dynamic networks & clathrin-mediated endocytosis

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