CELB40060

Membrane Trafficking in Animal

Cells

Prof. Jeremy C. Simpson

Lecture 6
Endocytos
is
 Today’s lecture ...

Endocytic mechanisms
- phagocytosis
- macropinocytosis
- caveolin-mediated endocytosis
- other non-clathrin-based systems
- clathrin-mediated endocytosis

Formation of clathrin-coated pits

Cargo selection in clathrin-mediated endocytosis

Membrane curvature in clathrin-mediated

endocytosis

Scission and coat dissociation in clathrin-dependent

endocytosis
 The plasma
membrane

- defines the outer boundary of each living cell

-provides basic mechanical rigidity on which protein structures can be built, allowing cells to
communicate with one another, transport solutes between the cytoplasm and extracellular environment,
and capture and internalise nutrients

-needs to consist of sufficiently flexible components such that its shape can be easily and rapidly
changed, for example during endocytosis

-up until the early 1990s it was thought that all endocytic events utilised the clathrin coat, however now we
know that in fact there are multiple clathrin-independent pathways of internalisation in cells
 Endocytic
mechanisms
(Clathrin-independent
carriers / GPI-enriched
early endosomal
compartments)

Doherty and McMahon (2007). Mechanisms of endocytosis. Ann. Rev. Biochem.

 Endocytic
mechanisms

Doherty and McMahon (2007). Mechanisms of endocytosis. Ann. Rev. Biochem.

 Endocytic mechanisms
- phagocytosis : specialised form of endocytosis for internalisation of very large particles such as
bacteria

- macropinocytosis : internalisation of large amounts of fluid and growth factors (and viruses)

- clathrin-dependent endocytosis : internalisation of specific receptors and extracellular

molecules

- caveolin-dependent endocytosis : internalisation of specific lipid-associated molecules

- caveolin- and clathrin-independent pathways : internalisation of membrane and small amounts

of fluid

Mayor and Pagano (2007). Pathways of clathrin-independent endocytosis. Nat. Rev. Mol. Cell Biol.
 Phagocytosis
-phagocytosis is a specialised form of endocytosis for internalisation of very large particles such as
bacteria and dead cells

-in animals phagocytosis is mainly carried out by specialised cells called ‘phagocytes’, for example macrophages
and neutrophils (types of white blood cell)

- ingested particles enter the ‘phagosome’, the diameter of which is determined by the particle itself

- phagocytosis is triggered by the activation of cell surface receptors, linked to the phagocytic machinery

-cellular extensions termed ‘pseudopods’ engulf the particle. The phagocytic process is driven by actin, the small
GTPase Rho and specific phosphinositides

- phagosomes are internalised into the cell where they fuse with lysosomes, forming phagolysosomes
 Macropinocytosis

- macropinocytosis refers to the generation of large endocytic vesicles (up to 5 μm

in diameter). This process is distinct from phagocytosis as it is independent
of the presence of particles, the membrane is not in close proximity to the
internalised molecules, and large amounts of non-specific fluids are
endocytosed

-it is regulated by activation of receptor tyrosine kinases, such as the

epidermal growth factor receptor (EGFR) and platelet-derived growth factor
receptor (PDGFR), leading to a global increase in actin polymerisation at the
cell surface

-this results in plasma membrane ruffling and the formation of lamellipodia or

blebs over the entire surface of the cell
10 µm

- the molecular mechanism is not fully understood, although the kinase PAK1
and the C-terminal-binding protein-1 / brefeldinA-ADP ribosylated substrate
(CtBP1/BARS) proteins are essential for closure of the macropinocytic cup

PAK1 and CtBP1/BARS

 Caveolin-dependent
endocytosis
- caveolae are present in most cell types, appearing as deeply invaginated flask-shaped structures

- they are believed to form from plasma membrane microdomains (lipid rafts), areas enriched in
cholesterol,
glycosphingolipids and GPI-anchored proteins

-their major structural proteins are caveolins, unusual integral membrane proteins that insert a
hydrophobic loop into the cytoplasmic leaflet of the plasma membrane

- the large GTPase dynamin is needed for caveolae to pinch off from the plasma membrane
 Caveolin-dependent
endocytosis
- caveolae pinch off and deliver their cargo to an endosome-like compartment termed the caveosome

- unlike conventional coat proteins, caveolin remains associated with the membrane

-caveolin-dependent pathways are known to be utilised by toxins, such as cholera toxin, and viruses,
such as SV40 and papilloma virus

Parton and Simons (2007) Nat Rev Mol Cell Biol

8:185-194
 Clathrin-dependent
endocytosis
- clathrin-dependent (receptor-mediated) endocytosis is the best characterised internalisation pathway

