Dynamic networks

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Dynamic networks clathrin-mediated
endocytosis
Eva Schmid (LMB Cambridge)
Gerrit Praefcke (now at Cologne)
Marijn Ford (now at UC Davis)
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What is a Hub? Are they static? Why have them?
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At the synapse speed and fidelity are important
to ensure the quantal nature and reliability of
synaptic vesicle exocytosis
speed
fidelity

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Fidelity?
What is
Exo
Endo
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Clathrin-mediated endocytosis
The overall process is a series of linear steps
but at the same time it is a series of
simultaneous micro-reactions (e.g. cargo
recruitment, membrane invagination and coat
assembly occurring in parallel)
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Clathrin-mediated endocytosis
The overall process is a series of linear steps
but at the same time it is a series of
simultaneous micro-reactions (e.g. cargo
recruitment, membrane invagination and coat
assembly occurring in parallel)
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Clathrin-mediated endocytosis
The overall process is a series of linear steps
but at the same time it is a series of
simultaneous micro-reactions (e.g. cargo
recruitment, membrane invagination and coat
assembly occurring in parallel) Involving
clathrin, adaptors (AP2) and at least 20
different accessory proteins
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The endocytic interactome
Accessory Proteins
(over 20 different proteins bind to the AP2
a-appendage)
Hubs
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The AP2 hub binds to accessory proteins via it
appendage domains
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The a-appendage two independent binding sites
W840
Top Site
Peptide containing an FxDxF motif Binds with an
affinity of 4.6mM
F740
Side Site
Peptide containing a WVxF motif Binds with an
affinity of 0.7mM
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Endocytic accessory proteins have a similar
overall structure
Structured domains

• Proteinprotein interaction domains
• with no obvious tertiary structure
• Contain multiple motifs, short amino acid
  sequences,
• Please dont call them unstructured domains as
  they may have some secondary structure!!

Motif domains
Epsin
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Motif domains
Structured domains
AP2 a-motifs
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Eps15 affinity for the a-appendage
Eps15 Motif Domain
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Eps15 affinity for the a-appendage
contains 15 repeats of the sequence DPF
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Eps15 affinity for the a-appendage

2-3 sites of 16mM
1 site of 20nM
So not all 15 motifs are available for
simultaneous interactions
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Eps15 affinity for the a-appendage
2-3 sites of 16mM
1 site of 20nM
16mM
16mM
16mM
20nM

• From mutagenesis we know that the 20nM affinity
  is due to occupation of both top and side sites
  of one appendage
• Thus this is a novel way to gain high affinity
  yet a readily reversible interaction ie. 2
  linear peptides linked by a flexible linker

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Eps15 affinity for the a-appendage
2-3 sites of 16mM
1 site of 20nM

• Eps15 with its simultaneous interactions with 4
  appendage domains could help to cluster AP2s at
  sites of endocytosis

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Motif domains are not unstructured and linear
But neither are they stable globular
domains. They are designed to package motifs in
an efficient manner, such that when one motif is
occupied then further motifs are exposed
Motif
Motif domain
a-appen- dage
structural cooperativity in motif binding
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This low structural stability means that these
motif domains can search a wide range of space
for potential ligands
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This low structural stability means that these
motif domains can search a wide range of space
for potential ligands
Like a fishing line with lots of hooks
But for entropic and statistical reasons the
domain will prefer a more compact fold And thus
the hooks will gather ligands back to the core
folded domains
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Motifdomain interactions

• A novel way to gain relatively high affinity and
  yet reversibility
• Give rise to dynamic instability (a necessary
  characteristic of many cellular processes)
• Allow cross-linking/multimerisation of binding
  targets
• Efficient packaging of many different
  interactions surfaces
• Multiple interactions that filter noise
• A way to search space and draw ligands to a point

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The network behaviour makes sense..

• Clathrin is an organising hub, not a protein
  recruitment hub. This ensures that empty clathrin
  cages do not form in the absence of membranes and
  cargo
• AP2 does not self assemble, and only weakly binds
  to cargo. This ensures that cargo recruitment,
  membrane bending and polymerisation are tightly
  coupled.

