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Dynamic networks &
clathrin-mediated endocytosis
Gerrit Praefcke (now at Cologne)
Marijn Ford(now at UC Davis)
Eva Schmid(LMB Cambridge)
What is a Hub? Are they static?
Why have them?
At the synapse speed and fidelity are important to ensure the quantal nature
and reliability of synaptic vesicle exocytosis
speed fidelity
ExoEndo
Fidelity?What is
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) andat least 20 different accessory proteins
The endocytic interactome
Hubs
Accessory Proteins (over 20 different proteins bind to the AP2 -appendage)
The AP2 hub binds to accessory proteinsvia it appendage domains
The -appendage:two independent binding sites
Top Site
Side Site
W840
F740
Peptide containing an FxDxF motifBinds with an affinity of 4.6M
Peptide containing a WVxF motifBinds with an affinity of 0.7M
Motifdomains
Structureddomains
•Protein:protein interaction domains
•with no obvious tertiary structure
•Contain multiple motifs, short amino acid sequences,
•Please don’t call them ‘unstructured domains’ as they may have some secondary structure!!
Endocytic accessory proteins have a similar overall structure
Epsin
Motifdomains
Structureddomains
AP2 -motifs
Eps15 affinity for the -appendage
3 x EH UIM
Eps15 Motif Domain
Eps15 affinity for the -appendage
3 x EH UIM
contains 15 repeats of the sequence DPF
Eps15 affinity for the -appendage
3 x EH UIM
So not all 15 motifs are available for simultaneous interactions
+
1 site of 20nM
2-3 sites of 16M
Eps15 affinity for the -appendage
1 site of 20nM
2-3 sites of 16M
•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
20nM
16M 16M 16M
Eps15 affinity for the -appendage
1 site of 20nM
2-3 sites of 16M
•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
‘structural cooperativity’ in motif binding
Motif domain -appen- dage
Motif
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 foldAnd thus the hooks will gather ligands back to the core folded domains
•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
Motif:domain interactions
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
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).
(feed forward and competitive loops)
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
AP2in solution
Recruitment of AP2to membrane
and concentration Clathrin polymerisation
The clathrin hub
Amph WxxW 3 adaptor hinge LLDLD
Miele et al 2004Ter Haar et al 2002
Changing hubs gives directionality
AP2in solution
Recruitment of AP2to membrane
and concentration Clathrin polymerisation
•Only on self-polymerisation does clathrin become a hub
Clathrin binding to the -appendage displaces ligands, pushing accessory proteins to the edge of a clathrin-coated pit (appendage assembly zones)
Clathrin
-appendage
Clathrin binding to the -appendage displaces ligands, pushing accessory proteins to the edge of a clathrin-coated pit (appendage assembly zones)
Clathrin terminal domain
Clathrin binding to the -appendage displaces ligands, pushing accessory proteins to the edge of a clathrin-coated pit (appendage assembly zones)
Clathrin binding to the -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…
•Sequential displacement of core and accessory proteins (affinity matures to avidity matures to matricity)
•The process is pulled forward from the end
ATPGTP
AP2
AAP2 adaptors sense
lipids, cargo,accessory proteins
and other cargo adaptors
A Network view of clathrin-coated vesicle formation
AP2
BBuilding the cage: AP2
network hub is stabilizedthrough crosslinking by
accessory proteins
AP2
CClathrin is recruited andpolymerisation stabilises
the forming vesicle. AP2 loses its position as a hub.
Clathrin is the neworganising hub
AP2
DDynamin and other late interacting partners (like uncoating factors) start to function
The point of no return.
AP2
EEnergy is used to re-prime the system for a new start.
Changing hubs gives directionality
AP2in solution
Recruitment of AP2to 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
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……
•A short path-length gives an immediate response•To put a time delay in the response an additional interaction step is added
Cage formation
Vesicle scissionUncoating and repriming of molecules
Time
Early and late events can be predicted…
1
2
3 3’
This view maintains that:Overexpression of a pathway hub will have little phenotypeUnderexpression of a pathway hub will have a major phenotype
Overexpression of an accessory node will have a major phenotypeUnderexpression of an accessory node will have little phenotype
Hubs
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