CCVs (clathrin coated vesicles) to internalise the entire cell surface

1-2 hrs

Main route for receptor-mediated endocytosis

clathrin-coated vesicles

Broad types of physiological ligands

• Serum transport proteins and antibodies

• Growth factor receptors

Examples of Serum transport proteins and antibodies

• transferrin

• low-density lipoprotein (LDL) and receptor

• maternal IgG

Growth factor receptors examples

• insulin receptors

• EGF receptors

• growth hormone receptor

Types of pathological ligands which hijack clathrin-coated vesicles for receptor-mediated endocytosis

• viruses

• toxins

Examples of viruses that hijack clathrin-mediated endocytosis machinery

• Sars-CoV-2

• Adenovirus

• HIV

Examples of toxins that hijack clathrin-mediated endocytosis machinery

• Diphtheria toxin

• Pseudomonas toxin

• Cholera toxin

LDL (low density lipoprotein) function

transport of cholesterol esters in the blood

LDL uptake

taken up into cells by receptor mediated endocytosis to supply cholesterol to cells for membrane synthesis

No. of molecules of cholesterol esters in LDL core

1500

No. of free cholesterol in LDL core

370

No. of triacylglycerols in LDL core

185

3 main parts of the LDL particle

• apolipoprotein B

• hydrophobic LDL core

• phospholipid monolayer

Purpose of phospholipid monolayer (of LDL)

allows LDL to associate with aqueous environment of the blood

Purpose of apolipoprotein B

Recognised by the LDL receptor

2 extracellular parts of the LDL receptor

• ligand-binding domain

• β-propeller domain

Cytosol pH

7.0-7.4

Endosome pH

5.5-6.5

Role of lower pH in endosome (compared to cytosol)

promotes the dissociation and release of LDL particle from the receptor inside the endosome

Effect of endosome pH on LDL receptor

surface of β-propeller domain becomes positively charged allowing it to bind to the ligand-binding arm (causing dissociation and release of the LDL particle)

Signal for endocytosis of LDL receptor into CCVs

NPXY (Asn, Pro, anything, Tyr)

Mutation found in patient with familial hypercholesteremia

single amino acid substitution from Y → C

Effect of ‘JD’ mutation on LDL receptor endocytosis

• LDL binds to receptor but does not get internalised

• LDL and receptor stay in the plasma membrane

• accumulation of LDL in blood vessels

• results in hypercholesteremia which leads to vascular disease

Endocytosis signals and respective proteins

• Tyr-X-X-θ — Transferrin receptor

• NPXY — LDL receptor

• LL — CD4

Cytosolic partner for internalisation of endocytic signals

AP2 adaptor proteins

Receptor-mediated endocytosis of LDL

• LDL particles bind to receptors incorporated into CCPs

• uncoating of vesicle

• uncoated vesicle fuses with early endosome

• the endosome pH allows LDL to dissociate, and its receptor returns to plasma membrane (recycling for more LDL binding)

• matures into late endosome

• fuses with lysosome to form endolysosome

• LDL particles break up into cholesterol (+ fatty acids, peptides, amino acids)

• free cholesterol released into cytosol to be used by the cell

Acid hydrolases within lysosomes

• nucleases

• proteases

• glycosidases

• lipases

• phosphatases

• sulfatases

• phospholipases

Transferrin

soluble protein that carries iron in the blood

Transferrin Receptor function

delivery of transferring with bound iron into cells by receptor mediated endocytosis

Transferrin-receptor cycle

• ApoTransferrin binds to Fe³⁺ extracellularly, forming Diferric Transferrin

• Diferric transferrin binds to its receptor and enters the cell by clathrin coated pits

• DMT1 is also incorporated into clathrin coated pits

• Once inside, CCV uncoats and fuses with the endosome

• Iron dissociates due to the lower pH, Apotransferrin remains bound to receptor

• Iron escapes the endosome through DMT1

• Apotransferrin and receptor returned to cell surface

DMT1

divalent metal ion transporter protein essential to enable iron to exit the endosome when inside the cell

Endocytosis of epidermal growth factor (EGF) receptor

• upon binding, EGFR dimerises

• becomes phosphorylated and activated on Tyr residues

• recruited signalling proteins relay downstream signals

• endocytosis effectively terminates the signals

Inactivation of EGFR

• EGFR is ubiquitinylated

• then endocytosed by clathrin coated vesicle

• fusion with early endosome

• pinching off (sequestration) of early endosomes to form a multivesicular body with ubiquitin tail in the internal vesicle

• fusion with lysosome containing enzymes to degrade receptor and ligand as well as internal vesicles

ESCRT

Endosome Sorting Complex Required for Transport

Sars-Cov2 endosomal entry

• Spike protein recognised by ACE2 receptor

• binds to ACE2 receptor enabling clathrin-mediated endocytosis

• endosome acidification activates the protease, Cathepsin L

• cathepsin L cleaves S1 and S2 of the spike protein

• revealing a fusion peptide allowing the virus to fuse with the endosome membrane and escape into the cytosol

• viral RNA uncoats and hijacks nucleus to replicate

Potential therapeutics against viral endosomal entry

• ACE2 mimetics

• therapeutic antibodies

• vaccine-elicited antibodies

• hydroxychloroquine and chloroquine drugs

Transcytosis of maternal IgG across the intestinal epithelium of neonates

• IgG binds to receptor in the intestinal lumen

• CCP brings receptor and ligand into cell

• endosome transported across the epithelial cell

• higher pH on the opposite end of the cell allows fusion with membrane and dissociation of IgG from its receptor

• receptor is recycled

Example of recycled endosomes acting as storage molecules

• endosome can act as a store of glucose transporters

• when insulin receptors are activated signals are sent to these endosomes

• causing delivery of more glucose transporters to the cell surface to take up more glucose

Pathways for degradation in lysosomes

• phagocytosis

• endocytosis

• macropinocytosis

• autophagy

Macropinocytosis pathway

• activation of signalling receptor

• activation of Ras and Rac GTPases to mediate actin rearrangement

• formation of plasma membrane protrusion (ruffle)

• protrusion becomes long and fuses with another part of the plasma membrane

• forming a macropinosome which contains large volume of extracellular fluid (and whatever nutrients are in it)

Cell which carries out phagocytosis

macrophages

Phagocytosis pathway

• antibodies bind to bacterium

• antibodies bind to Fc receptor

• binding to receptor stimulates actin localisation at the membrane to form membrane protrusions which surround around the bacterium

• protrusions engulf and internalise bacterium into a phagosome

• phagosome fuses with lysosome to form phagolysosome

• digestive enzymes destroy foreign particle

• any indigestible material is released outside the cell

Mediators of actin localisation and polymerisation at the membrane in phagocytosis/macropinocytosis

Rho family GTPases

Mechanisms of pathogen escape from phagocytosis

• phagosome escape

• proteolysis prevention

• phagolysosome survival

Phagosome escape example

Listeria monocytogenes produces toxins to lyse the phagosome and escape into the cytosol

Prevention of proteolysis example

Mycobacterium tuberculosis prevents the formation of a phagolysosome

Phagolysosome survival example

Coxiella burnetti produces chemicals causing the cell to adjust conditions within the phagolysosome so it can survive