Transcript Document

Dynamic networks
&
clathrin-mediated
endocytosis
Eva Schmid
(LMB Cambridge)
Marijn Ford
Gerrit Praefcke
(now at UC Davis)
(now at Cologne)
What is a Hub?
Are they static?
Why have them?
fidelity are
At the synapse speed and fidelity
important to ensure the quantal nature
and reliability of synaptic vesicle
exocytosis
What is
Endo
Fidelity?
Exo
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)
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)
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
The endocytic interactome
Accessory Proteins
(over 20 different
proteins bind to the AP2
a-appendage)
Hubs
The AP2 hub binds to accessory proteins
via it appendage domains
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
Endocytic accessory proteins have a similar overall structure
Structured
domains
•Protein:protein
interaction domains
•with no obvious tertiary
structure
Motif
domains
Epsin
•Contain multiple motifs,
short amino acid
sequences,
•Please don’t call them
‘unstructured domains’ as they
may have some secondary
structure!!
Structured
domains
Motif
domains
AP2 a-motifs
Eps15 affinity for the a-appendage
3 x EH
UIM
Eps15 Motif
Domain
Eps15 affinity for the a-appendage
3 x EH
UIM
contains 15 repeats
of the sequence DPF
Eps15 affinity for the a-appendage
UIM
+
2-3 sites of 16mM
1 site of 20nM
So not all 15 motifs are available for simultaneous interactions
Eps15 affinity for the a-appendage
2-3 sites of 16mM
16mM
1 site of 20nM
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
16mM
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
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-appendage
‘structural cooperativity’ in motif binding
This low structural stability means that these motif domains
can search a wide range of space for potential ligands
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
Motif:domain 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
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.
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).
Building the network around AP2….
There are 4 potential ligand interaction sites on each
AP2 complex
Thus 4 potential ligand interaction sites on each AP2
complex. Does this make it a HUB?
Thus 4 potential ligand interaction sites on each AP2
complex. Does this make it a HUB? No
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
Changing hubs gives directionality
Recruitment of AP2
to membrane
and concentration
AP2
in solution
Clathrin polymerisation
The clathrin hub
Miele et al 2004
Ter Haar et al 2002
Amph WxxW
b3 adaptor hinge
LLDLD
Changing hubs gives directionality
Recruitment of AP2
to membrane
and concentration
Clathrin polymerisation
AP2
in solution
•Only on self-polymerisation does clathrin
become a hub
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
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
Clathrin binding to the b -appendage displaces
ligands, pushing accessory proteins to the edge of a
clathrin-coated pit (appendage assembly zones)
Clathrin binding to the b -appendage displaces
ligands, pushing accessory proteins to the edge of a
clathrin-coated pit (appendage assembly zones)
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
How clathrin-coated pits mature…
affinity
avidity
matricity
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
A Network view of clathrin-coated vesicle formation
A
AP2 adaptors sense
lipids, cargo,
accessory proteins
and other cargo adaptors
AP2
B
Building the cage: AP2
network hub is stabilized
through crosslinking by
accessory proteins
AP2
C
Clathrin is recruited and
polymerisation stabilises
the forming vesicle.
AP2 loses its position as a hub.
Clathrin is the new
organising hub
AP2
D
Dynamin and other late
interacting partners (like
uncoating factors) start to
function
The point of no return.
AP2
E
Energy is used to reprime the system for a
new start.
AP2
Changing hubs gives directionality
Recruitment of AP2
to membrane
and concentration
Clathrin polymerisation
AP2
in solution
•Only on self-polymerisation does clathrin
become a hub
•Note: in a clathrin-coated pit one has a snapshot of the network at several different stages
AP2 hubs and clathrin hubs co-exist at the
same time, but spatially separated
In a coated-pit there may even be the beginning
stages of uncoating, as the lipid phosphatase
begins to work under the clathrin lattice
This means that fluorescent imaging will
frequently not have the resolution to deduce the
time dependence of recruitment
But… we can deduce this information from
the path-length in the network……
Early and late events can be predicted…
1
2
Vesicle scission
3
3’
Cage formation
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
T
i
m
e
This view maintains that:
Overexpression of a pathway hub will have little phenotype
Underexpression 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