- the clathrin coat protein is found at the TGN, endosomes and plasma membrane

- at the plasma membrane, discrete areas of clathrin accumulation called ‘clathrin-coated pits’ can beobserved

- clathrin-coated pits invaginate cargo, and form clathrin-coated vesicles (CCVs)

-in cultured animal cells ca. 2,500 CCVs leave the plasma membrane every minute, and it has been
estimated that within 1 hour an area the size of entire plasma membrane can be internalised

EM has revealed that clathrin-coated pits and

CCVs have a basket-like appearance,
resulting from the geometry of how the clathrin
subunits assemble
 Clathrin-dependent
endocytosis
- cargo is selected by binding to specific cell surface cargo receptors

-these receptors can become clustered as a result of their cytoplasmic tails binding to adaptor proteins
(adaptins). At the cell surface, the main adaptin complex is AP-2

- the adaptors in turn recruit clathrin from the cytoplasm

-this cycle of clathrin recruitment continues until the membrane is deformed into a vesicle shape completely
coated with clathrin

-the CCV buds away from the plasma membrane, the clathrin coat is rapidly lost, and the naked transport
vesicle fuses with an early endosome
 Clathrin-dependent
endocytosis
- CCVs have the appearance of regular polyhedral cages (pentagons and hexagons)

- they can spontaneously assemble in vitro, without the presence of membranes or cargo

-each clathrin subunit consists of three large and three small protein chains, that together form a three-
legged structure called a‘triskelion’

-in order for a clathrin-coated pit to become a CCV, the large GTPase dynamin is required to
bind to the neck of the forming vesicle

-dynamin recruits other proteins to the neck which eventually destabilise the interacting lipid bilayers
until they fuse. Blocking dynamin function prevents release of the CCV
Role of
phosphoinositides
 Cargo selection in clathrin-dependent
endocytosis
-AP-2 has a low affinity for membranes, but this is increased by binding to PI(4,5)P2 and
transmembrane cargo, causing a local nucleation to be initiated

-AP-2 subunits recognise internalisation motifs on cargo, in particular YXXφ, [DE]XXXL[LI],

FXNPXY, and polyubiquitination

- as clathrin starts to be assembled, CLASPs (clathrin-associated sorting proteins) such as epsins are
also recruited

-as assembly progresses the AP-2-mediated interactions become tighter, restricting the mobility of
the cargo laterally in the membrane

Traub (2009). Nature Reviews Molecular Cell Biology

10:583-595.
Cargo selection in clathrin-dependent
endocytosis

Traub (2009). Nature Reviews Molecular Cell Biology

10:583-595.
 Membrane curvature in clathrin-dependent
endocytosis - a variety of additional machinery molecules are
required to sculpture the membrane at various points in
the clathrin- dependent endocytosis cycle

- in the early stages of pit formation, following

PI(4,5)P2
binding, ‘wedge’-shaped molecules such as epsin
promote membrane curvature by insertion of a sort
amphipathic helix into the cytoplasmic leaflet of the
membrane,
- proteins containing BAR domains , such as
amphiphysin, bind to membranes that are already
curved, and help to stabilise the structure

- clathrin itself also contributes to membrane curvature

-the GTPase dynamin, and actin and actin-binding

proteins also contribute to the final stages of neck
constriction and bud formation

Doherty and McMahon (2007). Mechanisms of

endocytosis. Ann. Rev. Biochem. 78:857-902.
Scission and coat dissociation in clathrin-dependent
endocytosis
- the large GTPase dynamin is rapidly recruited to coated vesicles as AP-2 and clathrin
assembly nears completion. Mutant forms of dynamin, or addition of GTPγS, result in the
production of elongated necks with the vesicle unable to detach from the membrane

- it is thought that hydrolysis of GTP by dynamin causes the

GTPase to contract and twist. This contraction brings the
opposing membranes into close proximity until ultimately they
fuse

- recently a role for actin in the scission event has also been proposed

-one myosin motor may act to pull the dynamin ring towards the
membrane, while another pulls the vesicle towards the cytoplasm

- the phophatase synaptojanin and the kinase GAK (cyclin G associated

protein
kinase) are also found to be associated with the emerging vesicle

-synaptojanin converts PI(4,5)P2 to PI(4)P, likely reducing the interaction of

AP-2 with the membrane, allowing uncoating to rapidly follow

Ungewickell and Hinrichsen (2007). Endocytosis: clathrin-mediated

 Key take home
point

Pathways from the cell surface to internal

membranes are highly specialised and regulated
to ensure that the cell can accurately control the
type and amount of molecules it needs to
internalise