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Properties of endocytic and other biological
networks
(feed forward and competitive loops)
Noise reduction Low affinity interactions ensure
that processes are only activated on coincidence
of several signals Information processing The
multimeric state of the AP2 hub allows it to bind
multiple ligands according to their relative
affinities and concentrations. Thus the hub
integrates information. The competition between
AP2 and clathrin also means that there is a
sensing of the commitment along the endocytic
pathway (the process gestalts).
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Building the network around AP2.
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There are 4 potential ligand interaction sites on
each AP2 complex
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Thus 4 potential ligand interaction sites on each
AP2 complex. Does this make it a HUB?
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Thus 4 potential ligand interaction sites on each
AP2 complex. Does this make it a HUB? No
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It is the concentration of AP2s on the membrane
that gives it the ability to bind multiple
partners according to affinities and
concentrations
AP2 hub zone
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Changing hubs gives directionality
Recruitment of AP2 to membrane and concentration
Clathrin polymerisation
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The clathrin hub
Miele et al 2004 Ter Haar et al 2002
b3 adaptor hinge LLDLD
Amph WxxW
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Changing hubs gives directionality
Recruitment of AP2 to membrane and concentration
Clathrin polymerisation

• Only on self-polymerisation does clathrin become
  a hub

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Clathrin binding to the b -appendage displaces
ligands, pushing accessory proteins to the edge
of a clathrin-coated pit (appendage assembly
zones)
Clathrin
b -appendage
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Clathrin binding to the b -appendage displaces
ligands, pushing accessory proteins to the edge
of a clathrin-coated pit (appendage assembly
zones)
Clathrin terminal domain
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Clathrin binding to the b -appendage displaces
ligands, pushing accessory proteins to the edge
of a clathrin-coated pit (appendage assembly
zones)
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Clathrin binding to the b -appendage displaces
ligands, pushing accessory proteins to the edge
of a clathrin-coated pit (appendage assembly
zones)
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How clathrin-coated pits mature
affinity
avidity
matricity

• Sequential displacement of core and accessory
  proteins (affinity matures to avidity matures to
  matricity)
• The process is pulled forward from the end

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How clathrin-coated pits mature
affinity
avidity
matricity
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How clathrin-coated pits mature
ATP GTP

• Sequential displacement of core and accessory
  proteins (affinity matures to avidity matures to
  matricity)
• The process is pulled forward from the end

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A Network view of clathrin-coated vesicle
formation
A AP2 adaptors sense lipids, cargo, accessory
proteins and other cargo adaptors
AP2
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B Building the cage AP2 network hub is
stabilized through crosslinking by accessory
proteins
AP2
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C Clathrin is recruited and polymerisation
stabilises the forming vesicle. AP2 loses its
position as a hub. Clathrin is the
new organising hub
AP2
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D Dynamin and other late interacting partners
(like uncoating factors) start to function The
point of no return.
AP2
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E Energy is used to re-prime the system for a new
start.
AP2
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Changing hubs gives directionality
Recruitment of AP2 to membrane and concentration
Clathrin polymerisation

• Only on self-polymerisation does clathrin become
  a hub
• Note in a clathrin-coated pit one has a
  snap-shot of the network at several different
  stages

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AP2 hubs and clathrin hubs co-exist at the same
time, but spatially separated
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In a coated-pit there may even be the beginning
stages of uncoating, as the lipid phosphatase
begins to work under the clathrin lattice
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This means that fluorescent imaging will
frequently not have the resolution to deduce the
time dependence of recruitment
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But we can deduce this information from the
path-length in the network
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Early and late events can be predicted
Time
1
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Cage formation
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Vesicle scission
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Uncoating and repriming of molecules

• A short path-length gives an immediate response
• To put a time delay in the response an additional
  interaction step is added

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This view maintains that Overexpression of a
pathway hub will have little phenotype Underexpre
ssion of a pathway hub will have a major
phenotype Overexpression of an accessory node
will have a major phenotype Underexpression of
an accessory node will have little phenotype
Hubs